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12/6 - Seminar (organic): Michael Rabinowitz, Janssen Pharmaceutical Research & Development, LLC

Michael Rabinowitz
Research Fellow and Team Leader
Janssen Pharmaceutical Research & Development, LLC
Host: David MacMillan

Structure based design and biological evaluation of benzimidazole HIF prolyl hydroxylase inhibitors for the treatment of anemia

The discovery in 1991 of the oxygen-dependent transcription factor HIF as a key mediator of the mammalian response to hypoxia, and the subsequent identification in 2001 that oxygen-dependent iron-containing hydroxylases (PHD, Prolyl Hydroxylase Domain-containing protein) mediate the degradation of HIF, has led to the hypothesis that the beneficial effects of low oxygen levels (enhanced erythropoiesis, preconditioned ischemia protection, improved metabolic function, etc.) may be promoted pharmacologically with a small molecule PHD inhibitor.

Using structure based design for specific compounds employing both literature crystal structures and ligand docking, we have identified certain glycine amide heterocyclic analogs of 2-oxoglutarate that competitively and potently inhibit prolyl hydroxylases (IC50 < 100 nM) in vitro and stabilize HIF protein and stimulate EPO production in whole cell cultures. We also have shown that simple divalent metal chelators (such as certain 8-hydroxyquinolines and 1,10-phenanthrolines) functionally inhibit PHD enzymes, but via a non-competitive process that likely involves general iron chelation. By setting up primary functional assays for both PHD inhibition as well as iron chelation, we created a compound analysis and progression path that rapidly separated mechanism-based inhibitors from general iron chelators allowing us to pursue only the former.

Our initial work produced eight distinct chemotypes, of which a thiadiazole series displayed PHD inhibition activity (IC50 = 60 uM) with poor Fe chelation. An x-ray co-crystal structure with PHD2 showed certain ligand-protein interactions: chelation of the bound Fe atom and a salt bridge between the inhibitor and Arg383. Comparison with interactions of PHD2 and the known inhibitor [(1-chloro-4-hydroxyisoquinoline-3-carbonyl)-amino]acetic acid showed that the relatively weak activity of certain compounds in this series could be improved by incorporating the hydrogen-bond interaction and additional van der Waals interactions evident in the crystal structure of [(1-chloro-4-hydroxyisoquinoline-3-carbonyl)-amino]acetic acid. Replacement of the glycine amide with certain heterocycles resulted in marked improvement in both enzyme IC50 as well as whole cell ED50 potencies.

Further studies resulted in the identification of specific benzimidazole compounds that showed improved activity over the related benzothiadiazole analogs, but without the addition of a hydroxyl group, which has proven important in nearly all other literature inhibitor series in picking up a key hydrogen bond interaction with Tyr303. The structural basis for the potent binding of this series to the PHD2 enzyme in the absence of this key interaction will be discussed. Optimization for enzyme potency and whole cell activity resulted in selected compounds with good pharmacokinetic properties that were highly efficacious acute stimulators of EPO production in vivo as well as potent promoters of hemoglobin production upon chronic oral dosing in rodents.

In this talk, the above x-ray structure analysis program, including new x-ray co-crystal structures, resulting compounds, and associated data will be presented as a new approach in the treatment of anemia.