Hypoxia‐selective allosteric destabilization and degradation of a receptor protein kinase: potential prophylaxis by H‐SAAD/Ds

Abstract

Development of competitive inhibitors that target the ATP-binding cleft of protein kinases has been a major pursuit of academic and industrial laboratories for decades. However, due to the number (500+) of protein kinases and the similarity in structure of the ATP -Mg2+-complex binding site, disproportionately few inhibitors have reached clinics and emerged from trials considering the substantial investment of time, money and effort. As a result, inhibitors that bind at sites other than the substrate-binding pocket, allosterically acting small molecules, have gained increasing attention as innovative alternatives that are not constrained by the limitations inherent to typical competitive inhibitors of protein kinases. Our dissection of the structural basis of the gain-of-function by a shared mutation in a bone morphogenetic protein (BMP) receptor kinase (ACVR1/ALK2), causative of a rare but highly debilitating bone disorder, indicated that the active site was rendered accessible to a broader range of polypeptide substrates. Thus we hypothesized that sites other than the ATP-binding cleft could be targets for therapeutic intervention by pharmacological chaperones that buffered the gain in plasticity and activity imparted by the rare but shared mutation. To test the hypothesis, an in silico screen of a subset of the UCSF ZINC library of commercially available, drug-like small molecules was conducted, targeting a doughnut-shaped surface pocket flanking the adenine-binding hinge segment or back wall of the ATP-binding cleft on one side and the mutated helix-loop-helix regulatory element on the other. Unexpectedly, none of the selected subset of hits tested in vitro detectably diminished the gain of activity. However, a family of compounds was identified that substantially destabilized the recombinant kinase protein in a pH- and concentration-dependent (non-linear) manner, as shown orthogonally by native PAGE and by differential scanning fluorimetry (thermofluor). The related BMPRII kinase, which forms a heterodimeric complex with ALK2 kinase, was relatively insensitive. Another binding partner, FKBP12, was essentially inert to compound-induced destabilization under any condition tested providing additional evidence of specificity. The family of compounds was disparate with respect to scaffolds, but contained common putative warhead groups: terminal aromatic heterocyclic rings with sp2-hybridized nitrogen atoms with a hydrogen bond-accepting lone pair of electrons extending out in the plane of the ring. In reliably docked poses, the lone pair of the warheads was juxtaposed to the terminal guanidino group of an arginine sidechain that presented five hydrogen-bonding donors (NH, 2 NH2) from the protein side of the targeted allosteric site. Because the disorder is triggered by trauma to and inflammation in soft tissues, the dependency of destabilization activity on lower pH serendipitously endows the family of compounds with selective efficacy, potentially allowing for a prophylactic drug that could be administered life-long. Therapeutics that inhibit through degradation of target proteins are an emerging field. Selective Estrogen Receptor Degraders (SERDs; Genentech) are currently marketed as hormone-receptor positive breast cancer therapeutics. PROTACs (proteolysis-targeting chimeras), though preclinical, show great promise as selective degraders of over-active proteins implicated in the pathology of diseases and disorders. Support or Funding Information Basic science and drug development research support provided to JCG by the NIH and a family foundation: Mechanisms of BMP Receptor Kinase Dysregulation in Skeletal Dysplasias (NIH NIAMS 1 R03 AR056838-03) and two Cali Family Developmental Research Grants (administered by the Center for Research in FOP & Related Disorders, Penn SOM). C28 H-SAAD/D docked in the allosteric pocket. Potential for five different ARG258 NH H-bonds depicted by dashes. Putative pH-dependent effect mediated by HIS318 imidazole with backbone of 258 shown with single red dashed line. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.