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Francine Acher

Publications and source records attributed to Francine Acher.

12 recordsLinked to original sources
PubMed2006

Amino-pyrrolidine tricarboxylic acids give new insight into group III metabotropic glutamate receptor activation mechanism.

Like most class C G-protein-coupled receptors, metabotropic glutamate (mGlu) receptors possess a large extracellular domain where orthosteric ligands bind. Crystal structures revealed that this domain, called Venus FlyTrap (VFT), adopts a closed or open conformation upon agonist or antagonist binding, respectively. We have described amino-pyrrolidine tricarboxylic acids (APTCs), including (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (FP0429), as new selective group III mGlu agonists. Whereas FP0429 is an almost full mGlu4 agonist, it is a weak and partial agonist of the closely related mGlu8 subtype. To get more insight into the activation mechanism of mGlu receptors, we aimed to elucidate why FP0429 behaves differently at these two highly homologous receptors by focusing on two residues within the binding site that differ between mGlu4 and mGlu8. Site-directed mutagenesis of Ser157 and Gly158 of mGlu4 into their mGlu8 homologs (Ala) turned FP0429 into a weak partial agonist. Conversely, introduction of Ser and Gly residues into mGlu8 increased FP0429 efficacy. Docking of FP0429 in mGlu4 VFT 3D model helped us characterize the role of each residue. Indeed, mGlu4 Ser157 seems to have an important role in FP0429 binding, whereas Gly158 may allow a deeper positioning of this agonist in the cavity of lobe I, thereby ensuring optimal interactions with lobe II residues in the fully closed state of the VFT. In contrast, the presence of a methyl group in mGlu8 (Ala instead of Gly) weakens the interactions with the lobe II residues. This probably results in a less stable or a partially closed form of the mGlu8 VFT, leading to partial receptor activation.

Animals↗
PubMed2006

Design and synthesis of APTCs (aminopyrrolidinetricarboxylic acids): identification of a new group III metabotropic glutamate receptor selective agonist.

A new family of mGlu receptor orthosteric ligands called APTCs was designed and synthesized using a parallel chemistry approach. Amongst 65 molecules tested on mGlu4, mGlu6 and mGlu8 subtypes, (2S,4S)-4-amino-1-[(E)-3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid (8a06-FP0429) has been shown to be a full mGlu4 agonist and a partial mGlu8 agonist. In addition, 8a06 was shown to be selective versus group I and II mGlu subtypes. A possible explanation for this efficacy difference is proposed by docking experiment performed with molecular model of the receptor.

Amination↗
PubMed2005

Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators.

The recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation.

Actins↗
PubMed2005

Virtual screening workflow development guided by the "receiver operating characteristic" curve approach. Application to high-throughput docking on metabotropic glutamate receptor subtype 4.

The "receiver operating characteristic" (ROC) curve method is a well-recognized metric used as an objective way to evaluate the ability of a given test to discriminate between two populations. This facilitates decision-making in a plethora of fields in which a wrong judgment may have serious consequences including clinical diagnosis, public safety, travel security, and economic strategies. When virtual screening is used to speed-up the drug discovery process in pharmaceutical research, taking the right decision upon selecting or discarding a molecule prior to in vitro evaluation is of paramount importance. Characterizing both the ability of a virtual screening workflow to select active molecules and the ability to discard inactive ones, the ROC curve approach is well suited for this critical decision gate. As a case study, the first virtual screening workflow focused on metabotropic glutamate receptor subtype 4 (mGlu4R) agonists is reported here. Six compounds out of 38 selected and tested in vitro were shown to have agonist activity on this target of therapeutic interest.

Binding Sites↗
PubMed2004

Molecular determinants of ligand selectivity in a vertebrate odorant receptor.

The identification of the chemical structure of an odorant by the vertebrate olfactory system is thought to occur through the combinatorial activity from multiple receptors, each tuned to recognize different chemical features. What are the molecular determinants underlying the selectivity of individual odorant receptors for their cognate ligands? To address this question, we performed molecular modeling and site-directed mutagenesis on the ligand-binding region of two orthologous amino acid odorant receptors belonging to the "C family" of G-protein-coupled receptors in goldfish and zebrafish. We identified the critical ligand-receptor interactions that afford ligand binding as well as selectivity for different amino acids. Moreover, predictions regarding binding pocket structure allowed us to alter, in a predictable manner, the receptor preferences for different ligands. These results reveal how this class of odorant receptor has evolved to accommodate ligands of varying chemical structure and further illuminate the molecular principles underlying ligand recognition and selectivity in this family of chemosensory receptors.

Amino Acids↗
PubMed2004

The heptahelical domain of GABA(B2) is activated directly by CGP7930, a positive allosteric modulator of the GABA(B) receptor.

