Luc De Vries

Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

μ-opioid and 5-HT1A receptors heterodimerize and show signalling crosstalk via G protein and MAP-kinase pathways

μ-opioid receptors have been shown to form heterodimers with several G protein coupled receptors involved in pain regulation such as α(2A)-adrenergic and neurokinin 1 receptors. Because the 5-HT(1A) receptor is also involved in pain control, we investigated whether it can interact with the μ-opioid receptor in cell lines. Using epitope-tagged μ-opioid and 5-HT(1A) receptors, we show that both receptors can co-immunoprecipate when expressed in the same cells. This physical interaction was corroborated by a Bioluminescence Resonance Energy Transfer signal between the μ-opioid receptor fused to Renilla luciferase and the 5-HT(1A) receptor fused to the Green Fluorescent Protein. Consistent with the presence of functional heterodimers, the μ-opioid receptor activated a Gα(o) protein covalently fused to the 5-HT(1A) receptor in membrane preparations as well as a Gα(15) protein fused to the 5-HT(1A) receptor in living cells. We demonstrate that both receptors can coexerce control of the ERK1/2 pathway: for example, μ-opioid receptor-induced ERK1/2 phosphorylation was selectively desensitized by 5-HT(1A) receptor activation. Although 5-HT(1A) and μ-opioid receptors were capable to internalize in response to their own activation, they were ineffective to induce the co-internalization of their partners. Thus, we show a functional heterodimerization of μ-opioid and 5-HT(1A) receptors in cell lines, a complex that might play a role in the control of pain in vivo. These results also support the potential therapeutic action of 5-HT(1A) agonists against nociceptive processes.

Endogenous RGS proteins facilitate dopamine D2S receptor coupling to Gαo proteins and Ca2+ responses in CHO-K1 cells

The role of RGS proteins on dopaminergic D2S receptor (D2SR) signalling was investigated in Chinese hamster ovary (CHO)‐K1 cells, using recombinant RGS protein‐ and PTX‐insensitive Gαo proteins. Dopamine‐mediated [35S]GTPγS binding was attenuated by more than 60% in CHO‐K1 D2SR cells coexpressing a RGS protein‐ and PTX‐insensitive GαoGly184Ser:Cys351Ile protein versus cells coexpressing a similar amount of PTX‐insensitive GαoCys351Ile protein. Dopamine‐agonist‐mediated Ca2+ responses were dependent on the coexpression with a GαoCys351Ile protein and were fully abolished upon coexpression with a GαoGly184Ser:Cys351Ile protein. These results suggest that interactions between the Gαo protein and RGS proteins are involved in efficient D2SR signalling.

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation.

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC(50) value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100-150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.

Effects of a new PAR1 antagonist, F 16618, on smooth muscle cell contraction.

This study evaluated the effects of two PAR1 antagonists on vessels contracted by SFLLR. ER 121958 antagonized the SFLLR-induced contraction on rat denuded superior mesenteric artery and pig coronary artery in a non-competitive manner (IC(50) values were 22 [7.5-43.6] nM and 2.9 [2.09-4.02] nM, respectively). F 16618 inhibited the SFLLR-induced superior mesenteric arteries and coronary arteries contraction in a competitive manner (pA(2) values of 7.3 and 6.2, respectively). PAR1 antagonists do not affect vessel resting tension or haemodynamic parameters in anaesthetized rats. Thus, PAR1 antagonists could have beneficial effects against vasospasm due to vessel injury.

