Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists

Abstract

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC 50 values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms. (Journal of Biomolecular Screening 2009:679-689)

Supergenomic Network Compression and the Discovery of EXP1 as a Glutathione Transferase Inhibited by Artesunate

“…These assays revealed competitive inhibition (Figure S5E) with a half-maximal inhibitory concentration (IC 50 ) of 2.05 nM (Figure 4C and 5E). Non-specific inhibition was excluded through stoichiometry controls (Habig et al, 2009): after reducing enzyme concentration tenfold, from 100 nM to 10 nM, potency changed by only ∼25% and remained within error margins (Figure 5F). A control through liquid chromatography-mass spectrometry (LC-MS) confirmed an ART-dependent suppression of EXP1-mediated GSH-hematin adduct formation down to spontaneous levels (Figure 5F inset and S5F).…”

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…[43,87] In many more cases, detergents are included in the assay buffer for the primary screen right away, [56] which obviates the need for a separate counterscreen, even if this motivation is rarely mentioned explicitly. [30,88] In contrast, demonstrating stoichiometric inhibition by measuring potency at different concentrations of enzyme [25] typically is used later in the flowchart, [52] where the limited number of compounds allows for dose-response experiments. The same holds for direct measurements of compound aggregation which often are used in conjunction with other experimental methods, e.g.…”

“…[49] Another technique to detect aggregation of a compound is nuclear magnetic resonance (NMR), which either analyzes the signal from the compound itself [50] or uses the conformational distortion of the human La antigen as a surrogate readout in what is called ALARM NMR. [18,51] An indirect approach compares compound potency at different concentrations of enzyme [25,52]; bellshaped dose-response curves in cellular assays are another warning signal. One of several possible explanations is the failure of colloidal aggregates to enter intact cells.…”

“…Although stoichiometric binding has so far been difficult to measure in cellular systems and the term non-stoichiometric inhibition was originally coined in a biochemical context, [25] hit lists from cellular screening in practice can be affected by similar issues as those from biochemical screens. Therefore, this review extrapolates to cellular assays wherever appropriate and contains an additional section specifically discussing cellular target engagement.…”

A Label-Free LC/MS/MS-Based Enzymatic Activity Assay for the Detection of Genuine Caspase Inhibitors and SAR Development

“…Such a detergent dependence [12][13][14] and a steep Hill slope 19 have been proposed as markers of aggregation-based nonstoichiometric inhibition. Although there has been some recent debate on the usefulness and accuracy of detergent sensitivity as a criterion to eliminate artifactual inhibitors, 20 a Hill coefficient well above 1 in the absence of detergent combined with complete abrogation of activity in the pre sence of ionic or nonionic detergent strongly suggests that 1 is not a stoichiometric inhibitor of Csp-3. In the case of Csp-6, the detergent effect was not as pronounced as for Csp-3, but a high Hill coefficient coupled with unusual inhibition curves such as partial inhibition indicated suspicious behavior.…”