Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin-and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.Signals mediated by BMP ligands serve diverse roles throughout the life of vertebrates. During embryogenesis, the dorsoventral axis is established by BMP signaling gradients formed by the coordinated expression of ligands, receptors, co-receptors and soluble antagonists 1-3 . Excess BMP signaling causes ventralization (an expansion of ventral structures at the expense of dorsal structures) whereas diminished BMP signaling causes dorsalization (an expansion of dorsal structures at the expense of ventral structures) 1,3,4 . BMPs are key regulators of gastrulation,

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues 1-4 and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2)5. Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I Correspondence should be addressed to P.B.Y. (pbyu@partners.org). 8 Present address:

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1R206H) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1R206H mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1R206H causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP.

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. We identified BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH bearing mutations in BMPR-II. In vivo, we report the spontaneous generation of PAH in a mouse model bearing a heterozygous knock-in of a human BMPR-II mutation, R899X. Administration of BMP9 reversed established PAH in Bmpr2+/R899X mice, as well as in models of disease developed in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a novel therapeutic strategy for PAH.

A structure-activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo [1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo [1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g. plasma t 1/2 = 1.6 h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.Bone morphogenetic proteins (BMPs) are a group of > 25 protein ligands that comprise a subset of the transforming growth factor β (TGF-β) family. BMPs modulate a multitude of biological processes, including bone and cartilage formation during embryogenesis. 1a However, they are also intimately involved with numerous nonosteogenic developmental and physiological processes throughout adulthood as well as several pathological conditions. BMPs bind to two classes of cell surface bone morphogenetic protein receptors (BMPR-I and BMPRII). 1a The BMPR-I receptor class consists of three receptor types, activin receptor-like kinase-2 (ALK-2 or ActR-IA), ALK-3 (BMPR-IA) and ALK-6 (BMPR-IB). The BMPR-II receptor class is comprised of three receptor types, BMPR-II, ActR-IIA and ActR-IIB. Binding of BMPs results in the formation of heterotetrameric complexes containing two type I and two *To whom correspondence should be addressed: Phone: 617-768-8640, Fax: 617-768-8606, E-mail: gcuny@rics.bwh.harvard.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Recently, dorsomorphin, 1, 7a, 11, 12 was discovered as an inhibitor of SMAD 1/5/8 phosphorylation by BMP type 1 receptors (ALK2, 3, and 6) utilizing a phenotypic screen to identify compounds that perturb embryonic dorsoventral axis formation. Furthermore, this inhibition was shown to decrease BMP-regulated hepatic hepcidin gene transcription, leading to increased iron levels in vivo. 7a However, 1 only demonstrated moderate inhibition of SMAD 1/5/8 phosphorylation by BMPR-I with an IC50 ~ 0.5 µM and lacks metabolic stability. Herein, we describe the results of a structu...

Fibrodysplasia ossificans progressiva (FOP), a congenital heterotopic ossification (HO) syndrome caused by gain-of-function mutations of bone morphogenetic protein (BMP) type I receptor ACVR1, manifests with progressive ossification of skeletal muscles, tendons, ligaments, and joints. In this disease, HO can occur in discrete flares, often triggered by injury or inflammation, or may progress incrementally without identified triggers. Mice harboring an Acvr1 knock-in allele recapitulate the phenotypic spectrum of FOP, including injury-responsive intramuscular HO and spontaneous articular, tendon, and ligament ossification. The cells that drive HO in these diverse tissues can be compartmentalized into two lineages: an Scx tendon-derived progenitor that mediates endochondral HO of ligaments and joints without exogenous injury, and a muscle-resident interstitial Mx1 population that mediates intramuscular, injury-dependent endochondral HO. Expression of Acvr1 in either lineage confers aberrant gain of BMP signaling and chondrogenic differentiation in response to activin A and gives rise to mutation-expressing hypertrophic chondrocytes in HO lesions. Compared to Acvr1, expression of the man-made, ligand-independent ACVR1 mutation accelerates and increases the penetrance of all observed phenotypes, but does not abrogate the need for antecedent injury in muscle HO, demonstrating the need for an injury factor in addition to enhanced BMP signaling. Both injury-dependent intramuscular and spontaneous ligament HO in Acvr1 knock-in mice were effectively controlled by the selective ACVR1 inhibitor LDN-212854. Thus, diverse phenotypes of HO found in FOP are rooted in cell-autonomous effects of dysregulated ACVR1 signaling in nonoverlapping tissue-resident progenitor pools that may be addressed by systemic therapy or by modulating injury-mediated factors involved in their local recruitment.

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.

Bone morphogenetic protein (BMP) ligands signal by binding the BMP type II receptor (BMPR2) or the activin type II receptors (ActRIIa and ActRIIb) in conjunction with type I receptors to activate SMADs 1, 5, and 8, as well as members of the mitogen-activated protein kinase family. Loss-of-function mutations in Bmpr2 have been implicated in tumorigenesis and in the etiology of primary pulmonary hypertension. Because several different type II receptors are known to recognize BMP ligands, the specific contribution of BMPR2 to BMP signaling is not defined. Here we report that the ablation of Bmpr2 in pulmonary artery smooth muscle cells, using an ex vivo conditional knock-out (Cre-lox) approach, as well as small interfering RNA specific for Bmpr2, does not abolish BMP signaling. Disruption of Bmpr2 leads to diminished signaling by BMP2 and BMP4 and augmented signaling by BMP6 and BMP7. Using small interfering RNAs to inhibit the expression of other BMP receptors, we found that wild-type cells transduce BMP signals via BMPR2, whereas BMPR2-deficient cells transduce BMP signals via ActRIIa in conjunction with a set of type I receptors distinct from those utilized by BMPR2. These findings suggest that disruption of Bmpr2 leads to the net gain of signaling by some, but not all, BMP ligands via the activation of ActRIIa. Bone morphogenetic protein (BMP)1 signals regulate embryonic tissue patterning and organogenesis, as well as the remodeling of mature tissues (1). BMPs, like other transforming growth factor ␤ superfamily ligands, induce apposition of type I and type II receptors to cause phosphorylation of type I receptors. Activated BMP type I receptors phosphorylate the BMP-responsive SMAD proteins 1, 5, and 8, leading to their nuclear translocation and the regulation of target gene transcription. The Id (inhibitor of differentiation) gene family is an important target of BMP signals (2-5), serving to regulate the differentiation and proliferation of a variety of mature and embryonic cell lineages, including vascular smooth muscle and endothelium (2, 6 -8). BMP ligands may also trigger the activation of MAP kinases via SMAD-independent signaling pathways (9 -11).Three type II receptors, BMPR2 and the activin type II receptors ActRIIa and ActRIIb (12-17), can pair with three different type I receptors, ActRIa/ALK2, BMPRIa/ALK3, and BMPRIb/ALK6 (13, 18 -23), to transduce BMP signals. These various BMP receptors have distinct temporal and spatial expression in tissues and have varying affinities for each of more than 15 known BMP molecules (1, 24). Although BMPR2, ActRIIa, and ActRIIb can each bind BMP ligands (12-15, 21, 22, 25), the preferred ligand-receptor complexes used by cells for BMP signaling are not known.In this study, we examined the signaling mediated by two structurally distinct classes of BMP ligands that are known to be expressed in vascular smooth muscle and endothelial cells and that regulate their function (6, 26 -30). The first class of BMP ligands includes the 92% homologous BMP2 and BMP4, considere...