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AMRI - Drug Discovery Solutions

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About AMRI - Drug Discovery Solutions

Divison of: AMRI

AMRI is a global contract research and manufacturing organization that has been working with the pharmaceutical and biotechnology industries to improve patient outcomes and quality of life. With locations in North America, Europe and Asia, we provide a complete suite of capabilities in drug discovery,... Show more »

AMRI is a global contract research and manufacturing organization that has been working with the pharmaceutical and biotechnology industries to improve patient outcomes and quality of life. With locations in North America, Europe and Asia, we provide a complete suite of capabilities in drug discovery, development and manufacturing. We’ve got it down to an exact science.

We care about the quality and success of your projects as if they were our own. No matter the size of your project or the complexity of the challenge, our experts pinpoint and execute the precise solution to hit your target. From molecule to molecule, batch to batch, your product will benefit from our ingenuity.

Drug Discovery Solutions

Capitalize on the value of integrated drug discovery centers of excellence that combine scientific expertise and leading-edge technology. We offer comprehensive discovery biology, synthetic and medicinal chemistry, DMPK and bioanalytical services for successful hit-to-lead-to-candidate selection. You can also leverage our novel business models, such as global insourcing, to meet your capacity and timeline demands. Our approach ensures a seamless transition across every step of your project.

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Our Services (22)


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ADME/DMPK Studies

Drug Metabolism and Pharmacokinetics
Price on request

AMRI’s DMPK scientists are highly experienced in providing the following standard assays to characterize and prioritize hits and support their optimization to preclinical and clinical candidates.

Aqueous solubility, kinetic
Incubation of test compound introduced into an aqueous buffer (e.g. PBS, saline, simulated gastric or... Show more »

AMRI’s DMPK scientists are highly experienced in providing the following standard assays to characterize and prioritize hits and support their optimization to preclinical and clinical candidates.

Aqueous solubility, kinetic
Incubation of test compound introduced into an aqueous buffer (e.g. PBS, saline, simulated gastric or intestinal fluid, etc.) as a concentrated solution in DMSO for 1 hour, followed by measurement of dissolved concentration via LC/MS/MS or UV/Vis absorbance detection against a calibration curve. Final DMSO concentration in the solubility assay medium is 1%.

Aqueous solubility, shake flask
Overnight incubation of test compound powder in an aqueous buffer (e.g. PBS, saline, simulated gastric or intestinal fluid, etc.), followed by measurement of dissolved concentration via LC/MS/MS or UV/Vis absorbance detection against a calibration curve.

LogP and LogD7.4 determination
Partition coefficient of test compound measured in water – octanol system. The assay is conducted in a 96-well plate format using an automated workstation and chemiluminescent nitrogen detection.

Chemical stability in aqueous media
Incubation of test compound in an aqueous buffer (e.g. PBS, saline, simulated gastric or intestinal fluid, etc.) at ambient temperature, followed by LC/MS analysis to measure remaining compound at specified time points (8 time points over 72 h).

Metabolic clearance
Incubation of test compound with mammalian liver microsomes, S9 fractions, or cryopreserved hepatocytes in the presence of cofactor regeneration system (as appropriate), followed by LC/MS analysis to measure remaining test compound at specified time points (6 time points). Includes calculation of intrinsic clearance, half-life, and hepatic extraction ratio. The assay is also available in a single time point format.

Blood and plasma stability
Incubation of test compound in fresh whole blood or plasma, followed by LC/MS analysis to measure remaining compound at specified time points (5 time points over 90 min).

CYP inhibition, LC/MS detection
Incubation of test compound with human liver microsomes in the presence of CYP-specific probe substrates, followed by LC/MS/MS analysis to measure percent inhibition of corresponding CYP isoforms. IC50 values are determined from percent inhibition vs test compound concentration curves, using eight concentrations of the test compound. CYP isoforms include 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. CYP3A4 is tested using two probe substrates (testosterone and midazolam).

CYP time-dependent inhibition, LC/MS detection
Incubation of test compound with human liver microsomes in the presence and in the absence of NADPH for a specified period of time. After the preincubation, appropriate CYP isoform-specific probe substrates and NADPH (for preincubations without NADPH) are added to the incubation mixture, and the IC50 values are measured in both (+)NADPH and (-) NADPH preincubations. The IC50 shift is calculated as the ratio of IC50 values measured in the presence and in the absence of NADPH in the preincubation. CYP isoforms include 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. CYP3A4 is tested using two probe substrates (testosterone and midazolam).

CYP time-dependent inhibition, kinact/Ki determination
Incubation of test compound with human liver microsomes in the presence of NADPH with sample collection from the incubation mixture at 6 time points. The remaining activity of individual CYP enzymes is measured using isoform-specific probe substrates with LC/MS/MS detection to determine the rate constant of CYP inactivation (kobs). The experiment is repeated at 6 concentrations of the test compound to measure the dependence of kobs on inactivator concentration. Inhibition constants Ki (equilibrium binding) and kinact (irreversible CYP inactivation) are determined from the kinetic data using a non-linear regression analysis. CYP isoforms include 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. CYP3A4 is tested using two probe substrates (testosterone and midazolam).

CYP phenotyping using specific CYP inhibitors
Incubation of test compound with microsomes and cofactor regeneration system, both in the presence and absence of specific chemical inhibitors for individual CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4), followed by LC/MS/MS analysis to measure remaining test compound. CYP isoforms responsible for metabolism of test compound are identified from percent of metabolized test compound in the presence of corresponding specific CYP inhibitors.

CYP phenotyping using recombinant human CYP isoforms
Incubation of test compound with a panel of seven major recombinant human CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and cofactor regeneration system, followed by LC/MS/MS analysis to measure metabolism of test compound. CYP isoforms responsible for metabolism of test compound are identified from percent of metabolized test compound in the presence of corresponding isoforms.

CYP induction
Incubation of test compound with cryopreserved plateable human hepatocytes from three individual donors. Upon incubation, CYP1A2, CYP2B6, and CYP3A4 enzyme activities are measured using specific probe substrates with LC/MS/MS detection to determine increase in enzyme activity compared to the vehicle control (fold induction).

Hepatotoxicity, HepG2 cells
Incubation of HepG2 cells at 10 different concentrations of the test compound for 48 h and 72 h at 37⁰C, followed by cell lysis and measurement of ATP levels using luminescence detection.

Hepatotoxicity, human hepatocytes
Incubation of plateable human hepatocytes at eight different concentrations of the test compound for 24 h at 37 oC, followed by cell lysis and measurement of ATP levels using luminescence detection.

hErg blockade
Automated patch-clamp technique to measure hERG conductance using HEK293 cells stably transfected with the human ERG gene, at 5 concentrations of test compound.

Mini-Ames fluctuation assay
Mutagenicity potential evaluation using four Salmonella typhimurium bacterial strains, including TA98, TA100, TA1535, and TA1537, in the presence and absence of S9 liver fraction from Aroclor-induced rat.

Ames test
Mutagenicity potential evaluation in agar plates using Salmonella typhimurium bacterial strains, including TA98, TA100, TA102, TA1535, TA1537, and Escherichia coli WP2, in the presence and absence of S9 liver fraction from Aroclor-induced rat.

Formation of reactive metabolites
Incubation of the test compound with liver microsomes in the presence of glutathione or KCN as a trapping reagent. After metabolic transformation of the test compound in the presence of a trapping reagent, formation of corresponding conjugates with possible reactive metabolites is evaluated by LC/MS/MS analysis.

MDR1-MDCK and Caco2 permeability
Passive and active transport evaluation across a monolayer of MDR1-MDCK or Caco2 cells in two directions (apical to basolateral and reverse). Permeability and efflux ratio are determined based on measurement of compound concentration in receiving compartment by LC/MS analysis. Ability of test compound to act as a P-gp inhibitor or P-gp substrate can also be evaluated.

PAMPA permeability
Passive transport evaluation across membrane impregnated with phospholipid. Permeability (Pe) is determined based on measurement of relative compound concentrations in donor and receiving compartments via UV/Vis absorbance or LC/MS analysis.

Plasma protein binding, equilibrium dialysis
Equilibration of test compound dissolved in plasma against buffer across semi-permeable membrane, followed by LC/MS analysis to measure compound concentrations in both compartments.

Blood/plasma partitioning
Incubation of test compound in blood, followed by removal of red blood cells by centrifugation. Concentration of test compound in plasma is measured by LC/MS analysis, and the RBC-to-plasma ratio (KRBC/PL) is calculated based on comparison with compound concentration measured in a separate reference incubation in spiked plasma.

Metabolite profiling/species comparison
Identification of metabolites of test compounds in incubations with liver microsomes, S9 fractions, or cryopreserved hepatocytes. Includes required method development and determination of metabolite molecular weights and relative abundance of metabolites based on HPLC/UV and/or HPLC/MS/MS peak areas, and structural characterization of metabolites by high-resolution MS/MS.

Antibody-Drug Conjugate Analysis
• Characterization of Antibody-Drug Conjugate (ADC) using Sciex TripleTOF 6600 System.
• Determination of drug-to-antibody ratio (DAR).
• Analysis of both intact and reduced ADC.
• For reduced ADC analysis, ADC is incubated with DTT at 37 °C for 30 minutes.

Discovery Bioanalytical Services
• Express, standard, and advanced LC/MS/MS bioanalytical methods designed to support specific stages of drug discovery.
• Measurement of drug concentrations in complex biological matrices, including plasma, whole blood, urine, bile, and various organs and tissues.
• Highly sensitive analysis (as low as pg/mL levels) using top of the line instrumentation.
• Pharmacokinetic modeling and calculation of pharmacokinetic parameters.
• Preformulation development for animal dosing.
• In vivo animal dosing and sample collection available via our partners.

Synthesis of Metabolites
• Screening of wide variety of biocatalytic systems to identify the most efficient way to synthesize target metabolites. Biocatalysts include mammalian liver microsomes and S9 fractions, human recombinant CYP isoforms, and microorganisms from a proprietary AMRI collection that mimic mammalian metabolism.
• Streamlined testing of experimental conditions to achieve the highest synthetic yield of target metabolites, including concentrations of reaction components, pH, temperature, reaction time, and other relevant reaction parameters.
• Optimization of volumetric productivity of metabolite synthesis to achieve the most cost-effective production of target metabolites.
• Development of metabolite isolation and purification protocols, typically using solid phase extraction and preparative HPLC.

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Biochemistry & Molecular Biology

Price on request

AMRI provides a suite of molecular biology-based discovery services to provide support to clients who need novel tools developed for their research and development projects. These can be offered as stand-alone services or as part of more comprehensive research projects. Services include the routine generation and testing of... Show more »

AMRI provides a suite of molecular biology-based discovery services to provide support to clients who need novel tools developed for their research and development projects. These can be offered as stand-alone services or as part of more comprehensive research projects. Services include the routine generation and testing of expression constructs including site-directed mutagenesis along with gene knock-down and knock-in studies using standard transfection or viral-mediated transduction of expression vectors, reporter constructs, siRNA, shRNA or CRISPR/Cas9 targeting elements.

The following is a selection of the services provided:

• Molecular Cloning

• Mammalian Cell Expression

• Recombinant adenoviral, Lentiviral and Retroviral Production and Transduction

• Cell Line Development

• siRNA and shRNA-mediated Knock-down Studies

• CRISPR/Cas9-mediated Knock-out or Knock-in Studies

Molecular Cloning

• Generate expression constructs for mammalian, insect, yeast and E. coli;
• Subclone to different vector;
• Construct design with different tags;
• Site-directed mutagenesis.

Mammalian Cell Transfection

AMRI scientists can prepare expression vectors using standard recombinant biology techniques into vectors supplied by clients or use client expression vectors to use as templates for mutagenesis and transfection into mammalian cell lines for transient or stable expression. Positive transfections will be verified by cell sorting, Western blot or ELISA and can be validated for functional assays if needed. The lines can be shipped back to the client or used in-house for downstream functional assays or phenotyping using high content imaging studies.

