A new class of small molecule inhibitor of BMP signaling

Abstract

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.

Figure 1. Identification of a novel 2-aminopyridine inhibitor of ALK2.

(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 Table S1. (B) In vitro 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.

Figure 2. Kinome-wide selectivity of K02288 and LDN-193189.

(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 Table S2. (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.; www.cellsignal.com).

Figure 3. Structural basis for inhibitor binding to ALK2.

(A) Structure of the ALK2-K02288 complex. Inset shows the interactions of K02288 in the ATP pocket. Hydrogen bonds are shown as dotted lines. (B) ITC measurements showed ALK2 bound K02288 with KD = 7.9 nM. (C) Comparison of the binding modes of K02288 (green) and LDN-193189 (yellow) in ALK2. Both inhibitors formed hydrogen bonds to the hinge residue H286, but other interactions were divergent, including water-mediated hydrogen bonds to K235 and E248, respectively. (D) A spacefill representation of K02288 shown against a surface mesh view of the ATP pocket highlights the shape complementarity in ALK2.

Figure 4. K02288 selectively inhibits BMP signaling.

(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.

Figure 5. K02288 induces dorsalization of zebrafish embryos.

(A) Brightfield photographs of 26 hours old Tg(BRE:mRFP) 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. . (C) Western blot for mRFP in extracts prepared from Tg(BRE:mRFP) 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.

Figure 6. K02288 does not inhibit vasculature development.

(Top panels) Brightfield photographs of 48 hours old Tg(fli1a:eGFP) 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.

Figure 7. The K02288 scaffold is similar to an anti-malarial compound.

(A) The 2-aminopyridine scaffold of K02288. (B) A similar initial hit 1 was identified in a recent anti-malarial screen and optimized to the more divergent lead 2 in development of the pre-clinical candidate MMV390048 .