Discovery of a novel class of benzimidazoles as highly effective agonists of bone morphogenetic protein (BMP) receptor signaling

Increasing or restoring Bone Morphogenetic Protein receptor signaling is an effective therapy for conditions such as bone fracture and pulmonary arterial hypertension. However, direct use of recombinant BMPs has encountered significant obstacles. Moreover, synthetic, full agonists of BMP receptor signaling have yet to be identified. Here, we report the discovery of a novel class of indolyl-benzimidazoles, synthesized using a one-pot synthetic methodology, which appear to mimic the biochemical and functional activity of BMPs. The first-in-series compounds, SY-LB-35 and SY-LB-57, stimulated significant increases in cell number and cell viability in the C2C12 myoblast cell line. Cell cycle analysis revealed that these compounds induced a shift toward proliferative phases. SY-LB-35 and SY-LB-57 stimulated canonical Smad and non-canonical PI3K/Akt, ERK, p38 and JNK intracellular signaling pathways, similar to BMP2-stimulated responses. Importantly, increases in Smad phosphorylation and cell viability were dependent on type I BMP receptor activity. Thus, these compounds robustly activate intracellular signaling in a BMP receptor-dependent manner and may signify the first known, full agonists of BMP receptor signaling. Moreover, discovery of small molecule activators of BMP pathways, which can be efficiently formulated and targeted to diseased or damaged areas, could potentially substitute recombinant BMPs for treatment of BMP-related pathologies.

via the canonical Smad pathway of transcriptional regulators [16][17][18] . Multiple Smad-independent or non-canonical signaling pathways have also been identified downstream of BMP receptor activation. Some pathways, like the Smad pathway, regulate gene expression but other pathways elicit diverse effects on non-nuclear targets, such as the actin cytoskeleton [19][20][21] .
In an attempt to synthesize an effective small molecule agonist of BMP signaling pathways, several small molecules that either enhance or synergistically improve BMP activity were considered in our synthetic design for new classes of molecules ( Fig. 1) 13,[22][23][24] . PD407824 (1), a carbazole derivative, was reported as a sensitizer of subthreshold levels of BMP4 in C2C12 myoblast cells and human embryonic stem cells. However, this sensitization appears to be the result of an indirect mechanism independent of BMP receptors 22 . Benzoxazoles and benzimidazoles (compounds 3-5) are classes of small molecules identified by high-throughput screening (HTS) using a luciferease reporter 13 . Compound 3 (a benzoxazole), in particular, increased the phosphorylated forms of Smad1/5/8 (p-Smads) and led to the activation of BMP target genes, such as Id1 and Id3 13 . This compound (3, sb4) appears to allosterically activate the type I BMP receptor as Smad-dependent signaling was observed even in the presence of an extracellular inhibitor of BMP signaling or a type I BMP receptor inhibitor. Compounds 4 and 5 are benzimidazole class molecules and were shown to enhnace BMP signaling as well 13 . Finally, Roussel and coworkers reported two unique classes of small molecules as potential activators of BMP signaling 23,24 . Once again, a HTS led to the identification of two natural chalcones (isoliquiritigenin and 4'hydroxychalcone) and three synthetic compounds that target BMP signaling. These compounds induced the phosphorylation of Smad1/5/8 in a dose dependent manner 23,24 .
Careful assessment of these previously reported small molecules identified specific heterocyclic scaffolds, such as indole, benzoxazole and benzimidazole, that may be characterized as useful chemical space to explore. Indeed, the Yoganathan lab has been investigating the biochemistry and biology of structurally diverse benzimidazoles and established a library of small molecules 25,26 . Based on the heterocylic scaffolds in such BMP signaling modulators as compounds 1, 3, 4 and 5 (Fig. 1), two distinct indolyl-benzimidazoles (compounds 10 and 11; Fig. 2) were designed and synthesized as structurally novel, small molecules for our initial studies. Moreover, the Yoganathan lab is poised to generate a larger library of similar small molecules for further exploration of the structure-activity relationships of indolyl-benzimidazole derivatives.