Recombinant Adenoviral, Lentiviral and Retroviral Production and Transduction

AMRI scientists, working in a BSL2 certified laboratory, can prepare adenoviral, lentiviral and retroviral particles for research (non-GLP) studies. Vectors can be constructed or client vectors can be used to transfect into packaging lines and particles will be harvested for downstream purification, concentration and titering. The purified and titered viral particles can be used in-house for cell line development and model creation using immortalized cell lines or primary cells of choice. The particles can also be shipped back to the client for further study.
Cell Line Development

AMRI provides cell line engineering in the form of traditional stable cell line generation and CRISPR/Cas9-mediated knock-out or knock-in lines. Parental cell lines can be generated from cells obtained from repositories or from client internal stocks. Standard lipofection, electroporation or PEI can be used to introduce the expression or targeting vectors to the line of choice. Adenoviral, lentiviral or retroviral expression systems can also be used. Positive transfections will be verified by cell sorting, Western blot or ELISA and can be validated for functional assays if needed.

siRNA or shRNA-mediated Knock-down Studies

AMRI can help design, validate and test siRNA or shRNA strategies ranging from single gene knock-down approaches to siRNA or shRNA libraries for screening the effects of gene knock-down on cellular processes. Constructs can be delivered using standard transfection, electroporation or viral-mediated transduction. The knock-down will be validated using mRNA expression measurements, western blots, ELISA or immunofluorescence and the phenotypes resulting from the knockdown can be examined using a full array of high-throughput and high-content phenotypic readouts.

CRISPR/Cas9-mediated Knock-out or Knock-in Studies

AMRI can help design, validate and test CRISPR/Cas9 strategies ranging from single gene knock-down approaches to CRISPR libraries for screening the effects of gene knock-down on cellular processes. Cas9 expressing lines can be generated to help facilitate the process. A variety delivery methods can be tested to ensure optimal delivery of the targeting constructs to immortalized cell lines or primary cells. Next Generation Sequencing will be used to confirm the gene editing at the genomic level and the expression level, localization of function of the edited gene product will be validated using mRNA expression measurements, western blots, ELISA or immunofluorescence and the phenotypes resulting from the editing can be examined using a full array of high-throughput and high-content phenotypic readouts.

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Computer Aided Drug Design

Price on request

Discovery consulting services:
Experienced drug discovery scientists with extensive multidisciplinary backgrounds can provide consulting services to assist clients in selection of chemical starting points, screening and other critical decision points as new projects are initiated.

Virtual Screening and... Show more »

Discovery consulting services:
Experienced drug discovery scientists with extensive multidisciplinary backgrounds can provide consulting services to assist clients in selection of chemical starting points, screening and other critical decision points as new projects are initiated.

Virtual Screening and Docking:
Structure-guided docking and scoring to identify chemical substances for purchase and testing. Fixed fees on simple docking and selection. Unique approaches to selection of compounds ensures that chemical space is adequately covered and that new chemical matter for a target is found.

Protein Modeling:
Development of homology models from crystal structures and active site models emphasizing the protein-ligand interactions. New methods have been developed to simplify modeling of protein multimeric complexes.

Binding Site Analysis
Evaluation and prioritization of protein binding sites based on druggability. Analysis of binding site interactions and solvation for guiding ligand design. Identification of transient/induced fit subpockets for novel ligand design strategies.

Compound Selectivity
Virtual selectivity panels for ranking synthesis targets. Identification of relevant binding regions for increasing selectivity. Mutant-targeted design strategies.

Synthesis Strategies
Chemical series diversification that maintains validated ligand-protein interactions. Evaluation of client synthesis targets for prioritization of resources. Design of multiple chemical series (back-ups) via SAR directed scaffold hopping.

Fragment Libraries
Target-specific parallel synthesis libraries designed via virtual screening of fragment libraries. Client specific fragments, chemistries, and diversity filters can be applied.

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Compound Library Design

Price on request

We offer high-quality, cost-effective custom library synthesis services for both large, diverse libraries and focused libraries. Our capabilities and technologies include:

• Dedicated team specific for scaffold and template design that includes property filters using CADD resources, parallel production, parallel purification,... Show more »

We offer high-quality, cost-effective custom library synthesis services for both large, diverse libraries and focused libraries. Our capabilities and technologies include:

• Dedicated team specific for scaffold and template design that includes property filters using CADD resources, parallel production, parallel purification, analytics and computational support

• High-throughput purification via auto-prep, mass-triggered, high-performance liquid chromatography (APMS from Waters, six instruments) and high-throughput analytics using multichannel UPLC-MS (from Waters, three instruments), enabling us to analyze up to 10,000 compounds per month

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Compound Library Synthesis

Price on request

We offer high-quality, cost-effective custom library synthesis services for both large, diverse libraries and focused libraries. Our capabilities and technologies include:

• Dedicated team specific for scaffold and template design that includes property filters using CADD resources, parallel production, parallel purification,... Show more »

We offer high-quality, cost-effective custom library synthesis services for both large, diverse libraries and focused libraries. Our capabilities and technologies include:

• Dedicated team specific for scaffold and template design that includes property filters using CADD resources, parallel production, parallel purification, analytics and computational support

• High-throughput purification via auto-prep, mass-triggered, high-performance liquid chromatography (APMS fromWaters, six instruments) and high-throughput analytics using multichannel UPLC-MS (from Waters, three instruments), enabling us to analyze up to 10,000 compounds per month

• 8 years of experience in library synthesis projects. Successfully delivered several libraries with varying library size from 100 to 76000 compounds.

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Drug Discovery

Price on request

Capitalize on the value of integrated drug discovery centers of excellence that combine scientific expertise and leading-edge technology. We offer comprehensive discovery biology, synthetic and medicinal chemistry, DMPK and bioanalytical services for successful hit-to-lead-to-candidate selection. You can also leverage our novel... Show more »

Capitalize on the value of integrated drug discovery centers of excellence that combine scientific expertise and leading-edge technology. We offer comprehensive discovery biology, synthetic and medicinal chemistry, DMPK and bioanalytical services for successful hit-to-lead-to-candidate selection. You can also leverage our novel business models, such as global insourcing, to meet your capacity and timeline demands. Our approach ensures a seamless transition across every step of your project.

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High Content Screening

Price on request

AMRI offers a range of multiplexed imaging-based assays to simultaneously assess cell viability, physiology and morphology, using the PerkinElmer Opera Phenix automated confocal system and Incucyte Zoom live cell imaging system. Live cell imaging allows the impact of compounds on cellular physiology over time in 2D and 3D. Imaging... Show more »

AMRI offers a range of multiplexed imaging-based assays to simultaneously assess cell viability, physiology and morphology, using the PerkinElmer Opera Phenix automated confocal system and Incucyte Zoom live cell imaging system. Live cell imaging allows the impact of compounds on cellular physiology over time in 2D and 3D. Imaging assays can also be multiplexed with traditional endpoint measures of cell viability to further enrich the data obtained from screening campaigns. High content analysis also allows unbiased phenotypic screening to use for compound library characterization and drug repurposing studies. Combining high content screening with CRISPR or siRNA libraries links genotype to phenotype and allows the creation of models of mutation-based human disease.
Available readouts include:

• Cell viability
• Cell morphology
• Apoptosis
• Cell proliferation
• Cytoskeletal disruption
• Histone modifications
• Mitochondrial function
• Oxidative stress
• Neurite outgrowth
• T-cell mediated cytoxicity

Below are brief descriptions of specific high content screening assays provided by AMRI. The list is not comprehensive and we would be happy to provide a quote for your specific needs.

2D & 3D High Content Imaging and Analysis- Neurite Outgrowth

AMRI Scientists will isolate E18 primary rat neurons and plate in 384-well plates. Cultures of neuron enriched, astrocyte enriched or mixed cortical cultures are available. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. Neurite outgrowth will be determined in live cultures using an Incucyte Zoom or in fixed and immunostained cultures using a PerkinElmer Opera Phenix. Neurite features will be analyzed using validated routines and compared to DMSO treated and positive control treated wells.

2D High Content Assay- Cell Viability

AMRI Scientists will evaluate the effects of compounds on cell viability in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Viability will evaluated using fluorescent dyes that specifically label dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Proliferation

AMRI Scientists will evaluate the effects of compounds on cell proliferation in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Proliferation will be assessed by Edu incorporation and co-staining with an antibody against phosphorylated H3. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Apoptosis

AMRI Scientists will evaluate the effects of compounds on apoptosis in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Apoptosis will evaluated using fluorescent dyes that specifically mark apoptotoic cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Cytoskeletal Disruption

AMRI Scientists will evaluate the effects of compounds on the cytoskeleton in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Cytoskeletal disruption will evaluated using fluorescent dyes that bind to either tubulin or actin. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Histone Modifications

AMRI Scientists will evaluate the effects of compounds on histone phosphorylation and/or methylation in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Specific phosphorylation of histones will be assessed using an immunostaining approach with an antibody against phosphorylated or methylated histones. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Mitochondrial Morphology

AMRI Scientists will evaluate the effects of compounds on mitochondrial membrane potential in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Viability will evaluated using fluorescent dyes that specifically label dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Mitochondrial Morphology

AMRI Scientists will evaluate the effects of compounds on mitochondrial morphology in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. The effect of compound treatment on mitochondrial and cellular morphology will evaluated using fluorescent dyes that specifically label mitochondria and dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D High Content Assay- Oxidative Stress

AMRI Scientists will evaluate the effects of compounds on induction or suppression of oxidative stress in live cells using dyes that measure specific reactive oxygen and nitrogen species in various cellular compartments. These assays can also be multiplexed with other high content assays to measure a range of diverse cellular physiolgical states upon compound treatment. Cells will be plated in 384-well pates and cultured in the presence of test compounds for the desired amount of time. Kinetic measures are availabe to look for acute and chronic effects on oxidative stress. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

2D & 3D High Content Assay- T-cell Mediated Cytotoxicity

AMRI Scientists will evaluate the effects of compounds on induction or suppression of T-cell mediated cytotoxicity in mixed cultures containing cancer cell lines and human PBMCs or isolated T-cell populations. Cancer cells will be fluorescently labeled and grown as monolayers or spheroids prior to addition of immune cells. The viability of the cancer cells can be monitored in live imaging or fixed endpoint experiments. T-cell counting and phenotyping is available with conjugated antibodies against surface markers. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

High Throughput, High Content Screening

AMRI’s cell biologists, automation specialists and in vitro pharmacologists work closely together as teams and with their client to ensure appropriate design and execution of high-throughput, high content screening campaigns. Screens are conducted on PerkinElmer CellExplorer systems equipped with controlled environment sample and assay plate storage systems, acoustic dispensing (Labcyte Echo 555) and imaging on a PerkinElmer Opera Phenix high content imager. Data is analyzed and visualized by PerkinElmer Signals for Screening and SpotFire applications.

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High Throughput Screening (HTS)

Price on request

Our world-class high-throughput screening capabilities include compound and sample management, CellExplorer high-throughput robotic screening and various work station screening platforms, state-of-the-art liquid handling, diverse detection technologies (including MALDI-PharmaPulse MS for label free and Opera Phenix for phenotypic... Show more »

Our world-class high-throughput screening capabilities include compound and sample management, CellExplorer high-throughput robotic screening and various work station screening platforms, state-of-the-art liquid handling, diverse detection technologies (including MALDI-PharmaPulse MS for label free and Opera Phenix for phenotypic / high content screens) and advanced data informatics. AMRI’s innovative instrumentation and highly experienced assay development, automation and high throughput screening scientists deliver high efficiency for screening novel targets.

*384-well low volume and 1536-well formats are supported for increased screening speed, reduction of compound and reagent requirements, and significantly lower overall HTS costs.

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In vitro Pharmacology Studies

Price on request

AMRI’s scientific team includes specialists in receptor pharmacology, enzyme kinetics and compound testing schema design. Compound testing funnels and progression criteria are established to identify and aid the development of compounds with the desired mechanism of action (agonist / antagonist, inhibitor, allosteric modulator,... Show more »

AMRI’s scientific team includes specialists in receptor pharmacology, enzyme kinetics and compound testing schema design. Compound testing funnels and progression criteria are established to identify and aid the development of compounds with the desired mechanism of action (agonist / antagonist, inhibitor, allosteric modulator, etcetera), selectivity and ADMET-PK profile for their intended therapeutic purpose. Hit-to-Lead and Lead optimization campaigns are supported by rapid generation of SAR-quality data providing shorter, more effective design-synthesis-test cycles during the optimization process. Full transparency to assay performance statistics, raw data and processed data is provided.

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Medicinal Chemistry

Price on request

AMRI’s medicinal chemistry team has deep experience in the development and advancement of compounds from screening hits through preclinical development. Working on over 200 drug discovery programs, AMRI has generated more than 85 pre-clinical and clinical candidates, 240 issued patents and dozens of additional filings. The AMRI... Show more »

AMRI’s medicinal chemistry team has deep experience in the development and advancement of compounds from screening hits through preclinical development. Working on over 200 drug discovery programs, AMRI has generated more than 85 pre-clinical and clinical candidates, 240 issued patents and dozens of additional filings. The AMRI team has a wealth of lead discovery and lead development experience in a variety of therapeutic areas including infectious diseases, CNS, oncology, ophthalmic, pain, immunology/inflammation, metabolic and respiratory disorders. The depth of expertise, experience and capability provided by AMRI offers a significant, competitive advantage for our partners.

AMRI’s medicinal chemistry staff is well-versed in the synthesis of all chemical classes, including, but not limited to:

Asymmetric Synthesis, Alkaloids, Amino Acids, Carbocycles, Eicosanoid Analogs, Heterocycles, Natural Products, Opioids, Retinoic Acids, Rifalizil Analogs, Aromatics, Nucleosides, Peptides, Cyclic Peptides, Peptidomimetics, Porphyrins, Carbohydrates, Polysaccharides, Steroids, Tetracyclines, Vitamin D Analogs, Macrocycles

The laboratories at all AMRI sites are state-of-the-art, as is the equipment and analytical instrumentation The staff possess excellent chromatographic separation/purification skills, as well as a high level of expertise in the use of MS, LC/MS, and NMR. Clients often seek out AMRI for their most challenging chemistry because of AMRI chemists’ skilled design of synthetic routes and expert execution. Within the past five years, AMRI has invested in the Supercritical Fluid Chromatography (SFC) technology, with capabilities to conduct analytical, semi-preparative and preparative separations. The medicinal chemistry team at all sites has access to this technology for chiral analysis and preparative separations of enantiomers and/or diastereomers

Augmenting AMRI’s medicinal chemistry efforts, we provide computer-aided design, ADMET profiling, assay development and screening.

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Bioconjugation/Chemical Crosslinking

Price on request

A leading provider of protein expression, purification & analysis services to the biopharmaceutical industry, healthcare foundations and government institutions. AMRI has earned a reputation for excellent service and solving historically challenging protein expression and purification goals. AMRI can take your project directly... Show more »

A leading provider of protein expression, purification & analysis services to the biopharmaceutical industry, healthcare foundations and government institutions. AMRI has earned a reputation for excellent service and solving historically challenging protein expression and purification goals. AMRI can take your project directly from gene reference to final process and purified product with as much (or little) direction as you may wish to provide. Each project is customized to meet the specific client’s needs and can be initiated from gene synthesis, or we can express your target protein from a construct designed or supplied by you. At the conclusion of each project, we deliver a complete certificate of analysis (CofA) for the protein delivered.
Protein Expression, Conjugation and Analysis Services provided include:
• Expression construct design and evaluation.
• Custom cloning.
• Expression in: bacteria; yeast; insect cell; and mammalian cell systems.
• Co-expression optimization.
• Methods / process development and optimization.
• Low pressure liquid chromatography / AKTA Avant / AKTA Pure / FPLC.
• Preparative High Pressure Liquid Chromatography (Prep-HPLC) / Waters Autopurification System.
• Single cycle purification / production of up to 250 milligrams of high purity final product / protein / polypeptide.
• Multi-cycle purification / production of up to 5 gram of high purity product / protein / polypeptide.
• Purification by ion exchange (IEX), immobilized metal anion (IMAC), affinity (AC), size exclusion (SEC), hydrophobic interaction (HIC) and reverse phase (RPC) chromatographies and any combination thereof.
• Isotope labeling (bacterial expression only) and production of C13, N15 labelled purified protein.
• Antibody-Drug Conjugate (ADC) production, optimization & analysis:
-Optimization of conjugation reaction conditions
-Analysis of conjugation reaction products
-Drug-Antibody Ratio (DAR)
-Percent conversion to desired product
-Aggregation state
-Impurity /byproduct identification
-Bioactivity
-Design and optimization of drug-linker compounds.
-Non-GxP production of ADCs to client specifications of DAR, endotoxin content, aggregate content,
purity, quantity and formulation for in vivo proof of concept studies (non-human).

• Protein analysis by one or more of the following: SDS-PAGE (purity); Western blot (identity); LAL assay (endotoxin concentration); analytical SEC-MALS-RI (aggregation profile & purity), mass spectroscopy by LC-MS, LC-MS/MS, LC-ToF, MALDI-ToF or MALDI-Tof/ToF (purity, heterogeneity &/or DAR); specific activity; and X-ray crystallography.

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Protein Services

Price on request

A leading provider of protein expression, purification & analysis services to the biopharmaceutical industry, healthcare foundations and government institutions. AMRI has earned a reputation for excellent service and solving historically challenging protein expression and purification goals. AMRI can take your project directly... Show more »

A leading provider of protein expression, purification & analysis services to the biopharmaceutical industry, healthcare foundations and government institutions. AMRI has earned a reputation for excellent service and solving historically challenging protein expression and purification goals. AMRI can take your project directly from gene reference to final process and purified product with as much (or little) direction as you may wish to provide. Each project is customized to meet the specific client’s needs and can be initiated from gene synthesis, or we can express your target protein from a construct designed or supplied by you. At the conclusion of each project, we deliver a complete certificate of analysis (CofA) for the protein delivered.

Protein Expression, Conjugation and Analysis Services provided include:
• Expression construct design and evaluation
• Custom cloning
• Expression in: bacteria; yeast; insect cell; and mammalian cell systems
• Co-expression optimization
• Methods / process development and optimization
• Low pressure liquid chromatography / AKTA Avant / AKTA Pure / FPLC
• Preparative High Pressure Liquid Chromatography (Prep-HPLC) / Waters Autopurification System
• Single cycle purification / production of up to 250 milligrams of high purity final product / protein / polypeptide
• Multi-cycle purification / production of up to 5 gram of high purity product / protein / polypeptide
• Purification by ion exchange (IEX), immobilized metal anion (IMAC), affinity (AC), size exclusion (SEC), hydrophobic interaction (HIC) and reverse phase (RPC) chromatographies and any combination thereof
• Isotope labeling (bacterial expression only) and production of C13, N15 labelled purified protein
• Antibody-Drug Conjugate (ADC) production, optimization & analysis:
-Optimization of conjugation reaction conditions
-Analysis of conjugation reaction products
-Drug-Antibody Ratio (DAR)
-Percent conversion to desired product
-Aggregation state
-Impurity /byproduct identification
-Bioactivity
-Design and optimization of drug-linker compounds.
-Non-GxP production of ADCs to client specifications of DAR, endotoxin content, aggregate content,
purity, quantity and formulation for in vivo proof of concept studies (non-human)

• Protein analysis by one or more of the following: SDS-PAGE (purity); Western blot (identity); LAL assay (endotoxin concentration); analytical SEC-MALS-RI (aggregation profile & purity), mass spectroscopy by LC-MS, LC-MS/MS, LC-ToF, MALDI-ToF or MALDI-Tof/ToF (purity, heterogeneity &/or DAR); specific activity; and X-ray crystallography

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Drug Discovery

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Structural Biology

Price on request

AMRI provides a comprehensive portfolio of protein expression, crystallography, modeling and computer aided drug design services based on the expertise of highly experienced crystallographers and computational chemists. These services include:
• Expression construct design and evaluation
• Co-expression optimization
• Protein... Show more »

AMRI provides a comprehensive portfolio of protein expression, crystallography, modeling and computer aided drug design services based on the expertise of highly experienced crystallographers and computational chemists. These services include:
• Expression construct design and evaluation
• Co-expression optimization
• Protein purification and analysis
• Protein crystallization condition screening and optimization
• De novo structure determination
• Compound - protein co-crystallization and protein crystal soaking
• Protein–Ligand, Protein-Protein and Protein-DNA complex structure elucidation
• Structure analysis, modeling and Computer Aided Drug Design
• Rational drug design

X-Ray Crystallography

AMRI’s team of experienced protein crystallographers have a combined experience of > 50 years solving the structures of both membrane-associated and cytoplasmic proteins as evidenced by their respective publication records. The team is tightly integrated within AMRI’s protein expression, conjugation and analysis group and with all aspects of AMRIs integrated discovery service offerings including medicinal chemistry, computational chemistry, discovery biology and in vitro pharmacology. With expertise in protein-drug and protein-protein complex co-crystallization we offer comprehensive solutions for macromolecular crystallization studies, structural biology, structure-based / rational drug design and biophysical study.
Protein Crystallography Services provided include:
• Design, expression, purification and analysis of protein variants for crystallography.
• High throughput crystallization screening.
• Membrane protein crystallization screening.
• De novo structure determination.
• Compound - protein co-crystallization and protein crystal soaking.
• Protein–Ligand, Protein-Protein and Protein-DNA complex structure elucidation.
• Structure analysis, modeling and Computer Aided Drug Design.

Computer Aided Drug Discovery

AMRI’s Computer Aided Drug Discovery Team provides a wide range of computational analysis and prediction services to the biopharmaceutical industry. These include:

• Protein Modeling:
o Development of homology models from crystal structures and active site models emphasizing the protein-ligand interactions.
o Modeling of multimeric protein complexes.
• Binding Site Analysis:
o Evaluation and prioritization of protein binding sites based on druggability.
o Analysis of binding site interactions and solvation for guiding ligand design.
o Identification of transient/induced fit sub-pockets for novel ligand design strategies.
• Virtual Screening and Docking:
o Structure-guided docking and scoring to identify chemical substances for purchase and testing.
o Identification of relevant binding regions for increasing selectivity.
o Mutant-targeted design strategies.
o 3D pharmacophore searches and high-throughput docking of database of > 100 million compounds.
• Compound library design
o Target-focused compound libraries.
o Quality diversity libraries
• Chemical library analysis for assessments of quality and relative value.
• Lead Selection and Optimization
o Hit list analysis for prioritization of molecules with desired drug-like and DMPK properties.
o Hypothesis-driven, rational compound design based on target protein structure, binding model or ligand-based models.
o Development and application of quantitative structure activity relationships (QSARs)
o Computer modeling of DMPK properties including CYP inhibition and metabolism.
o Development of lead series using fragment-based design, hit-to-lead approaches including de novo, lead merging, evolution

Protein Expression, Conjugation & Analysis

A leading provider of protein expression, purification & analysis services to the biopharmaceutical industry, healthcare foundations and government institutions. AMRI has earned a reputation for excellent service and solving historically challenging protein expression and purification goals. AMRI can take your project directly from gene reference to final process and purified product with as much (or little) direction as you may wish to provide. Each project is customized to meet the specific client’s needs and can be initiated from gene synthesis, or we can express your target protein from a construct designed or supplied by you. At the conclusion of each project, we deliver a complete certificate of analysis (CofA) for the protein delivered.
Protein Expression, Conjugation and Analysis Services provided include:
• Expression construct design and evaluation.
• Custom cloning.
• Expression in: bacteria; yeast; insect cell; and mammalian cell systems.
• Co-expression optimization.
• Methods / process development and optimization.
• Low pressure liquid chromatography / AKTA Avant / AKTA Pure / FPLC.
• Preparative High Pressure Liquid Chromatography (Prep-HPLC) / Waters Autopurification System.
• Single cycle purification / production of up to 250 milligrams of high purity final product / protein / polypeptide.
• Multi-cycle purification / production of up to 5 gram of high purity product / protein / polypeptide.
• Purification by ion exchange (IEX), immobilized metal anion (IMAC), affinity (AC), size exclusion (SEC), hydrophobic interaction (HIC) and reverse phase (RPC) chromatographies and any combination thereof.
• Isotope labeling (bacterial expression only) and production of C13, N15 labelled purified protein.
• Antibody-Drug Conjugate (ADC) production, optimization & analysis:
o Optimization of conjugation reaction conditions.
o Analysis of conjugation reaction products
 Drug-Antibody Ratio (DAR)
 Percent conversion to desired product
 Aggregation state
 Impurity /byproduct identification
 Bioactivity
o Design and optimization of drug-linker compounds.
o Non-GxP production of ADCs to client specifications of DAR, endotoxin content, aggregate content, purity, quantity and formulation for in vivo proof of concept studies (non-human).
• Protein analysis by one or more of the following: SDS-PAGE (purity); Western blot (identity); LAL assay (endotoxin concentration); analytical SEC-MALS-RI (aggregation profile & purity), mass spectroscopy by LC-MS, LC-MS/MS, LC-ToF, MALDI-ToF or MALDI-Tof/ToF (purity, heterogeneity &/or DAR); specific activity; and X-ray crystallography.

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Drug Discovery

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Synthetic Chemistry

Price on request

For more than 25 years, AMRI has provided expertise in synthetic and medicinal chemistry, driven by the goal of discovering compounds for progression into human clinical trials. We deliver expert solutions for complex discovery challenges.

Our complete suite of diverse chemistry capabilities includes:

For more than 25 years, AMRI has provided expertise in synthetic and medicinal chemistry, driven by the goal of discovering compounds for progression into human clinical trials. We deliver expert solutions for complex discovery challenges.

Our complete suite of diverse chemistry capabilities includes:

  • Integration with discovery biology and pharmacology to support hit-to-lead identification through candidate selection
  • Structure activity relationships (SARs)/structure property relationships (SPRs) and novel target development guided by computer-aided drug design (CADD)Route design
  • Custom synthesisParallel synthesis
  • Library design and synthesis, including AMRI’s Compound Library Consortium (diverse compound library)
  • Asymmetric synthesis
  • Chiral separations (analytical and preparative supercritical fluid chromatography)
  • Metabolite synthesis
  • Controlled substance and high-potency synthesis
  • Antibody drug conjugate (ADC) expertise for novel therapeutics
  • Complex chemistries including eicosanoids, polysaccharides and challenging reactions
  • Global facilities in Albany and Hyderabad, India (Hyderabad Research Centre, Pvt. Ltd./AMRHRC)
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Drug Discovery

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X-ray Crystallography

Price on request

AMRI’s team of experienced protein crystallographers have a combined experience of > 50 years solving the structures of both membrane-associated and cytoplasmic proteins as evidenced by their respective publication records. The team is tightly integrated within AMRI’s protein expression, conjugation and analysis group and with all... Show more »

AMRI’s team of experienced protein crystallographers have a combined experience of > 50 years solving the structures of both membrane-associated and cytoplasmic proteins as evidenced by their respective publication records. The team is tightly integrated within AMRI’s protein expression, conjugation and analysis group and with all aspects of AMRIs integrated discovery service offerings including medicinal chemistry, computational chemistry, discovery biology and in vitro pharmacology. With expertise in protein-drug and protein-protein complex co-crystallization we offer comprehensive solutions for macromolecular crystallization studies, structural biology, structure-based / rational drug design and biophysical study.
Protein Crystallography Services provided include:
• Design, expression, purification and analysis of protein variants for crystallography.
• High throughput crystallization screening.
• Protein crystallization screening including membrane proteins.
• De novo structure determination.
• Compound - protein co-crystallization and protein crystal soaking.
• Protein–Ligand, Protein-Protein and Protein-DNA complex structure elucidation.
• Structure analysis, modeling and Computer Aided Drug Design.

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Drug Discovery

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Cell-Based Compound Screening

Price on request

Cell Biology Discovery Services

AMRI provides a comprehensive portfolio of cell-based discovery services for the identification and optimization of novel compounds targeting human diseases including cancer, neurodegenerative and immune system disorders. These include development of custom cell lines and assays,... Show more »

Cell Biology Discovery Services

AMRI provides a comprehensive portfolio of cell-based discovery services for the identification and optimization of novel compounds targeting human diseases including cancer, neurodegenerative and immune system disorders. These include development of custom cell lines and assays, implementation of client-developed assays and compound testing for hit identification and lead optimization. The following is a selection of some of the assay models and services provided:

• Cell Line Development

• FLIPR-based assays

• High content imaging based assays / phenotypic assays

• In-Cell Western based assays

• Gene reporter assays

• Fluorescence, FRET and HTRF assays

• MSD and flow cytometry based assays

• MALDI PharmaPulse and LC-MS/MS based label free assays

• Radiosubstrate uptake and radioisotope incorporation based assays

• Cytotoxicity testing against cancer cell lines (2D & 3D)

• Cytotoxicity testing against normal cells (2D)

• Cytotoxicity testing against HepG2 cells (2D & 3D)

• Mitotoxicity testing against HepG2 cells (Glu/Gal, 2D & 3D)

• Inflammatory response testing with PBMCs

• Inflammatory response testing with select T-Cells

• T-cell progenitor differentiation testing

• T-cell response testing

• Neurotoxicity evaluation in primary rat neurons

• Co-culture of PBMCs with cancer cells (2D & 3D)

• Stem cell differentiation testing

• Multiplexed, Quantitative, Biomarker Expression Testing

More traditional cell-based assay formats that are supported include Calcium Flux / FLIPR, membrane potential, protein translocation / HCS, ELISA, MSD, radioligand binding and radiosubstrate incorporation.

Cell Line Development

AMRI provides cell line engineering in the form of traditional stable cell line generation and CRISPR/Cas9-mediated knock-out or knock-in lines. Parental cell lines can be generated from cells obtained from repositories or from client internal stocks. Standard lipofection, electroporation or PEI can be used to introduce the expression or targeting vectors to the line of choice. Adenoviral, lentiviral or retroviral expression systems can also be used. Positive transfections will be verified by cell sorting, Western blot or ELISA and can be validated for functional assays if needed.

High Throughput Screening and Lead Optimization

Automated cell-based readouts are provided by state-of-the-art high content imaging (HCI) and analysis systems, including the PerkinElmer Opera Phenix and Incucyte Zoom; and non-imaging readers for assays dependent on fluorescence (FI, TRF, HTRF, FRET, TR-FRET, LanthaScreen, Alamar Blue), luminescence (examples ADP-Glo, CellTiter-Glo and Steady-Glo) and AlphaScreen, which are conducted on PerkinElmer Envision readers upgraded with the enhanced luminescence detection, TRF laser and AlphaScreen HTS options; or for assays dependent upon the detection of a radioligand or uptake of a radiosubstrate which are readout using PerkinElmer Microbeta2 reader technology.

AMRI’s cell biologists, automation specialists and in vitro pharmacologists work closely together as teams and with their client to ensure appropriate design and execution of high-throughput screening campaigns. This collaborative philosophy is applied to all phases of preclinical candidate development including hit prioritization, hit-to-lead optimization, lead optimization and preclinical candidate selection. As a consequence clients retain full oversight and control of their programs while benefiting from the experience and recommendations of biologists and chemists from an organization which has developed >100 preclinical and clinical candidates.

Below are brief descriptions of specific cell-based assays provided by AMRI. The list is not comprehensive and we would be happy to provide a quote for your specific needs.

Cytotoxicity Testing Against Cancer Cell Lines- 2D & 3D

AMRI Scientists will culture the cancer cell line of choice and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each will be compared to DMSO treated wells and validated cytotoxic agents.

AMRI Scientists will culture 3D spheroids using a cancer cell line of choice and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in six replicates as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of spheroids in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo 3D. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

Cytotoxicity Testing Against Normal Cells- 2D

AMRI Scientists will culture normal human dermal fibroblasts and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

Cytotoxicity Testing Against HepG2 Cells- 2D & 3D

AMRI Scientists will culture HepG2 cells and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

AMRI Scientists will culture 3D spheroids using HepG2 cells and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in six replicates as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of spheroids in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo 3D. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

Mitotoxicity Testing Against HepG2 Cells (Glu/Gal) - 2D & 3D

AMRI Scientists will culture HepG2 cells grown in glucose and galactose containing media and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo. Each well will be compared to DMSO treated wells and validated cytotoxic agents. Compound effects on mitochondrial function will be deduced by comparison of cytotoxicity data generated from HepG2 cultures grown in media with glucose versus galactose.

AMRI Scientists will culture 3D spheroids using HepG2 cells grown in both glucose and galactose containing media and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in six replicates as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of spheroids in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo 3D. Each well will be compared to DMSO treated wells and validated cytotoxic agents. Compound effects on mitochondrial function will be deduced by comparison of cytotoxicity data generated from HepG2 cultures grown in media with glucose versus galactose.

Inflammatory Response Testing with PBMCs

AMRI Scientists will test your compounds for pro- or anti-inflammatory responses in commercially obtained human PBMCs purified from healthy donors. PBMCs will be received either fresh or frozen and incubated with test compounds for the desired amount of time. Culture supernatants will be collected and assayed for the presence of inflammatory cytokines using MSD multiplex assays. Alternatively, PBMCs will be treated with pro-inflammatory agents and compounds will be tested for their ability to inhibit pro-inflammatory cytokine production. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

Inflammatory Response Testing with Select T-cell Populations

AMRI Scientists will test your compounds for pro- or anti-inflammatory responses in commercially obtained T-cell populations isolated from healthy donors. T-cells can also be induced into select differentiated states such as Th17 or Treg using established induction protocols. T cells will be incubated with test compounds for the desired amount of time. Culture supernatants will be collected and assayed for the presence of inflammatory cytokines using MSD multiplex assays. Alternatively, PBMCs will be treated with pro-inflammatory agents and compounds will be tested for their ability to inhibit pro-inflammatory cytokine production. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

T-cell Progenitor Differentiation Testing

AMRI Scientists will test your compounds for effects on T-cell differentiation from common projenitor cells such as CD4+ cells. CD4+ T-cells will be induced towards the phenotype of choice using established protocols in the presence of test compounds. Cells will be phenotyped by FACS with validated antibodies against cell surface or intracellular markers. T cells will be incubated with test compounds throughout the differentiation protocols. Culture supernatants will be collected and assayed for the presence of marker cytokines using MSD multiplex assays or ELISA. Cells from the same wells will be subjected to FACS to allow for a comprehensive phenotypic analysis. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

T-cell Response Testing

AMRI Scientists will test your compounds for effects on T-cell function in commercially sourced T-cells from healthy donors. The effects of compounds on T-cell function such as proliferation, cyokine secretion, differentiation potential or cytolytic activity can be assessed. Cells will be phenotyped by FACS with validated antibodies against cell surface or intracellular markers. Culture supernatants will be collected and assayed for the presence of marker cytokines using MSD multiplex assays or ELISA. Cells from the same wells will be subjected to FACS to allow for a comprehensive phenotypic analysis. Populations of PBMCs or T-cells can be grown in co-culture studies to examine T-cell mediated cytotoxicity. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

Neurotoxicity Evaluation in Primary Rat Neurons

AMRI Scientists will isolate E18 primary rat neurons and plate in 384-well plates. Cultures of neuron enriched, astrocyte enriched or mixed cortical cultures are available. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or by measuring LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each will will be compared to DMSO treated wells and validated neurotoxic agents.

Co-Culture of PBMCs with Cancer Cells – 2D & 3D

AMRI Scientists will evaluate the effects of stimulated human PBMCs in co-culture with cancer cell lines grown as 2D monolayers and 3D spheroids over time. Fluorescently labelled immune cells will be stimulated using IL-2 and CD3/CD28 coated Dynabeads before addition in culture with transfected NucLight HCT116 cancer cells. The IncuCyte live cell analysis imaging software will be used to track immune cell cytotoxicity over time in the co-culture by the acquisition of fluorescent images. Confocal microscopy images will also be attained at chosen time points using the PerkinElmer Opera Phenix confocal imaging system at optimized instrument settings.

Stem Cell Differentiation Testing

AMRI Scientists will evaluate protocols or compound treatment on stem cell differentiation from common projenitors including hematopoietic stem cells, neuronal projenitor cells or induced-pluripotent stem cells. Cells will be subjected to phentoypic analysis to assess the effects of compounds or culture conditions on stem cell differentiation.

Multiplexed, Quantitative, Biomarker Expression Testing

AMRI Scientists will utilize multiple approaches to identify the presence and/or localization of biomarkers of interest in cells where appropriatly specific antibodies are available. Four channel confocal imaging of cells on the PerkinElmer Opera Phenix allows simultaneous detection and localization of protein targets. Multichannel flow cytometry allows the simultaneous detection of cell surface and intracellular markers in cells. Multiplexed MesoScale Discovery (MSD) assays can be used to detect multiple biomarkers of interest in biological fluids, cellular lysates and conditioned media supernatants.

High Content Imaging and Analysis
AMRI offers a range of multiplexed imaging-based assays to simultaneously assess cell viability, physiology and morphology, using the PerkinElmer Opera Phenix automated confocal system and Incucyte Zoom live cell imaging system. Live cell imaging allows the impact of compounds on cellular physiology over time in 2D and 3D. Imaging assays can also be multiplexed with traditional endpoint measures of cell viability to further enrich the data obtained from screening campaigns. High content analysis also allows unbiased phenotypic screening to use for compound library characterization and drug repurposing studies. Combining high content screening with CRISPR or siRNA libraries links genotype to phenotype and allows the creation of models of mutation-based human disease.
Available readouts include:

• Cell viability
• Cell morphology
• Apoptosis
• Cell proliferation
• Cytoskeletal disruption
• Histone modifications
• Mitochondrial function
• Oxidative stress
• Neurite outgrowth
• T-cell mediated cytoxicity

2D & 3D High Content Imaging and Analysis- Neurite Outgrowth
AMRI Scientists will isolate E18 primary rat neurons and plate in 384-well plates. Cultures of neuron enriched, astrocyte enriched or mixed cortical cultures are available. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. Neurite outgrowth will be determined in live cultures using an Incucyte Zoom or in fixed and immunostained cultures using a PerkinElmer Opera Phenix. Neurite features will be analyzed using validated routines and compared to DMSO treated and positive control treated wells.
2D High Content Assay- Cell Viability
AMRI Scientists will evaluate the effects of compounds on cell viability in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Viability will evaluated using fluorescent dyes that specifically label dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Proliferation
AMRI Scientists will evaluate the effects of compounds on cell proliferation in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Proliferation will be assessed by Edu incorporation and co-staining with an antibody against phosphorylated H3. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Apoptosis
AMRI Scientists will evaluate the effects of compounds on apoptosis in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Apoptosis will evaluated using fluorescent dyes that specifically mark apoptotic cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Cytoskeletal Disruption
AMRI Scientists will evaluate the effects of compounds on the cytoskeleton in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Cytoskeletal disruption will evaluated using fluorescent dyes that bind to either tubulin or actin. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Histone Modifications
AMRI Scientists will evaluate the effects of compounds on histone phosphorylation and/or methylation in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Specific phosphorylation of histones will be assessed using an immunostaining approach with an antibody against phosphorylated or methylated histones. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Mitochondrial Morphology
AMRI Scientists will evaluate the effects of compounds on mitochondrial membrane potential in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Viability will evaluated using fluorescent dyes that specifically label dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Mitochondrial Morphology

AMRI Scientists will evaluate the effects of compounds on mitochondrial morphology in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. The effect of compound treatment on mitochondrial and cellular morphology will evaluated using fluorescent dyes that specifically label mitochondria and dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Oxidative Stress

AMRI Scientists will evaluate the effects of compounds on induction or suppression of oxidative stress in live cells using dyes that measure specific reactive oxygen and nitrogen species in various cellular compartments. These assays can also be multiplexed with other high content assays to measure a range of diverse cellular physiological states upon compound treatment. Cells will be plated in 384-well pates and cultured in the presence of test compounds for the desired amount of time. Kinetic measures are availabe to look for acute and chronic effects on oxidative stress. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D & 3D High Content Assay- T-cell Mediated Cytotoxicity

AMRI Scientists will evaluate the effects of compounds on induction or suppression of T-cell mediated cytotoxicity in mixed cultures containing cancer cell lines and human PBMCs or isolated T-cell populations. Cancer cells will be fluorescently labeled and grown as monolayers or spheroids prior to addition of immune cells. The viability of the cancer cells can be monitored in live imaging or fixed endpoint experiments. T-cell counting and phenotyping is available with conjugated antibodies against surface markers. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

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Antibacterial Screening

Price on request

Antimicrobial Screening Services & Capabilities

AMRI provides a comprehensive portfolio of antimicrobial discovery services for the identification and optimization of antibacterial and antifungal agents. These include high throughput screening utilizing aerobic, microaerobic or anaerobic conditions; MIC, synergy, time-kill... Show more »

Antimicrobial Screening Services & Capabilities

AMRI provides a comprehensive portfolio of antimicrobial discovery services for the identification and optimization of antibacterial and antifungal agents. These include high throughput screening utilizing aerobic, microaerobic or anaerobic conditions; MIC, synergy, time-kill kinetic and resistance testing; hit-to-lead and lead optimization chemistry; in vitro ADMET and DMPK testing; and a complete portfolio of protein expression and assay development capabilities for generation of the materials and assays required to establish target-based biochemical or cellular assays. The antimicrobial testing services offered by AMRI and range of clinical isolates available for testing are briefly summarized below.

• Minimum Inhibitory Concentration (MIC) testing (bacterial and fungal)
• Minimum Bacterial Cidal Concentration (MBC) testing
• Antibiotic Synergy (Checkerboard) testing (bacterial and fungal)
• Time Kill Kinetic testing
• Mutation Rate and Mutation Frequency testing
• Mutation Prevention Concentration (MPC) testing
• Resistance development determination (serial passage)
• Resistance mechanism identification
• Mammalian cytotoxicity testing, standard methods
• Mammalian cytotoxicity testing, High Content Imaging methods
• Mechanism of Action determination
• Efflux pump inhibition
• Antibiotic uptake testing
• High Throughput Antimicrobial Screening (HTS)

MIC Determination, Standard Method
Minimum Inhibitory Concentrations (MICs) are determined using a modified CLSI microbroth dilution assay protocol (document M7), including clinical isolates representing both current and developing antibiotic resistance in a 384-well microplate format which can accommodate up to 8 reference antibiotics or 8 test compounds, in triplicate, with a 12-point, 1:2 dilution series (plus appropriate controls). The CLSI guidelines result in a cutoff determination for the MIC; an exact value can be extrapolated if needed. A serum binding MIC assay can also be performed as discussed in CLSI document M26. Standardized MIC test panels, comprising up to sixty (60) ESKAPE class; thirty (30) Neisseria gonorrhaeae; and ten (10) fungal, clinically relevant isolates are available for those requiring immediate MIC testing encompassing various resistance phenotypes and strains sourced from both Europe and North America. Bespoke MIC panels tailored to match client-specific needs such as species composition, resistance phenotypes and panel size can be generated as needed from AMRI’s extensive collection of recent clinical isolates and then used for rapid sample testing.

MIC Determination, Custom Methods
MICs may also be determined using custom conditions to answer specific questions. For example, by inclusion of serum (to detect the impact of plasma protein binding on antibiotic activity) or salmon sperm DNA (to eliminate DNA interchalators). Custom high content imaging assays combining readouts of efficacy vs. intracellular bacteria and toxicity on the mammalian host cell can be provided.

Minimum Bactericidal Concentration
MBCs are determined after the MIC assay to calculate cidal activity.

Antibiotic Synergy (Checkerboard) Testing
Compounds are tested as nine (9) point, two-fold serial dilutions across the assay plate in combination with an seven (7) point, two-fold serial dilution of the second antibiotic (typically a standard of care antibiotic) down the assay plate. In addition each assay plate includes two (2) columns for control wells of uninhibited growth (no addition of test article) and for drug free sterile control wells (Any clinical isolate from AMRI’s collection may be used for the assay. Test articles are first MIC tested against the strain selected to determine an appropriate range of test concentrations for the synergy test. Fractional Inhibitory Concentration values (FICA and FICB) and FIC index are calculated for each combination of compounds and clinical isolate using the following standard equations for this analysis:
FIC index = FICA+FICB
FICA= MIC of compound A in combination/MIC of compound A alone
FICB= MIC of compound B in combination/MIC of compound B alone
Synergy will be defined as a FIC index value of ≤0.5. Indifference or no interaction will be defined as a FIC index value of >0.5 and <4. Antagonism will be defined as a FIC index value of >4. When the FIC index value is within the range of 0.5 – 1, the combination is considered to be non-synergistic or additive. Results are provided in tabular form.

Time Kill Kinetics
Time Kill Kinetics are determined using various time intervals of the pre -determined MIC over a 24 hour period. This assay can be performed with any of the organisms listed in tables 2 and 3. The time-kill curve is beneficial in identifying possible problems with resistance development or compound degradation over the 24 hour testing period.

Mutation Rate and Frequency
Mutation rate and frequency assays are available to further determine resistance development towards a specific compound. The mutation rate assay is performed following the protocol from Luria et al. (1943) and Foster (2006).

MPC Determination
Mutation Prevention Concentration (MPC) assays are available to determine concentrations at which resistance mutations are unable to develop. It is the lowest drug concentration required to block the growth of the least susceptible cell present in high density bacterial populations. Testing is conducted using large bacterial inocula (≥ 109 cfu/mL).

Resistance Development Determination
Isolated resistant colonies from the Time-kill Kinetics or the Mutation Rate and Frequency studies may be used to determine the potential for resistance development over time and multiple generations. The concentration of test antibiotic is increased in step with the increase in resistance. Solid and liquid media based assays are available.

Resistance mechanism identification
Resistant and non-resistant isolates may be analyzed by RT-PCR or whole genome sequencing to detect expression of resistance-associated genes or changes in target protein (or RNA) sequence associated with resistance. Secondary experiments employing molecular biology, biochemical or biophysical methods may also be undertaken to confirm resistance mechanism.

Cytotoxicity Assay, Standard Methods
Use of a mammalian liver cell line (HepG2) or mammalian normal fibroblast (NHDF) to determine in vitro therapeutic index and exclude overtly toxic compounds. Standard cytotoxicity detection methods employ MTT or Celltiter-Glo.

Cytotoxicity Assay, High Content Imaging Methods
A variety of live cell and fixed cell multi-parametric high content imaging assays are available. Custom assays combining readouts of efficacy vs. intracellular bacteria and toxicity on the mammalian host cell can be provided.

Mechanism of Action Determination
While there is no standard assay, AMRI has extensive experience in determining the mechanism of action of antimicrobial compounds and has developed custom assays to best suit the targets of interest.

Efflux Pump Inhibition
Two detection methods are offered to measure inhibition of drug efflux pumps, ethidium bromide uptake (measure accumulation in the cells) and LC-MS/MS (measure depletion from minimal media and/or accumulation in cells). The latter method may be tailored to monitor a variety of analytes including the components of a combination treatment. Assay is validated for E. coli, P. aeruginosa, and S. aureus (additional strains may be validated as needed).

Antibiotic Uptake
LC-MS/MS is used to measure the accumulation of the antibiotic in the cellular component of the culture after 30 minutes to 1 hour of exposure. As an alternative the depletion of the antibiotic from the media component of the culture may be determined.

High Throughput Antimicrobial Screening
AMRI has developed and conducted a number of natural product sample and small molecule based anti-infective screens. Programs requiring screens against biosafety level 3 organisms or under micro-aerobic conditions can be accommodated. The examples listed below are representative of the non-proprietary detection systems utilized to date and were conducted in single point, 384-well plate format:

  • 280,000 sample natural product screens against S. aureus and E. coli utilizing absorbance / turbidity;
  • 100,000 sample synthetic molecule screens against clinically relevant targets / pathogens (8) utilizing absorbance / turbidity or fluorescence;
  • 100,000 natural product sample screen against fungal pathogens (4) utilizing turbidity or luminescence;
  • 160,000 sample synthetic molecule whole cell anti-virulence screen utilizing luminescence;
    AMRI can provide a highly curated diversity library of 105,000 synthetic compounds to support high throughput screens conducted at AMRI or use any pre-plated natural product sample or synthetic compound collection you may wish to provide for a screen.

Clinical Isolates Available for Testing
The AMRI clinical isolate collection of > 500 isolates includes antibiotic susceptible and resistant isolates for each clinically relevant microbial species. The collection provides comprehensive coverage of current and developing clinical pathogens and antibiotic resistance profiles (blaNDM, blaKPC, blaOXA, blaCTX, blaVIM and mcr-1) from both North America and Europe and new isolates are regularly sourced. A rapid response MIC test panel comprising sixty (60) ESKAPE class clinical isolates and encompassing various resistance phenotypes and strains sourced from Europe and North America is available for those requiring immediate MIC testing.
Testing against custom selections of clinical isolates is also provided with next day testing an option after a custom panel has been established. Bespoke MIC testing panels can be assembled from the AMRI clinical isolate collection as required to meet specific client needs within two to four weeks for panels consisting of assays requiring aerobic or micro-aerobic conditions, respectively. The clinical isolate collection currently includes:
• Gram-positive pathogens (MRSA, S. pneumoniae, and VRE, 80 strains currently available).
• Gram-negative pathogens (424 strains available)
• Carbapenemase-producing MDR Enterobacteriaceae (164 representatives)
• P. aeruginosa (76 representatives)
• A. baumannii (68 representatives)
• N. gonorrhoeae (51 representatives)
• A diverse collection of enteric pathogens (30)
• Drug resistant Candida species (32 representatives)
• Pathogenic filamentous fungi (2 representatives)

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Cell-Based Assays

Price on request

Cell Biology Discovery Services

AMRI provides a comprehensive portfolio of cell-based discovery services for the identification and optimization of novel compounds targeting human diseases including cancer, neurodegenerative and immune system disorders. These include development of custom cell lines and assays, implementation... Show more »

Cell Biology Discovery Services

AMRI provides a comprehensive portfolio of cell-based discovery services for the identification and optimization of novel compounds targeting human diseases including cancer, neurodegenerative and immune system disorders. These include development of custom cell lines and assays, implementation of client-developed assays and compound testing for hit identification and lead optimization. The following is a selection of some of the assay models and services provided:

• Cell Line Development

• FLIPR-based assays

• High content imaging based assays / phenotypic assays

• In-Cell Western based assays

• Gene reporter assays

• Fluorescence, FRET and HTRF assays

• MSD and flow cytometry based assays

• MALDI PharmaPulse and LC-MS/MS based label free assays

• Radiosubstrate uptake and radioisotope incorporation based assays

• Cytotoxicity testing against cancer cell lines (2D & 3D)

• Cytotoxicity testing against normal cells (2D)

• Cytotoxicity testing against HepG2 cells (2D & 3D)

• Mitotoxicity testing against HepG2 cells (Glu/Gal, 2D & 3D)

• Inflammatory response testing with PBMCs

• Inflammatory response testing with select T-Cells

• T-cell progenitor differentiation testing

• T-cell response testing

• Neurotoxicity evaluation in primary rat neurons

• Co-culture of PBMCs with cancer cells (2D & 3D)

• Stem cell differentiation testing

• Multiplexed, Quantitative, Biomarker Expression Testing

More traditional cell-based assay formats that are supported include Calcium Flux / FLIPR, membrane potential, protein translocation / HCS, ELISA, MSD, radioligand binding and radiosubstrate incorporation.

Cell Line Development

AMRI provides cell line engineering in the form of traditional stable cell line generation and CRISPR/Cas9-mediated knock-out or knock-in lines. Parental cell lines can be generated from cells obtained from repositories or from client internal stocks. Standard lipofection, electroporation or PEI can be used to introduce the expression or targeting vectors to the line of choice. Adenoviral, lentiviral or retroviral expression systems can also be used. Positive transfections will be verified by cell sorting, Western blot or ELISA and can be validated for functional assays if needed.

High Throughput Screening and Lead Optimization

Automated cell-based readouts are provided by state-of-the-art high content imaging (HCI) and analysis systems, including the PerkinElmer Opera Phenix and Incucyte Zoom; and non-imaging readers for assays dependent on fluorescence (FI, TRF, HTRF, FRET, TR-FRET, LanthaScreen, Alamar Blue), luminescence (examples ADP-Glo, CellTiter-Glo and Steady-Glo) and AlphaScreen, which are conducted on PerkinElmer Envision readers upgraded with the enhanced luminescence detection, TRF laser and AlphaScreen HTS options; or for assays dependent upon the detection of a radioligand or uptake of a radiosubstrate which are readout using PerkinElmer Microbeta2 reader technology.

AMRI’s cell biologists, automation specialists and in vitro pharmacologists work closely together as teams and with their client to ensure appropriate design and execution of high-throughput screening campaigns. This collaborative philosophy is applied to all phases of preclinical candidate development including hit prioritization, hit-to-lead optimization, lead optimization and preclinical candidate selection. As a consequence clients retain full oversight and control of their programs while benefiting from the experience and recommendations of biologists and chemists from an organization which has developed >100 preclinical and clinical candidates.

Below are brief descriptions of specific cell-based assays provided by AMRI. The list is not comprehensive and we would be happy to provide a quote for your specific needs.

Cytotoxicity Testing Against Cancer Cell Lines- 2D & 3D

AMRI Scientists will culture the cancer cell line of choice and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each will be compared to DMSO treated wells and validated cytotoxic agents.

AMRI Scientists will culture 3D spheroids using a cancer cell line of choice and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in six replicates as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of spheroids in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo 3D. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

Cytotoxicity Testing Against Normal Cells- 2D

AMRI Scientists will culture normal human dermal fibroblasts and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

Cytotoxicity Testing Against HepG2 Cells- 2D & 3D

AMRI Scientists will culture HepG2 cells and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

AMRI Scientists will culture 3D spheroids using HepG2 cells and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in six replicates as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of spheroids in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo 3D. Each well will be compared to DMSO treated wells and validated cytotoxic agents.

Mitotoxicity Testing Against HepG2 Cells (Glu/Gal) - 2D & 3D

AMRI Scientists will culture HepG2 cells grown in glucose and galactose containing media and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo. Each well will be compared to DMSO treated wells and validated cytotoxic agents. Compound effects on mitochondrial function will be deduced by comparison of cytotoxicity data generated from HepG2 cultures grown in media with glucose versus galactose.

AMRI Scientists will culture 3D spheroids using HepG2 cells grown in both glucose and galactose containing media and plate in 384-well plates. Once cultures are established, compounds will be delivered to the wells in six replicates as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of spheroids in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo 3D. Each well will be compared to DMSO treated wells and validated cytotoxic agents. Compound effects on mitochondrial function will be deduced by comparison of cytotoxicity data generated from HepG2 cultures grown in media with glucose versus galactose.

Inflammatory Response Testing with PBMCs

AMRI Scientists will test your compounds for pro- or anti-inflammatory responses in commercially obtained human PBMCs purified from healthy donors. PBMCs will be received either fresh or frozen and incubated with test compounds for the desired amount of time. Culture supernatants will be collected and assayed for the presence of inflammatory cytokines using MSD multiplex assays. Alternatively, PBMCs will be treated with pro-inflammatory agents and compounds will be tested for their ability to inhibit pro-inflammatory cytokine production. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

Inflammatory Response Testing with Select T-cell Populations

AMRI Scientists will test your compounds for pro- or anti-inflammatory responses in commercially obtained T-cell populations isolated from healthy donors. T-cells can also be induced into select differentiated states such as Th17 or Treg using established induction protocols. T cells will be incubated with test compounds for the desired amount of time. Culture supernatants will be collected and assayed for the presence of inflammatory cytokines using MSD multiplex assays. Alternatively, PBMCs will be treated with pro-inflammatory agents and compounds will be tested for their ability to inhibit pro-inflammatory cytokine production. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

T-cell Progenitor Differentiation Testing

AMRI Scientists will test your compounds for effects on T-cell differentiation from common projenitor cells such as CD4+ cells. CD4+ T-cells will be induced towards the phenotype of choice using established protocols in the presence of test compounds. Cells will be phenotyped by FACS with validated antibodies against cell surface or intracellular markers. T cells will be incubated with test compounds throughout the differentiation protocols. Culture supernatants will be collected and assayed for the presence of marker cytokines using MSD multiplex assays or ELISA. Cells from the same wells will be subjected to FACS to allow for a comprehensive phenotypic analysis. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

T-cell Response Testing

AMRI Scientists will test your compounds for effects on T-cell function in commercially sourced T-cells from healthy donors. The effects of compounds on T-cell function such as proliferation, cyokine secretion, differentiation potential or cytolytic activity can be assessed. Cells will be phenotyped by FACS with validated antibodies against cell surface or intracellular markers. Culture supernatants will be collected and assayed for the presence of marker cytokines using MSD multiplex assays or ELISA. Cells from the same wells will be subjected to FACS to allow for a comprehensive phenotypic analysis. Populations of PBMCs or T-cells can be grown in co-culture studies to examine T-cell mediated cytotoxicity. Compounds will be tested in duplicate as 10-point, 3-fold dilution series and compared against DMSO and control compound treated cells.

Neurotoxicity Evaluation in Primary Rat Neurons

AMRI Scientists will isolate E18 primary rat neurons and plate in 384-well plates. Cultures of neuron enriched, astrocyte enriched or mixed cortical cultures are available. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. At the termination of the experiment, the viability of cultures in each well will be determined by measuring ATP levels in lysed cells using CellTiterGlo or by measuring LDH levels in culture supernatants. Culture viability can also be tested using MTT or AlamarBlue readouts. Each will will be compared to DMSO treated wells and validated neurotoxic agents.

Co-Culture of PBMCs with Cancer Cells – 2D & 3D

AMRI Scientists will evaluate the effects of stimulated human PBMCs in co-culture with cancer cell lines grown as 2D monolayers and 3D spheroids over time. Fluorescently labelled immune cells will be stimulated using IL-2 and CD3/CD28 coated Dynabeads before addition in culture with transfected NucLight HCT116 cancer cells. The IncuCyte live cell analysis imaging software will be used to track immune cell cytotoxicity over time in the co-culture by the acquisition of fluorescent images. Confocal microscopy images will also be attained at chosen time points using the PerkinElmer Opera Phenix confocal imaging system at optimized instrument settings.

Stem Cell Differentiation Testing

AMRI Scientists will evaluate protocols or compound treatment on stem cell differentiation from common projenitors including hematopoietic stem cells, neuronal projenitor cells or induced-pluripotent stem cells. Cells will be subjected to phentoypic analysis to assess the effects of compounds or culture conditions on stem cell differentiation.

Multiplexed, Quantitative, Biomarker Expression Testing

AMRI Scientists will utilize multiple approaches to identify the presence and/or localization of biomarkers of interest in cells where appropriatly specific antibodies are available. Four channel confocal imaging of cells on the PerkinElmer Opera Phenix allows simultaneous detection and localization of protein targets. Multichannel flow cytometry allows the simultaneous detection of cell surface and intracellular markers in cells. Multiplexed MesoScale Discovery (MSD) assays can be used to detect multiple biomarkers of interest in biological fluids, cellular lysates and conditioned media supernatants.

