SM04690, A potential disease-modifying treatment for knee osteoarthritis, functions through inhibition of CLK2 and DYRK1A, novel molecular regulators of Wnt signaling, chondrogenesis, and inflammation

Abstract

Purpose: In the synovial joint, Wnt pathway upregulation contributes to osteoarthritis (OA) through increasing osteocyte differentiation, cartilage thinning, and inflammation. SM04690, a novel small molecule, has previously demonstrated OA disease modifying potential through Wnt pathway inhibition in vitro and in vivo. Further studies haveelucidated a novel mechanism of action for SM04690, leading to Wnt pathway inhibition, chondrocyte differentiation, and anti-inflammatory activity. Methods: Wnt pathway activity was measured using a cell-based luciferase reporter assay controlled by a TCF/LEF promoter in SW480 colon cancer cells, a mutant cell line exhibiting active Wnt signaling. A kinome screen (318 kinases) and biochemical kinase assays were performed. SM04690 effects on protein phosphorylation of serine and arginine rich splicing factor (SRSF) proteins, Sirt1, and FoxO1, in hMSCs, chondrocytes and synovial fibroblasts were measured using Western blot. SM04690 and siRNA knockdown effects on chondrogenic and Wnt pathway gene expression were measured using nCounter® gene expression panels (NanoString Technologies), and effects on LPS-induced inflammatory cytokines (IL-6, IL-8, TNF-a) expression in BEAS-2B cells were measured by qPCR and ELISA. In vivo, SM04690 (0.1 mg, 0.3 mg, 1.0 mg) pharmacodynamic effects were evaluated in rat knee OA models: (1) An inflammatory monosodium iodoacetate (MIA) injection-induced knee OA model and (2) a surgical anterior cruciate ligament transection with partial medial meniscectomy (ACLTþpMMX) model, followed by single intra-articular SM04690 or vehicle injection. Knee cartilage was isolated at Days 10 and 35, and phosphorylation and expression of SRSF proteins, NF-kB, STAT3, and Sirt1 measured by Western blot. Statistical analyses used one-way ANOVA for multiple group comparisons and t-tests for comparison between two groups. Results: SM04690 was a potent (EC 50 ¼11nM) inhibitor of Wnt signaling. Biochemical assays identified cdc-like kinases (CLKs) and dualspecificity tyrosine kinase (DYRK1A) as molecular targets of SM04690. SM04690 potently inhibited CLK-mediated phosphorylation of SRSF proteins compared with DMSO control. Separately, SM04690 inhibited DYRK1A-mediated phosphorylation of Sirt1 and FoxO1 resulting in increased levels of total and nuclear FoxO1 compared with DMSO.

Comparative Efficacy and Selectivity of Pharmacological Inhibitors of DYRK and CLK Protein Kinases

“…IC 50 values of 12.3 and 350 nM were reported for CLK1 and CLK2 . Surprisingly, INDY was reported as inactive by Deshmukh et al (IC 50 > 10 μM). Like harmine, INDY stimulated the proliferation of human neural progenitor cells .…”

“…Lorecivivint was initially identified in a cell-based reporter assay for Wnt pathway activity and is being developed for the treatment of osteoarthritis of the knee (currently in clinical phase 3), of the hip, and of the shoulder, , as well as for the treatment of degenerative disc disease . It turned out to be a potent inhibitor of CLK2, DYRK1A, and DYRK1B (IC 50 = 5.8, 26.9, and 41.2 nM, respectively) but also of GSK3β, HIPK1, and HIPK2 (IC 50 = 37.8, 33.2, and 16.8 nM, respectively) . In our hands, lorecivivint was most potent on CLK2, DYRK1A, and DYRK1B (IC 50 = 13.6, 48, and 89 nM, respectively) but much less potent on GSK3β (IC 50 = 641 nM).…”

Development of Cdc2-like Kinase 2 Inhibitors: Achievements and Future Directions

“…Alternative splicing is a critical molecular mechanism of gene expression in which pre-mRNA transcripts from a single gene are spliced into multiple isoforms. , CLKs are involved in the assembly of spliceosomes and are implicated in both constitutive and alternative splicing control mechanisms and the selection of splicing sites. − They consist of four characterized isoforms in mammals (namely, CLK1, CLK2, CLK3, and CLK4) and belong to the CMGC group of kinases which include cyclin-dependent kinases (CDKs), CLKs, mitogen-activated protein kinase (MAPK), GSK3, serine–arginine-rich protein kinases (SRPK), and dual-specificity tyrosine phosphorylation-regulated kinases (DYRK). , The overexpression of CLK proteins localizes to nuclear speckles, where splicing factors are concentrated and affects slicing site selection of pre-mRNA . Several small-molecule inhibitors of the CLK family have been reported to control mRNA splicing. , …”

“…32 Several small-molecule inhibitors of the CLK family have been reported to control mRNA splicing. 33,34 Among the CLK family, CLK2 has been recently reported to be a promising drug target for various diseases. 6,35 Hence, there is a demand for developing CLK2 modulators on various disease settings.…”