Development of BMP type I receptor kinase inhibitors for the treatment of fibrodysplasia ossificans progressiva and the study of the BMP signaling pathway

[摘要] The BMP signaling pathway is essential for embryonic development and the maintenance of tissue homeostasis. Dysregulated BMP signaling, both loss and gain-of-function, has been demonstrated in the pathogenesis of diseases including cancer, atherosclerosis, anemia and particularly hereditary disorders such as pulmonary arterial hypertension, hereditary hemorrhagic telangiectasia, and fibrodysplasia ossificans progressiva (FOP). FOP is a rare and disabling condition caused by a highly recurrent mutation in the ACVR1 gene encoding the BMP type I receptor activin-like kinase 2 (ALK2), characterized by the progressive heterotopic ossification (HO) of skeletal muscle and connective tissue leading to widespread joint immobilization, with significant morbidity and premature mortality. There are currently no effective treatments for FOP. The goal of this thesis is to develop and characterize highly selective BMP type I receptor inhibitors targeting ALK2 for the treatment of FOP. Despite the high degree of structural homology between all the BMP and TGF-[beta] type I receptors, I hypothesized that potent and selective inhibitors targeting a single BMP type I receptor, ALK2, could be developed based on a previously identified pyrazolo[1,5-a]pyrimidine core scaffold. I screened a library of pyrazolo[1,5-a]pyrimidine derivatives in a high throughout sensitive radiometric assay of BMP and TGF-[beta] type I receptor kinase activities. I identified a derivative with a unique chemical moiety (5-quinoline) that demonstrated high selectivity for ALK2, but with lower potency than the parent molecule. We synthesized a new 5-quinoline derivative with increased potency and selectivity for ALK2 over the other BMP type I receptors and greatly improved selectivity against the TGF--[beta] type I receptors. I used this highly selective compound to examine ALK2-mediated BMP signaling in vitro and demonstrated in vivo efficacy in two mouse models of HO. In a complementary approach, we generated a library of novel BMP type I receptor inhibitors based on the 2-aminopyridine core scaffold. I developed a structure activity relationship to determine the key structural elements responsible for potency and selectivity. We identified a several novel derivative compounds with improved potency and selectivity for ALK2 over the parent. We successfully used this set of derivatives to address a specific question in FOP biology, of whether ATP-competitive kinase inhibitors exert differential activity against wild-type or diverse FOP-causing ALK2 mutants. Finally, in our SAR of pyrazolopyrimidine compounds, we identified a highly potent inhibitor of both BMP and TGF-[beta] type I receptor activity. I characterized the ability of this compound to inhibit ligand-induced BMP and TGF-[beta] signaling in a variety of cell culture models, as well as inhibit the activity of individual type I receptors. We then used this compound to examine the contribution of individual BMP and TGF-[beta] receptors to signal transduction. We used the broad activity of this inhibitor to limit signaling of all endogenous BMP and TGF-[beta] type I receptors in cells, while reconstituting the activity of specific type I receptors using engineered, inhibitor-resistant mutant receptor kinases which we developed by modifying gatekeeper residues critical for interactions with inhibitor. These mutant receptor kinases demonstrated preserved basal and ligand-mediated signaling functions which were unaffected by inhibitor. These results demonstrate proof-of-principle of a system for examining the function of individual receptors of this pathway in isolation. The work presented in this thesis advances the development of novel BMP type I receptor kinase inhibitors of high selectivity and potency which could serve as important tools for the study of BMP signaling and as therapies for diseases of excessive BMP signaling such as FOP. Development of highly potent and selective inhibitors of ALK2 offers the hope of rational disease modifying therapy for the treatment of FOP.

[发布机构] Massachusetts Institute of Technology