Abstract
Most enzymes act on more than a single substrate. There is frequently a need to block the production of a single pathogenic outcome of enzymatic activity on a substrate but to avoid blocking others of its catalytic actions. Full blocking might cause severe side effects because some products of that catalysis may be vital. Substrate selectivity is required but not possible to achieve by blocking the catalytic residues of an enzyme. That is the basis of the need for "Substrate Selective Inhibitors" (SSI), and there are several molecules characterized as SSI. However, none have yet been designed or discovered by computational methods. We demonstrate a computational approach to the discovery of Substrate Selective Inhibitors for one enzyme, Prolyl Oligopeptidase (POP) (E.C 3.4.21.26), a serine protease which cleaves small peptides between Pro and other amino acids. Among those are Thyrotropin Releasing Hormone (TRH) and Angiotensin-III (Ang-III), differing in both their binding (Km) and in turnover (kcat). We used our in-house "Iterative Stochastic Elimination" (ISE) algorithm and the structure-based "Pharmacophore" approach to construct two models for identifying SSI of POP. A dataset of ~1.8 million commercially available molecules was initially reduced to less than 12,000 which were screened by these models to a final set of 20 molecules which were sent for experimental validation (five random molecules were tested for comparison). Two molecules out of these 20, one with a high score in the ISE model, the other successful in the pharmacophore model, were confirmed by in vitro measurements. One is a competitive inhibitor of Ang-III (increases its Km), but non-competitive towards TRH (decreases its Vmax).
Highlights
Inhibitors of excess activities of proteins, mainly of enzymes, form a major group of clinical drugs
We demonstrate a computational approach to the discovery of Substrate Selective Inhibitors for one enzyme, Prolyl Oligopeptidase (POP) (E.C 3.4.21.26), a serine protease which cleaves small peptides between Pro and other amino acids
In collaboration with a research group that excels in studying that specific enzyme we found that two candidates out of a set of twenty that we picked out of 1.8 million molecules by filtering through computer models —are selective to one substrate vis-a-vis the other
Summary
Inhibitors of excess activities of proteins, mainly of enzymes, form a major group of clinical drugs. Most of these therapeutics cause side effects. The common explanation of side effects is that drugs interact with more than a single targeted protein due to similarity among enzyme families. Effective blocking of an enzyme’s active site might result in interfering with activities of other enzymes which are essential to the balance and viability of the biological system (called "off targets" or "anti-targets"). Due to the increase in the understanding of the commonalities in enzyme mechanisms and of enzyme "families", assay panels of proteins have become available in order to examine the selectivity of candidate inhibitor drugs.
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