Target Identification of a novel small molecule for treating joint degeneration

Abstract

Purpose: Degenerative joint diseases, including osteoarthritis and intervertebral disc degeneration (IDD), are associated with a loss of proteoglycans, presumably attributed to an imbalanced matrix synthesis and degradation [1,2]. Our ongoing study revealed a novel small molecule, termed as KVTS8, that is capable of promoting proteoglycans production in both chondrocytes and nucleus pulposus cells (NPCs). Pilot study in mouse tail disc model supported its function in alleviating IDD progression. Defining the target of KVTS8 may understand its mechanism of action. Drug affinity responsive target stability (DARTS) has been used to identify small molecule targets without a need of chemical modification [3]. In this study, we hypothesized that KVTS8 could bind and modulate the function of specific proteins that respond to proteoglycans degradation in IDD. We aimed to identify the targets for KVTS8 by DARTS and characterize the interaction under perturbation by interleukin-1 alpha (IL-1 α). Methods: DARTS: Protein lysates were extracted from mouse chondrogenic cell line (ATDC5) and incubated with KVTS8 (10nM and 100nM) or 1% DMSO for 1 h at 4°C, followed by limited proteolysis by pronase (1:10,000: 1μg/ml; 1:3,000: 3.3μg/ml) for 30 min at room temperature. SDS loading buffer was added to quench the digestion, and 10-20 μg of each sample were separated by a 10% SDS-PAGE and visualized by coomassie stain. Expression of potential targets was further assessed by immunoblotting. LC-MS/MS: Distinguished bands from coomassie stain were cut into slices and subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis as previous described [4], and peptides were identified via Thermo Proteome discoverer software and against Mouse UniProt FASTA. Triplicates were set for each condition. In vitro assay: Alginate-human NPCs were conditioned by IL-1α (10ng/ml) for 3 days, followed by combined treatment with either KVTS8 (10nm and 100nM) or vehicle (0.1% DMSO) for another 7 days. Expression changes of FlnB was assessed by immunoblotting. Results: DARTS strategy is based on the capacity of small molecules to modify the susceptibility of its protein interactors to the proteolytic cleavages. We demonstrated strong protected bands at molecular weight (MW) >245 kDa in the KVTS8-treated digests of ATDC5 cell protein extracts (Fig. 1A), whereas no detectable difference was found in the sample that underwent mock digestion. Intriguingly, bands appeared at lower MW (≈245 kDa) when pronase concentration increased to 1:3,000. MS analysis for the aforementioned bands identified a list of putative protein partners (Fig. 1B). Filamin-b (FlnB) was selected as a potential target on the basis of its high abundance and reproducibility (3 readings over 3 independent MS analysis). Immunoblotting confirmed a specific enrichment of FlnB by KVTS8 in a dose-dependent manner (Fig. 1C). IL-1α is capable of activating proteoglycans catabolism and implicated in joint degeneration [5]. While we detected two forms of FlnB in human NPCs, IL-1α NPCs resulted in a shift of the higher MW form to a lower MW form (Fig. 2). This shift was suppressed by a combined treatment of KVTS8 in a dose-dependent manner. Conclusions: Deficiency of FlnB was reported to cause skeleton malformation in mouse along with loss of hyaline cartilage and nucleus pulposus in the intervertebral discs [6]. Our findings suggested FlnB as a binding target of KVTS8 and its regulation under inflammatory condition. The two molecular forms of FlnB may be related to enzymatic cleavage, such as by ASB2 [7], or alternative splicing [8]. The functional implication of FlnB-KVTS8 interaction in proteoglycans metabolism and joint degeneration warrant further investigation. Overall, our findings identify FlnB as KVTS8 binding target and reveal its potential function in regulating joint homeostasis and degeneration.