Herein, this study presents the synthetic approach and initial pharmacological evaluation of two new indolylbenzimidazole compounds (SY-LB-35 and SY-LB-57). The one-pot synthesis 27 combined with the low cost and stability of these compounds is an advance over other benzimidazole synthesis methods. The novel compounds showed signs of toxicity only at high mircomolar concentrations. SY-LB-35 and SY-LB-57 stimulated large increases in cell viability, which is likely due to stimulation of proliferation, given that these compounds also increased cell number with respect to control and caused shifts towards the S and G2/M phases of the cell cycle. These compounds stimulated robust increases in phospho(p)-Smad1/5/8 (p-Smad), p-Akt, p-ERK and p-p38 levels. Moreover, inhibition of type I BMP receptor activity blocked increases in p-Smad and cell viability induced by SY-LB-35 and SY-LB 57. Taken together, these data suggest that the novel indolyl-benzimidazoles, SY-LB-35 and SY-LB-57, are the first reported small molecules with true BMP receptor agonist activity.

Results
Development of a new one-pot synthesis procedure. The two indolyl-benzimidazoles (SY-LB-35 and SY-LB-57) were prepared using a highly efficient synthetic approach as described in Fig. 2 27 . The first step in the synthesis led to the conversion of indole-2-carboyxlic acid to the corresponding aryl amide. The use of DMF as solvent provided the needed solubility and the high boiling point of DMF allowed us to perform the reflux in the second step of the synthesis. The formation of amide was confirmed by TLC and LC/MS analysis. Without isolating the amide, the synthesis continued with the conversion of the amide to the corresponding benzimidazole using an HBTU-promoted protocol. The final compounds were obtained in high yield (78%-92%) after a two-step purification sequence, which included a silica gel column chromatography and recrystallization. Both compounds were fully characterized using NMR spectroscopy and mass spectrometry (see Supplemental Materials and Supplemental Figs. S1 and S2).

SY-LB-35 and SY-LB-57 significantly increase the cell viability of C2C2 cells.
To first determine how these novel compounds effect the health of C2C12 cells and to establish the range of subtoxic concentrations for future studies, cell viability assays were carried out. Serum-starved C2C12 cells were exposed to increasing concentrations (0.01-1000 µM) of SY-LB-35 and SY-LB-57 for 24 h. Exposure to Triton X-100 (125 µM) is toxic to cells and was used as a negative control for cell viability. Following the 24-h exposure period, the MT Cell viability substrate was added to the cultures. Only viable cells can reduce the substrate and, thereby, generate a luminescent signal. Any decrease in luminescence was considered an indication of cell death or toxicity. In contrast, increases in luminescence signals are typically due to proliferation of cells in the treated cultures.
In response to treatment of C2C12 cells with SY-LB-35, the highest concentrations (100 µM and 1 mM) significantly decreased cell viability compared with control (30% decrease; ***p < 0.001 and 80% decrease; ****p < 0.0005, respectively; Fig. 3A). At lower concentrations (0.01 µM to 10 µM), no decreases in cell viability were observed indicating that SY-LB-35 was well tolerated by C2C12 cells. Instead, statistically significant increases in cell viability were observed following treatment with concentrations less than 100 µM compared to control (0.01 µM, 150% over control; ****p < 0.0005; 0.1 µM and 1 µM, 150% and 120% over control, respectively; ***p < 0.001; Fig. 3A). A non-linear curve analysis was carried out to calculate the IC 50 value allowing determination of the concentration of SY-LB-35 at which the cell viability is reduced by 50%. The IC 50 value for SY-LB-35 in C2C12 cells was 401.06 µM (Fig. 3B). These results indicate that concentrations of SY-LB-35 from 0.01 to 10 µM are non-toxic and can be used for future experiments.
