Mechanism of Destabilization of BMP Receptor Kinase by Hypoxia-dependent Degraders

Objectives: An NIH R01 proposal to further develop novel therapeutics (hypoxia-selective ALK2/BMPR allosteric destabilizers/degraders, H-SAAD/Ds) for blocking post-traumatic heterotopic ossification (HO) received negative reviews (BCMB/MSFA) principally due to skepticism that destabilizing effects were specific. One argued H-SAAD/Ds were acting globally similar to detergents, not binding and interacting at a targeted pocket. To validate results of computer docking and subsequent wet-bench analyses, a highly related, yet less compound- and pH-sensitive BMP receptor kinase (BMPRII) was systematically substituted at residues in the allosteric hub (αC-β4) hypothesized to play key roles in destabilization.
Methods: BMPRII variant constructs were produced by site-directed mutagenesis or as synthetic cDNAs. Recombinant proteins (cytoplasmic kinase) were expressed in E. coli and purified to near-homogeneity via engineered N-terminal poly-histidine tags and two-step affinity chromatography (forward/reverse). The most active H-SAAD/D, C24THI, was diluted from 50 mM water-soluble disodium-salt stocks. pH- and compound-dependent effects on stability were analyzed qualitatively by native/non-denaturing PAGE and quantitatively by thermal shift assay/thermofluor with a qPCR instrument. Midpoints of thermal unfolding were determined from calculated derivatives of melting curves.
Results: In thermofluor assays of pH-dependent stability, BMPRII E253R kinase was as stable as wildtype, but in native gels with compound, more sensitive, consistent with the hypothesized role of the “shoestring” arginine residue as the warhead-interacting trigger of destabilization. Above neutrality, Y314H variant was also as stable as wildtype, but increasingly unstable below, consistent with the hypothesized role of the ALK-specific histidine as the hypoxia-selective “pH-switch”. In native gels, double-variant BMPRII approached ALK2-like pH- and compound-dependent sensitivity.
Conclusions: Further evidence obtained here provided support for the specificity of the mechanism of action, site of interaction and roles of residues at the key conformational hub. Thus, development of next-generation H-SAAD/Ds inducing degradation of BMP receptors to block post-traumatic HO appears worthwhile with respect to time, effort and costs.