Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

Inhibitory effects of sesquiterpene lactones from the Indonesian marine sponge Lamellodysidea cf. herbacea on bone morphogenetic protein-induced osteoblastic differentiation.

A new unique sesquiterpene lactone, bicyclolamellolactone A (1), was isolated together with two known monocyclofarnesol-type sesquiterpenes, lamellolactones A (2) and B (3), from the Indonesian marine sponge Lamellodysidea sp. (cf. L. herbacea). The planar structure of 1 was assigned based on its spectroscopic data (1D and 2D NMR, HRESIMS, UV, and IR spectra). The relative and absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism spectra in combination with NOESY correlations. Compounds 1-3 inhibited bone morphogenic protein (BMP)-induced alkaline phosphatase activity in mutant BMP receptor-carrying C2C12 cells with IC50 values of 51, 4.6, and 20 µM, respectively.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2.

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1+ osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3.

Discovery of a Fungal Multicopper Oxidase That Catalyzes the Regioselective Coupling of a Tricyclic Naphthopyranone To Produce Atropisomers.

Atropisomeric dinapinones A1 and A2 (DPA1 and DPA2) were isolated from a culture of Talaromyces pinophilus FKI-3864. Monapinone coupling enzyme (MCE), which dimerizes naphthopyranone monapinone A (MPA), was purified from a cell-free extract of T. pinophilus FKI-3864. MCE regioselectively dimerizes MPA at the 8,8'-positions to synthesize the atropisomers DPA1 and DPA2 in a ratio of approximately 1:2.5 without a cofactor. The optimal pH value and temperature for MCE were 4.0 and 50 °C, and the apparent Km and Vmax values for MPA were (72.7±23.2) µm and (1.21±0.170) µmol min-1 mg-1 protein. The MCE polypeptide is significantly homologous with multicopper oxidases. Heterologous expression of MCE and functional analysis confirmed that MCE catalyzes the regioselective coupling reaction of MPA to produce DPA. No fungal multicopper oxidase has previously been reported to catalyze regioselective intermolecular oxidative phenol coupling to produce naphthopyranone atropisomers.

Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-ß1 (TGF-ß1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

The new seriniquinone glycoside by biological transformation using the deep sea-derived bacterium Bacillus licheniformis KDM612.

Seriniquinone was isolated as a melanoma-selective anti-cancer agent from a culture broth of the marine-derived bacterium Serinicoccus marinus CNJ927 in 2014. It targets the unique small protein, dermcidin, which affects the drug resistance of cancer cells. Due to its significant activity against cancer cells, particularly melanoma, and its unique target, seriniquinone has been developed as a new pharmacophore. However, it has the disadvantage of poor solubility in drug discovery research, which needs to be resolved. A new seriniquinone glycoside (1) was synthesized by the biological transformation of seriniquinone using the deep sea-derived bacterium Bacillus licheniformis KDM612. Compound 1 exhibited selective anti-cancer activity against melanoma, similar to seriniquinone, and was 50-fold more soluble in DMSO than seriniquinone.

TLE3, transducing-like enhancer of split 3, suppresses osteoblast differentiation of bone marrow stromal cells.

In senile osteoporosis the balance of adipogenesis and osteoblastogenesis in bone marrow stromal cells (BMSCs) is disrupted so that adipogenesis is increased with respect to osteoblastogenesis, and as a result, bone mass is decreased. While the molecular mechanisms controlling the balance between osteoblastogenesis and adipogenesis are of great interest, the exact nature of the signals regulating this process remains to be determined. In general, adipogenesis is a reciprocal relationship with osteoblastogenesis in BMSCs. Recently transducin-like enhancer of split 3 (TLE3), was reported to enhance adipogenesis in pre adipocytes. However, the effect of TLE3 on osteoblast differentiation of BMSCs is completely unknown. Here we report that TLE3 not only enhances adipocyte differentiation in BMSCs but also suppresses osteoblast differentiation. Firstly we examined the expression and localization of TLE3. We found that TLE3 is expressed in the nucleus of bone marrow stromal cells and that over-expression of TLE3 induced adipocyte differentiation and suppressed ALP activity induced by treatment with BMP2 in these cells. In contrast, adipocyte differentiation was decreased and ALP activity increased when endogenous TLE3 was knocked down by shRNA in BMSCs. To examine the mechanism by which TLE3 is able to suppress osteoblast differentiation, we focused on Runx2, a transcription factor essential for osteoblast differentiation. We found that TLE3 strongly suppressed ALP activity and OSE2-luciferase activity induced by Runx2 and this repression of Runx2 by TLE3 occurs via HDACs because treatment with TSA, a class I and II HDAC inhibitor, rescued this repression. In conclusion, we identify TLE3 as a suppressor of BMSC differentiation in osteoblast lineage cells in vitro. Our data suggest that TLE3 activity may be a key in balancing adipocyte and osteoblast differentiation in the adult bone marrow microenvironment.