The gamma-aminobutyric acid, type B (GABA(B)) receptor is well recognized as being composed of two subunits, GABA(B1) and GABA(B2). Both subunits share structural homology with other class-III G-protein-coupled receptors. They are composed of two main domains: a heptahelical domain (HD) typical of all G-protein-coupled receptors and a large extracellular domain (ECD). Although GABA(B1) binds GABA, GABA(B2) is required for GABA(B1) to reach the cell surface. However, it is still not demonstrated whether the association of these two subunits is always required for function in the brain. Indeed, GABA(B2) plays a major role in the coupling of the heteromer to G-proteins, such that it is possible that GABA(B2) can transmit a signal in the absence of GABA(B1). Today only ligands interacting with GABA(B1) ECD have been identified. Thus, the compounds acting exclusively on the GABA(B2) subunit will be helpful in analyzing the specific role of this subunit in the brain. Here, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABA(B) receptor. We showed that it activates the wild type GABA(B) receptor but with a low efficacy. The GABA(B2) HD is necessary for this effect, although one cannot exclude that CGP7930 could also bind to GABA(B1). Of interest, CGP7930 could activate GABA(B2) expressed alone and is the first described agonist of GABA(B2). Finally, we show that CGP7930 retains its agonist activity on a GABA(B2) subunit deleted of its ECD. This demonstrates that the HD of GABA(B2) behaves similar to a rhodopsin-like receptor, because it can reach the cell surface alone, can couple to G-protein, and be activated by agonists. These data open new strategies for studying the mechanism of activation of GABA(B) receptor and examine any possible role of homomeric GABA(B2) receptors.

Allosteric Regulation↗
PubMed2003

Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors.

Although agonists bind directly in the heptahelical domain (HD) of most class-I rhodopsin-like G protein coupled receptors (GPCRs), class-III agonists bind in the extracellular domain of their receptors. Indeed, the latter possess a large extracellular domain composed of a cysteine-rich domain and a Venus flytrap module. Both the low sequence homology and the structural organization of class-III GPCRs raised the question of whether or not the HD of these receptors functions the same way as rhodopsin-like GPCRs. Here, we show that the HD of metabotropic glutamate receptor 5 (mGlu(5)) displays the same agonist-independent constitutive activity as the wild-type receptor. Moreover, we show that the noncompetitive antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine hydrochloride] and the positive allosteric modulator DFB (3,3'-difluorobenzaldazine) act as inverse agonist and full agonist, respectively, on the mGlu(5) HD in the absence of the extracellular domain. This finding illustrates that, like rhodopsin-like receptors, the HD of mGluRs can constitutively couple to G proteins and be negatively and positively regulated by ligands. These data show that the HD of mGluRs behave like any other class-I GPCRs in terms of G protein coupling and regulation by various types of ligands.

Allosteric Regulation↗
PubMed2003

A conserved region of human vitamin K-dependent carboxylase between residues 393 and 404 is important for its interaction with the glutamate substrate.

Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their gamma-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu394 is similar in all known carboxylases, suggesting that the region is functionally important. To test this hypothesis we made the following mutant enzymes: W390A, Y395A, S398A, W399A, and H404A. We purified the enzymes and corrected the activity measurements for active enzyme concentration. Carboxylases W390A, S398A, and H404A had activities similar to that of wild type; however, Y395A and W399A had lower activities than did wild type. In the following descriptions we include our previously reported results for L394R. Kinetic studies with the substrate FLEEL, revealed Km values of 0.5 (wild type), 6.5 (L394R), 15 (Y395A), and 24 (W399A) mm. The kcat values relative to wild type were 51% (L394R), 1% (Y395A), and 2% (W399A). The kcat/Km values were 24-fold (L394R) and >2000-fold lower for Y395A and W399A than for wild-type carboxylase. Inhibition of FLEEL carboxylation by the competitive inhibitor, Boc-mEEV, gave Ki values of 0.013 (wild type), 1.4 (L394R), 2.1 (Y395A), and >5 (W399A) mm. The Y395A propeptide affinity was similar to that of wild type, but those of L394R and W399A were 16-22-fold less than that of wild type. Results of kinetic studies with a propeptide-containing substrate were consistent with results of propeptide binding and FLEEL kinetics. Although propeptide and vitamin K binding in some mutants were affected, our data provide compelling evidence that glutamate recognition is the primary function of the conserved region around Leu394.

Amino Acid Sequence↗
PubMed2002

Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists.

Ca2+, pheromones, sweet taste compounds, and the main neurotransmitters glutamate and gamma-aminobutyric acid activate G protein-coupled receptors (GPCRs) that constitute the GPCR family 3. These receptors are dimers, and each subunit has a large extracellular domain called a Venus flytrap module (VFTM), where agonists bind. This module is connected to a heptahelical domain that activates G proteins. Recently, the structure of the dimer of mGlu1 VFTMs revealed two important conformational changes resulting from glutamate binding. First, agonists can stabilize a closed state of at least one VFTM in the dimer. Second, the relative orientation of the two VFTMs in the dimer is different in the presence of glutamate, such that their C-terminal ends (which are connected to the G protein-activating heptahelical domain) become closer by more than 20 A. This latter change in orientation has been proposed to play a key role in receptor activation. To elucidate the respective role of VFTM closure and the change in orientation of the VFTMs in family 3 GPCR activation, we analyzed the mechanism of action of the mGlu8 receptor antagonists ACPT-II and MAP4. Molecular modeling studies suggest that these two compounds prevent the closure of the mGlu8 VFTM because of ionic and steric hindrance, respectively. We show here that the replacement of the residues responsible for these hindrances (Asp-309 and Tyr-227, respectively) by Ala allows ACPT-II or MAP4 to fully activate the receptors. These data are consistent with the requirement of the VFTM closure for family 3 GPCR activation.

Amino Acid Sequence↗
PubMed2002

Characteristics and composition of the vitamin K-dependent gamma-glutamyl carboxylase-binding domain on osteocalcin.

Two different sites on vitamin K-dependent gamma-glutamyl carboxylase (VKC) are involved in enzyme-substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-K(m) substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the gamma-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.

Amino Acid Motifs↗
PubMed2002

The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these offer for drug development.

Animals↗