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

ABSTRACT

Introduction: Non-stoichiometric inhibition summarizes different mechanisms by which low-molecular weight compounds can reproducibly inhibit high-throughput screening (HTS) and other lead finding assays without binding to a structurally defined site on their molecular target. This disqualifies such molecules from optimization by medicinal chemistry, and therefore their rapid elimination from screening hit lists is essential for productive and effective drug discovery.

Areas covered: This review covers recent literature that either investigates the various mechanisms behind non-stoichiometric inhibition or suggests assays and readouts to identify them. In addition, combination of the various methods to distill promising molecules out of raw primary hit lists step-by-step is considered. Emerging technologies to demonstrate target engagement in cells are also discussed.

Expert opinion: Over the last few years, awareness of non-stoichiometric inhibitors within screening libraries and HTS hit lists has considerably increased, not only in the pharmaceutical industry but also in the academic drug discovery community. This has resulted in a variety of methods to detect and handle such compounds. These range from in silico approaches to flag suspicious compounds, and counterassays to measure non-stoichiometric inhibition, to biophysical methods that positively demonstrate stoichiometric binding. In addition, novel technologies to verify target engagement within cells are becoming available. While still a time- and resource-consuming nuisance, non-stoichiometric inhibitors therefore do not fundamentally jeopardize the discovery of low molecular weight lead and drug candidates. Rather, they should be viewed as a manageable issue that with appropriate expertise can be overcome through integration of the above-mentioned approaches.

Keywords:

• Non-stoichiometric inhibition
• integrated lead finding
• HTS (high-throughput screening)
• PAINS (pan-assay interfering compounds)
• false-positive
• frequent hitter
• compound aggregation
• redox cycling
• counterscreen
• orthogonal assay
• binding assay
• target engagement

Acknowledgments

The fruitful collaboration and stimulating discussions with numerous Novartis colleagues in both project teams and line functions are deeply appreciated as they were essential to get a grip on non-stoichiometric inhibitors in our screening hit lists. Particularly valuable was critical reading of and input to the section on in silico analysis by Steffen Renner. Special thanks for reviewing of the manuscript with regard to English usage are due to Sandra Jacob.

ORCID

Martin Klumpp http://orcid.org/0000-0003-3373-9605