To further investigate how SY-LB-35 and SY-LB-57 cause increases in cell viability, the total cell number was determined following a 24-h treatment of C2C12 cells with 0.01-10 µM SY-LB-35 or SY-LB-57. The live cell counts demonstrated that SY-LB-35 and SY-LB-57 increased cell numbers significantly compared with cultures grown in Serum-Starvation (SS) medium alone (Fig. 4, ***p < 0.001; **p < 0.01; *p < 0.05). For the concentrations tested, SY-LB-35 increased the number of cells by an average of approximately 40% (Fig. 4A) and SY-LB-57 enhanced cell numbers to an average increase of 70% compared to the control (Fig. 4B). Taken together, these
To establish that SY-LB-35 and SY-LB-57 also stimulate the translocation of phosphorylated Smad to the nucleus, immunofluorescent labeling was performed. Serum-starved C2C12 cells grown on poly-D-lysine (PDL)coated coverslips were stimulated with BMP2 (50 ng/mL), SY-LB-35 (1 µM) or SY-LB-57 (1 µM) for 30 min. The cultures were fixed and labelled with anti-p-Smad antibodies (red) and DAPI (blue), which stains the nuclei (Fig. 5B-E). In control, unstimulated C2C12 cells, low levels of diffuse, cytoplasmic p-Smad labeling were detected (Fig. 5B). Treatment of C2C12 cells with SY-LB-35 (Fig. 5D) and SY-LB-57 (Fig. 5E) induced the translocation of p-Smad into the nucleus similar to that induced by BMP2 (Fig. 5C). Thus, like BMP2, SY-LB-35 and SY-LB-57 not only strongly stimulated Smad phosphorylation but also prompted p-Smad nuclear translocation. To investigate whether the novel benzimidazoles directly regulate PI3K enzymatic activity, serum-starved C2C12 cells were stimulated with 50 ng/mL BMP2, 10 µM SY-LB-35, or 10 µM SY-LB-57 for 15 min and whole cell lysates were prepared. PI3K enzyme was immunoprecipitated and processed in a PI3K ELISA (Fig. 7B). A standard curve of known concentrations of PIP 3 versus absorbance was plotted and used to determine the amount of PIP 3 generated from a PIP 2 substrate by active PI3K enzyme immunoprecipitated from the treated cell samples (Supplemental Fig. S7). The enzyme activity is expressed as the amount of product (PIP 3 ) generated in each sample by the PI3K enzyme per minute of reaction. The results of PI3K ELISA assay revealed that the amount of the PIP 3 produced by PI3K enzymatic activity was significantly higher in BMP2-(30%), SY-LB-35-(26%) and SY-LB 57-treated samples (26%) compared to control, untreated cells (****p < 0.0005; Fig. 7B) confirming the direct activation of PI3K in response to SY-LB-35 and SY-LB-57.

SY-LB-35 and SY-LB
To resolve whether the SY-LB-35 or SY-LB-57 compound requires type I BMP receptor activity for the induction of cell viability in C2C12 cells, cell viability assays were carried out in the presence or absence of 10 µM DM. After 24 h, the presence of DM led to complete inhibition of the cell viability increases stimulated by SY-LB-35 and SY-LB-57 compared to cell viability responses in the absence of DM (****p < 0.0005; ***p < 0.001; **p < 0.01; *p < 0.05; Fig. 10B,C). Thus, the novel benzimidazoles, SY-LB-35 and SY-LB-57, appear to stimulate both canonical BMP signaling and robust increases in cell viability through a process dependent on the activity of type I BMP receptors.

