A Three-Part, Randomised Study to Investigate the Safety, Tolerability, Pharmacokinetics and Mode of Action of BC 007, Neutraliser of Pathogenic Autoantibodies Against G-Protein Coupled Receptors in Healthy, Young and Elderly Subjects.

BACKGROUND AND OBJECTIVE: BC 007 is a substance with a novel and innovative mode of action for the first-time causal treatment of chronic heart failure, associated with the occurrence of autoantibodies against the ß1-adrenoceptor, and other diseases of mostly the heart and vascular system, being accompanied by the occurrence of functionally active agonistic autoantibodies against G-protein-coupled receptors (fGPCR-AAb). The proposed mechanism of action of BC 007 is the neutralisation of these pathogenic autoantibodies which stimulate the respective receptor. To evaluate the safety, tolerability, pharmacokinetics and mode of action of BC 007, single intravenous infusions of increasing concentration were given to healthy young males and healthy elderly autoantibody-negative and autoantibody-positive participants of both sexes. METHODS: This study was subdivided into three parts. Part A was a single-centre, randomised, double-blind, placebo-controlled safety and tolerability study including healthy young male autoantibody-negative Whites (N = 23) and Asians (N = 1), testing doses of 15, 50 and 150 mg BC 007 (Cohorts 1-3) and elderly male and female Whites (N = 8), testing a dose of 150 mg BC 007 (Cohort 4), randomly assigned in a 3:1 ratio to BC 007 or placebo. Open-label Part B included fGPCR-AAb-positive subjects (50 and 150 mg BC 007, Cohorts 1 and 2, respectively). Open-label Part C included fGPCR-AAb-positive subjects for testing doses of 300, 450, 750, 1350 mg and 1900 mg BC 007. Lower doses were either given as an infusion or divided into a bolus plus infusion up to a dose of 300 mg followed by a constant bolus of 150 mg up to a dose of 750 mg, while at doses of 1350 mg and 1900 mg it was a slow infusion with a constant infusion rate. Infusion times increased with increasing dose from 20 min (15, 50 or 150 mg) to 40 min (300, 450 or 750 mg), 75 min (1350 mg) and 105 min (1900 mg). RESULTS: The mean observed BC 007 area under the concentration-time curve (AUC0-24) increased with increasing dose in a dose proportional manner (slope estimate of 1.039). No serious adverse events were observed. Drug-related adverse events were predominantly the expected mild-to-moderate increase in bleeding time (aPTT), beginning with a dose of 50 mg, which paralleled the infusion and returned to normal shortly after infusion. fGPCR-AAb neutralisation efficiency increased with increasing dose and was achieved for all subjects in the last cohort. CONCLUSION: BC 007 is demonstrated to be safe and well tolerated. BC 007 neutralised fGPCR-AAb, showing a trend for a dose-response relationship in elderly healthy but fGPCR-AAb-positive subjects. CLINICALTRIALS. GOV REGISTRATION NUMBER: NCT02955420.

Small molecules dorsomorphin and LDN-193189 inhibit myostatin/GDF8 signaling and promote functional myoblast differentiation.

GDF8, or myostatin, is a member of the TGF-ß superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescued myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis.

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5ß recruitment at the cytocortex.

BACKGROUND: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. RESULTS: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5ß to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5ß or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. CONCLUSIONS: Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5ß signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2-induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.

SMAD versus non-SMAD signaling is determined by lateral mobility of bone morphogenetic protein (BMP) receptors.

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFß superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.

Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva.

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation.

Bone extracellular matrix consists of a network of proteins in which growth factors, like bone morphogenetic protein 2 (BMP-2), are embedded and released upon matrix turnover and degradation. Recombinant human (rh)BMP-2 shows promise in enhancing bone fracture repair, although issues regarding finding a suitable delivery system still limit its extensive clinical use. The aim of this study is to determine which cell activities are triggered by the presentation of immobilized rhBMP-2. For this purpose gold surfaces were first decorated with a self-assembled monolayer consisting of a hetero-bifunctional linker. rhBMP-2 was covalently bound to the surfaces via this linker and used to investigate the cellular responses of C2C12 myoblasts. We show that covalently immobilized rhBMP-2 (iBMP-2) initiates short-term signaling events. Using a BMP-responsive reporter gene assay and western blotting to monitor phosphorylation of Smad1/5/8 we prove that iBMP-2 activates BMP-dependent signal transduction. Furthermore, we demonstrate that iBMP-2 suppresses myotube formation and promotes the osteoblast phenotype in C2C12 cells. The bioactivity of surface-bound rhBMP-2 presented in this study is not due to its release into the medium. As such, our simple approach paves the way for the controlled local presentation of immobilized growth factors, limiting degradation while still maintaining biological activity.

PP2A regulates BMP signalling by interacting with BMP receptor complexes and by dephosphorylating both the C-terminus and the linker region of Smad1.

Phosphorylation of Smads is a crucial regulatory step in the signal transduction pathway initiated by bone morphogenetic proteins (BMPs). Although the dephosphorylation events terminating the pathway in the nucleus have been characterized, little is known about the dephosphorylation of Smads in the cytoplasm. In a proteomic screen for proteins interacting with the BMP type-II receptor, we found the regulatory Bbeta subunit of PP2A. PP2A is one of the major serine/threonine phosphatases involved in cell-cycle regulation and signal transduction. Here, we present data showing that the Bbeta subunit of PP2A interacts with both BMP type-I and type-II receptors. Furthermore, we demonstrate that several B subunits can associate with the BMP type-II receptor, independently of the kinase activity of the receptor and the catalytic subunit of PP2A. By contrast, the PP2A catalytic subunit is required for PP2A function at the receptor complex. This function of PP2A is the dephosphorylation of Smad1, mainly in the linker region. PP2A-mediated dephosphorylation of the BMP-Smad linker region leads to increased nuclear translocation of Smads and overall amplification of the BMP signal. Although other phosphatases identified within the BMP pathway are all shown to inhibit signalling, PP2A is the first example for a signalling stimulatory phosphatase within this pathway.

Different routes of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling.

Endocytosis is important for a variety of functions in eukaryotic cells, including the regulation of signaling cascades via transmembrane receptors. The internalization of bone morphogenetic protein (BMP) receptor type I (BRI) and type II (BRII) and its relation to signaling were largely unexplored. Here, we demonstrate that both receptor types undergo constitutive endocytosis via clathrin-coated pits (CCPs) but that only BRII undergoes also caveola-like internalization. Using several complementary approaches, we could show that (i) BMP-2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in nonraft regions, (ii) continuation of Smad signaling resulting in a transcriptional response requires endocytosis via the clathrin-mediated route, and (iii) BMP signaling leading to alkaline phosphatase induction initiates from receptors that fractionate into cholesterol-enriched, detergent-resistant membranes. Furthermore, we show that BRII interacts with Eps15R, a constitutive component of CCPs, and with caveolin-1, the marker protein of caveolae. Taken together, the localization of BMP receptors in distinct membrane domains is prerequisite to their taking different endocytosis routes with specific impacts on Smad-dependent and Smad-independent signaling cascades.