The Beijer Laboratory, Science for Life Laboratory, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden.
Pivot Park Screening Centre, Kloosterstraat 9, 5349 AB Oss, The Netherlands.
Int J Mol Sci. 2024 Apr 6;25(7):4084. doi: 10.3390/ijms25074084.
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 IC 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 IC 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.
我们的目标是鉴定新的化学起点，可以进一步优化为胰岛素调节氨肽酶 (IRAP) 的小分子抑制剂，并作为潜在的未来临床认知增强剂。为此，我们进行了一次超大规模的、约 40 万个具有药物特性的小分子化合物化学多样性文库的高通量筛选活动。我们开发了三种生化和一种生物物理测定法，以实现大规模筛选和命中初筛。筛选漏斗设计为与高密度微孔板兼容，采用两种酶抑制测定法，分别使用荧光或吸光度读数。由于 IRAP 是一种锌依赖性酶，因此剩余的活性化合物在主要测定法中进一步进行评估，尽管添加了锌离子。用锌重新筛选证实了大多数化合物的抑制活性，强调了一种非锌依赖性作用机制。此外，使用互补的生物物理热移位测定法确认了靶标结合，其中引起正/负热移位的化合物被认为是真正的结合物。基于生化活性、靶标结合和药物特性的初筛，选择了 50 个合格的命中化合物，其中 32 个化合物的 IC 低于 3.5 µM。尽管羟肟酸占据主导地位，但仍发现了具有生化活性和靶标结合的多样化化学型，包括非羟肟酸化合物。最有效的化合物 (QHL1) 经重新合成，其抑制 IC 被确认为 320 nM。在这些化合物中，确定了 20 种新的化合物结构类别，为开发独特的 IRAP 抑制剂提供了许多新的起点。正在对先导化合物进行详细的表征和优化，同时考虑羟肟酸和其他多样化的结构，以进行进一步探索。
