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Background
Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor.
Results
Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.
Conclusions
Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.
An important kinetic parameter for drug efficacy is the residence time of a compound at a drug target, which is related to the dissociation rate constant koff. For the essential antimycobacterial target InhA, this parameter is most likely governed by the ordering of the flexible substrate binding loop (SBL). Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes. To assess the structural differences of the complexes from a dynamic point of view, molecular dynamics (MD) simulations with a total sampling time of 3.0 µs were performed for three ligand-bound and two ligand-free (perturbed) InhA systems. The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families. Based on their different occurrence frequencies in the simulated systems, the conformational preferences could be linked to structural differences of the respective ligands to reveal important determinants of residence time. The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA. The analyses revealed potential directions for avoiding this conformational change and, thus, hindering rapid dissociation: (1) an anchor group in 2'-position of the B-ring for scaffold stabilization, (2) proper occupation of the hydrophobic pocket, and (3) the introduction of a barricade substituent in 5'-position of the diphenyl ether B-ring.
The title compound, C\(_{12}\)H\(_{9}\)N\(_{3}\)O, is an intermediate in the synthesis of the muscarinic M2 receptor antagonist AFDX-384. The seven-membered ring adopts a boat conformation and the dihedral angle between the planes of the aromatic rings is 41.51 (9)°. In the crystal, molecules are linked into [001] chains of alternating inversion dimers formed by pairs of N-H・・・O hydrogen bonds and pairs of N-H・・・N hydrogen bonds. In both cases, R\(_{2}\)\(^{2}\)(8) loops are generated.