Researchers, if your compound

• Has strong noncompetitive inhibition
• Has an unusually steep dose response curve
• Exhibits time dependent inhibition
• Does not have clear structure-activity relationships

It’s not your next big hit. It could be an aggregator instead.

These are some general pointers for flagging out false positives when screening for new hits (B. Shiochet PMID: 16793529). As PK scientists, this is also highly applicable to us, especially in screening drug-drug interactions, where microsomes or recombinant CYP enzymes are commonly used. As both microsomes and recombinant CYPs present as micelles or free floating proteins in solution, they are also susceptible to this aggregation phenomenon, resulting in compounds being falsely flagged as potent inhibitors of CYPs.

Aggregators cause inhibition by causing enzymes and proteins in the reaction solution to clump together, making the enzymes unable to catalyze new reactions.  This in turn results in a strong inhibition, even when the compound in question does not impact the enzyme’s active site either via direct binding (competitive inhibition) or influencing the active site conformation by binding elsewhere on the protein (noncompetitive inhibition).

In reality, however, CYP enzymes are embedded on intracellular rough endoplasmic reticulum, making them much less susceptible to aggregation occurring when kept within intact cells and organs.

These aggregates can be identified via dynamic light scattering which helps us detect the actual clumps in solution, or via the addition of a mild detergent, which can break up the aggregate, allowing the enzyme to regain activity.

Understanding how to detect aggregators, which are in vitro artefacts, is thus important in order that we do not wrongly classify enzyme inhibitors, and thus drug-drug interactions.

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