Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors.

Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes.

Exploring the active conformation of cyclohexane carboxylate positive allosteric modulators of the type 4 metabotropic glutamate receptor.

The active conformation of a family of metabotropic glutamate receptor subtype 4 (mGlu4 ) positive allosteric modulators (PAMs) with the cyclohexane 1,2-dicarboxylic scaffold present in cis-2-(3,5-dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041) was investigated by testing structurally similar six-membered ring compounds that have a locked conformation. The norbornane and cyclohexane molecules designed as mGlu4 conformational probes and the enantiomers of the trans diastereomer were computationally characterized and tested in mGlu4 pharmacological assays. The results support a VU0155041 active conformation, with the chair cyclohexane having the aromatic amide substituent in an axial position and the carboxylate in an equatorial position. Moreover, the receptor displays enantiomeric discrimination of the chiral PAMs. The constructed pharmacophore characterized a highly constrained mGlu4 allosteric binding site, thus providing a step forward in structure-based drug design for mGlu4 PAMs.

Potent aminocyclitol glucocerebrosidase inhibitors are subnanomolar pharmacological chaperones for treating gaucher disease.

Amino-myo-inositol derivatives have been found to be potent inhibitors of glucocerebrosidase (GCase), the ß-glucosidase enzyme deficient in Gaucher disease (GD). When tested using lymphoblasts derived from patients with GD homozygous for N370S or L444P mutations, the compounds enhanced GCase activity at very low concentrations. The most potent inhibitor, (1R,2S,3R,4S,5S,6R)-5-(nonylamino)-6-(nonyloxy)cyclohexane-1,2,3,4-tetraol had a K(i) of 1 nM using isolated enzyme and an IC(50) of 4.3 nM when assayed in human fibroblast cell culture. This aminocyclitol produced maximum increases of GCase activities of 90% in N370S lymphoblasts at 1 nM and 40% in L444P at 0.01 nM following a three-day incubation. In addition to inhibitory potency, this compound has the permeability, subcellular distribution, and cell metabolism characteristics that are important for use as a pharmacological chaperone. It is a remarkable finding that picomolar concentrations of aminocyclitols are sufficient to enhance activity in the L444P variant, which produces a severe neuronopathic form of GD without clinical treatment.

PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP:phosphocholine cytidylyltransferase activity.

In the human malaria parasite Plasmodium falciparum, the synthesis of the major and essential membrane phospholipid, phosphatidylcholine, occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation (SDPM) pathways, which are fueled by host choline, serine, and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes, P. falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) and choline-phosphate transferase (PfCEPT). We identified a novel class of phospholipid mimetics, which inhibit the growth of P. falciparum as well as Leishmania and Trypanosoma species. Metabolic analyses showed that one of these compounds, PG12, specifically blocks phosphatidylcholine biosynthesis from both the CDP-choline and SDPM pathways via inhibition of PfCCT. In vitro studies using recombinant PfCCT showed a dose-dependent inhibition of the enzyme by PG12. The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo.

Integrated synthetic, pharmacological, and computational investigation of cis-2-(3,5-dichlorophenylcarbamoyl)cyclohexanecarboxylic acid enantiomers as positive allosteric modulators of metabotropic glutamate receptor subtype†4.

2-(3,5-Dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (1) is a potent and selective positive allosteric modulator of metabotropic glutamate receptor subtype 4 (mGluR4). The activity of 1 was reported to reside in the cis diastereomer with equal potency between its enantiomeric forms (Niswender et al., Mol. Pharmacol. 2008, 74, 1345-1358). In the present study, the asymmetric synthesis of each of the cis enantiomers was performed, and their activities were compared with that of the racemic trans. In our assays, the cis enantiomers differ in potency, with one of them (1R,2S) higher and the other (1S,2R) lower than the racemic trans. High-level quantum chemical calculations were carried out to characterize the structures of minimum energy in all-isomer conformational space as well as particular intermediates between conformational transitions. Computational analysis identified structural features of 1 that can play a role in mGluR4 functionality and establish the basis for subsequent work, in which molecular chirality constructed on conformations derived from those found for the active (1R,2S) enantiomer can provide new ideas for drug discovery. Comparison between experimental and theoretical circular dichroism spectra confirmed both the absolute configuration of the (1R,2S) compound and its calculated most stable conformation, thereby supporting experimental and theoretical work.

2-aminohydroxamic acid derivatives as inhibitors of Bacillus cereus phosphatidylcholine preferred phospholipase C PC-PLC(Bc).

Phosphatidylcholine preferring phospholipase C (PC-PLC) is an important enzyme that plays a key role in a variety of cellular events and lipid homoeostases. Bacillus cereus phospholipase C (PC-PLC(Bc)) has antigenic similarity with the elusive mammalian PC-PLC, which has not thus far been isolated and purified. Therefore the discovery of inhibitors of PC-PLC(Bc) is of current interest. Here, we describe the synthesis and biological evaluation of a new type of compounds inhibiting PC-PLC(Bc). These compounds have been designed by evolution of previously described 2-aminohydroxamic acid PC-PLC(Bc) inhibitors that block the enzyme by coordination of the zinc active site atoms present in PC-PLC(Bc) [Gonzalez-Roura, A.; Navarro, I.; Delgado, A.; Llebaria, A.; Casas, J. Angew. Chem. Int. Ed.2004, 43, 862]. The new compounds maintain the zinc coordinating groups and possess an extra trimethylammonium function, linked to the hydroxyamide nitrogen by an alkyl chain, which is expected to mimic the trimethylammonium group of the phosphatidylcholine PC-PLC(Bc) substrates. Some of the compounds described inhibit the enzyme with IC(50)'s in the low micromolar range. Unexpectedly, the most potent inhibitors found are those that possess a trimethylammonium group but have chemically blocked the zinc coordinating functionalities. The results obtained suggest that PC-PLC(Bc) inhibition is not due to the interaction of compounds with the phospholipase catalytic zinc atoms, but rather results from the inhibitor cationic group recognition by the PC-PLC(Bc) amino acids involved in choline lipid binding.

Practical synthesis of (-)-1-amino-1-deoxy-myo-inositol from achiral precursors.

A new synthesis of enantiomerically pure 1-amino-1-deoxy-myo-inositol is reported. The route described employs p-benzoquinone, an achiral compound, as the starting material to give conduritol B tetraacetate in three steps. Kinetic resolution of this compound using a palladium catalyst with a chiral ligand allows access to a conduritol B tetraester in high enantiomeric excess. This compound is transformed into tetrabenzyl conduritol B epoxide, which is regioselectively opened with azide to give the key azidocyclitol. Final transformation into (-)-1-amino-1-deoxy-myo-inositol hydrochloride is achieved in four synthetic steps. This sequence allows the synthesis of this compound in high enantiomeric purity in a semi-preparative scale.