Determining the Mechanism of Action
A mechanism of action (MOA) is a specific biochemical interaction through which small molecule substances exert their pharmacological effect.

Discovery teams employ high-throughput screening (HTS) to test large libraries of compounds and pinpoint those that act through a targeted mechanism of action. To determine a single compound’s mechanism of action, discovery teams expose the compound to a battery of ion channels, enzymes, transporter molecules and receptors.

Small Molecule Actions
Small molecules can interact with the following target sites in a cell:

• Receptors (e.g., Benzodiazepine, Kainic Acid, Mifepristone, Terazosin)
• Ion channels (e.g., Astemizole, DLAP5.Na, Felodipine, Amlodipine)
• Enzymes (e.g., Triacsin-C, Phenylbutyrate Na, Necrostatin-1, Resveratrol)
• Transporter molecules

The majority of small molecules exert their effect by interacting with receptors located on the cell surface or within the cytoplasm or nucleus. Receptors are macromolecules that are involved in chemical signaling between and within cells. There are multiple ways a ligand can engage a receptor:

• Full agonist: a ligand that binds with high affinity and full efficacy to produce a maximal effect
• Partial agonist: a ligand that binds with high affinity and partial efficacy and produces a sub-maximal effect regardless of small molecule concentration (full receptor occupancy)
• Inverse agonist: a ligand that binds with high affinity and negative efficacy a response opposite to that of the agonist
• Antagonist: a ligand blocks activity at the receptor

A small molecule’s ability to affect a given receptor is related to the small molecule’s affinity and intrinsic efficacy. In turn, a small molecule’s affinity and efficacy are determined by its chemical structure.

In some cases, a molecule comprises two enantiomers with differing biological activity. Thalidomide is an example of a small molecule with stereoisomers – they have the same molecular formula and the atoms form the same connections. However, the enantiomers differ in the way they are arranged in space. Thalidomide is a chiral molecule, or asymmetric, in that the two halves cannot be superimposed.²¹ Other examples of chiral molecules include: glyceraldehyde, betamethasone, amphetamine, alanine, and lactate.

Thalidomide was prescribed to pregnant women in the 1960s and caused spontaneous abortions and severe birth defects. One thalidomide isomer carried the efficacy of the drug, while the other carried the teratogenic effects. Modern drug discovery and development considers the importance of chirality, in part, as a result of research on thalidomide’s two enantiomers. Beginning in 1992, US Food and Drug Administration (FDA) identified and provided guidance for chiral-specific issues such as appropriate manufacturing controls, product stability, pharmacokinetic evaluations, and quantifications.²² FDA Guidance mandated that a chiral drug’s composition must be known when it is used in pharmacological, toxicological, or clinical studies.²³

The Chemical Basis for Drug-Receptor Interactions
Drugs can interact with receptors through a variety of chemical interactions including:

• Electrostatic interactions (ionic bond, hydrogen bonds, Van der Waals forces) – the most common mechanism
• Hydrophobic interactions (important for lipid soluble drugs)
• Covalent bonds (e.g. phenoxybenzamine binding to α-adrenergic receptors) – least common
• Stereospecific interactions (>50% of drugs exist as stereoisomers and interact stereospecifically with receptors. (e.g. S (-) Carvedilol binds to both α-adrenoceptors and β-adrenergic receptors, whereas R(+) Carvedilol binds selectively to α-adrenergic receptors)

Summary
In summary, discovery teams assess a small molecule’s MOA, or they begin with a targeted MOA and find a molecule to match. Teams must consider chirality of the molecule and run assays to ensure that the characteristics of the enantiomers are known. Single enantiomer or achiral products comprise most newly approved drugs.²⁴ Small molecules can act at the receptor site in a variety of ways that activate or deactivate the receptor.