Cinzia Bizzarri

Targeting C5a: Recent Advances in Drug Discovery

Activation of complement via the innate and adaptive immune system is vital to the bodys defences in fighting infections. Unregulated complement activation is likely to play a crucial role in the pathogenesis of several diseases including psoriasis, adult (acute) respiratory distress syndrome (ARDS), bullous pemphigoid (BP), rheumatoid arthritis (RA) and ischemia-reperfusion (I/R) injury. The 74 amino acid peptide C5a is released after complement activation at sites of inflammation and is a potent chemoattractant for neutrophils, basophils, eosinophils, leukocytes, monocytes and macrophages. Recombinant proteins and humanized anti-C5 antibodies have been recently developed for blocking specific proteins of the complement system bringing renewed attention towards complement inhibition. Pharmacological studies have been highlighting the complement fragment C5a as an interesting target for the management of complement mediated diseases. Specific inhibition of C5a biological activity could gain therapeutic benefit without affecting the protective immune response. In the last few years several peptide and non-peptide antagonists of C5a have been discovered and tested in relevant pharmacological models; the availability of orally active compounds is rapidly helping to delineate the precise role of C5a in immunoinflammatory disorders. Moreover, mutagenesis data for the C5a/C5a receptor (C5aR) couple make C5aR a valuable model for mechanistic studies of peptidergic G-protein coupled receptors (GPCRs). The aim of this review is to outline the recent advances in C5a inhibition, especially highlighting the value of a multidisciplinary integrated approach in drug discovery.

Blockade of the chemokine receptor CXCR2 ameliorates adjuvant-induced arthritis in rats

Chemokine receptors CXCR1 and CXCR2 may mediate influx of neutrophils in models of acute and chronic inflammation. The potential benefits of oral administration of a CXCR1/2 inhibitor, DF 2162, in adjuvant‐induced polyarthritis (AIA) were investigated.

IL-1β primes IL-8-activated human neutrophils for elastase release, phospholipase D activity, and calcium flux

Interleukin‐8 (IL‐8), the prototype of the α (i.e., C‐X‐C branch) chemokine family, induced elastase release in a concentration‐dependent manner (50‐1000 ng/mL) in cytochalasin B‐treated human polymorphonuclear leukocytes (PMNs). This response was potentiated about twofold if PMNs were preexposed to interleukin‐1β (IL‐1β) at concentrations that were by themselves inactive. The effect of IL‐1β was clearly observed after 5 min and was maximal after a 30‐min preincubation of the cells. The effect was present over the whole active concentration range of IL‐8 and was completely blocked by the presence of IL‐1 receptor antagonist. Priming of elastase release by IL‐1β was not associated with a change in receptor number or affinity for IL‐8. On the contrary, it was correlated with priming of phospholipase D activity and calcium flux activated by IL‐8. Preincubation of the cells with ethanol and/or La3+ inhibited IL‐8‐induced degranulation, suggesting that activation of phospholipase D and increase of [Ca2+]i were important for this response. In contrast, ethanol and La3+ did not decrease the priming effect of IL‐1β. IL‐8 and IL‐1β have been shown to be released by the same cell types and may be concomitantly present at sites of inflammation, giving rise to an amplification of the inflammatory response.

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.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor

BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL‐8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential.

Selective inhibition of interleukin-8-induced neutrophil chemotaxis by ketoprofen isomers.

Although it is commonly accepted that the anti-inflammatory effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is mainly associated to their ability to inhibit the cyclooxygenase (COX) enzyme system, several results indicate that non-COX mechanisms could be important in the therapeutical effect of these drugs. The aim of this study was to define if NSAIDs could exert, at least in part, their anti-inflammatory effect by inhibiting the activities of human polymorphonuclear leukocytes (PMNs) triggered by chemotactic stimuli and, if so, to understand the relationship of this effect with COX inhibition. A unique opportunity to dissociate the inhibition of prostaglandin (PG) synthesis from other therapeutical properties of NSAIDs is constituted by ketoprofen isomers being the S-isomer 100 time more potent than R-isomer on COX inhibition. Our results show that R- and S-ketoprofen, independently of their potency as PG inhibitors, proved very efficacious in selective inhibition of interleukin-8 (IL-8) chemotaxis. Inhibition of IL-8 chemotaxis was not restricted to ketoprofen isomer as it could be observed also with drugs belonging to different classes of NSAIDs and it was obtained at drug concentration superimposable to plasma levels after therapeutic administration in patients. Reduction of IL-8 migration by ketoprofen isomers was paralleled by selective inhibition of PMN response in terms of intracellular calcium concentration ([Ca(2+)]i) increase and extracellular signal regulated kinase(ERK)-2 activation, two intracellular mediators reported to be critical for PMN activities. It is concluded that inhibition of IL-8 chemotaxis could represent a new clinical target for ketoprofen isomers and, in fact, contribute to the anti-inflammatory activity of NSAIDs.

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.

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

Significance Persistent pain in inflammatory and neuropathic conditions is often refractory to conventional analgesic therapy, with most patients suffering with unrelieved pain and serious treatment-related side effects. There is still a tremendous need to identify novel therapeutics for pain control with innovative biological mechanisms and minimal side effects. In this paper we challenge the hypothesis that a conserved structural motif across the G protein-coupled receptor family plays a regulatory role in the negative modulation of receptor activation and use a multidisciplinary approach to the rational drug design and characterization of a novel potent allosteric inhibitor of the C5a anaphylatoxin receptor (C5aR), thus providing a new promising avenue for the improvement of pharmacotherapy of chronic pain. 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.

Characterization and Properties of Dominant-negative Mutants of the Ras-specific Guanine Nucleotide Exchange Factor CDC25Mm

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25Mm, are described. In vitro, the mutant GEFW1056E and GEFT1184Eproteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21 ras , and strongly reduce affinity of the nucleotide-free ras·GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras·GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEFW1056Emutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

G Protein-Linked Receptors in the Thyroid

The FRTL5 cell line has the advantage that its hormonal activation leads to important and measurable thyroid function such as the transport of iodide and the iodination of thyroglobulin. Secondly, the coexistence in the plasma membrane of these cells of several physiologically relevant receptors (TSH, alpha 1-adrenergic, M1 and M2 muscarinic, insulin, IGF1) coupled to at least three transducing enzymes (adenylyl cyclase, PLC, PLA2) gives the possibility to analyze the interaction among second messengers in the cell activation process. This has allowed us and others to show that in the case of the iodide efflux regulation at least two second messengers (Ca++ and arachidonic acid) mediate the adrenergic stimulation, whereas the TSH activation of the same phenomenon probably uses other signals in addition to Ca++ and arachidonic acid. Growth is mostly regulated by TSH, that activates the adenylyl cyclase by a mechanism that may involve the modulation of the availability of Gi. TSH is also able to regulate an endogenous ADP ribosyl transferase in FRTL5. This could be a novel mechanism of cell regulation by this hormone, but the role of this phenomenon in the physiological action of TSH is still unclear.