Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief.

Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the "minor pocket," previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR(-/-) mice compared with WT mice. Furthermore, treatment of C5aR(-/-) mice with DF2593A did not produce any further antinociceptive effect compared with C5aR(-/-) mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.

Allosteric inhibitors of chemoattractant receptors: opportunities and pitfalls.

Given the central role of chemokines in infection, inflammation and immunity, chemokine receptors are a prime target for pharmacological intervention, and more so after the recent approval of chemokine receptor inhibitors for HIV. Allosteric inhibitors offer a largely unexploited opportunity to interfere with and modulate chemokine receptor activation and signaling. In addition to characterizing binding mode as a first step to understanding the specific mechanism underlying drug action, allosteric inhibitors pose new questions concerning different phases in drug discovery and pharmacological characterization, including the identification of appropriate screening tests, the evaluation of inhibitory effects on different signaling pathways and the implications of agonist- and signaling pathway-dependent inhibition for overall in vivo efficacy.

Structure-Activity Relationship of novel phenylacetic CXCR1 inhibitors.

We reported recently the Structure-Activity Relationship (SAR) of a class of CXCL8 allosteric modulators. They invariably share a 2-arylpropionic moiety so far considered a key structural determinant of the biological activity. We show the results of recent SAR studies on a novel series of phenylacetic derivatives supported by a combined approach of mutagenesis experiments and conformational analysis. The results suggest novel insights on the fine role of the propionic/acetic chain in the modulation of CXCL8 receptors.

ELR+ CXC chemokines and their receptors (CXC chemokine receptor 1 and CXC chemokine receptor 2) as new therapeutic targets.

ELR+ CXC chemokines, by direct interaction with their cell surface receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2), are believed to be crucially involved in the direct migration and activation of leukocytes. ELR+ CXC chemokines are supposed to play a key role in several inflammatory diseases and this makes ELR+ CXC chemokines and their receptors attractive therapeutic targets. The first aim of this review is to discuss the potential pathological role of ELR+ CXC chemokines in different pathologies, including ulcerative colitis (UC), ischaemia/reperfusion injury (RI), bronchiolitis obliterans syndrome (BOS) and tumor progression. Moreover, the most recently described inhibitors of ELR+ CXC chemokines and their therapeutic indications will be reviewed. Finally, the mode of action and the potential therapeutical use of reparixin, a new potent and selective inhibitor of CXCR1/2 activity, and its chemical derivatives are also discussed.

Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2.

Chemokines CXCL8 and CXCL1 play a key role in the recruitment of neutrophils at the site of inflammation. CXCL8 binds two membrane receptors, CXCR1 and CXCR2, whereas CXCL1 is a selective agonist for CXCR2. In the past decade, the physiopathological role of CXCL8 and CXCL1 has been investigated. A novel class of small molecular weight allosteric CXCR1 inhibitors was identified, and reparixin, the first drug candidate, is currently under clinical investigation in the prevention of ischemia/reperfusion injury in organ transplantation. Reparixin binding mode to CXCR1 has been studied and used for a computer-assisted design program of dual allosteric CXCR1 and CXCR2 inhibitors. In this paper, the results of modeling-driven SAR studies for the identification of potent dual inhibitors are discussed, and three new compounds (56, 67, and 79) sharing a common triflate moiety have been selected as potential leads with optimized pharmacokinetic characteristics.

Development of a systemically-active dual CXCR1/CXCR2 allosteric inhibitor and its efficacy in a model of transient cerebral ischemia in the rat.

The chemokine receptors CXCR1 and CXCR2 present on polymorphonuclear neutrophils (PMN), bind the chemokine CXC ligand 8 (CXCL8)/interleukin-8 (IL-8), and have a key role in PMN recruitment in inflammation. Based on the structure of reparixin, a small-molecular-weight allosteric inhibitor of CXCR1, we designed a dual inhibitor of CXCR1 and CXCR2 with a longer in vivo half-life, DF2156A. This molecule inhibited human and rat PMN migration in response to CXCR1 and CXCR2 ligands and showed an elimination half-life following i.v. administration, of 19 hours. In a rat model of cerebral ischemia/reperfusion induced by temporary (90 min) middle cerebral artery (MCA) occlusion, DF2156A (8 mg-kg, i.v., at the time of reperfusion) decreased the PMN infiltrate, infarct size and significantly improved neurological function. These results indicate that CXCR1/CXCR2 and their ligands have a role in the inflammatory component of cerebral ischemia, and that these pathways represent an important pharmacological target.

Requirements for the different cysteines in the chemotactic and desensitizing activity of human thioredoxin.

Thioredoxin (Trx) is a protein disulfide oxidoreductase that can be secreted and act as a chemoattractant for leukocytes. Like chemokines, it causes desensitization of monocytes against its chemotactic activity and that of monocyte chemoattractant protein-1 (MCP-1). To investigate the role of the redox properties of Trx, and particularly of some of its five cysteines, in its chemotactic and desensitizing action, we tested different mutants, including Trx80, a truncated form, and various mutants lacking specific cysteines: Trx C62S/C73S and the redox-inactive mutant Trx C32S/C35S. Of the mutants, only Trx80 maintained the chemotactic activity of wild-type Trx toward both monocytes and polymorphonuclear neutrophils, all of them desensitized monocytes against wild-type Trx or MCP-1, but not chemotactic peptide formyl-methionyl-leucil peptide. These data indicate that different redox-active cysteines are important for Trx chemotactic action, whereas its desensitizing action does not have these requirements, suggesting a redox-independent mechanism.

2-Arylpropionic CXC chemokine receptor 1 (CXCR1) ligands as novel noncompetitive CXCL8 inhibitors.

The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We report here molecular modeling studies showing a putative interaction site of 1 in the TM region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimization of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia reperfusion injury.

Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2.

Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.

Thioredoxin specifically cross-desensitizes monocytes to MCP-1.

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.

Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis.

The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis.

Noncompetitive allosteric inhibitors of the inflammatory chemokine receptors CXCR1 and CXCR2: prevention of reperfusion injury.

The chemokine CXC ligand 8 (CXCL8)/IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors.