High-throughput measurements of bone morphogenetic protein/bone morphogenetic protein receptor interactions using biolayer interferometry.

Bone morphogenetic proteins (BMPs) are an important family of growth factors playing a role in a large number of physiological and pathological processes, including bone homeostasis, tissue regeneration, and cancers. In vivo, BMPs bind successively to both BMP receptors (BMPRs) of type I and type II, and a promiscuity has been reported. In this study, we used biolayer interferometry to perform parallel real-time biosensing and to deduce the kinetic parameters (ka, kd) and the equilibrium constant (KD) for a large range of BMP/BMPR combinations in similar experimental conditions. We selected four members of the BMP family (BMP-2, 4, 7, 9) known for their physiological relevance and studied their interactions with five type-I BMP receptors (ALK1, 2, 3, 5, 6) and three type-II BMP receptors (BMPR-II, ACTR-IIA, ACTR-IIB). We reveal that BMP-2 and BMP-4 behave differently, especially regarding their kinetic interactions and affinities with the type-II BMPR. We found that BMP-7 has a higher affinity for the type-II BMPR receptor ACTR-IIA and a tenfold lower affinity with the type-I receptors. While BMP-9 has a high and similar affinity for all type-II receptors, it can interact with ALK5 and ALK2, in addition to ALK1. Interestingly, we also found that all BMPs can interact with ALK5. The interaction between BMPs and both type-I and type-II receptors in a ternary complex did not reveal further cooperativity. Our work provides a synthetic view of the interactions of these BMPs with their receptors and paves the way for future studies on their cell-type and receptor specific signaling pathways.

BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma.

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

Integrated In Silico-In Vitro Identification and Optimization of Bone Morphogenic Protein-2 Armpit Epitope as Its Antagonist Binding Site.

Bone morphogenic protein-2 (BMP-2) is the most documented member of BMP family and plays a crucial role in bone formation and growth. In this study, we systematically analyze and compare the complex crystal structures and interaction properties of BMP-2 with its cognate receptors BMPR-I/BMPR-II and with its natural antagonist crossveinless-2 (CV-2) using an integrated in silico-in vitro strategy. It is found that the antagonist-binding site is not fully overlapped with the two receptor-binding sites on BMP-2 surface; the antagonist can competitively disrupt BMP-2-BMPR-II interaction using a blocking-out-of-site manner, but has no substantial influence on BMP-2-BMPR-I interaction. Here, the antagonist-binding site is assigned as a new functional epitope armpit to differ from the traditional conformational epitope wrist and linear epitope knuckle at receptor-binding sites. Structural analysis reveals that the armpit comprises three sequentially discontinuous, structurally vicinal peptide segments, separately corresponding to a loop region and two ß-strands crawling on the protein surface. The three segments cannot work independently when splitting from the protein context, but can restore binding capability to CV-2 if they are connected to a single peptide. A systematic combination of different-length polyglycine linkers between these segments obtains a series of designed single peptides, from which several peptides that can potently interact with the armpit-recognition site of CV-2 with high affinity and specificity are identified using energetic analysis and fluorescence assay; they are expected to target BMP-2-CV-2 interaction in a self-inhibitory manner.

Palmitoylation of BMPR1a regulates neural stem cell fate.

Neural stem cells (NSCs) generate neurons and glial cells throughout embryonic and postnatal brain development. The role of S-palmitoylation (also referred to as S-acylation), a reversible posttranslational lipid modification of proteins, in regulating the fate and activity of NSCs remains largely unknown. We used an unbiased screening approach to identify proteins that are S-acylated in mouse NSCs and showed that bone morphogenic protein receptor 1a (BMPR1a), a core mediator of BMP signaling, is palmitoylated. Genetic manipulation of S-acylated sites affects the localization and trafficking of BMPR1a and leads to altered BMP signaling. Strikingly, defective palmitoylation of BMPR1a modulates NSC function within the mouse brain, resulting in enhanced oligodendrogenesis. Thus, we identified a mechanism regulating the behavior of NSCs and provided the framework to characterize dynamic posttranslational lipid modifications of proteins in the context of NSC biology.

Core and biological motif of self-assembling peptide nanofiber induce a stronger electrostatic interaction than BMP2 with BMP2 receptor 1A.

Recent studies suggest that nanotopography can trigger colocalization of integrins and bone morphogenetic protein 2 (BMP2) receptors (e.g., BMPR1A), thereby leading to osteogenesis. In this study, the bone marrow homing peptide 1 (BMHP1) motif was bound to a self-assembling peptide core to form a hydrogel-based nanofiber (R-BMHP1). The docking and molecular dynamic study revealed that the R-BMHP1 sequence induced a stronger electrostatic interaction than BMP2 through arginines in the RADA core sequence and through lysine24 in the BMHP1 motif with BMPR1A. Notably, decrease of polar solvation binding energy will enhance the total binding energy and increases bone regeneration even more than BMP2 The enhanced osteogenesis and bone repair potential of R-BMHP1 nanofiber might be related to its chemical interaction with BMPR1A, which triggered downstream signal transduction through osteogenic genes overexpression in osteo-differentiated mesenchymal stem cells (MSCs), as well as implanted critical-sized bone defects in rats. Following that, calcium deposition occurred by osteoblast-like cells, ALP activity increased in osteodifferentiation MSCs and rat serum, and calcium density improved in bone defects (X-ray). The nanofiber was biocompatible and enhanced the cell viability of MSCs, without multinuclear cell infiltration into the defect site. Taking everything into account, not only does nanotopography induce osteogenesis through colocalization of BMPRs and integrins, but also R-BMHP1 nanofibers (considering their chemical structure) induce cell proliferation, osteogenesis, and bone repair through strong electrostatic interaction with BMPR1A and downstream signaling. The entire outcome of this study manifests the plausibility of R-BMHP1 for spine and spinal cord injury repair.

Correlating interfacial water dynamics with protein-protein interaction in complex of GDF-5 and BMPRI receptors.

GDF-5 mediated signal transduction regulating chondrogenesis and skeletogenesis involves three different type-I receptors viz. Act-RI, BMPRIA and BMPRIB. BMPRIA and BMPRIB generally shows temporal and spatial co-expression but some spatially different expression pattern has also been observed. BMPRIA receptor is the key receptor implicated in BMP signalling during osteogenesis and is expressed in osteoblasts during the course of bone formation. However, BMPRIB appears to be primarily expressed in mesenchymal pre-cartilage condensations and also found in differentiated osteoblast and chondrocytes. The extracellular pH affects bone cell function and it is experimentally known that mineralization of bone is affected by shift of pH in cultured osteoblast. Here we report the effect of pH on dynamics of water present at the interface of GDF-5:BMPRIA and GDF-5:BMPRIB and binding interaction energy of these complexes. Water dynamics at different pH was analysed using residence time and hydrogen bond relaxation kinetics. pH influences the interaction energy between GDF-5 and BMPRIA and BMPRIB receptors indicating the electrostatic environment modulating the activity of two receptors. This pH dependence of interaction energy is further supported by similar behaviour of hydrogen bond existence of buried water molecules at the interface. In contrast to this the slow and fast exchanging water molecules do not show similar pH dependence of hydrogen bonding relaxation kinetics. Hence; we conclude that only buried water molecule at the interface influences the protein-protein interaction and the electrostatic environment of the extracellular fluid might decide the specificity of the two receptors.

Strontium doping promotes bioactivity of rhBMP-2 upon calcium phosphate cement via elevated recognition and expression of BMPR-IA.

Preserving and improving osteogenic activity of bone morphogenetic protein-2 (BMP-2) upon implants remains one of the key limitations in bone regeneration. With calcium phosphate cement (CPC) as model, we have developed a series of strontium (Sr)-doped CPC (SCPC) to address this issue. The effects of fixed Sr on the bioactivity of recombinant human BMP-2 (rhBMP-2) as well as the underlying mechanism were investigated. The results suggested that the rhBMP-2-induced osteogenic activity was significantly promoted upon SCPCs, especially with a low amount of fixed Sr (SrCO3 content <10wt%). Further studies demonstrated that the Sr-induced enhancement of bioactivity of rhBMP-2 was related to an elevated recognition of bone morphogenetic protein receptor-IA (BMPR-IA) to rhBMP-2 and an increased expression of BMPR-IA in C2C12 model cells. As a result, the activations of BMP-induced signaling pathways were different in C2C12 cells incubated upon CPC/rhBMP-2 and SCPCs/rhBMP-2. These findings explicitly decipher the mechanism of SCPCs promoting osteogenic bioactivity of rhBMP-2 and signify the promising application of the SCPCs/rhBMP-2 matrix in bone regeneration implants.

BMP-2 and BMP-9 binding specificities with ALK-3 in aqueous solution with dynamics.

Signal ligands of the transforming growth factor-ß (TGF-ß) superfamily include the bone morphogenetic proteins (BMPs). BMPs bind to type I and type II serine-threonine kinase receptors and trigger the transphosphorylation cascade, wherein the active type II receptor phosphorylates the inactive type I receptor. This process further activates the cytoplasmic effectors of the pathway, such as SMAD proteins, which are homologs of both the Drosophila protein MAD (mothers against decapentaplegic) and the Caenorhabditis elegans protein SMA (small body size). Even though biological and medicinal studies have been performed on these complex species, we currently do not know the underlying molecular mechanisms of the signal ligand interactions with the receptors. Detailed understanding of these interactions increases our knowledge about these proteins, and also can provide the lacking information for successful mutation experiments. This study focuses on the computational analysis of binding affinities and structural binding specificities of two different types of BMPs (BMP-2 and BMP-9) to the activin receptor-like kinases (ALK-3) in solution. For studying the binding characteristics of BMP-2 or BMP-9 with ALK-3 in aqueous solution, we performed extensive molecular dynamics simulations coupled with thermodynamic calculations. The calculated thermodynamic properties show that the BMP-2/ALK-3 complex is thermodynamically more stable than a possible BMP-9/ALK-3 species in aqueous solution. The binding free energies indicate that ALK-3 preferably binds to BMP-2 instead of BMP-9. The structural analysis shows that ALK-3 binding with BMP-2 occurs in a perfectly symmetry pathway, whereas this symmetry is lost for possible ALK-3 interactions with BMP-9. The Phe49 to Val70 loop region of BMP-2 presents strong inter-molecular interactions with ALK-3. On the other hand, BMP-9 presents weaker interactions with ALK-3 via a non-continuous sequence. ALK-3-binding region of BMP-2 corresponds to the region predicted to be flexible by our intrinsic disorder analysis, whereas the related region of BMP-9 is expected to be noticeably less flexible. This study proposes that mutating the BMP-9 with the partial Phe49 to Val70 sequence of BMP-2 can help to increase the reactivity of BMP-9 towards stable ALK-3 binding, which in turn has the potential to develop new signaling pathways for improving the formation of tissues and to prevent or treat severe diseases. Furthermore, this study also demonstrates the usefulness of theoretical physical chemistry tools, such as molecular dynamics simulations and the ProtMet simulation software package in the structural characterization of the TGF-ß superfamily proteins.

The receptor genes PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in the pearl oyster Pinctada fucata.

Mounting evidence suggests that TGFß/BMP signaling pathway is most likely involved in shell biomineralization in molluscs, but the function of pathway receptors is poorly studied. Here, we cloned and identified two homologous BMP receptor genes, PfBMPR1B and PfBAMBI, from the pearl oyster Pinctada fucata. Real-time quantitative PCR and in situ hybridization revealed that these genes were expressed in mantle edge and pallial, specifically located at the outer epithelia. Knockdown of PfBMPR1B by RNA interference (RNAi) significantly decreased the expression levels of matrix protein (MP) genes and induced the abnormal ultrastructure of prismatic and nacreous layers. Conversely, knockdown of PfBAMBI significantly increased the expression levels of a portion of MP genes and induced the overgrowth of nacreous layer crystals. In the RNAi and shell notching experiments, MP gene expressions were competitively regulated by PfBMPR1B and PfBAMBI. In addition, the receptor inhibitor LDN193189 reduced the expression levels of MP genes in mantle primary cells and larvae, and induced abnormal D-shaped shell formation during larval development. Collectively, these results clearly show that PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in P. fucata. Our study therefore provides the direct evidence that BMP receptors participate in mollusc biomineralization.

New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing.

STUDY QUESTION: Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? SUMMARY ANSWER: WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. WHAT IS KNOWN ALREADY: POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study performed on 69 women affected by POI. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. LIMITATIONS, REASONS FOR CAUTION: It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WIDER IMPLICATIONS OF THE FINDINGS: WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest.

Long-Range Communication Network in the Type 1B Bone Morphogenetic Protein Receptor.

Protein-protein interactions are recognized as a fundamental phenomenon that is intimately associated with biological functions and thus are ideal targets for developing modulators for regulating biological functions. A challenge is to identify a site that is situated away from but functionally connected to the protein-protein interface. We employed bone morphogenetic proteins (BMPs) and their receptors as a model system to develop a strategy for identifying such a network of communication. Accordingly, using computational analyses with the COREX/BEST algorithm, we uncovered an overall pattern connecting various regions of BMPR-1B ectodomain, including the four conserved residues in the protein-protein interface. In preparation for testing the long-range effects of mutations of distal residues for future studies, we examined the extent of measurable perturbation of the four conserved residues by determination of the conformation and relative affinities of these BMPR-1B mutants for ligands BMP-2, -6, and -7 and GDF-5. Results suggest no significant structural changes in the receptor but do suggest that the four residues play different roles in defining ligand affinity and both intra- and intermolecular interactions play a role in defining ligand affinity. Thus, these results established two primary but necessary goals: (1) the baseline knowledge of perturbation of conserved interfacial residues for future reference and (2) the ability of the computational approach to identify the distal residues connecting to the interfacial residues. The data presented here provide the foundation for future experiments to identify the effects of distal residues that affect the specificity and affinity of BMP recognition. Protein-protein interactions are integral reactions in essentially all biological activities such as gene regulation and age-related development. Often, diseases are consequences of the alteration of these intermacromolecular interactions, which are thus recognized as a legitimate target for developing modulators for regulating biological functions. One approach is to design ligands that bind to the protein-protein interface. Another is to identify an allosteric site, an advantage of which is bypassing the potential challenge in competing for high-affinity interfacial interactions or a specific interface in a superassembly of multiple macromolecules. However, a challenge of this approach is identifying a site that is situated away from but functionally connected to the protein-protein interface.

Nanostructured hydroxyapatite surfaces-mediated adsorption alters recognition of BMP receptor IA and bioactivity of bone morphogenetic protein-2.

Highly efficient loading of bone morphogenetic protein-2 (BMP-2) onto carriers with desirable performance is still a major challenge in the field of bone regeneration. Till now, the nanoscaled surface-induced changes of the structure and bioactivity of BMP-2 remains poorly understood. Here, the effect of nanoscaled surface on the adsorption and bioactivity of BMP-2 was investigated with a series of hydroxyapatite surfaces (HAPs): HAP crystal-coated surface (HAP), HAP crystal-coated polished surface (HAP-Pol), and sintered HAP crystal-coated surface (HAP-Sin). The adsorption dynamics of recombinant human BMP-2 (rhBMP-2) and the accessibility of the binding epitopes of adsorbed rhBMP-2 for BMP receptors (BMPRs) were examined by a quartz crystal microbalance with dissipation. Moreover, the bioactivity of adsorbed rhBMP-2 and the BMP-induced Smad signaling were investigated with C2C12 model cells. A noticeably high mass-uptake of rhBMP-2 and enhanced recognition of BMPR-IA to adsorbed rhBMP-2 were found on the HAP-Pol surface. For the rhBMP-2-adsorbed HAPs, both ALP activity and Smad signaling increased in the order of HAP-Sin

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.

Discovery of a nanomolar inhibitor of lung adenocarcinoma in vitro.

Efficient methods for the preparation of 5'-substituted 5'-amino-5'-deoxy-N(6)-ureidoadenosine derivatives are described. Compounds were screened for antiproliferative activity against a panel of murine and human cell lines (L1210, CEM, and HeLa) and/or against the NCI-60. The most potent derivative inhibited the lung adenocarcinoma cell line NCI-H522 at low nanomolar concentrations (GI50 = 9.7 nM).

Sulfated glycosaminoglycans exploit the conformational plasticity of bone morphogenetic protein-2 (BMP-2) and alter the interaction profile with its receptor.

Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.

Anti-wrinkle effect of bone morphogenetic protein receptor 1a-extracellular domain (BMPR1a-ECD).

Bone morphogenetic proteins (BMPs) have diverse and important roles in the proliferation and differentiation of adult stem cells in our tissues. Especially, BMPs are well known to be the main inducers of bone formation, by facilitating both proliferation and differentiation of bone stem cells. Interestingly, in skin stem cells, BMPs repress their proliferation but are indispensable for the proper differentiation into several lineages of skin cells. Here, we tested whether BMP antagonists have an effect on the prevention of wrinkle formation. For this study we used an in vivo wrinkle-induced mouse model. As a positive control, retinoic acid, one of the top anti-wrinkle effectors, showed a 44% improvement compared to the non-treated control. Surprisingly, bone morphogenetic protein receptor 1a extracellular domain (BMPR1a-ECD) exhibited an anti-wrinkle effect which was 6-fold greater than that of retinoic acid. Our results indicate that BMP antagonists will be good targets for skin or hair diseases.

Structure of the Alk1 extracellular domain and characterization of its bone morphogenetic protein (BMP) binding properties.

Bone morphogenetic proteins (BMPs) are secreted signaling proteins - they transduce their signals by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homologue Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the ß-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the ß4-ß5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the ß4-ß5 loop, and to lesser extent ß1-ß2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9/Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3 but is rotated by 40 deg. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the ß4-ß5 loop that contribute significantly to binding and are nonconservatively substituted relative to the corresponding residues in Alk3.

Odontogenic ameloblasts-associated protein (ODAM), via phosphorylation by bone morphogenetic protein receptor type IB (BMPR-IB), is implicated in ameloblast differentiation.

To elucidate the function of the odontogenic ameloblast-associated protein (ODAM) in ameloblasts, we identified more than 74 proteins that interact with ODAM using protoarray. Of the identified proteins, bone morphogenetic protein receptor type-IB (BMPR-IB) was physiologically relevant in differentiating ameloblasts. ODAM and BMPR-IB exhibited similar patterns of expression in vitro, during ameloblast differentiation. ODAM and BMPR-IB interacted through the C-terminus of ODAM, which resulted in increased ODAM phosphorylation in the presence of bone morphogenetic protein 2 (BMP-2). Immunoprecipitation assays using Ser-Xaa-Glu (SXE) mutants of ODAM demonstrated that the phosphorylation of ODAM by BMPR-IB occurs at this motif, and this phosphorylation is required for the activation of MAPKs. ODAM phosphorylation was detected in ameloblasts during ameloblast differentiation and enamel mineralization in vitro and involved in the activation of downstream factors of MAPKs. Therefore, the BMP-2-BMPR-IB-ODAM-MAPK signaling cascade has important roles in ameloblast differentiation and enamel mineralization. Our data suggest that ODAM facilitates the progression of tooth development in cooperation with BMPR-IB through distinct domains of ODAM.

Synthesis, SAR, and preliminary mechanistic evaluation of novel antiproliferative N6,5'-bis-ureido- and 5'-carbamoyl-N6-ureidoadenosine derivatives.

We have developed efficient methods for the preparation of N(6),5'-bis-ureidoadenosine derivatives and their 5'-carbamoyl-N(6)-ureido congeners. Treatment of 5'-azido-5'-deoxy-N(6)-(N-alkyl or -arylurea)adenosine derivatives (6a-d) with H(2)/Pd-C or Ph(3)P/H(2)O, followed by N-methyl-p-nitrophenylcarbamate gave N(6),5'-bis-ureido products 7a-d in 49-78% yield. Analogous derivatives in the 5'-carbamoyl-N(6)-ureido series were prepared by treatment of 2',3'-bis-O-TBS-adenosine (11) with N-methyl-p-nitrophenylcarbamate followed by acylation with appropriate isocyanates which gave 13a-d in 45-69% yield. A more versatile route for obtaining potentially vast libraries of compounds from both series was achieved by treatment of 5'-N-methylureido- or 5'-N-methylcarbamoyladenosine derivatives with ethylchlorformate to give N(6)-ethoxycarbonyl derivatives (9 and 14) in 55-63% yields, respectively. Simple heating of 9 or 14 in the presence of primary alkyl- or arylamines gave the corresponding N(6),5'-bis-ureido- or 5'-carbamoyl-N(6)-ureidoadenosine derivatives in good yields (33-72% and 39-83%; 10a-e and 15a-e, respectively). Significant antiproliferative activities (IC(50)≈4-10 µg/mL) were observed for a majority of the N(6),5'-bis-ureido derivatives, whereas the 5'-carbamoyl-N(6)-ureido derivatives were generally less active (IC(50) >100 µg/mL). A 2',3'-O-desilylated derivative (5'-amino-5'-deoxy-5'-N-methylureido-N(6)-(N-phenylcarbamoyl)adenosine, 16) was shown to inhibit binding of 16 of 441 protein kinases to immobilized ATP-binding site ligands by 30-40% in a competitive binding assay at 10 µM. Compound 16 was also shown to bind to bone morphogenetic protein receptor 1b (BMPR1b) with a Kd=11.5 ± 0.7 µM.