Identification of small-molecules targeting CXCR2 function and signaling

2013-07-30Chemokine receptor, CXCR2, and its ligands are an essential component of the immune system that mediates the trafficking of neutrophils to sites of infection. It is also important in angiogenesis by activating and coordinating the assembly of endothelial cells. Given its roles in the immune system and angiogenesis, CXCR2 has been implicated in a number of inflammation-mediated diseases such as chronic obstructive pulmonary diseases (COPD) and various types of cancer. CXCR2 inhibition has shown promising anti-inflammatory effects in the clinics as well as anti-cancer effects in several in vivo animal models. And within the last two decades, several classes of small-molecules targeting CXCR2 has been developed and advanced onto clinical trials. ❧ However, current CXCR2 inhibitors are limited to four major chemical classes of compounds and are by far scarce compared to other clinically relevant therapeutic targets. Thus, we sought to identify additional classes of CXCR2 inhibitors using cell-based, functional assays rather than conventional competitive ligand binding assays. We also capitalized on the literature of previously described CXCR2 inhibitors and developed a pharmacophore model to identify important chemical features of CXCR2 inhibitors and to perform in silico screening of large databases of compounds. ❧ We have identified several classes of compounds that modulate CXCR2 signaling and cellular functions in CXCR2-expressing cells. These compounds are chemically different from any reported CXCR2 inhibitors. Mechanistic studies of these compounds showed that they differentially regulate CXCR2-mediated G-protein and β-arrestin1/2 signaling. For example, CX4338 inhibited CXCR2-mediated β-arrestin1/2 recruitment, while enhancing CXCR2-mediated G-protein signaling (calcium and ERK1/2 activation). Selective inhibition of CXCR2/β-arrestin1/2 association by CX4338 was sufficient to reduce cell migration and LPS-induced PMN migration. We also discovered CX797 in our screening endeavors using a cell-based cyclic AMP assay. CX797 inhibited the effects of CXCL8 on forskolin-induced cyclic AMP signaling, while enhancing CXCR2/β-arrestin2 association and up-regulating total CXCR2 expression. CX797 also inhibited CXCL8-mediated cell migration and cell proliferation in CXCR2-expressing cell lines. ❧ Lastly, using a pharmacophore model, we identified CX25 as a potent inhibitor of CXCR2 in the Tango assay. However, these class of compounds did not show much selectivity amongst different chemokine receptors and inhibited cancer cell proliferation in a CXCR2-independent manner. CX4152 was also identified from the pharmacophore screen and exhibit similar chemical features as CX25. Though CX4152 was not as active as CX25 against CXCR2, it showed 8-fold selectivity for CXCR2 over CXCR4 (another chemokine receptor). Mechanistic studies suggest that CX4152 inhibit CXCR2/β-arrestin-2 association via CXCR2 down-regulation. Treatment with CX4152 reduced surface CXCR2 expression within 10 minutes in a dose and time dependent manner. Taken together, compounds identified from our screens have differential effects on CXCR2 signaling and provide several novel avenues to mechanistically modulate CXCR2 function and signaling