A New Class of Small Molecule Inhibitor of BMP Signaling

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development

Structure–Activity Relationship of 3,5-Diaryl-2-aminopyridine ALK2 Inhibitors Reveals Unaltered Binding Affinity for Fibrodysplasia Ossificans Progressiva Causing Mutants

There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure–activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-β type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG

Mediators of lifestyle behaviour changes in obese pregnant women. Secondary analyses from the DALI lifestyle randomised controlled trial

A better understanding of what drives behaviour change in obese pregnant overweight women is needed to improve the effectiveness of lifestyle interventions in this group at risk for gestational diabetes (GDM). Therefore, we assessed which factors mediated behaviour change in the Vitamin D and Lifestyle Intervention for GDM Prevention (DALI) Lifestyle Study. A total of 436 women, with pre-pregnancy body mass index ≥29 kg/m 2 , ≤19 + 6 weeks of gestation and without GDM, were randomised for counselling based on motivational interviewing (MI) on healthy eating and physical activity, healthy eating alone, physical activity alone, or to a usual care group. Lifestyle was measured at baseline, and at 24–28 and 35–37 weeks of gestation. Outcome expectancy, risk perception, task self-efficacy and social support were measured at those same time points and considered as possible mediators of intervention effects on lifestyle. All three interventions resulted in increased positive outcome expectancy for GDM reduction, perceived risk to the baby and increased task self-efficacy. The latter mediated intervention effects on physical activity and reduced sugared drink consumption. In conclusion, our MI intervention was successful in increasing task self-efficacy, which was related to improved health behaviours

Investigation of Kinase Activation in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.This thesis is not currently available on ORA

K02288 induces dorsalization of zebrafish embryos.

<p>(A) Brightfield photographs of 26 hours old <i>Tg(BRE:mRFP)</i> transgenic embryos treated with DMSO or varying doses of K02288 from the 8- to 16-cell stage. Severity of the dorsalization correlated with the dose of K02288. Very strong dorsalized phenotypes were observed with 8–10 µM K02288. (B) The phenotypes of the embryos shown in A were classified according to Kishimoto et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062721#pone.0062721-Kishimoto1" target="_blank">[49]</a>. (C) Western blot for mRFP in extracts prepared from <i>Tg(BRE:mRFP)</i> embryos treated in a parallel experiment. Loss of mRFP protein was evident at 8–10 µM K02288. As a control, the effects of dorsomorphin (DM) and LDN-193189 (LDN) on mRFP expression are also shown. Protein loading control is shown with the MCM6 blot.</p

Diffraction data and refinement statistics.

a<p>Values in brackets show the statistics for the highest resolution shells.</p>b<p>P/L/O indicate protein, ligand molecules presented in the active sites, and other (water and solvent molecules), respectively.</p>c<p>rms indicates root-mean-square.</p

Identification of a novel 2-aminopyridine inhibitor of ALK2.

<p>(A) Schematic summary of a thermal shift assay screen using recombinant ALK2 kinase domain. A novel 2-aminopyridine hit K02288 was identified with an affinity for ALK2 intermediate between dorsomorphin and LDN-193189. Complete screening data are shown in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062721#pone.0062721.s002" target="_blank">Table S1</a>. (B) <i>In vitro</i> kinase assays showed K02288 specificity for ALK1,2,3,6 over ALK4,5. IC₅₀ measurements were performed in triplicate at the Km value of ATP. (C) ActRIIA kinase inhibition was determined using the Kinase-Glo® assay (Promega). IC₅₀ measurements were performed in duplicate at the Km value of ATP. (D) Summary of IC₅₀ measurements in all experiments.</p

K02288 selectively inhibits BMP signaling.

<p>(A) K02288 and LDN-193189 inhibited BMP4 induced Smad1/5/8 phosphorylation in C2C12 cells. Phosphorylated Smads and total Smads were detected by Western blot. (B) BRE-luciferase assay in C2C12 cells showing the dose dependent inhibition of the BMP4 response. Cells were treated with LDN-193198 or K02288 at the indicated concentrations prior to BMP4 stimulation. Y-axis displays the ratio of Firefly/Renilla activity from three independent experiments each performed in triplicate ± S.E.M. (C) K02288 and LDN-193189 potently inhibited BMP6-induced Smad1/5/8 phosphorylation in C2C12 cells, but had no effect on TGF-β-induced phosphorylation of Smad2. Activin A-induced P-Smad2 in HEK293 cells was weakly inhibited by K02288 and LDN-193189.</p

K02288 does not inhibit vasculature development.

<p>(Top panels) Brightfield photographs of 48 hours old <i>Tg</i>(<i>fli1a</i>:<i>eGFP)</i> embryos treated with DMSO or chemical inhibitors from 12 hours post fertilization. Embryos were manually dechorionated after bud stage before treatment. (Center panels) Same view under UV light for visualization of eGFP expression in the vasculature. Dorsomorphin and LDN-193189 treatment resulted in intersomitic vessel (ISV) formation defects, consistent with their known inhibition of VEGF signaling. (Lower panels) Higher magnification views of representative embryos and phenotype summary. The most severe phenotypes were observed with dorsomorphin. No effects on ISV formation were observed with K02288 treatment.</p

Kinome-wide selectivity of K02288 and LDN-193189.

<p>(A) Some 200 human kinases were individually ranked according to their enzymatic inhibition by K02288 or LDN-193189 present at 0.1 or 1 µM concentration (screening performed by Nanosyn). Complete screening data are shown in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062721#pone.0062721.s003" target="_blank">Table S2</a>. (B) Percent inhibition values for each kinase in the presence of 1 µM K02288 were plotted against those using 1 µM LDN-193189. There is little correlation between those kinases inhibited by K02288 and by LDN-193189. Overall, fewer kinases were inhibited by K02288. (C) Kinome tree visualization of inhibitor profiling showing the appearance of target family clusters (illustration reproduced courtesy of Cell Signaling Technology, Inc.; <a href="http://www.cellsignal.com" target="_blank">www.cellsignal.com</a>).</p