Inhibition of ALK2 with bicyclic pyridyllactams.

Activin receptor-like kinase 2 (ALK2) has been implicated as a key target in multiple rare diseases. Herein, we describe the design of a novel bicyclic lactam series of potent and selective ALK2 inhibitors. This manuscript details an improvement in potency of two orders of magnitude from the initial bicyclic structure as well as a two-fold improvement in cellular potency from the original monocyclic inhibitor. Furthermore, we provide a detailed strategy for progressing this project in the future.

Partial inhibition of activin receptor-like kinase 4 alleviates bladder fibrosis caused by bladder outlet obstruction.

The bladder undergoes profound structural alterations after bladder outlet obstruction (BOO), characterized by hypertrophy of the bladder wall and accumulation of extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) has been found to promote fibrosis of the bladder induced by partial bladder outlet obstruction (pBOO). Activin receptor-like kinase 4 (ALK4) is a downstream receptor of the TGF-ß superfamily. However, the role of the ALK4-Smad2/3 pathway in the pathogenesis of bladder fibrosis caused by pBOO remains unknown. This study focused on learning the role of ALK4 in the process of bladder fibrosis caused by pBOO. The pBOO mice models showed that ALK4 expression was found to upregulate in the wild-type bladder 6 weeks after pBOO compared to control group. Then, mice with heterozygous knockout of the ALK4 gene (ALK4+/-) were generated. Histological analysis and Western blot (WB) results showed significant suppression of collagen expression in the bladders of ALK4+/- mice after pBOO compared with WT mice. WB also showed that ALK4+/- mice demonstrated significant suppression of phosphorylated Smad2/3 (p-Smad2/3) expression in the bladder 6 weeks after pBOO but not of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase or protein 38 (p-ERK, p-JNK, p-P38) expression. This effect might have occurred through partial inactivation of the Smad2/3 signaling pathway. In vitro, ALK4 overexpression promoted collagen production in cultured BSMCs and activated the Smad2/3 signaling pathway. Taken together, our results demonstrated that ALK4 insufficiency alleviated bladder fibrosis in a mouse model of pBOO partly by suppressing Smad2/3 activity.

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.

MOMENTUM: momelotinib vs danazol in patients with myelofibrosis previously treated with JAKi who are symptomatic and anemic.

Hallmark features of myelofibrosis (MF) are cytopenias, constitutional symptoms and splenomegaly. Anemia and transfusion dependency are among the most important negative prognostic factors and are exacerbated by many JAK inhibitors (JAKi). Momelotinib (MMB) has been investigated in over 820 patients with MF and possesses a pharmacological and clinical profile differentiated from other JAKi by inhibition of JAK1, JAK2 and ACVR1. MMB is designed to address the complex drivers of iron-restricted anemia and chronic inflammation in MF and should improve constitutional symptoms and splenomegaly while maintaining or improving hemoglobin in JAKi-naive and previously JAKi-treated patients. The MOMENTUM Phase III study is designed to confirm and extend observations of safety and clinical activity of MMB.

Lay abstract The most important features of myelofibrosis (MF) are low blood cell counts and symptoms including tiredness, night sweats and itching, along with increased size of the spleen, which may cause a feeling of fullness and pain. Low red blood cell counts (anemia) may mean regular blood transfusions are needed and this is one of the signs MF is getting worse. Drugs called JAK inhibitors (JAKi) are available to treat MF, but can have a side effect of making blood cell counts lower. Momelotinib (MMB) is a different type of JAKi to the ones currently available, and is an experimental drug for MF. MMB is designed to treat symptoms and spleen like other JAKi, but also to improve blood cell counts. MMB has already been given to more than 820 patients with MF in other clinical studies. Some of the patients in these studies had been treated with different JAKi before, and others got MMB as their first JAKi treatment. The MOMENTUM Phase III study is designed to collect more information on the safety and effectiveness of MMB in MF.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Design, synthesis and biological evaluation of 2-arylaminopyrimidine derivatives bearing 1,3,8-triazaspiro[4,5]decan-4-one or piperidine-3-carboxamide moiety as novel Type-I1/2 ALK inhibitors.

Aiming to develop novel Type-I1/2 inhibitors of ALK to overcome extensive resistance mutations, especially the L1196M mutation surrounding the ATP pocket, two series of 2-arylaminopyrimidine derivatives (11a-f and 22a-t) were designed based on scaffold hopping. The extensive structural elaboration discovered compound 22o which possessed excellent IC50 values of 0.06 and 0.23 µM against ALK-positive Karpas299 and H2228 cell lines, respectively. Meanwhile, 22o displayed encouraging inhibitory potency in the ALKWT (2.5 nM) and ALKL1196M (6.5 nM) enzymatic assays. Furthermore, the AO/EB and Hoechst 33258 assays illustrated 22o could induce cell apoptosis in a dose-dependent manner. Eventually, the molecular docking of 22o with ALK clearly presented the vital interactions within the active site, which was in accordance with Type-I1/2 inhibitor binding mode.

SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. METHODS: The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4iΔEC). RESULTS: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. CONCLUSIONS: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents.

