Jean-Luc Galzi

TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Delta(9)-tetrahydrocannabiorcol

TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. Here we show that the antinociceptive effects of spinal and systemic administration of acetaminophen (paracetamol) are lost in Trpa1(-/-) mice. The electrophilic metabolites N-acetyl-p-benzoquinoneimine and p-benzoquinone, but not acetaminophen itself, activate mouse and human TRPA1. These metabolites also activate native TRPA1 and, as a consequence, reduce voltage-gated calcium and sodium currents in primary sensory neurons. The N-acetyl-p-benzoquinoneimine metabolite L-cysteinyl-S-acetaminophen was detected in the mouse spinal cord after systemic acetaminophen administration. In the hot-plate test, intrathecal administration of N-acetyl-p-benzoquinoneimine, p-benzoquinone and the electrophilic TRPA1 activator cinnamaldehyde produced antinociception that was lost in Trpa1(-/-) mice. Intrathecal injection of a non-electrophilic cannabinoid, Δ(9)-tetrahydrocannabiorcol, also produced TRPA1-dependent antinociception in this test. Our study provides a molecular mechanism for the antinociceptive effect of acetaminophen and discloses spinal TRPA1 activation as a potential pharmacological strategy to alleviate pain.

Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels

The nicotinic acetylcholine receptor (nAChR) ~ is involved in chemo-electrical transduction at the neuromuscular junction and at cholinergic synapses of the central nervous system. At the motor endplate, invasion of the motor nerve ending by an action potential causes the release in the synaptic cleft of a brief pulse of acetylcholine (ACh), whose local concentration reaches 0. I to 1 mM (Katz & Miledi, 1977) for less than 1 msec (see also Clements et al., 1992 in the case of GluR). ACh diffuses through the cleft and binds to the nAChR present in the postsynaptic membrane, where it triggers the all-ornone opening of cation-selective ion channels through which Na+/K + ions flow passively. When depolarization reaches a threshold, muscle contraction occurs. In the cleft, ACh concentration rapidly declines to background levels (10 .9 M) as a consequence of diffusion and degradation by ACh esterase (Kuffler & Yoshikami, 1975; Katz & Miledi, 1977).

Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site-directed mutagenesis

Three aromatic amino acids, Tyr92, Trp148 and Tyr187 belonging to three separate domains of the α7‐subunit of neuronal nicotinic acetylcholine receptor were mutated to phenylalanine, and the electrophysiological response of the resulting mutant receptors analyzed in the Xenopus oocyte expression system. All mutations significantly decreased the apparent affinities for acetylcholine and nicotine, and to a lesser extent, those for the competitive antagonists dihydro‐β‐erythroidine and α‐bungarotoxin. Other properties investigated, such as the voltage dependency of the ion response as well as its sensitivity to the open channel blocker QX222, were not significantly changed, indicating that the mutations affected selectively the recognition of cholinergic ligands by the receptor protein. The maximal rates for the rapid desensitization process were slightly modified, suggesting that the contribution of Tyr92, Trp148 and Tyr187 to the binding area might differ in the various conformations of the nicotinic receptor. Other mutations at nearby positions (S94N, W153F, G151D and G82E) did not affect the properties of the electrophysiological response. These data point to the functional significance of Tyr92, Trp148 and Tyr187 in the binding of cholinergic ligands and ion channel activation of the nicotinic receptor, thus supporting a multiple loop model [(1990) J. Biol. Chem. 265, 10430–10437] for the ligand binding area.

Small Neutralizing Molecules to Inhibit Actions of the Chemokine CXCL12

The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer‐based assay to screen a G‐protein‐coupled receptor‐focused library of fluorescent compounds on the human EGFP‐tagged apelin receptor. This led to isolated E339–3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339–3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water‐deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.—Iturrioz, X., Alvear‐Perez, R., De Mota, N., Franchet, C., Guillier, F., Leroux, V., Dabire, H., Le Jouan, M., Chabane, H., Gerbier, R., Bonnet, D., Berdeaux, A., Maigret, B., Galzi J.‐L., Hibert, M., Llorens‐Cortes, C. Identification and pharmacological properties of E339–3D6, FASEB J. 24, 1506–1517 (2010). www.fasebj.org

A novel, conformation-specific allosteric inhibitor of the tachykinin NK2 receptor (NK2R) with functionally selective properties

The orthosteric agonist neurokinin A (NKA) interacts with the tachykinin NK2 receptors (NK2Rs) via an apparent sequential binding process, which stabilizes the receptor in at least two different active conformations (A1L and A2L). The A1L conformation exhibits fast NKA dissociation kinetics and triggers intracellular calcium elevation;the A2L conformation exhibits slow NKA dissociation kinetics and triggers cAMP production. The new compound LPI805 is a partial and noncompetitive inhibitor of NKA binding to NK2Rs. Analysis of NKA dissociation in the presence of LPI805 suggests that LPI805 decreases the number of NKA‐NK2R complexes in A2L conformation while increasing those in the A1L conformation. Analysis of signaling pathways of NK2Rs shows that LPI805 dramatically inhibits the NKA‐induced cAMP response while slightly enhancing the NKA‐induced calcium response. Analysis of NKA association kinetics reveals that LPI805 promotes strong and specific destabilization of the NKA‐NK2R complexes in the A2L conformation whereas access of NKA to the A1L conformations is unchanged. Thus, to our knowledge, LPI805 is the first example of a conformation‐specific allosteric antagonist of a G‐protein‐coupled receptor. This work establishes the use of allosteric modulators in order to promote functional selectivity on certain agonist‐receptor interactions.–Maillet E. L., Pellegrini, N., Valant, C., Bucher, B., Hibert, M., Bourguignon J‐J., Galzi J‐L. A novel, conformation‐specific allosteric inhibitor of the tachykinin NK2 receptor (NK2R) with functionally selective properties. FASEB J. 21, 2124–2134 (2007)

Stratification of the channel domain in neurotransmitter receptors

Analyses of the ionic pore of ligand-gated ion channels at the amino acid level reveal a structural and functional stratification of the M2 channel domain. Mutations in the equatorial and outer regions affect channel gating, whereas mutations of other amino acid rings alter ionic permeability or selectivity.

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N‐terminal extracellular moiety of the receptor and (iii) to set up a fluorescence‐based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N‐terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino‐acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy‐pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor‐ligand complexes, relative donor (EGFP)‐acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino‐terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence‐based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.

Subcellular Compartmentalization of Activation and Desensitization of Responses Mediated by NK2 Neurokinin Receptors

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nm to μm agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.

A Pivotal Role for CXCL12 Signaling in HPV-Mediated Transformation of Keratinocytes: Clues to Understanding HPV-Pathogenesis in WHIM Syndrome

The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.