Individuals with fibrodysplasia ossificans progressiva are born with malformations of the great toes and develop a heterotopic skeleton during childhood because of an identical heterozygous mutation in the glycine-serine activation domain of ACVR1, a bone morphogenetic protein type I receptor. Substitution of adenine for guanine at nucleotide 617 replaces an evolutionarily conserved arginine with histidine at residue 206 of ACVR1 in all classically affected individuals, making this one of the most highly conserved disease-causing mutations in the human genome. To better understand the molecular constraints and physiological implications of this mutation, we performed in silico modeling of wild-type and mutant ACVR1. In both the wild-type ACVR1 model and template crystal structures (TβRI), the conserved arginine appears to form a salt bridge with an invariant aspartate residue. Although lysine, a conservative substitution in BMPRIA and BMPRIB, can be readily accommodated, histidine at residue 206 (like in fibrodysplasia ossificans progressiva) would participate in a salt bridge with the aspartate only at decreased intracellular pH and with extensive structural rearrangement. Protein modeling predicts that substitution with histidine, and only histidine, creates a pH-sensitive switch within the activation domain of the receptor that leads to ligand-independent activation of ACVR1 in fibrodysplasia ossificans progressiva.

Introduction: Protein kinases are a major target for small-molecule drug development. However, relatively few compounds are free of off-target toxicity and reach the clinic. Because the 500-plus kinases share conserved ATP-binding clefts, the site targeted by competitive inhibitors, generation of specific therapeutics remains a nearly intractable challenge. Areas covered: Inducing degradation, instead of inhibition by occupancy-driven drugs, is an emerging strategy that offers the long-sought specificity, as well as mechanistic benefits. Currently approved inhibitors require steady-state binding and leave proteins intact for interactions in multi-protein complexes. After a general background about induced protein degradation, perspectives on protein kinases are provided. Expert opinion: Induced degradation by state-of-the-art compounds (proteolysis-targeting chimeras, PROTACs) has been shown for protein kinases, albeit in early pre-clinical stages. Further work is required to expand the number of enzymes that could be exploited to direct proteins for degradation by ubiquitylation. In addition, despite the simple modularity of the chimeras, generation of hits will require empirical approaches due to the role of protein–protein interactions and distribution of tagging sites. However, given the advantages of degradation, drug discovery efforts targeting protein kinases should increasingly shift toward generation and screening of inducers of degradation and away from occupancy-based inhibitors of old.

Bone morphogenetic protein (BMP) signaling is critical in skeletal development. Overactivation can trigger heterotopic ossification (HO) as in fibrodysplasia ossificans progressiva (FOP), a rare, progressive disease of massive HO formation. A small subset of FOP patients harboring the causative ACVR1R206H mutation show strikingly mild or delayed-onset HO, suggesting that genetic variants in the BMP pathway could act as disease modifiers. Whole-exome sequencing of one such patient identified BMPR1AR443C and ACVR2AV173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1R206H signaling. Overexpression of BMPR1AR443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2AV173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1R206H HEK cells decreased ligand-stimulated BMP signaling but did not affect dorsoventral patterning in Tg(ACVR1-R206Ha) zebrafish. ACVR2A knockdown decreased only AA-stimulated signaling in ACVR1R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1R206H HEK cells decreased basal and ligand-stimulated signaling, and co-knockdown/knockout (bmpr1aa/ab; acvr2aa/ab) decreased Tg(ACVR1-R206Ha) zebrafish ventralization phenotypes. Our functional studies showed that knockdown of wild-type BMPR1A and ACVR2A could attenuate ACVR1R206H signaling, particularly in response to AA, and that ACVR2AV173I unexpectedly increased ACVR1R206H-mediated signaling in zebrafish. These studies describe a useful strategy and platform for functionally interrogating potential genes and genetic variants that may impact the BMP signaling pathway. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Although structurally similar to type II counterparts, type I or activin receptor-like kinases (ALKs) are set apart by a metastable helix–loop–helix (HLH) element preceding the protein kinase domain that, according to a longstanding paradigm, serves passive albeit critical roles as an inhibitor-to-substrate-binding switch. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder activation of ACVR1/ALK2 leading to sporadic heterotopic bone deposition in patients presenting with fibrodysplasia ossificans progressiva, or FOP. To determine the protein structural–functional basis for the gain of function, R206H mutant, Q207D (aspartate-substituted caALK2) and HLH subdomain-truncated (208 Ntrunc) forms were compared to one another and the wild-type enzyme through in vitro kinase and protein–protein interaction analyses that were complemented by signaling read-out (p-Smad) in primary mouse embryonic fibroblasts and Drosophila S2 cells. Contrary to the paradigm, the HLH subdomain actively suppressed the phosphotransferase activity of the enzyme, even in the absence of FKBP12. Unexpectedly, perturbation of the HLH subdomain elevated kinase activity at a distance, i.e., allosterically, at the ATP-binding and polypeptide-interacting active site cleft. Accessibility to polypeptide substrate (BMP Smad C-terminal tails) due to allosterically altered conformations of type I active sites within heterohexameric cytoplasmic signaling complexes—assembled noncanonically by activin-type II receptors extracellularly—is hypothesized to produce a gain of function of the R206H mutant protein responsible for episodic heterotopic ossification in FOP.