Identification of a novel bone morphogenetic protein (BMP)-inducible transcript, BMP-inducible transcript-1, by utilizing the conserved BMP-responsive elements in the Id genes.

Bone morphogenetic proteins (BMPs) inhibit myogenesis and induce osteoblastic differentiation in myoblasts. They also induce the transcription of several common genes, such as Id1, Id2 and Id3, in various cell types. We have reported that a GC-rich element in the Id1 gene functions as a BMP-responsive element (BRE) that is regulated by Smads. In this study, we analyzed and identified BREs in the 5'-flanking regions of the mouse Id2 and Id3 genes. The core GGCGCC sequence was conserved among the BREs in the Id1, Id2 and Id3 genes and was essential for the response to BMP signaling via Smads. We found a novel BRE on mouse chromosome 13 at position 47,723,740-47,723,768 by searching for conserved sequences containing the Id1 BRE. This potential BRE was found in the 5'-flanking region of a novel gene that produces a non-coding transcript, termed BMP-inducible transcript-1 (BIT-1), and this element regulated the expression of this gene in response to BMP signaling. We found that BIT-1 is expressed in BMP target tissues such as the testis, brain, kidney and cartilage. These findings suggest that the transcriptional induction of the Ids, BIT-1 and additional novel genes containing the conserved BRE sequence may play an important role in the regulation of the differentiation and/or function of target cells in response to BMPs.

Synthesis and structure-activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: part 1.

In an effort to develop potent and selective inhibitors toward ACAT2, structure-activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). We have successfully developed novel PPPA derivatives with a 7-O-substituted benzoyl substituent that significantly exhibit more potent ACAT2 inhibitory activity and higher ACAT2 isozyme selectivity than 1.

Synthesis and structure-activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: part 3.

In an effort to develop potent and selective inhibitors toward ACAT2, structure-activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). In particular, we investigated the possibility of introducing appropriate 1,11-O-benzylidene and 7-O-substituted benzoyl moieties into PPPA (1). The new o-substituted benzylidene derivatives showed higher selectivity for ACAT2 than PPPA (1). Among them, 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7q and 1,11-O-o,o-dimethylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7z proved to be potent ACAT2 inhibitors with unprecedented high isozyme selectivity.

A blocking monoclonal antibody reveals dimerization of intracellular domains of ALK2 associated with genetic disorders.

Mutations in activin receptor-like kinase 2 (ALK2) can cause the pathological osteogenic signaling seen in some patients with fibrodysplasia ossificans progressiva and other conditions such as diffuse intrinsic pontine glioma. Here, we report that intracellular domain of wild-type ALK2 readily dimerizes in response to BMP7 binding to drive osteogenic signaling. This osteogenic signaling is pathologically triggered by heterotetramers of type II receptor kinases and ALK2 mutant forms, which form intracellular domain dimers in response to activin A binding. We develop a blocking monoclonal antibody, Rm0443, that can suppress ALK2 signaling. We solve the crystal structure of the ALK2 extracellular domain complex with a Fab fragment of Rm0443 and show that Rm0443 induces dimerization of ALK2 extracellular domains in a back-to-back orientation on the cell membrane by binding the residues H64 and F63 on opposite faces of the ligand-binding site. Rm0443 could prevent heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva that carries the human R206H pathogenic mutant.

Identification and functional analysis of Zranb2 as a novel Smad-binding protein that suppresses BMP signaling.

Smads 1/5/8 transduce the major intracellular signaling of bone morphogenetic proteins (BMPs). In the present study, we analyzed Smad1-binding proteins in HEK293T cells using a proteomic technique and identified the protein, zinc-finger, RAN-binding domain-containing protein 2 (ZRANB2). Zranb2 interacted strongly with Smad1, Smad5, and Smad8 and weakly with Smad4. The overexpression of Zranb2 inhibited BMP activities in C2C12 myoblasts in vitro, and the injection of Zranb2 mRNA into zebrafish embryos induced weak dorsalization. Deletion analyses of Zranb2 indicated that the serine/arginine-rich (SR) domain and the glutamine-rich domain were required for the inhibition of BMP activity and the interaction with Smad1, respectively. Zranb2 was found to be localized in the nucleus; however, the SR domain-deleted mutant localized to the cytoplasm. The knockdown of endogenous Zranb2 in C2C12 cells enhanced BMP activity. Zranb2 suppressed Smad transcriptional activity without affecting Smad phosphorylation, nuclear localization, or DNA binding. Taken together, these findings suggested that Zranb2 is a novel BMP suppressor that forms a complex with Smads in the nucleus.

Discovery of prescopranone, a key intermediate in scopranone biosynthesis.

