Rational Design of Drugs Targeting G-Protein-Coupled Receptors: A Structural Biology Perspective.

G protein-coupled receptors (GPCRs) play a key role in the transduction of extracellular signals to cells and regulation of many biological processes, which makes these membrane proteins one of the most important targets for pharmacological agents. A significant increase in the number of resolved atomic structures of GPCRs has opened the possibility of developing pharmaceuticals targeting these receptors via structure-based drug design (SBDD). SBDD employs information on the structure of receptor-ligand complexes to search for selective ligands without the need for an extensive high-throughput experimental ligand screening and can significantly expand the chemical space for ligand search. In this review, we describe the process of deciphering GPCR structures using X-ray diffraction analysis and cryoelectron microscopy as an important stage in the rational design of drugs targeting this receptor class. Our main goal was to present modern developments and key features of experimental methods used in SBDD of GPCR-targeting agents to a wide range of specialists.

Unusual Oligomeric Laccase-like Oxidases from Ascomycete Curvularia geniculata VKM F-3561 Polymerizing Phenylpropanoids and Phenolic Compounds under Neutral Environmental Conditions.

The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds (dihydroxybenzyl alcohol and hydroxytyrosol) from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.

The Laccase of Myrothecium roridum VKM F-3565: A New Look at Fungal Laccase Tolerance to Neutral and Alkaline Conditions.

Most of the currently known fungal laccases show their maximum activity under acidic environmental conditions. It is known that a decrease in the activity of a typical laccase at neutral or alkaline pH values is the result of an increase in the binding of the hydroxide anion to the T2/T3 copper center, which prevents the transfer of an electron from the T1 Cu to the trinuclear copper center. However, evolutionary pressure has resolved the existing limitations in the catalytic mechanism of laccase, allowing such enzymes to be functionally active under neutral/alkaline pH conditions, thereby giving fungi an advantage for their survival. Combined molecular and biochemical studies, homological modeling, calculation of the electrostatic potential on the Connolly surface at pH 5.0 and 7.0, and structural analysis of the novel alkaliphilic laccase of Myrothecium roridum VKM F-3565 and alkaliphilic and acidophilic fungal laccases with a known structure allowed a new intramolecular channel near the one of the catalytic aspartate residues at T2-copper atom to be found. The amino acid residues of alkaliphilic laccases forming this channel can presumably serve as proton donors for catalytic aspartates under neutral conditions, thus ensuring proper functioning. For the first time for ascomycetous laccases, the production of new trimeric products of phenylpropanoid condensation under neutral conditions has been shown, which could have a potential for use in pharmacology.

Gentisate 1,2-dioxygenase from the gram-positive bacteria Rhodococcus opacus 1CP: Identical active sites vs. different substrate selectivities.

Gentisate 1,2-dioxygenases belong to the class III ring-cleaving dioxygenases catalyzing key reactions of aromatic compounds degradation by aerobic microorganisms. In the present work, the results of complete molecular, structural, and functional investigations of the gentisate 1,2-dioxygenase (rho-GDO) from a gram-positive bacterium Rhodococcus opacus 1CP growing on 3-hydroxybenzoate as a sole source of carbon and energy are presented. The purified enzyme showed a narrow substrate specificity. Among fourteen investigated substrate analogues only gentisate was oxidized by the enzyme, what can be potentially applied in biosensor technologies. The rho-GDO encoding gene was identified in the genomic DNA of the R. opacus 1CP. According to phylogenetic analysis, the rho-GDO belongs to the group of apparently most recently acquired activities in bacterial genera Rhodococcus, Arthrobacter, Corynebacterium, Nocardia, Amycolatopsis, Comamonas, and Streptomyces. Homology modeling the rho-GDO 3D-structure demonstrates the composition identity of the first-sphere residues of the active site of rho-GDO and salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans (RCSB PDB: 2PHD), despite of their different substrate specificities. The phenomenon described for the first time for this family of enzymes supposes a more complicated mechanism of substrate specificity than previously imagined, and makes the rho-GDO a convenient model for a novel direction of structure-function relationship studies.

Conversion of 2-fluoromuconate to cis-dienelactone by purified enzymes of Rhodococcus opacus 1cp.

The present study describes the (19)F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.

A new modified ortho cleavage pathway of 3-chlorocatechol degradation by Rhodococcus opacus 1CP: genetic and biochemical evidence.

The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase.