Cell based and functional assays Flashcards

1
Q

What are the primary types of assays used in drug discovery?

A

1) Cell-based assays: Assess compound effects in living cells to understand biological activity.
2) Functional assays: Measure the biological activity of compounds, such as enzyme activity or receptor binding.

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2
Q

Why are cell-based assays important?

A

They provide a physiologically relevant environment, allowing researchers to assess cellular pathway effects, target engagement, off-target effects, and phenotypic changes such as cytotoxicity, proliferation, and apoptosis.

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3
Q

What are key examples of cell-based assays?

A

1) Cytotoxicity Assays: Measure cell viability (e.g., MTT/MTS, LDH release).
2) Proliferation Assays: Assess cell growth (e.g., BrdU incorporation).
3) Apoptosis Assays: Detect programmed cell death (e.g., Annexin V, caspase activity).
4) Reporter Gene Assays: Measure pathway activation (e.g., luciferase assay).
5) Phenotypic Assays: Study changes in morphology or differentiation.

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4
Q

How do reporter gene assays like the TCF-LEF luciferase assay work?

A

The assay monitors Wnt/β-catenin pathway activity. Wnt3a activates β-catenin, which enters the nucleus and interacts with TCF/LEF transcription factors to activate luciferase. Luminescence measures pathway activation, correlating with transcriptional activity.

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5
Q

What are target engagement assays, and give examples?

A

Target engagement assays confirm compound binding to intended targets. Examples include:
1) Cellular Thermal Shift Assay (CETSA): Measures protein stability upon binding.
2) NanoBRET: Detects molecular proximity through bioluminescence resonance energy transfer.

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6
Q

Describe CETSA and its application.

A

CETSA monitors protein stability upon compound binding. Protein aggregation is reduced when a compound binds, stabilizing the protein and increasing the AlphaLisa signal. It validates target engagement in live cells.

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7
Q

How does NanoBRET assess target engagement?

A

NanoBRET uses an energy transfer technique between a fluorescent tracer and NanoLuc fusion protein. Compound binding displaces the tracer, reducing the NanoBRET signal, which provides data on binding affinity, permeability, and residence time.

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8
Q

What are functional assays, and what do they measure?

A

Functional assays assess a compound’s biological effect on a target. Examples include:
1) Enzyme Activity Assays: Measure inhibition/activation (e.g., kinase or protease activity).
2) Receptor-Ligand Binding Assays: Quantify receptor binding affinity (e.g., radioligand binding).
3) GPCR Signaling Assays: Detect downstream effects (e.g., cAMP or β-arrestin recruitment).

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9
Q

What are examples of functional assay methods?

A

1) Kinase assays: Use labeled substrates to track phosphorylation.
2) Patch-clamp techniques: Measure ionic currents in ion channel assays.
3) Flow cytometry: Quantify surface markers using fluorescent antibodies.

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10
Q

What are the advantages and limitations of cell-based assays?

A

Advantages: Physiological relevance, pathway insights, broad applications.
Limitations: High cost, time-consuming, variability, and potential differences between in vitro and in vivo results.

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11
Q

What are the advantages and limitations of functional assays?

A

Advantages: Provide mechanistic insights, quantitative data, and high-throughput adaptability.
Limitations: Limited context (e.g., biochemical assays lack cellular relevance), potential for false positives/negatives, and assay interference.

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12
Q

How does high-throughput screening (HTS) function in drug discovery?

A

HTS involves testing large libraries of >100,000 compounds using automated systems to identify biologically active molecules. HTS generates leads for drug discovery pipelines and can help answer basic research questions.

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13
Q

What factors influence experimental design in assays?

A

Key factors include:
1) Biological system: Primary/native cells, engineered cell lines, or model organisms.
2) Assay type: Functional, phenotypic, reporter gene.
3) Readout: Fluorescence, luminescence, spectrophotometric, etc.
4) Optimization: Plate type, incubation times, buffer conditions, and assay robustness.

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14
Q

How do you optimize an assay for reliability?

A

Optimize parameters such as plate type (e.g., white/black, 96/384 wells), cell density, reagents, and incubation times. Evaluate DMSO tolerance, signal-to-background ratios, and statistical parameters like Z-prime for robustness.

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15
Q

What is the goal of DMSO tolerance testing in assays?

A

DMSO is a common solvent for compounds. Testing ensures the assay can tolerate DMSO concentrations up to 1% without affecting results, ensuring compound solubility does not interfere with assay performance.

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16
Q

What detection methods are used in assays?

A

Common methods include:
1) Fluorescence: Intensity, polarization, time-resolved.
2) Luminescence: Light emission for reporter gene assays.
3) Spectrophotometric: Measures absorbance or colorimetric changes.

17
Q

What are the key considerations when planning an assay?

A

1) Test multiple parameters simultaneously to save time.
2) Establish agonist curves to determine EC50.
3) Scale assays for higher throughput (e.g., transition from 384- to 1536-well plates).
4) Ensure standardization and validation of results.