High Content Imaging and Analysis
AMRI offers a range of multiplexed imaging-based assays to simultaneously assess cell viability, physiology and morphology, using the PerkinElmer Opera Phenix automated confocal system and Incucyte Zoom live cell imaging system. Live cell imaging allows the impact of compounds on cellular physiology over time in 2D and 3D. Imaging assays can also be multiplexed with traditional endpoint measures of cell viability to further enrich the data obtained from screening campaigns. High content analysis also allows unbiased phenotypic screening to use for compound library characterization and drug repurposing studies. Combining high content screening with CRISPR or siRNA libraries links genotype to phenotype and allows the creation of models of mutation-based human disease.
Available readouts include:

• Cell viability
• Cell morphology
• Apoptosis
• Cell proliferation
• Cytoskeletal disruption
• Histone modifications
• Mitochondrial function
• Oxidative stress
• Neurite outgrowth
• T-cell mediated cytoxicity

2D & 3D High Content Imaging and Analysis- Neurite Outgrowth
AMRI Scientists will isolate E18 primary rat neurons and plate in 384-well plates. Cultures of neuron enriched, astrocyte enriched or mixed cortical cultures are available. Once cultures are established, compounds will be delivered to the wells in quadruplicate as 10-point, 3-fold dilution series and incubated for the desired amount of time. Neurite outgrowth will be determined in live cultures using an Incucyte Zoom or in fixed and immunostained cultures using a PerkinElmer Opera Phenix. Neurite features will be analyzed using validated routines and compared to DMSO treated and positive control treated wells.
2D High Content Assay- Cell Viability
AMRI Scientists will evaluate the effects of compounds on cell viability in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Viability will evaluated using fluorescent dyes that specifically label dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Proliferation
AMRI Scientists will evaluate the effects of compounds on cell proliferation in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Proliferation will be assessed by Edu incorporation and co-staining with an antibody against phosphorylated H3. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Apoptosis
AMRI Scientists will evaluate the effects of compounds on apoptosis in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Apoptosis will evaluated using fluorescent dyes that specifically mark apoptotic cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Cytoskeletal Disruption
AMRI Scientists will evaluate the effects of compounds on the cytoskeleton in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Cytoskeletal disruption will evaluated using fluorescent dyes that bind to either tubulin or actin. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Histone Modifications
AMRI Scientists will evaluate the effects of compounds on histone phosphorylation and/or methylation in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Specific phosphorylation of histones will be assessed using an immunostaining approach with an antibody against phosphorylated or methylated histones. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Mitochondrial Morphology
AMRI Scientists will evaluate the effects of compounds on mitochondrial membrane potential in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. Viability will evaluated using fluorescent dyes that specifically label dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Mitochondrial Morphology

AMRI Scientists will evaluate the effects of compounds on mitochondrial morphology in cultured cell lines using an imaging based approach with automated image collection and analysis using a PerkinElmer Opera Phenix. Cells will be plated in 384-well plates and cultured in the presence of test compounds for the desired amount of time. Each compound will be tested in quadruplicate as 10-point, 3-fold dilution series. The effect of compound treatment on mitochondrial and cellular morphology will evaluated using fluorescent dyes that specifically label mitochondria and dead or dying cells. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D High Content Assay- Oxidative Stress

AMRI Scientists will evaluate the effects of compounds on induction or suppression of oxidative stress in live cells using dyes that measure specific reactive oxygen and nitrogen species in various cellular compartments. These assays can also be multiplexed with other high content assays to measure a range of diverse cellular physiological states upon compound treatment. Cells will be plated in 384-well pates and cultured in the presence of test compounds for the desired amount of time. Kinetic measures are availabe to look for acute and chronic effects on oxidative stress. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.
2D & 3D High Content Assay- T-cell Mediated Cytotoxicity

AMRI Scientists will evaluate the effects of compounds on induction or suppression of T-cell mediated cytotoxicity in mixed cultures containing cancer cell lines and human PBMCs or isolated T-cell populations. Cancer cells will be fluorescently labeled and grown as monolayers or spheroids prior to addition of immune cells. The viability of the cancer cells can be monitored in live imaging or fixed endpoint experiments. T-cell counting and phenotyping is available with conjugated antibodies against surface markers. Compound efficacy will be compared to DMSO treated and positive control compound treated cells.

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Assay Development

Price on request

AMRI scientists are experienced at developing biochemical and cell-based assays for a wide range of targets and target classes. The service includes evaluation of commercially available biological components (enzymes, cells or substrates), production of custom biological components as may be required, assay optimization and... Show more »

AMRI scientists are experienced at developing biochemical and cell-based assays for a wide range of targets and target classes. The service includes evaluation of commercially available biological components (enzymes, cells or substrates), production of custom biological components as may be required, assay optimization and performance testing. Detection technologies available at AMRI for assay development include, but are not limited to:

PerkinElmer Envision Multimode readers with TRF laser, enhanced luminescence and AlphaScreen HTS upgrades.

PerkinElmer Opera Phenix high content imager with 4 color laser, extended live cell and 3D imaging upgrades.

IncuCyte Zoom high content live cell imager. Two color version.

Molecular Devices FLIPR with temperature controlled stage for membrane potential and calcium flux assays.

Meso-Scale Discovery SectorImager 6000 for MSD-format assays

MALDI-PharmaPulse MS and LC-MS/MS for label free assays based on MS detection

Ge Lifesciences Biacore T200 SPR for label free assays based on SPR detection

PerkinElmer Microbeta2 for assays requiring the use of radioisotopically labelled materials.

Please request a quote for additional information.

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Biochemical Assays

Price on request

Assay Development and Testing Services

AMRI provides a comprehensive portfolio of assay development compound testing services for the identification and optimization of novel compounds targeting human diseases including cancer, neurodegenerative and immune system disorders. These include production of recombinant proteins and... Show more »

Assay Development and Testing Services

AMRI provides a comprehensive portfolio of assay development compound testing services for the identification and optimization of novel compounds targeting human diseases including cancer, neurodegenerative and immune system disorders. These include production of recombinant proteins and biochemical assays; development of custom cell lines and cellular assays; implementation of client-developed assays; and compound testing for hit identification and lead optimization. The following is a selection of some of the assay models and services provided:

• Recombinant protein production.

• Cell Line Development

• FLIPR-based assays

• High content imaging based assays / phenotypic assays

• In-Cell Western based assays

• Gene reporter assays

• Fluorescence, FRET, HTRF and luminescence assays (biochemical and cellular)

• MSD and flow cytometry based assays

• MALDI PharmaPulse and LC-MS/MS based label free assays (biochemical and cellular)

• SPR assays and studies (Biacore T200)

• Radiosubstrate uptake and radioisotope incorporation based assays (biochemical and cellular)

• High throughput screening

• Potency and selectivity testing in support of Hit-to-Lead and Lead optimization programs

• Mechanism of action studies

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Microbiology

Price on request

AMRI provides a comprehensive portfolio of antimicrobial discovery services for the identification and optimization of antibacterial and antifungal agents. These include high throughput screening utilizing aerobic, microaerobic or anaerobic conditions; MIC, synergy, time-kill kinetic and resistance testing; hit-to-lead and lead... Show more »

AMRI provides a comprehensive portfolio of antimicrobial discovery services for the identification and optimization of antibacterial and antifungal agents. These include high throughput screening utilizing aerobic, microaerobic or anaerobic conditions; MIC, synergy, time-kill kinetic and resistance testing; hit-to-lead and lead optimization chemistry; in vitro ADMET and DMPK testing; and a complete portfolio of protein expression and assay development capabilities for generation of the materials and assays required to establish target-based biochemical or cellular assays. The antimicrobial testing services offered by AMRI and range of clinical isolates available for testing are briefly summarized below.

• Minimum Inhibitory Concentration (MIC) testing (bacterial and fungal)
• Minimum Bacterial Cidal Concentration (MBC) testing
• Antibiotic Synergy (Checkerboard) testing (bacterial and fungal)
• Time Kill Kinetic testing
• Mutation Rate and Mutation Frequency testing
• Mutation Prevention Concentration (MPC) testing
• Resistance development determination (serial passage)
• Resistance mechanism identification
• Mammalian cytotoxicity testing, standard methods
• Mammalian cytotoxicity testing, High Content Imaging methods
• Mechanism of Action determination
• Efflux pump inhibition
• Antibiotic uptake testing
• High Throughput Antimicrobial Screening (HTS)

MIC Determination, Standard Method
Minimum Inhibitory Concentrations (MICs) are determined using a modified CLSI microbroth dilution assay protocol (document M7), including clinical isolates representing both current and developing antibiotic resistance in a 384-well microplate format which can accommodate up to 8 reference antibiotics or 8 test compounds, in triplicate, with a 12-point, 1:2 dilution series (plus appropriate controls). The CLSI guidelines result in a cutoff determination for the MIC; an exact value can be extrapolated if needed. A serum binding MIC assay can also be performed as discussed in CLSI document M26. Standardized MIC test panels, comprising up to sixty (60) ESKAPE class; thirty (30) Neissria gonorrheae; and ten (10) fungal, clinically relevant isolates are available for those requiring immediate MIC testing encompassing various resistance phenotypes and strains sourced from both Europe and North America. Bespoke MIC panels tailored to match client-specific needs such as species composition, resistance phenotypes and panel size can be generated as needed from AMRI’s extensive collection of recent clinical isolates and then used for rapid sample testing.

MIC Determination, Custom Methods
MICs may also be determined using custom conditions to answer specific questions. For example, by inclusion of serum (to detect the impact of plasma protein binding on antibiotic activity) or salmon sperm DNA (to eliminate DNA interchalators). Custom high content imaging assays combining readouts of efficacy vs. intracellular bacteria and toxicity on the mammalian host cell can be provided.

Minimum Bactericidal Concentration
MBCs are determined after the MIC assay to calculate cidal activity.

Antibiotic Synergy (Checkerboard) Testing
Compounds are tested as nine (9) point, two-fold serial dilutions across the assay plate in combination with an seven (7) point, two-fold serial dilution of the second antibiotic (typically a standard of care antibiotic) down the assay plate. In addition each assay plate includes two (2) columns for control wells of uninhibited growth (no addition of test article) and for drug free sterile control wells (Any clinical isolate from AMRI’s collection may be used for the assay. Test articles are first MIC tested against the strain selected to determine an appropriate range of test concentrations for the synergy test. Fractional Inhibitory Concentration values (FICA and FICB) and FIC index are calculated for each combination of compounds and clinical isolate using the following standard equations for this analysis:
FIC index = FICA+FICB
FICA= MIC of compound A in combination/MIC of compound A alone
FICB= MIC of compound B in combination/MIC of compound B alone
Synergy will be defined as a FIC index value of ≤0.5. Indifference or no interaction will be defined as a FIC index value of >0.5 and <4. Antagonism will be defined as a FIC index value of >4. When the FIC index value is within the range of 0.5 – 1, the combination is considered to be non-synergistic or additive. Results are provided in tabular form.

Time Kill Kinetics
Time Kill Kinetics are determined using various time intervals of the pre -determined MIC over a 24 hour period. This assay can be performed with any of the organisms listed in tables 2 and 3. The time-kill curve is beneficial in identifying possible problems with resistance development or compound degradation over the 24 hour testing period.

Mutation Rate and Frequency
Mutation rate and frequency assays are available to further determine resistance development towards a specific compound. The mutation rate assay is performed following the protocol from Luria et al. (1943) and Foster (2006).

MPC Determination
Mutation Prevention Concentration (MPC) assays are available to determine concentrations at which resistance mutations are unable to develop. It is the lowest drug concentration required to block the growth of the least susceptible cell present in high density bacterial populations. Testing is conducted using large bacterial inocula (≥ 109 cfu/mL).

Resistance Development Determination
Isolated resistant colonies from the Time-kill Kinetics or the Mutation Rate and Frequency studies may be used to determine the potential for resistance development over time and multiple generations. The concentration of test antibiotic is increased in step with the increase in resistance. Solid and liquid media based assays are available.

Resistance mechanism identification
Resistant and non-resistant isolates may be analyzed by RT-PCR or whole genome sequencing to detect expression of resistance-associated genes or changes in target protein (or RNA) sequence associated with resistance. Secondary experiments employing molecular biology, biochemical or biophysical methods may also be undertaken to confirm resistance mechanism.

Cytotoxicity Assay, Standard Methods
Use of a mammalian liver cell line (HepG2) or mammalian normal fibroblast (NHDF) to determine in vitro therapeutic index and exclude overtly toxic compounds. Standard cytotoxicity detection methods employ MTT or Celltiter-Glo.

Cytotoxicity Assay, High Content Imaging Methods
A variety of live cell and fixed cell multi-parametric high content imaging assays are available. Custom assays combining readouts of efficacy vs. intracellular bacteria and toxicity on the mammalian host cell can be provided.

Mechanism of Action Determination
While there is no standard assay, AMRI has extensive experience in determining the mechanism of action of antimicrobial compounds and has developed custom assays to best suit the targets of interest.

Efflux Pump Inhibition
Two detection methods are offered to measure inhibition of drug efflux pumps, ethidium bromide uptake (measure accumulation in the cells) and LC-MS/MS (measure depletion from minimal media and/or accumulation in cells). The latter method may be tailored to monitor a variety of analytes including the components of a combination treatment. Assay is validated for E. coli, P. aeruginosa, and S. aureus (additional strains may be validated as needed).

Antibiotic Uptake
LC-MS/MS is used to measure the accumulation of the antibiotic in the cellular component of the culture after 30 minutes to 1 hour of exposure. As an alternative the depletion of the antibiotic from the media component of the culture may be determined.

High Throughput Antimicrobial Screening
AMRI has developed and conducted a number of natural product sample and small molecule based anti-infective screens. Programs requiring screens against biosafety level 3 organisms or under micro-aerobic conditions can be accommodated. The examples listed below are representative of the non-proprietary detection systems utilized to date and were conducted in single point, 384-well plate format:

  • 280,000 sample natural product screens against S. aureus and E. coli utilizing absorbance / turbidity;
  • 100,000 sample synthetic molecule screens against clinically relevant targets / pathogens (8) utilizing absorbance / turbidity or fluorescence;
  • 100,000 natural product sample screen against fungal pathogens (4) utilizing turbidity or luminescence;
  • 160,000 sample synthetic molecule whole cell anti-virulence screen utilizing luminescence;
    AMRI can provide a highly curated diversity library of 105,000 synthetic compounds to support high throughput screens conducted at AMRI or use any pre-plated natural product sample or synthetic compound collection you may wish to provide for a screen.

Clinical Isolates Available for Testing
The AMRI clinical isolate collection of > 500 isolates includes antibiotic susceptible and resistant isolates for each clinically relevant microbial species. The collection provides comprehensive coverage of current and developing clinical pathogens and antibiotic resistance profiles (blaNDM, blaKPC, blaOXA, blaCTX, blaVIM and mcr-1) from both North America and Europe and new isolates are regularly sourced. A rapid response MIC test panel comprising sixty (60) ESKAPE class clinical isolates and encompassing various resistance phenotypes and strains sourced from Europe and North America is available for those requiring immediate MIC testing.
Testing against custom selections of clinical isolates is also provided with next day testing an option after a custom panel has been established. Bespoke MIC testing panels can be assembled from the AMRI clinical isolate collection as required to meet specific client needs within two to four weeks for panels consisting of assays requiring aerobic or micro-aerobic conditions, respectively. The clinical isolate collection currently includes:
• Gram-positive pathogens (MRSA, S. pneumoniae, and VRE, 80 strains currently available).
• Gram-negative pathogens (424 strains available)
• Carbapenemase-producing MDR Enterobacteriaceae (164 representatives)
• P. aeruginosa (76 representatives)
• A. baumannii (68 representatives)
• N. gonorrhoeae (51 representatives)
• A diverse collection of enteric pathogens (30)
• Drug resistant Candida species (32 representatives)
• Pathogenic filamentous fungi (2 representatives)

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Antimicrobial Activity Testing

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Antimicrobial Activity Testing Services

AMRI provides a comprehensive portfolio of antimicrobial discovery services for the identification and optimization of antibacterial and antifungal agents. These include high throughput screening utilizing aerobic, microaerobic or anaerobic conditions; MIC, synergy, time-kill kinetic and... Show more »

Antimicrobial Activity Testing Services

AMRI provides a comprehensive portfolio of antimicrobial discovery services for the identification and optimization of antibacterial and antifungal agents. These include high throughput screening utilizing aerobic, microaerobic or anaerobic conditions; MIC, synergy, time-kill kinetic and resistance testing; hit-to-lead and lead optimization chemistry; in vitro ADMET and DMPK testing; and a complete portfolio of protein expression and assay development capabilities for generation of the materials and assays required to establish target-based biochemical or cellular assays. The antimicrobial testing services offered by AMRI and range of clinical isolates available for testing are briefly summarized below.

• Minimum Inhibitory Concentration (MIC) testing (bacterial and fungal)
• Minimum Bacterial Cidal Concentration (MBC) testing
• Antibiotic Synergy (Checkerboard) testing (bacterial and fungal)
• Time Kill Kinetic testing
• Mutation Rate and Mutation Frequency testing
• Mutation Prevention Concentration (MPC) testing
• Resistance development determination (serial passage)
• Resistance mechanism identification
• Mammalian cytotoxicity testing, standard methods
• Mammalian cytotoxicity testing, High Content Imaging methods
• Mechanism of Action determination
• Efflux pump inhibition
• Antibiotic uptake testing
• High Throughput Antimicrobial Screening (HTS)

MIC Determination, Standard Method
Minimum Inhibitory Concentrations (MICs) are determined using a modified CLSI microbroth dilution assay protocol (document M7), including clinical isolates representing both current and developing antibiotic resistance in a 384-well microplate format which can accommodate up to 8 reference antibiotics or 8 test compounds, in triplicate, with a 12-point, 1:2 dilution series (plus appropriate controls). The CLSI guidelines result in a cutoff determination for the MIC; an exact value can be extrapolated if needed. A serum binding MIC assay can also be performed as discussed in CLSI document M26. Standardized MIC test panels, comprising up to sixty (60) ESKAPE class; thirty (30) Neissria gonorrheae; and ten (10) fungal, clinically relevant isolates are available for those requiring immediate MIC testing encompassing various resistance phenotypes and strains sourced from both Europe and North America. Bespoke MIC panels tailored to match client-specific needs such as species composition, resistance phenotypes and panel size can be generated as needed from AMRI’s extensive collection of recent clinical isolates and then used for rapid sample testing.

MIC Determination, Custom Methods
MICs may also be determined using custom conditions to answer specific questions. For example, by inclusion of serum (to detect the impact of plasma protein binding on antibiotic activity) or salmon sperm DNA (to eliminate DNA interchalators). Custom high content imaging assays combining readouts of efficacy vs. intracellular bacteria and toxicity on the mammalian host cell can be provided.

Minimum Bactericidal Concentration
MBCs are determined after the MIC assay to calculate cidal activity.

Antibiotic Synergy (Checkerboard) Testing
Compounds are tested as nine (9) point, two-fold serial dilutions across the assay plate in combination with an seven (7) point, two-fold serial dilution of the second antibiotic (typically a standard of care antibiotic) down the assay plate. In addition each assay plate includes two (2) columns for control wells of uninhibited growth (no addition of test article) and for drug free sterile control wells (Any clinical isolate from AMRI’s collection may be used for the assay. Test articles are first MIC tested against the strain selected to determine an appropriate range of test concentrations for the synergy test. Fractional Inhibitory Concentration values (FICA and FICB) and FIC index are calculated for each combination of compounds and clinical isolate using the following standard equations for this analysis:
FIC index = FICA+FICB
FICA= MIC of compound A in combination/MIC of compound A alone
FICB= MIC of compound B in combination/MIC of compound B alone
Synergy will be defined as a FIC index value of ≤0.5. Indifference or no interaction will be defined as a FIC index value of >0.5 and <4. Antagonism will be defined as a FIC index value of >4. When the FIC index value is within the range of 0.5 – 1, the combination is considered to be non-synergistic or additive. Results are provided in tabular form.

Time Kill Kinetics
Time Kill Kinetics are determined using various time intervals of the pre -determined MIC over a 24 hour period. This assay can be performed with any of the organisms listed in tables 2 and 3. The time-kill curve is beneficial in identifying possible problems with resistance development or compound degradation over the 24 hour testing period.

Mutation Rate and Frequency
Mutation rate and frequency assays are available to further determine resistance development towards a specific compound. The mutation rate assay is performed following the protocol from Luria et al. (1943) and Foster (2006).

MPC Determination
Mutation Prevention Concentration (MPC) assays are available to determine concentrations at which resistance mutations are unable to develop. It is the lowest drug concentration required to block the growth of the least susceptible cell present in high density bacterial populations. Testing is conducted using large bacterial inocula (≥ 109 cfu/mL).

Resistance Development Determination
Isolated resistant colonies from the Time-kill Kinetics or the Mutation Rate and Frequency studies may be used to determine the potential for resistance development over time and multiple generations. The concentration of test antibiotic is increased in step with the increase in resistance. Solid and liquid media based assays are available.

Resistance mechanism identification
Resistant and non-resistant isolates may be analyzed by RT-PCR or whole genome sequencing to detect expression of resistance-associated genes or changes in target protein (or RNA) sequence associated with resistance. Secondary experiments employing molecular biology, biochemical or biophysical methods may also be undertaken to confirm resistance mechanism.

Cytotoxicity Assay, Standard Methods
Use of a mammalian liver cell line (HepG2) or mammalian normal fibroblast (NHDF) to determine in vitro therapeutic index and exclude overtly toxic compounds. Standard cytotoxicity detection methods employ MTT or Celltiter-Glo.

Cytotoxicity Assay, High Content Imaging Methods
A variety of live cell and fixed cell multi-parametric high content imaging assays are available. Custom assays combining readouts of efficacy vs. intracellular bacteria and toxicity on the mammalian host cell can be provided.

Mechanism of Action Determination
While there is no standard assay, AMRI has extensive experience in determining the mechanism of action of antimicrobial compounds and has developed custom assays to best suit the targets of interest.

Efflux Pump Inhibition
Two detection methods are offered to measure inhibition of drug efflux pumps, ethidium bromide uptake (measure accumulation in the cells) and LC-MS/MS (measure depletion from minimal media and/or accumulation in cells). The latter method may be tailored to monitor a variety of analytes including the components of a combination treatment. Assay is validated for E. coli, P. aeruginosa, and S. aureus (additional strains may be validated as needed).

Antibiotic Uptake
LC-MS/MS is used to measure the accumulation of the antibiotic in the cellular component of the culture after 30 minutes to 1 hour of exposure. As an alternative the depletion of the antibiotic from the media component of the culture may be determined.

High Throughput Antimicrobial Screening
AMRI has developed and conducted a number of natural product sample and small molecule based anti-infective screens. Programs requiring screens against biosafety level 3 organisms or under micro-aerobic conditions can be accommodated. The examples listed below are representative of the non-proprietary detection systems utilized to date and were conducted in single point, 384-well plate format:

  • 280,000 sample natural product screens against S. aureus and E. coli utilizing absorbance / turbidity;
  • 100,000 sample synthetic molecule screens against clinically relevant targets / pathogens (8) utilizing absorbance / turbidity or fluorescence;
  • 100,000 natural product sample screen against fungal pathogens (4) utilizing turbidity or luminescence;
  • 160,000 sample synthetic molecule whole cell anti-virulence screen utilizing luminescence;
    AMRI can provide a highly curated diversity library of 105,000 synthetic compounds to support high throughput screens conducted at AMRI or use any pre-plated natural product sample or synthetic compound collection you may wish to provide for a screen.

Clinical Isolates Available for Testing
The AMRI clinical isolate collection of > 500 isolates includes antibiotic susceptible and resistant isolates for each clinically relevant microbial species. The collection provides comprehensive coverage of current and developing clinical pathogens and antibiotic resistance profiles (blaNDM, blaKPC, blaOXA, blaCTX, blaVIM and mcr-1) from both North America and Europe and new isolates are regularly sourced. A rapid response MIC test panel comprising sixty (60) ESKAPE class clinical isolates and encompassing various resistance phenotypes and strains sourced from Europe and North America is available for those requiring immediate MIC testing.
Testing against custom selections of clinical isolates is also provided with next day testing an option after a custom panel has been established. Bespoke MIC testing panels can be assembled from the AMRI clinical isolate collection as required to meet specific client needs within two to four weeks for panels consisting of assays requiring aerobic or micro-aerobic conditions, respectively. The clinical isolate collection currently includes:
• Gram-positive pathogens (MRSA, S. pneumoniae, and VRE, 80 strains currently available).
• Gram-negative pathogens (424 strains available)
• Carbapenemase-producing MDR Enterobacteriaceae (164 representatives)
• P. aeruginosa (76 representatives)
• A. baumannii (68 representatives)
• N. gonorrhoeae (51 representatives)
• A diverse collection of enteric pathogens (30)
• Drug resistant Candida species (32 representatives)
• Pathogenic filamentous fungi (2 representatives)

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