Discussion
There has been growing interest in identifying small molecules as useful modulators of BMP signaling. Classical approaches involve HTS of small molecule libraries to identify promising lead compounds. Based on the available literature, two privileged heterocyclic scaffolds that are components of reported BMP signaling modulators were identified as candidates for further exploration. PD407824 is a carbazole derivative and contains an indole structure 22 . A series of compounds reported by Bradford et al. contain a benzimidazole as the heterocyclic motif and shown to be essential for activity 13 . Compared to recombinant proteins, such synthetic small molecules provide several advantages as therapeutic agents. Small molecules are typically more stable, easily accessible via chemical synthesis, and pharmacological properties can be optimized via medicinal chemistry efforts. The previously reported small molecules identified as BMP-like show relatively low activity and fail to fully activate   www.nature.com/scientificreports/ BMP signaling. Thus, there is still a strong need to identify efficient BMP agonists that could replace approaches currently using less stable, inefficient, proteinaceous preparations of recombinant BMPs to treat BMP-related pathologies. Herein, the synthesis and evaluation of two novel compounds, SY-LB-35 and SY-LB-57 (Fig. 2), is reported. The design for these compounds came from the observation that indole and benzimidazole motifs are found in reported BMP modulators 13,[22][23][24] and are known to be privileged scaffolds in the drug discovery field 26,28,29 . A recently developed synthetic methodology within the Yoganathan lab provided an ideal opportunity to design and synthesize a hybrid molecule that contains the indole and benzimidazole scaffolds. These compounds were synthesized from commercially available carboxylic acid precursors and the synthesis was carried out in as high as 1 g scale. As the precursors are readily available and the synthesis can be executed in multi-gram scale, it is possible to do extensive biological evaluation both in vitro and in vivo. Since PD407824 (compound 1; Fig. 1) has a hydroxyl group at the 5-postion of the indole structure, we designed the two compounds to contain the same oxygenation pattern on the indole motif. SY-LB-35 has a hydroxyl group at the 5-position of the indole unit (compound 10; Fig. 2) and SY-LB-57 has a methoxy group at the same position on the indole motif (compound 11; Fig. 2). These two functionalities provide different physiochemical properties to these analogs and likely different types of intermolecular interactions to a potential protein target. It is expected that SY-LB-35 will be slightly more hydrophilic, in comparison to SY-LB-57. Additionally, having the hydroxyl handle on the indole unit (SY-LB-35) provides future opportunities to explore a larger library of compounds through medicinal chemistry efforts. Based on our own research, we have established a synthetic method that allows selective derivatization of the benzimidazole or the indole unit via N-alkylation chemistry for further investigation of these hybrid compounds 25 . Moreover, the initial biological evaluation revealed promising cell viability data, demonstrating that these new compounds are likely stimulating cell growth in C2C12 mouse myoblasts. A concentration-response analysis showed that these two compounds are essentially non-toxic to C2C12 cells at concentrations well above 100 µM. Indeed, the calculated IC 50 values for SY-LB-35 and SY-LB-57 are 401 µM and 808 µM, respectively. Previous efforts to stimulate BMP signaling pathways has led to the discovery of a few small molecules, which failed to fully activate the canonical Smad pathway. The BMP sensitizer, PD407824, did not induce Smad phosphorylation but, rather interestingly, reduced p-Smad levels in the absence of BMP. However, in the presence www.nature.com/scientificreports/ of BMP, PD407824 slightly increased p-Smad levels over BMP alone through an indirect mechanism involving inhibition of Smad2/3, which serve as R-Smads for TGFβ ligands 22 . HTS for agonists of BMP signaling introduced a series of compounds, typified by sb4, which increase p-Smad levels twofold over control compared to 11-fold for BMP4 in 30-min stimulation assays. Furthermore, sb4 appears to induce Smad phosphorylation through a mechanism that is independent of type I BMP receptors 13 .
In the current study, SY-LB-35 and SY-LB-57 at sub-micromolar concentrations stimulated robust increases in p-Smad levels in C2C12 cells after 30 min (100-250% increase over control; Fig. 5A). In comparison, BMP2 at a relatively high concentration of 50 ng/mL caused ~ 100% increase in level of phosphorylated Smads in the same cells demonstrating the efficacy of these novel indolyl-benzimidazoles. Moreover, inhibition of type I BMP receptor activity by DM indicated that Smad phosphorylation by these compounds relies on the activity of type I BMP receptors. Furthermore, immunofluorescence analysis of C2C12 cells treated with these compounds confirmed strong translocation of p-Smad into the nucleus, which was indistinguishable from the response to BMP2 (Fig. 5C-E).
SY-LB-35 and SY-LB-57 also significantly boosted the levels of p-Akt and induced cytoplasmic distribution of p-Akt in C2C12 cells (Fig. 6). Previous examination of type II BMP receptor-dependent regulation of PI3K/Akt activity revealed that BMP-evoked Akt phosphorylation was inhibited when ActRIIA expression was deficient 30 . Moreover, specific type II BMP receptor subunits, ActRIIA and BMPR2, were required for BMP-induced growth cone collapse in developing spinal neurons and for chemotaxis of monocytes 21,30 . In the present study, assessment of BMP signaling by SY-LB-35 and SY-LB-57 suggests that these compounds might induce Akt activation through type II BMP receptors as well. Taken together, these data strongly support the idea that SY-LB-35 and SY-LB-57, likely working through type I BMP receptors, can mirror the activity of recombinant BMPs to trigger BMP signaling pathways and, thus, appear to act as BMP agonists.
Type I BMP receptors initiate intracellular signaling by phosphorylating specific R-Smads, which ultimately leads to transcriptional regulation of target genes 18 . Stimulation of the PI3K/Akt pathway by BMPs has been biochemically and functionally linked to type II BMP receptor-dependent signaling 19,21,30 . How dimeric BMPs stimulate these distinct pathways through interaction with the tetrameric BMP receptor complex is an unanswered question in the BMP receptor field. www.nature.com/scientificreports/ Early evidence of small molecules activating BMP receptors was obtained from the study of immunosuppressants such as sirolimus (rapamycin) and tacrolimus (FK506), which appeared to liberate type I BMP receptors from inhibition by the immunophilin, FKBP12, and promote activation of BMP receptors intracellularly leading to activation of the Smad pathway 5,31 . Further efforts to identify small molecule agonists of BMPs resulted in development of sb-series compounds, which marginally enhanced BMP signaling 13 . sb4-mediated activation of the BMP pathway was resistant to inhibition by the endogenous BMP antagonist, noggin, or by type I BMP receptor inhibitors suggesting that sb4 works through a mechanism that does not involve type I BMP receptor activity 13 . Dependence on type I BMP receptor activity observed in this study suggests a more direct activation of BMP signaling by SY-LB-35 and SY-LB-57 resulting in increases in p-Smad levels on par with BMP-evoked activity. Inhibition of Smad signaling in the presence of DM showed that these indolyl-benzimidazole compounds may interact directly with type I BMP receptors or with a receptor-associated component like FKBP12. Moreover, pretreatment of C2C12 cells with DM not only decreased p-Smad levels but also completely blocked SY-LB-35-and SY-LB-57-induced increases in cell viability suggesting that these indolyl-benzimidazole compounds might functionally regulate BMP receptor activity. Furthermore, the ability of SY-LB-35 and SY-LB-57 to also stimulate robust phosphorylation of PI3K and Akt suggests that these compounds may regulate the activity of the whole tetrameric receptor complex. It will be important to determine whether and how these novel compounds interact with BMP receptor subunits. Moreover, given the differences in activity observed between SY-LB-35 and SY-LB-57 in Smad, Akt, p38 and JNK phosphorylation and in cell viability assays, it will be interesting to explore how derivatization of the indole or benzimidazole unit might influence the activity of type I or type II BMP receptor-dependent signaling pathways.
Treatment of C2C12 cells with either SY-LB-35 or SY-LB-57 for 15 min significantly upregulated the level of PI3K, Akt, ERK, p38 and JNK phosphorylation. The downstream effector molecules of the PI3K/Akt, ERK and p38 pathways are known to be involved in cellular proliferation [32][33][34] . Given the increases in cell viability, cell number as well as cell cycle shifts towards S and G2/M phases observed following 24-h treatment with SY-LB-35 and SY-LB-57, it is reasonable to infer that these compounds may mediate proliferation via stimulation of PI3K/ Akt, ERK or p38. BMPs are most well-known for their morphogenetic activities, which involves processes of growth, differentiation, and remodeling of tissues 1,2,4,16,18 . Indeed, SY-LB-35 and SY-LB-57 stimulated significant growth of C2C12 cells indicating the morphogenetic potential of these novel indolyl-benzimidazoles.
