An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

The Expansion of Heterotopic Bone in Fibrodysplasia Ossificans Progressiva Is Activin A-Dependent.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder that is characterized by episodic yet cumulative heterotopic ossification (HO) in skeletal muscles, tendons, and ligaments over a patient's lifetime. FOP is caused by missense mutations in the type I bone morphogenetic protein (BMP) receptor ACVR1. We have determined that the formation of heterotopic bone in FOP requires activation of mutant ACVR1 by Activin A, in part by showing that prophylactic inhibition of Activin A blocks HO in a mouse model of FOP. Here we piece together a natural history of developing HO lesions in mouse FOP, and determine where in the continuum of HO Activin A is required, using imaging (T2-MRI, µCT, 18 F-NaF PET/CT, histology) coupled with pharmacologic inhibition of Activin A at different times during the progression of HO. First, we show that expansion of HO lesions comes about through growth and fusion of independent HO events. These events tend to arise within a neighborhood of existing lesions, indicating that already formed HO likely triggers the formation of new events. The process of heterotopic bone expansion appears to be dependent on Activin A because inhibition of this ligand suppresses the growth of nascent HO lesions and stops the emergence of new HO events. Therefore, our results reveal that Activin A is required at least up to the point when nascent HO lesions mineralize and further demonstrate the therapeutic utility of Activin A inhibition in FOP. These results provide evidence for a model where HO is triggered by inflammation but becomes "self-propagating" by a process that requires Activin A. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.