Pharmacological evaluation of a series of smoothened antagonists in signaling pathways and after topical application in a depilated mouse model

The Hedgehog (HH) pathway has been linked to the formation of basal cell carcinoma (BCC), medulloblastoma, and other cancers. The recently approved orally active drugs vismodegib (GDC‐0449) and sonidegib (LDE–225) were not only efficacious for the treatment of advanced or metastatic BCC by antagonizing the smoothened (SMO) receptor, but also produced important side effects, limiting their use for less invasive BCC. Herein, we compared a large series of SMO antagonists, including GDC‐0449 and LDE‐225, the clinically tested BMS‐833923, CUR‐61414, cyclopamine, IPI‐926 (saridegib), itraconazole, LEQ‐506, LY‐2940680 (taladegib), PF‐04449913 (glasdegib), and TAK‐441 as well as preclinical candidates (PF‐5274857, MRT‐83) in two SMO‐dependent cellular assays and for G‐protein activation. We report marked differences in inhibitor potencies between compounds as well as a notable disparity between the G‐protein assay and the cellular tests, suggesting that classification of drugs is assay dependent. Furthermore, we explored topical efficacies of SMO antagonists on depilated mice using Gli1 and Ptch1 mRNA quantification in skin as biomarkers of the HH signaling inhibition. This topical model rapidly discriminated drugs in terms of efficacies and potencies for inhibition of both biomarkers. SMO antagonists showed also a large variation in their blood and skin partition, suggesting that some drugs are more favorable for topical application. Overall, our data suggested that in vitro and in vivo efficacious drugs such as LEQ‐506 and TAK‐441 may be of interest for topical treatment of less invasive BCC with minimal side effects.

Mutant 5-Hydroxytryptamine1A Receptor D116A Is a Receptor Activated Solely by Synthetic Ligands with a Rich Pharmacology

Like other biogenic amine G protein-coupled receptors, mutation of the conserved aspartatic residue into alanine at position 116 (D116A3.32) in the 5-hydroxytryptamine (5-HT)1A receptor greatly affects 5-HT binding and signal transduction. [3H]8-Hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and [3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) are capable to bind the 5-HT1A-D116A mutant and, using these radioligands, we show here that this mutation dramatically reduces the affinities of the selective 5-HT1A agonists N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylamino methyl]piperidine (F13640), 3-chloro-4-fluorophenyl-(4-fluorophenyl-4-{[(5-methyl-6 methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone (F13714), and 2-[5-[3-(4-methylsulfonylamino)benzyl-1,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694247) and that of 5-carboxamidotryptamine. Although to a lesser extent, the binding of buspirone, (+)-flesinoxan, (−)-pindolol, and (−)-8-OH-DPAT are also highly decreased. In contrast, affinities of the 5-HT1A ligands WAY100,635, spiperone, (−)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz {c,d}indole-6-carboxamide (LY228,729), and 1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl) piperazine (S14506) and the prototypical 5-HT1A agonist (+)-8-OH-DPAT are only slightly affected by the mutation, suggesting a moderate contribution of Asp116 to the binding pocket for these latter. Furthermore, LY228,729, S14506, and (+)-8-OH-DPAT induce a potent and efficacious coupling of the 5-HT1A-D116A receptor to G protein activation as measured by Ca2+ mobilization and guanosine 5′-O-(3-[35S]thio)triphosphate binding in Chinese hamster ovary cells as well as by G protein-coupled inwardly rectifying potassium channel current activation in Xenopus laevis oocytes. It is interesting that the selective 5-HT1A antagonist WAY100,635 shows potent partial agonist activity at the 5-HT1A-D116A mutant, whereas spiperone maintains its inverse agonist properties. The pharmacological approach reported here re-evaluates the binding and functional properties of the 5-HT1A-D116A receptor and describes for the first time this mutant as a receptor activated solely by synthetic ligands (RASSL), with a rich pharmacology. By bioengineering animal models incorporating this RASSL, one may further explore the role of 5-HT1A receptor signaling in the central nervous system as well as Gi protein-mediated signaling pathways in other tissues.

Expression of a truncated protein-tyrosine phosphatase mRNA in human lung.

Protein‐tyrosine phosphatases (PTPases) are becoming an important family of enzymes that might regulate key events in cell growth and transformation. While isolating a new member of this family via amplification of human lung cDNA by the polymerase chain reaction, we found a clone identical to but truncated at the 3′‐end of the coding region of human PTPase β (HPTPβ) mRNA. This difference in sequence is situated in the most conserved part of the catalytic domain of the enzyme. The expression level of the truncated form or HPTPβ mRNA in human lung was lower than its normal form