M3 HIT Identification Notes

HIT Identification

Learning Objectives

• Understand the difficulty of screening molecules to identify hits, leads, and ultimately drugs.

• Examine Lipinski's Rule of Five.

• Explore High Throughput Screening (HTS).

• Look at the use of NMR spectroscopy in identifying hits.

• Explore the use of Surface Plasmon Resonance (SPR) and Scintillation Proximity Assay (SPA) in identifying hits.

Screening Molecules for Hits

• Once a molecular target and its pathway are identified, screening small molecules becomes possible.

• The task is challenging due to the vast number of compounds.

• There are 70,000,000 compounds registered in the Chemical Abstract Service database.

• Choosing a starting point is crucial for efficient screening.

Lipinski's Rule of Five

• Helps identify molecules that are good candidates to become hits, leads, and drugs.

• States criteria for a molecule to be "drug-like":

  • Molecular weight less than 500.

  • Partition coefficient (log P) less than 5.

    • $\log P$: Measure of solubility in octanol and water mixture.

    • Higher ${log P}$ indicates greater solubility in the hydrophobic (octanol) phase.

    • Lower ${log P}$ indicates greater solubility in the hydrophilic (water) phase.

  • Less than 5 H-bond donors (electron-rich atoms with hydrogen).

  • Less than 10 H-bond acceptors (electronegative and electron-rich atoms).

  • Less than 10 rotatable single bonds (not involving hydrogen).

Sources of Molecules for Screening

• Natural product libraries:

  • Plants.

  • Animals.

  • Marine organisms.

  • Microbes.

  • Toxins and venoms (e.g., snakes).

• Synthetic compound libraries.

• Proprietary libraries:

  • Libraries that have undergone initial screening stages.

  • Made available in the drug discovery process.

Methods for Screening

• High Throughput Screening (HTS):

  • Physical.

  • Virtual (in silico).

• NMR Spectroscopy.

• Surface Plasmon Resonance (SPR).

• Scintillation Proximity Assay (SPA).

High Throughput Screening (Physical)

• Isolate and purify the molecular target (protein).

• Distribute it into 96-well plates.

• Determine which molecules from the library physically interact with the target.

Virtual High Throughput Screening

• Uses computer automation to identify molecules that can bind to the molecular target.

• Streamlines the process using selected characteristics.

High Throughput Screening: Ligand Displacement Assay

• Setup:

  • Protein in a well.

  • Labeled ligand bound to the protein.

  • Add the molecule under consideration.

• Measurement:

  • Displacement of the labeled ligand from the receptor.

• Indication:

  • Displacement suggests interaction between the molecule and the target.

NMR Spectroscopy

• Measures the affinity of the molecular target (receptor) for a molecule.

• Process:

  • Compound irradiated with a short pulse of energy.

  • Excites nuclei of specific atoms (hydrogen, nitrogen, carbon).

  • Excited nuclei relax back to ground state, emitting energy.

  • Relaxation time is measured.

• Principle:

  • Relaxation times of ligands bound to a macromolecule are shorter than when unbound.

Surface Plasmon Resonance (SPR) and Scintillation Proximity Assay (SPA)

• SPR measures the refractive index change upon binding of a ligand to the molecular target.

• SPA measures changes in light emission upon binding of a ligand to the molecular target.

• Both methods assess the presence or absence of binding.