Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization.

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

Fibrodysplasia ossificans progressiva: current concepts from bench to bedside.

Heterotopic ossification (HO) is a disorder characterised by the formation of ectopic bone in soft tissue. Acquired HO typically occurs in response to trauma and is relatively common, yet its aetiology remains poorly understood. Genetic forms, by contrast, are very rare, but provide insights into the mechanisms of HO pathobiology. Fibrodysplasia ossificans progressiva (FOP) is the most debilitating form of HO. All patients reported to date carry heterozygous gain-of-function mutations in the gene encoding activin A receptor type I (ACVR1). These mutations cause dysregulated bone morphogenetic protein (BMP) signalling, leading to HO at extraskeletal sites including, but not limited to, muscles, ligaments, tendons and fascia. Ever since the identification of the causative gene, developing a cure for FOP has been a focus of investigation, and studies have decoded the pathophysiology at the molecular and cellular levels, and explored novel management strategies. Based on the established role of BMP signalling throughout HO in FOP, therapeutic modalities that target multiple levels of the signalling cascade have been designed, and some drugs have entered clinical trials, holding out hope of a cure. A potential role of other signalling pathways that could influence the dysregulated BMP signalling and present alternative therapeutic targets remains a matter of debate. Here, we review the recent FOP literature, including pathophysiology, clinical aspects, animal models and current management strategies. We also consider how this research can inform our understanding of other types of HO and highlight some of the remaining knowledge gaps.

Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.

ALK4/5-dependent TGF-ß signaling contributes to the crosstalk between neurons and microglia following axonal lesion.

Neuronal activity is closely influenced by glia, especially microglia which are the resident immune cells in the central nervous system (CNS). Microglia in medicinal leech are the only cells able to migrate to the injury site within the 24 hours post-lesion. The microglia-neuron interactions constitute an important mechanism as there is neither astrocyte nor oligodendrocyte in the leech CNS. Given that axonal sprouting is impaired when microglia recruitment is inhibited, the crosstalk between microglia and neurons plays a crucial role in neuroprotection. The present results show that neurons and microglia both use ALK4/5 (a type of TGF-ß receptor) signaling in order to maintain mutual exchanges in an adult brain following an axonal injury. Indeed, a TGF-ß family member (nGDF) is immediately released by injured axons contributing to the early recruitment of ALK4/5+ microglia to the lesion site. Surprisingly, within the following hours, nGDF from microglia activates ALK4/5+ neurons to maintain a later microglia accumulation in lesion. Taken together, the results demonstrate that ALK4/5 signaling is essential throughout the response to the lesion in the leech CNS and gives a new insight in the understanding of this pathway. This latter is an important signal contributing to a correct sequential mobilization over time of microglia recruitment leading to axon regeneration.

Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors.

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.

Novel Quinazolinone Inhibitors of ALK2 Flip between Alternate Binding Modes: Structure-Activity Relationship, Structural Characterization, Kinase Profiling, and Cellular Proof of Concept.

Structure-activity relationship and crystallographic data revealed that quinazolinone-containing fragments flip between two distinct modes of binding to activin receptor-like kinase-2 (ALK2). We explored both binding modes to discover potent inhibitors and characterized the chemical modifications that triggered the flip in binding mode. We report kinase selectivity and demonstrate that compounds of this series modulate ALK2 in cancer cells. These inhibitors are attractive starting points for the discovery of more advanced ALK2 inhibitors.

Identification of small molecule inhibitors of ALK2: a virtual screening, density functional theory, and molecular dynamics simulations study.

Bone morphogenetic proteins (BMPs) are a family of more than 30 ligands and several receptors, such as activin like kinases (ALKs) and bone morphogenetic protein receptor (BMPR). Physiological significance of these proteins lies in their prominent role during homeostasis, apoptosis, tissue remodeling, embryonic patterning, and normal development. Fibrodysplasia ossificans progressive (FOP) is one among several other diseases caused by impaired BMP signaling. FOP is caused by the pathogenicity of activating mutation of ALK2. In order to treat FOP, a search for good inhibitors of ALK2 based on dorsomorphin and LDN substitution, which in essence is a ligand based search of inhibitors, is in progress. Contributing to this area of research we identified several lead molecules based on protein structure using virtual screening. After virtual screening of a huge library of small molecules and ab initio calculation of selected molecules for drug efficacy, we did molecular dynamic simulation of lead molecules and protein complexes. We identified five potential drug molecules that show very stable binding on the same binding site as LDN-213844. We also ranked these lead molecules based on MM/PBSA binding energy. This study provides a basis to think beyond the pyrimidine nucleus of dorsomorphin/LDN and design new chemical derivatives for effective treatment of FOP. Graphical abstract Small molecule inhibitors of ALK2.

Activin A Increases Human Trophoblast Invasion by Inducing SNAIL-Mediated MMP2 Up-Regulation Through ALK4.

CONTEXT: Activin A increases matrix metalloproteinase (MMP) 2 expression and cell invasion in human trophoblasts, but whether the expression of MMP2 is essential for the proinvasive effect of activin A has yet to be determined. Moreover, the identity of the activin receptor-like kinase (ALK; TGF-ß type I receptors) and downstream transcription factors (eg, SNAIL and SLUG) mediating the effects of activin on MMP2 expression and trophoblast cell invasion remains unknown. OBJECTIVE: To elucidate the role of MMP2 in activin A-induced human trophoblast cell invasion as well as the involvement of ALK4 and SNAIL. DESIGN: HTR8/SVneo immortalized human extravillous cytotrophoblast (EVT) cells and primary cultures of human first-trimester EVT cells were used as study models. Small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions. MAIN OUTCOME MEASURES: Levels of mRNA and protein were examined by reverse transcription-quantitative real-time PCR and Western blot, respectively. Cell invasiveness was measured by Matrigel-coated transwell assays. RESULTS: Treatment of HTR8/SVneo cells with activin A increased the production of SNAIL, SLUG, and MMP2 without altering that of MMP9, TIMP1, TIMP2, TWIST, RUNX2, ZEB1, or ZEB2. Similarly, activin A up-regulated the mRNA and protein levels of SNAIL and MMP2 in primary EVT cells. Knockdown of SNAIL attenuated activin A-induced MMP2 up-regulation in HTR8/SVneo and primary EVT cells. In HTR8/SVneo cells, activin A-induced production of SNAIL and MMP2 was abolished by pretreatment with the TGF-ß type I receptor (ALK4/5/7) inhibitor SB431542 or siRNA targeting ALK4, SMAD2/3, or common SMAD4. Likewise, knockdown of ALK4 or SMAD4 abolished the stimulatory effects of activin A on SNAIL and MMP2 expression in primary EVT cells. Importantly, activin A-induced HTR8/SVneo and primary EVT cell invasion were attenuated by siRNA-mediated depletion of ALK4 or MMP2. CONCLUSION: Activin A induces human trophoblast cell invasion by inducing SNAIL-mediated MMP2 expression through ALK4 in a SMAD2/3-SMAD4-dependent manner.

Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-ß type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling.