Rac and Cdc42 Inhibitors in Rheumatoid Arthritis Therapy

Abstract

Rheumatoid Arthritis (RA) is the most common autoimmune disorder characterized by chronic inflammation due to synovial fibroblast proliferation, neovascularization, leukocyte extravasation, and progressive joint destruction. Most RA medications focus on relieving symptoms of chronic inflammation, rather than prevent the inflammatory cells from attacking the joints and the underlying cartilage and bone. These initial processes have been shown to be under the regulation of key signaling proteins Rac and Cdc42. Therefore, the objective of this study was to demonstrate the effect of our patented Rac/Cdc42 inhibitors MBQ‐167 and EHop‐016 in inflammatory cells in vitro. Our hypothesis is that inflammatory cells in the synovium can be inhibited by Rac/Cdc42 inhibitors that have the potential as a RA therapeutics. To test this hypothesis, macrophage‐like cell lines WBC264‐9C human leukocytes or THP1 human macrophage were incubated with 0‐500 nM of MBQ‐167, lysed and subjected to Rac.GTP pulldown assays. Results showed that Rac activation was inhibited by 46‐56% at 250 nM and by 60‐77% at 500 nM MBQ‐167 for both cell lines. Total Rac levels were not significantly changed by MBQ‐167 in concentrations up to 500 nM. Moreover, preliminary results on synovial fibroblasts SW‐982 demonstrated inhibition of Rac activation in response to MBQ‐167 at 750 nM or higher. Next, macrophage‐like cells were treated with the Rac/Cdc42 inhibitors and tested for cell viability using a MTT assay. Results showed no effects on cell proliferation at high µM concentrations of Rac/Cdc42 inhibitors. Since Rac activation regulates cell migration, which is central to the inflammatory response in synovial joints, we tested the effect of MBQ‐167 in a wound healing assay. Incubation with MBQ‐167 at 250 or 500 nM inhibited macrophage migration. At 24h, 500 nM MBQ‐167 reduced macrophage cell migration by 80%. In conclusion, this data demonstrates that our Rac/Cdc42 inhibitors may be used to inhibit inflammatory cell action and their migration at sites of inflammation in RA, without affecting macrophage numbers, indicating no toxic effects in the immune system.