Biochemical Characterisation of Human Transglutaminase 4

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

Characterization of dopamine D2 receptor coupling to G proteins in postmortem brain of subjects with schizophrenia

Background Alterations of dopamine D1 (D1R) and D2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects. Methods G-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [35S]GTPγS-binding stimulation induced by increasing concentrations (10–10–10–3 M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration–response curves to NPA stimulation of [35S]GTPγS binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17). Results In caudate, [35S]GTPγS-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [35S]GTPγS-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 μM) and control (EC50H = 7.24 nM; EC50L = 15.14 μM) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters. Conclusions Feasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [35S]GTPγS-binding assays appears to be restricted to signalling through inhibitory Gi/o proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.

Over-expression of the Regulator of the Signaling of G Protein-Coupled Receptors RGS9-2 reduces the signaling elicited by the human histamine H3 receptor in HEK-293T cells

In HEK-293T cells transiently transfected with the human histamine H3 receptor (hH3R), we studied the effect of over-expressing the human RGS9-2 protein on H3R-mediated stimulation of [35S]-GTPγS binding and inhibition of forskolin-induced cAMP formation. Maximal specific binding (Bmax) of [3H]-N-methyl-histamine to cell membranes was 468 ± 12 and 442 ± 38 fmol/mg protein for HEK-293T-hH3R and HEK-293T-hH3R/hRGS9-2 cells, respectively, with dissociation constants (Kd) 2.57 nM and 3.38 nM. The H3R agonist immepip stimulated [35S]-GTPγS binding with similar potency and efficacy (Emax 146.3 ± 4.4 % and 150.0 ± 5.3 % of basal, pEC50 8.57 ± 0.26 and 9.00 ± 0.33, respectively), but was significantly less efficacious to inhibit forskolin-induced cAMP accumulation in HEK-293T-hH3R/hRGS9 cells (-19.2 ± 5.3 versus -37.7 ± 5.1 % in HEK-293T-hH3R cells) with no significant difference in potency (pEC50 9.60 ± 0.14 and 9.07 ± 0.29, respectively). These results indicate that in HEK-293T cells hRGS9-2 regulates hH3R445 signaling downstream G protein activation

Comparison of morphine, oxycodone and the biased MOR agonist SR-17018 for tolerance and efficacy in mouse models of pain

The mu opioid receptor-selective agonist, SR-17018, preferentially activates GTPγS binding over βarrestin2 recruitment in cellular assays. In mice, SR-17018 stimulates GTPγS binding in brainstem and produces antinociception with potencies similar to morphine. However, it produces much less respiratory suppression and mice do not develop antinociceptive tolerance in the hot plate assay upon repeated dosing. Herein we evaluate the effects of acute and repeated dosing of SR-17018, oxycodone and morphine in additional models of pain-related behaviors. In the mouse warm water tail immersion assay, an assessment of spinal reflex to thermal nociception, repeated administration of SR-17018 produces tolerance as does morphine and oxycodone. SR-17018 retains efficacy in a formalin-induced inflammatory pain model upon repeated dosing, while oxycodone does not. In a chemotherapeutic-induced neuropathy pain model SR-17018 is more potent and efficacious than morphine or oxycodone, moreover, this efficacy is retained upon repeated dosing of SR-17018. These findings demonstrate that, with the exception of the tail flick test, SR-17018 retains efficacy upon chronic treatment across several pain models.

The Intriguing Effects of Substituents in the N-Phenethyl Moiety of Norhydromorphone: A Bifunctional Opioid from a Set of “Tail Wags Dog” Experiments

(−)-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTPγS binding, forskolin-induced cAMP accumulation assay, and MOR-mediated β-arrestin recruitment assays). “Body” and “tail” interactions with opioid receptors (a subset of Portoghese’s message-address theory) were used for molecular modeling and simulations, where the “address” can be considered the “body” of the hydromorphone molecule and the “message” delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a δ/μ potency ratio of 1.2 in the ([35S]GTPγS) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.

pKa of opioid ligands as a discriminating factor for side effects

The non-selective activation of central and peripheral opioid receptors is a major shortcoming of currently available opioids. Targeting peripheral opioid receptors is a promising strategy to preclude side effects. Recently, we showed that fentanyl-derived μ-opioid receptor (MOR) agonists with reduced acid dissociation constants (pKa) due to introducing single fluorine atoms produced injury-restricted antinociception in rat models of inflammatory, postoperative and neuropathic pain. Here, we report that a new double-fluorinated compound (FF6) and fentanyl show similar pKa, MOR affinity and [35S]-GTPγS binding at low and physiological pH values. In vivo, FF6 produced antinociception in injured and non-injured tissue, and induced sedation and constipation. The comparison of several fentanyl derivatives revealed a correlation between pKa values and pH-dependent MOR activation, antinociception and side effects. An opioid ligand’s pKa value may be used as discriminating factor to design safer analgesics.

GPR31 and GPR151 are activated under acidic conditions

Recent studies have revealed that not only proton-sensing channels, but also one family of G protein-coupled receptors (GPCRs) comprising OGR1, GPR4, G2A and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31 and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31–Giα and GPR151–Giα fusion proteins in a narrow range between pH 6 and 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at pH ∼5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.