The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds

Abstract

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

Stabilization of the open conformation of Insulin-Regulated Aminopeptidase by a novel substrate-selective small molecule inhibitor

“…As a result, several efforts have been initiated to develop pharmacologically useful inhibitors of IRAP's aminopeptidase activity, in particular in the areas of cognition 7,8 and fibrosis 9,10 . IRAP inhibitors have been developed either by rational substrate-inspired design or after random chemical or virtual library screening and include pseudophosphinic peptide transition-state-analogues, aryl sulfonamides, benzopyran derivatives, diaminobenzoic acid derivatives, imidazopyridines and cyclic peptide analogues [11][12][13] . Several of these compounds have been shown to be active in cellular and in in vivo systems relating to IRAP biological activities, such as promoting the formation of functional dendritic spines in primary hippocampal neuron cultures 14 , regulation of acetylcholine-mediated vasoconstriction 15 and glucose tolerance in insulin-resistant Zucker fatty rats 16 .…”