Saracatinib is an efficacious clinical candidate for fibrodysplasia ossificans progressiva.

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-ß signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.

ALK2 inhibitors display beneficial effects in preclinical models of ACVR1 mutant diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.

Novel Quinazolinone Inhibitors of ALK2 Flip between Alternate Binding Modes: Structure-Activity Relationship, Structural Characterization, Kinase Profiling, and Cellular Proof of Concept.

Structure-activity relationship and crystallographic data revealed that quinazolinone-containing fragments flip between two distinct modes of binding to activin receptor-like kinase-2 (ALK2). We explored both binding modes to discover potent inhibitors and characterized the chemical modifications that triggered the flip in binding mode. We report kinase selectivity and demonstrate that compounds of this series modulate ALK2 in cancer cells. These inhibitors are attractive starting points for the discovery of more advanced ALK2 inhibitors.

Identification of small molecule inhibitors of ALK2: a virtual screening, density functional theory, and molecular dynamics simulations study.

Bone morphogenetic proteins (BMPs) are a family of more than 30 ligands and several receptors, such as activin like kinases (ALKs) and bone morphogenetic protein receptor (BMPR). Physiological significance of these proteins lies in their prominent role during homeostasis, apoptosis, tissue remodeling, embryonic patterning, and normal development. Fibrodysplasia ossificans progressive (FOP) is one among several other diseases caused by impaired BMP signaling. FOP is caused by the pathogenicity of activating mutation of ALK2. In order to treat FOP, a search for good inhibitors of ALK2 based on dorsomorphin and LDN substitution, which in essence is a ligand based search of inhibitors, is in progress. Contributing to this area of research we identified several lead molecules based on protein structure using virtual screening. After virtual screening of a huge library of small molecules and ab initio calculation of selected molecules for drug efficacy, we did molecular dynamic simulation of lead molecules and protein complexes. We identified five potential drug molecules that show very stable binding on the same binding site as LDN-213844. We also ranked these lead molecules based on MM/PBSA binding energy. This study provides a basis to think beyond the pyrimidine nucleus of dorsomorphin/LDN and design new chemical derivatives for effective treatment of FOP. Graphical abstract Small molecule inhibitors of ALK2.

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.

Development and validation of a phosphorylated SMAD ex vivo stimulation assay.

Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.

A novel small-molecule inhibitor of transforming growth factor beta type I receptor kinase (SM16) inhibits murine mesothelioma tumor growth in vivo and prevents tumor recurrence after surgical resection.

Malignant mesothelioma is an aggressive and lethal pleural cancer that overexpresses transforming growth factor beta (TGFbeta). We investigated the efficacy of a novel small-molecule TGFbeta type I receptor (ALK5) kinase inhibitor, SM16, in the AB12 syngeneic model of malignant mesothelioma. SM16 inhibited TGFbeta signaling seen as decreased phosphorylated Smad2/3 levels in cultured AB12 cells (IC(50), approximately 200 nmol/L). SM16 penetrated tumor cells in vivo, suppressing tumor phosphorylated Smad2/3 levels for at least 3 h following treatment of tumor-bearing mice with a single i.p. bolus of 20 mg/kg SM16. The growth of established AB12 tumors was significantly inhibited by 5 mg/kg/d SM16 (P < 0.001) delivered via s.c. miniosmotic pumps over 28 days. The efficacy of SM16 was a result of a CD8+ antitumor response because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice and (b) CD8+ T cells isolated from spleens of mice treated with SM16 showed strong antitumor cytolytic effects whereas CD8+ T cells isolated from spleens of tumor-bearing mice treated with control vehicle showed minimal activity. Treatment of mice bearing large tumors with 5 mg/kg/d SM16 after debulking surgery reduced the extent of tumor recurrence from 80% to <20% (P < 0.05). SM16 was also highly effective in blocking and regressing tumors when given p.o. at doses of 0.45 or 0.65 g/kg in mouse chow. Thus, SM16 shows potent activity against established AB12 malignant mesothelioma tumors using an immune-mediated mechanism and can significantly prevent tumor recurrence after resection of bulky AB12 malignant mesothelioma tumors. These data suggest that ALK5 inhibitors, such as SM16, offer significant potential for the treatment of malignant mesothelioma and possibly other cancers.

Identification of tocopherol-associated protein as an activin/TGF-beta-inducible gene in mast cells.

Previous studies have demonstrated that treatment with activin A and TGF-beta(1), members of the TGF-beta family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-beta(1)-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-beta family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-beta(1) at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with >1 muM SB431542, an inhibitor of activin and TGF-beta type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-beta(1), whereas <0.5 microM SB431542 effectively reduced TGF-beta(1)-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-beta-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-beta pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-beta occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-beta signaling.