BMPs regulate key processes during embryonic development, like formation of neural crest cells and patterning of the spinal cord and brain 2,4 . Emerging roles for BMPs have been recognized in maintaining adult tissue homeostasis and deficits in BMP-dependent signaling have been demonstrated to contribute to certain pathologies in adults 35 . Thus far, a few compounds have been introduced as positive regulators of BMP signaling and primarily act as enhancers or sensitizers increasing the responsiveness of the cells to BMPs or stabilizing Smad signling complexes 11,36,37 . In the current study, two novel chemical entities, SY-LB-35 and SY-LB-57, were introduced sharing an indolyl-benzimidazole core, which stimulated canonical and non-canonical BMP signaling pathways and faithfully mimicked recombinant BMP2 signaling activity in C2C12 cells. These compounds demonstrated substantial activation of the canonical Smad-dependent pathway and the Smad-independent, non-canonical PI3K/Akt pathway. Moreover, Smad activation by these compounds was blocked by inhibition of type I BMP receptor activity. Importantly, these compounds not only activated Smad signaling but appear to functionally mimic endogenous BMPs in cell viability and cell cycle assays.
Taken together, the data suggests that the SY-LB-35 and SY-LB-57 are efficient, non-selective activators of BMP receptor-dependent signaling pathways making them strong candidates for developing therapeutics that can robustly activate BMP receptor signaling in human disease conditions in which BMP signaling is defective or lost.

Materials and methods
Chemical synthesis. All chemicals and solvents were used without further purification. The two indolylbenzimidazoles were synthesized using a one-pot synthetic methodology (Fig. 2) based on previous studies 27 . Commercially available indole-2-carboxylic acid derivatives (compounds 6, 7) (1 equiv.) and N-diisopropylethylamine (DIPEA) (2.0 equiv.) were dissolved in 10 mL DMF and stirred for 10 min. Then, O-benzotriazol-1-yl N,N,N' ,N'-tetramethyluronium hexafluorophosphate (HBTU) (2.0 equiv.) was added, and the reaction mixture was stirred for another 10 min. Next, O-phenylenediamine (1.0 equiv.) was added and stirred for another 4 h to generate compounds 8 and 9. Thereafter, the reaction was heated under reflux for 6 h to produce the two indolyl-benzimidazoles (compounds 10, 11). Chemical reactions were analysed by thin layer chromatography (TLC) with silica gel G as the adsorbent (250 microns) on aluminium backed plates (Agela Technologies, Torrance, CA, USA) and Ultraviolet (UV) light at 254 nm or 365 nm for visualization purposes. The reaction vessel was cooled to room temperature (RT) and the reaction was diluted with water (100 mL) followed by extraction of the product using ethyl acetate (EtOAc). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was further purified by column chromatography using hexanes/ EtOAc in increasing polarity up to a 1:1 mixture. The fractions containing the desired product were concentrated and recrystallized in hexanes/EtOAc (1:1) to yield the final product. The compounds were characterized by 1D ( 1 H and 13 C) NMR using a Bruker 400 UltrashieldTM spectrometer (400 MHz) equipped with a z-axis gradient probe (Figures S1 and S2)  Determination of total protein concentration. The Amido-Schwarz TCA precipitation method was used to estimate the amount of total protein in whole cell lysates 38 . Briefly, whole cell lysates were diluted in dH 2 O, and total soluble protein was precipitated using 60% TCA (Trichloroacetic acid) and 1 M Tris/1% SDS. Precipitated protein was spotted onto 0.45 µm nitrocellulose membranes (Millipore, MA, USA), rinsed with 6% TCA and stained with 0.1% Amido Black in 45% methanol/10% acetic acid/45% dH 2 O. The membranes are rinsed at least 3 times in 90% methanol/2% acetic acid/8% dH 2 O until the membrane background was nearly white. The remaining stained protein was eluted from the membranes in 25 mM NaOH/0.05 M EDTA/50% ethanol. Absorbance of the eluted samples was measured at 630 nm using the Eppendorf BioSpectrometer Basic (Lake Forest, CA, USA). The total protein concentration in the whole cell lysates was determined using a BSA standard curve.
