Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H4 receptor.

BACKGROUND AND PURPOSE: The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182(5.46) in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98(3.36) in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98(3.36) by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. EXPERIMENTAL APPROACH: We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and ß-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. KEY RESULTS: VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and ß-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C98(3.36) prevented this covalent interaction. CONCLUSION AND IMPLICATIONS: VUF14480 is thought to bind covalently to the hH4 receptor-C98(3.36) residue and partially induce hH4 receptor-mediated G protein activation and ß-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor.

Serine and threonine residues bend alpha-helices in the chi(1) = g(-) conformation.

The relationship between the Ser, Thr, and Cys side-chain conformation (chi(1) = g(-), t, g(+)) and the main-chain conformation (phi and psi angles) has been studied in a selection of protein structures that contain alpha-helices. The statistical results show that the g(-) conformation of both Ser and Thr residues decreases their phi angles and increases their psi angles relative to Ala, used as a control. The additional hydrogen bond formed between the O(gamma) atom of Ser and Thr and the i-3 or i-4 peptide carbonyl oxygen induces or stabilizes a bending angle in the helix 3-4 degrees larger than for Ala. This is of particular significance for membrane proteins. Incorporation of this small bending angle in the transmembrane alpha-helix at one side of the cell membrane results in a significant displacement of the residues located at the other side of the membrane. We hypothesize that local alterations of the rotamer configurations of these Ser and Thr residues may result in significant conformational changes across transmembrane helices, and thus participate in the molecular mechanisms underlying transmembrane signaling. This finding has provided the structural basis to understand the experimentally observed influence of Ser residues on the conformational equilibrium between inactive and active states of the receptor, in the neurotransmitter subfamily of G protein-coupled receptors.

GRID/CPCA: a new computational tool to design selective ligands.

We present a computational procedure aimed at understanding enzyme selectivity and guiding the design of drugs with respect to selectivity. It starts from a set of 3D structures of the target proteins characterized by the program GRID. In the multivariate description proposed, the variables are organized and scaled in a different way than previously published methodologies. Then, consensus principal component analysis (CPCA) is used to analyze the GRID descriptors, allowing the straightforward identification of possible modifications in the ligand to improve its selectivity toward a chosen target. As an important new feature the computational method is able to work with more than two target proteins and with several 3D structures for each protein. Additionally, the use of a 'cutout tool' allows to focus on the important regions around the active site. The method is validated for a total number of nine structures of the three homologous serine proteases thrombin, trypsin, and factor Xa. The regions identified by the method as being important for selectivity are in excellent agreement with available experimental data and inhibitor structure-activity relationships.

Factor Xa: simulation studies with an eye to inhibitor design.

Factor Xa is a serine protease which activates thrombin and plays a key regulatory role in the blood-coagulation cascade. Factor Xa is at the crossroads of the extrinsic and intrinsic pathways of coagulation and, hence, has become an important target for the design of anti-thrombotics (inhibitors). It is not known to be involved in other processes than hemostasis and its binding site is different to that of other serine proteases, thus facilitating selective inhibition. The design of high-affinity selective inhibitors of factor Xa requires knowledge of the structural and dynamical characteristics of its active site. The three-dimensional structure of factor Xa was resolved by X-ray crystallography and refined at 2.2 A resolution by Padmanabhan and collaborators. In this article we present results from molecular dynamics simulations of the catalytic domain of factor Xa in aqueous solution. The simulations were performed to characterise the mobility and flexibility of the residues delimiting the unoccupied binding site of the enzyme, and to determine hydrogen bonding propensities (with protein and with solvent atoms) of those residues in the active site that could interact with a substrate or a potential inhibitor. The simulation data is aimed at facilitating the design of high-affinity selective inhibitors of factor Xa.

Computer based screening of compound databases: 1. Preselection of benzamidine-based thrombin inhibitors.

We present a computational protocol which uses the known three-dimensional structure of a target enzyme to identify possible ligands from databases of compounds with low molecular weight. This is accomplished by first mapping the essential interactions in the binding site with the program GRID. The resulting regions of favorable interaction between target and ligand are translated into a database query, and with UNITY a flexible 3D database search is performed. The feasibility of this approach is calibrated with thrombin as the target. Our results show that the resulting hit lists are enriched with thrombin inhibitors compared to the total database.

A theoretical study concerning the mode of interaction of the histamine H2-agonist dimaprit.

A theoretical study was performed to elucidate the mode of interaction of the histamine H2-agonist dimaprit with the histamine H2-receptor. For this purpose receptor mapping techniques, including ab initio energy calculations, geometry optimizations and molecular electrostatic potential calculations (MEPs), have been used. The characteristics of dimaprit were compared to those of histamine for which the points of interaction with the H2-receptor are known, as well as its bioactive conformation. In this comparative study two possible models for the interaction of dimaprit with the H2-receptor were considered. In one model the two nitrogen atoms of the isothiourea moiety of dimaprit play an essential role in the recognition of the ligand by the receptor and have the same function as the nitrogen atoms of the imidazole ring of histamine; in the second model this role is fulfilled by a sulphur and a nitrogen atom of the same isothiourea moiety. The comparison to histamine was based on geometrical resemblance as well as on similarity in MEPs. Also the conformational energy of dimaprit in the two interaction models was considered. Results of the investigations reveal that the isothiourea moiety of dimaprit most probably interacts with the histamine H2-receptor through the sulphur and nitrogen atom, the first atom acting as a proton acceptor and the second one as a proton donor. Subsequently, three analogues of dimaprit, namely SK&F 91487, SK&F 91488 and SK&F 92054, were studied. It was possible to explain their pharmacological behavior within the proposed model.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine receptors: subclasses and specific ligands.

In this review the three main types of histamine receptors are discussed together with their specific ligands. For the classical H1-receptors much emphasis is put on the mechanism by which the receptor is stimulated. For the H1- and H2-receptor the review includes information on the several models available for establishing agonistic or antagonistic activity. In the section on the H3-receptor the ligands are discussed as well as the possible physiological role of this receptor. In the final paragraphs some less well defined activities are presented.

Studies on the active molecular species of the H2 receptor antagonists cimetidine and mifentidine.

The N'-(4-1H-imidazol-4-ylphenyl)formamidines were recently introduced as a new class of active H2 antagonists; the authors of the compounds (Donetti et al. of de Angeli, Italy) have suggested that these compounds interact with the H2 receptor through their monocations. This is at variance with the model proposed for cimetidine by the SK&F (Smith Kline & French, UK) group who proposed the neutral molecule as the species active at the H2 receptor. In the present study we have investigated the issue whether the neutral or charged species is the active one by measuring the pA2 values of mifentidine and cimetidine at different pH values. Changing the pH will influence the species equilibria of both compounds and thereby affect their activity. The activity changes measured for both compounds are consistent with the proposition that cimetidine as well as mifentidine elicit their activity through their neutral species.