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Bis-Heteroaryl Pyrazoles: Identification of Orally Bioavailable Inhibitors of Activin Receptor-Like Kinase-2 (R206H).

Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.

Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.

Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling.

Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors.

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.

Identification of novel ALK2 inhibitors and their effect on cancer cells.

Bone morphogenetic protein 9 (BMP9), a member of the TGF-ß superfamily, is considered a regulator of glucose homeostasis as well as a neuronal differentiation factor. BMP9 induces phosphorylation of Smad1/5 through activin receptor-like kinase 1 and 2 (ALK1 and ALK2). Recently, many studies have shown that BMP9 contributes to tumorigenesis, and aberrant ALK2 expression is involved in many diseases. To investigate the role of BMP9-ALK2 signaling in cancer cells, we used TF-1 cells that require granulocyte-macrophage colony-stimulating factor (GM-CSF) for cell proliferation. BMP9 promoted the proliferation of TF-1 cells in media lacking GM-CSF. TF-1 cells overexpressing ALK2 resulted in the autophosphorylation of Smad1/5, leading to consequent increase in cell growth. Through high-throughput screening (HTS), we found two ALK2-specific inhibitors, KRC203 and KRC360, with IC50 values of 0.9 nM and 0.3 nM. These compounds were more potent and specific for the inhibition of ALK2 when compared to LDN193189. In cell-based assays, these compounds effectively inhibited the proliferation and migration of cancer cells induced by ALK2 and BMP9. Therefore, we propose that our compounds are promising candidates for the treatment of cancer or diseases with abnormal ALK2 or BMP9 signaling.

Forward genetic screening identifies a small molecule that blocks Toxoplasma gondii growth by inhibiting both host- and parasite-encoded kinases.

The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.

Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer.

BACKGROUND: Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear. METHODS AND RESULTS: We found a homozygous deletion of the activin A receptor, type IB (ACVR1B) gene in 2 out of 8 PC cell lines using array-comparative genomic hybridization, and the absence of ACVR1B mRNA and protein expression was confirmed in these 2 cell lines. Activin A stimulation inhibited cellular growth and increased the phosphorylation level of SMAD2 and the expression level of p21CIP1/WAF1 in the Sui66 cell line (wild-type ACVR1B and SMAD4 genes) but not in the Sui68 cell line (homozygous deletion of ACVR1B gene). Stable ACVR1B-knockdown using short hairpin RNA cancelled the effects of activin A on the cellular growth of the PC cell lines. In addition, ACVR1B-knockdown significantly enhanced the cellular growth and colony formation abilities, compared with controls. In a xenograft study, ACVR1B-knockdown resulted in a significantly elevated level of tumorigenesis and a larger tumor volume, compared with the control. Furthermore, in clinical samples, 6 of the 29 PC samples (20.7%) carried a deletion of the ACVR1B gene, while 10 of the 29 samples (34.5%) carried a deletion of the SMAD4 gene. Of note, 5 of the 6 samples with a deletion of the ACVR1B gene also had a deletion of the SMAD4 gene. CONCLUSION: We identified a homozygous deletion of the ACVR1B gene in PC cell lines and clinical samples and proposed that the deletion of the ACVR1B gene may mediate an aggressive cancer phenotype in PC. Our findings provide novel insight into the role of the activin signal in PC.

Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium.

The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.

Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare.

BACKGROUND: Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50-150 nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation. METHODS: Follicle contents were collected from mares at mid-estrous (~35 mm, before induction of follicular maturation) and pre-ovulatory follicles (30-34 h after induction of follicular maturation). A real time PCR screen was conducted to reveal significant differences in the presence of exosomal miRNAs isolated from mid-estrous and pre-ovulatory follicles, and according to bioinformatics analysis these exosomal miRNAs are predicted to target members belonging to the TGFB superfamily, including ACVR1 and ID2. Granulosa cells from pre-ovulatory follicles were cultured and treated with exosomes isolated from follicular fluid. Changes in mRNA and protein were measured by real time PCR and Western blot. RESULTS: ACVR1 mRNA and protein was detected in granulosa cells at mid-estrous and pre-ovulatory stages, and real time PCR analysis revealed significantly lower levels of ID2 (an ACVR1 target gene) in granulosa cells from pre-ovulatory follicles. Exposure to exosomes from follicular fluid of mid-estrous follicles decreased ID2 levels in granulosa cells. Moreover, exosomes isolated from mid-estrous and pre-ovulatory follicles contain ACVR1 and miR-27b, miR-372, and miR-382 (predicted regulators of ACVR1 and ID2) were capable of altering ID2 levels in pre-ovulatory granulosa cells. CONCLUSIONS: These data indicate that exosomes isolated from follicular fluid can regulate members of the TGFB/BMP signaling pathway in granulosa cells, and possibly play a role in regulating follicle maturation.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

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.

Pathogenic mutation of ALK2 inhibits induced pluripotent stem cell reprogramming and maintenance: mechanisms of reprogramming and strategy for drug identification.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies.

Synthesis and structure-activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of dorsomorphin: the discovery of ML347 as an ALK2 versus ALK3 selective MLPCN probe.

A structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.