A key intermediate in scopranone biosynthesis, prescopranone, accumulated in the mycelium of Streptomyces avermitilis SUKA carrying the biosynthetic gene cluster for scopranone lacking the sprT encoding the monooxygenase. The structure of prescopranone was elucidated by NMR and other spectral data. Prescopranone consists of a 2-pyranone ring with two atypical scoop-like moieties (1-ethyl-1-propenyl and 2-ethylbutyl groups), which was deduced as a product of the modular polyketide syntheses encoded by sprA, sprB, and sprC. Prescopranone inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity in a BMP receptor mutant cell line.

Screening for microbial potentiators of neutral lipid degradation in CHO-K1 cells.

A cell-based assay was conducted to screen microbial culture broths for potentiators of neutral lipid degradation in Chinese Hamster Ovary K1 cells. A total of 5,363 microbial cultures from fungi and actinomycetes were screened in this assay. Brefeldin A (1) from fungal cultures was found to promote the degradation of triacylglycerol (TG) with an EC50 of 2.6 µM. Beauveriolides I (2), III (3), beauverolides A (4), B (5), and K (6) from fungal cultures showed potentiating effect on cholesteryl ester (CE) degradation with EC50s ranging from 0.02 to 0.13 µM. Among these compounds, 2 and 6 exhibited the strongest activities (EC50, 0.02 µM). From actinomycete cultures, oxohygrolidin (7) (EC50 for TG and CE, > 1.7 and 0.8 µM, respectively) and hygrolidin (8) (EC50 for TG and CE, 0.08 and 0.004 µM, respectively) promoted degradation of CE more preferably than TG.

Dual roles of smad proteins in the conversion from myoblasts to osteoblastic cells by bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic ossification in muscle tissues. Constitutively activated mutants of a bone morphogenetic protein (BMP) receptor, ALK2, have been identified in patients with FOP. Recently, a novel ALK2 mutation, L196P, was found in the most benign case of FOP reported thus far. In the present study, we examined the biological activities of ALK2(L196P) in vitro. Over-expression of ALK2(L196P) induced BMP-specific activities, including the suppression of myogenesis, the induction of alkaline phosphatase activity, increased BMP-specific luciferase reporter activity, and increased phosphorylation of Smad1/5 but not Erk1/2 or p38. The activities of ALK2(L196P) were higher than those of ALK2(G356D), another mutant ALK2 allele found in patients with FOP and were equivalent to those of ALK2(R206H), a typical mutation found in patients with FOP. ALK2(L196P) was equally or more resistant to inhibitors in comparison to ALK2(R206H). These findings suggest that ALK2(L196P) is an activated BMP receptor equivalent to ALK2(R206H) and that ALK2(L196P) activity may be suppressed in vivo by a novel molecular mechanism in patients with this mutation.

Canonical Wnts and BMPs cooperatively induce osteoblastic differentiation through a GSK3beta-dependent and beta-catenin-independent mechanism.

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of beta-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3beta activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3beta-dependent but beta-catenin-independent mechanism.

A new diketopiperazine-like inhibitor of bone morphogenetic protein-induced osteoblastic differentiation produced by marine-derived Aspergillus sp. BFM-0085.

A new diketopiperazine-like compound, designated protuboxepin K (1), was isolated together with the known structurally related protuboxepin A (2) from culture broth of the marine-derived fungal strain Aspergillus sp. BFM-0085 isolated from a sediment sample of Tokyo Bay. The structure of protuboxepin K was elucidated by spectroscopic data, including 1D and 2D NMR. Compounds 1 and 2 inhibited bone morphogenetic protein (BMP)-induced alkaline phosphatase activity with IC50 values of 4.7 and 25.2 µM, respectively, in mutant BMP receptor-carrying C2C12(R206H) cells.

Smad4-dependent transforming growth factor-ß family signaling regulates the differentiation of dental epithelial cells in adult mouse incisors.

Teeth consist of two major tissues, enamel and dentin, which are formed during development by epithelial and mesenchymal cells, respectively. Rodent incisors are useful experimental models for studying the molecular mechanisms of tooth formation because they are simultaneously growing in not only embryos but also adults. Members of the transforming growth factor-ß (TGF-ß) family regulate epithelial-mesenchymal interactions through an essential coactivator, Smad4. In the present study, we established Smad4 conditional knockout (cKO) mice and examined phenotypes in adult incisors. Smad4 cKO mice died with severe anemia within one month. Phosphorylated Smad1/5/9 and Smad2/3 were detected in epithelial cells in both control and Smad4 cKO mice. Disorganized and hypoplastic epithelial cells, such as ameloblasts, were observed in Smad4 cKO mice. Moreover, alkaline phosphatase expression and iron accumulation were reduced in dental epithelial cells in Smad4 cKO mice. These findings suggest that TGF-ß family signaling through Smad4 is required for the differentiation and functions of dental epithelial cells in adult mouse incisors.