M4 Lead Identification and Cell-Based Assays

Lead Identification

Learning Objectives

  • Develop a cell-based assay based on the identified disease target and molecular target.

  • Examine features of cell-based assays.

  • Utilize cell-based assays to screen hits and convert them into leads (HITs to leads).

From Hits to Leads

  • The previous module detailed techniques for measuring interactions between small molecules and molecular targets.

  • Molecules that interact with the target are called "hits."

  • Hits vary in their strength of interaction with the target.

  • Screening 50,000 compounds might yield 0.1% positives (compounds that interact with the molecular target).

  • This could result in about 500 compounds to screen, which is still a large number.

  • Reducing This Number:

    • Categorizing compounds into classes.

    • Screening those with binding strength above a certain threshold.

Developing a Cell-Based Assay

  • When developing a bioassay:

    • It should be cell-based.

    • It should be simple to perform (few steps).

    • It should be based on a measurable biological activity of the target molecule.

    • Critically, it should be able to be miniaturized.

Cystic Fibrosis Example

  • Target disease: Cystic Fibrosis (CF)

  • Target molecule: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, a chloride channel.

  • CFTR Mutations:

    • Class 1: No protein synthesized.

    • Class 2: Truncated protein synthesized.

    • Class 3: Protein synthesized with mutation, stuck in the ER.

    • Class 4 & 5: Other types of mutations

    • Class II mutations are found in nearly 80% of CF patients.

Details of Class II Mutation (Delta F508)

  • CFTR is made in the ER.

  • There is a deletion at residue number 508 (delta F508 - deletion of phenylalanine at position 508).

  • This deletion causes the protein to misfold and be retained in the ER.

  • Only 5% of the protein reaches the plasma membrane.

  • Crucially, the 5% that reaches the membrane is fully functional.

Developing a Cell-Based Assay for CFTR

  • Start with messenger RNA (mRNA).

  • Use RT-PCR to clone the wild-type and delta F508 forms of the protein.

  • Clone these into mammalian expression vectors.

  • Transfect these vectors into epithelial cell lines.

  • Plate cells expressing either wild-type or delta F508 CFTR in a 96-well plate.

  • Measure chloride flux across the membrane in the presence or absence of identified hits.

  • Hits that stimulate CFTR activity become leads.

  • Stimulating CFTR activity implies promoting trafficking of CFTR from the ER to the membrane.