A New Class of Small Molecule Inhibitor of BMP Signaling

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development

Studies on bone matrix glycoproteins. Incorporation of [1-(14)C]glucosamine and plasma [(14)C]glycoprotein into rabbit cortical bone

The radioactively labelled constituents present in bone matrix were compared 12 days after injection of either [(14)C]glucosamine or plasma [(14)C]glycoprotein. Both precursors are utilized in the synthesis of organic matrix by bone tissue. Cortical bone from animals injected with [(14)C]glucosamine contains radioactivity derived from glucosamine and plasma glycoproteins and all glycoprotein fractions are labelled. Plasma [(14)C]glycoprotein labels the less acidic glycoproteins to a greater extent than the more acidic components. An antibody has been raised against the less-acidic-glycoprotein fraction of bone. The latter contains a glycoprotein of α-mobility that appears to be concentrated specifically in bone tissue and which is present also in plasma. This α-glycoprotein accounts for a large proportion of the components labelled and retained in bone matrix after [(14)C]glucosamine injection

Investigation of Kinase Activation in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.This thesis is not currently available on ORA

Patients with primary osteoarthritis show no change with ageing in the number of osteogenic precursors

The variation in marrow colony forming unit-fibroblastic (CFU-F) number in 59 patients (14-87 years of age) undergoing corrective surgery (14 controls; 14-48 years of age) or hip arthroplasty for primary osteoarthritis (45 OA; 46-87 years of age) was examined to determine whether marrow CFU-F, derived from marrow stromal fibroblastic stem cells, are maintained with the development of primary osteoarthritis (OA). Total colony number, colony size as well as alkaline phosphatase-positive colonies were determined. The mean fibroblast colony forming efficiency from the whole patient group was 2.4 x 10(-5) +/- 1.4 x 10(-5). Ageing had no effect on the colony forming efficiency or on the alkaline-phosphatase-positive colony forming efficiency, irrespective of gender. Thus precursor cells with the potential for osteogenic differentiation are maintained in OA with ageing. However, colony size showed a significant reduction with age, implying altered proliferation potential of osteogenic progenitors with ageing. This ageing effect may not be as significant in OA as in the rest of the population as bone mineral density is often preserved in osteoarthritis. As there is no apparent deficit in primitive progenitor cells, this preservation may be the result of altered regulation of osteoprogenitor activity in OA