PI3K ELISA assay. C2C12 cells were seeded in 35-mm dishes at 7 × 10 4 cells/mL and incubated at 37 °C in 5% CO 2 to reach 80% confluency. The cells were starved for 16-18 h and then stimulated with BMP2 (50 ng/mL) as positive control and SY-LB-35 and SY-LB-57 at 10 µM for 15 min at 37 °C. Cells (~ 10 6 ) per treatment were collected to prepare whole cell lysates followed by exposure to three freeze/thaw cycles in liquid nitrogen. The samples were centrifuged and PI3K enzyme was immunoprecipitated with a mouse monoclonal anti-PI3K p55 γ antibody ((E-9), Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at 4 °C with gentle rocking and the immune www.nature.com/scientificreports/ complexes were collected overnight on a rocking platform at 4 °C with Dynabeads™ Protein A (Life Technologies, Carlsbad, CA, USA). The next day, the immunoprecipitated PI3K enzyme was collected by centrifugation processed for a PI3K ELISA (Echelon Biosciences, Salt Lake, City, UT, USA) according to the manufacturer's instructions. Standard solutions of PIP 3 (0, 0.22, 0.67, 2, 6, 18, and 54 pmol) were added to kinase reactions using recombinant PI3K enzyme (Echelon Biosciences, Salt Lake, City, UT, USA) and used to generate a standard curve for production of PIP 3 . The optical density of the PIP 3 standards and the reactions containing immunoprecipitated PI3K from treated samples was measured at 450 nm.
Cell cycle analysis. C2C12 cells (10 mL) were seeded into 100 mm dishes at 1 × 10 6 cells/mL, grown to 80% confluency, serum starved for 16 h and treated with BMP2 (50 ng/mL), SY-LB-35 or SY-LB-57 (0.01-10 µM) for 24 h. Equal numbers (~ 10 6 ) of cells, from each treatment counted as above were collected, fixed with 4% paraformaldehyde (PFA) for 10 min at RT and pelleted at 300 g for 5 min. Cell pellets were washed with Stain buffer (BD Pharmingen, San Diego, CA, USA) followed by permeabilization with 90% ice-cold methanol for 15 min at 4 °C. The cells were again washed with Stain buffer and the pelleted cells were stained with BD Pharmingen Propidium Iodide/RNase Staining Buffer (0.5 mL/10 6 cells) for 30 min at RT while protected from light. Following two more washes, the pellet was resuspended in 225 µL Stain buffer and the cells were divided into 3 microcentrifuge tubes (75 µL each) for analysis with the Amnis ® Flowsight Imaging Flow Cytometer (Austin, TX, USA). IDEAS ® software (version 6.2, Seattle, WA, USA) was used to analyse the data files.
Immunofluorescent labeling. In a 24-well plate, 12 mm glass coverslips were coated for 1 h at RT with 500 µL PDL (0.1 mg/mL/ 0.1 M Boric acid), washed 4 times with cell culture-grade distilled water and sterilized under UV light for 30 min. C2C12 cells (500 µL) were seeded onto the PDL-coated coverslips at a density of 1 × 10 5 cells/mL, grown to 80% confluency and starved in SS medium for 2 h. Next, the cells were treated with 50 ng/mL BMP2 or 1 µM SY-LB-35 or SY-LB-57 for 15 min (for p-Akt labelling) or 30 min (for p-Smad labelling). Cells were then fixed using ice-cold 4% PFA/PBS for 10 min at RT. Next, the coverslips were washed with ice-cold 1X PBS, transferred to a humidified chamber and incubated (100 µL/coverslip) in Permeabilization solution (0.1% Triton X-100/PBS) for 15 min, then Blocking Buffer (10% heat inactivated goat serum/ 0.3% Triton X-100/PBS) for 30 min. Anti-p-Smad antibodies in Antibody Dilution Buffer (1% BSA/0.1% Tween 20/PBS) were added for 1 h and the coverslips were washed 4 times with 1X PBS. The Cy3-conjugated goat anti-rabbit IgG secondary antibodies (Jackson Immuno Research Labs, West Grove, PA, USA) in Antibody Dilution Buffer were added for 1 h followed by 4 washes with 1X PBS and once with dH 2 O. Finally, the coverslips were mounted on glass microscope slides in Vectashield Mounting Medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Images were obtained using a Zeiss Axioplan 200 M upright fluorescent microscope, Axi-oCam HRm digital camera and AxioVision 4.8.2.0 software.

Data availability
All data generated or analyzed during this study are included in this article and its supplementary information files.