The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors

New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS‐790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N‐terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X‐ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand‐soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.     

Discovery of new potent protein arginine methyltransferase 5 (PRMT5) inhibitors by assembly of key pharmacophores from known inhibitors

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been put into phase I clinical trial. In the present study, a series of candidate molecules were designed by combining key pharmacophores of formerly reported PRMT5 inhibitors. The in vitro PRMT5 inhibitory testing of compound 4b14 revealed an IC₅₀ of 2.71?μM, exhibiting high selectivity over PRMT1 and PRMT4 (>70-fold selective). As expected, 4b14 exhibited potent anti-proliferative activity against a panel of leukemia and lymphoma cells, including MV4-11, Pfeiffer, SU-DHL-4 and KARPAS-422. Besides, 4b14 showed significant cell cycle arrest and apoptosis-inducing effects, as well as reduced the cellular symmetric arginine dimethylation level of SmD3 protein. Finally, affinity profiling analysis indicated that hydrophobic interactions, π-π stacking and cation-π actions made the major contributions to the overall binding affinity. This scaffold provides a new chemical template for further development of better lead compounds targeting PRMT5.     

Sirtuin deacetylases: A new target for melanoma management

Melanoma continues to cause more deaths than any other skin cancer, necessitating the development of new avenues of treatment. One promising new opportunity comes in the form of mechanism-based therapeutic targets. We recently reported the overexpression and delocalization of the class III histone deacetylase SIRT1 in melanoma, and demonstrated that its small molecule inhibition via Tenovin-1 decreased cell growth and viability of melanoma cells, possibly by a p53 mediated induction of p21. Here, we support our data using additional SIRT inhibitors, viz. Sirtinol and Ex-527, which suggests possible benefits of concomitantly inhibiting more than one Sirtuin for an effective cancer management strategy. This “Extra View” paper also includes a discussion of our results in the context of similar recent and concurrent studies. Furthermore, we expand upon our findings in an analysis of new research that may link the cellular localization and growth effects of SIRT1 with the PI3K signaling pathway.     

KEY,LOCK, and LOCKSMITH: Complementary hydropathic map predictions of drug structure from a known receptor-receptor structure from known drugs

Three new routines (LOCK. KEY and LOCKSMITH) for the program HINT (Hydrophobic interactions) are described and demonstrated. The KEY routine uses receptor structure to model the hydropathic profile of the ideal substrate for the receptor. The LOCK routine uses substrate or drug structure to model the hydropathic character of the receptor. LOCKSMITH is an algorithm designed to highlight the significant hydropathic features from a collection of agents. Ten allosteric modifiers of hemoglobin that have been characterized biologically and with X-ray diffraction to determine their protein binding sites/conformations illustrate the KEY and LOCKSMITH routines: The LOCKSMITH composite map correctly identifies the structural features and conformation of the more active modifiers. In addition, many hydropathic features of the “ideal” drug predicted by the KEY map overlap with actual structural features of the most active hemoglobin allosteric modifiers.     

Evolution, structure and function of mitochondrial carriers: a review with new insights

The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins.     

Application of a new method for GWAS in a related case/control sample with known pedigree structure: identification of new loci for nephrolithiasis

In contrast to large GWA studies based on thousands of individuals and large meta-analyses combining GWAS results, we analyzed a small case/control sample for uric acid nephrolithiasis. Our cohort of closely related individuals is derived from a small, genetically isolated village in Sardinia, with well-characterized genealogical data linking the extant population up to the 16(th) century. It is expected that the number of risk alleles involved in complex disorders is smaller in isolated founder populations than in more diverse populations, and the power to detect association with complex traits may be increased when related, homogeneous affected individuals are selected, as they are more likely to be enriched with and share specific risk variants than are unrelated, affected individuals from the general population. When related individuals are included in an association study, correlations among relatives must be accurately taken into account to ensure validity of the results. A recently proposed association method uses an empirical genotypic covariance matrix estimated from genome-screen data to allow for additional population structure and cryptic relatedness that may not be captured by the genealogical data. We apply the method to our data, and we also investigate the properties of the method, as well as other association methods, in our highly inbred population, as previous applications were to outbred samples. The more promising regions identified in our initial study in the genetic isolate were then further investigated in an independent sample collected from the Italian population. Among the loci that showed association in this study, we observed evidence of a possible involvement of the region encompassing the gene LRRC16A, already associated to serum uric acid levels in a large meta-analysis of 14 GWAS, suggesting that this locus might lead a pathway for uric acid metabolism that may be involved in gout as well as in nephrolithiasis.     

Language emergence: clues from a new bedouin sign

A sign language has emerged among three generations of deaf people and their families in a Bedouin community in the Negev desert. This newly reported case sheds light on the minimal environmental social factors required to generate a language.     

Evolution of insect mushroom bodies: old clues,new insights

The mushroom bodies are a morphologically diverse sensory integration and learning and memory center in the brains of various invertebrate species, of which those of insects are the best described. Insect mushroom bodies are composed of numerous tiny intrinsic neurons (Kenyon cells) that form calyces with their dendrites and a pedunculus and lobes with their axons. The identities of conserved Kenyon cell subpopulations and the correlations between morphological and functional specializations of the mushroom bodies are just beginning to be elucidated, providing insight into mechanisms of mushroom body evolution. Comparisons of mushroom body organization in different insect lineages reveal trends in the evolution of subcompartments correlated with the elaboration, reduction, acquisition or loss of Kenyon cell subpopulations. Furthermore, these changes often appear correlated with variation in type and strength of afferent input and in behavioral ecology. These and other features of mushroom body organization suggest a striking convergence with mammalian cortex, with Kenyon cell subpopulations displaying evolutionary modularity in a manner reminiscent of cortical areas.     

Redoxal as a new lead structure for dihydroorotate dehydrogenase inhibitors: a kinetic study of the inhibition mechanism

Mitochondrial dihydroorotate dehydrogenase (DHOdehase; EC 1.3.99.11) is a target of anti-proliferative, immunosuppressive and anti-parasitic agents. Here, redoxal, (2,2'-[3,3'-dimethoxy[1, 1'-biphenyl]-4,4'-diyl)diimino]bis-benzoic acid, was studied with isolated mitochondria and the purified recombinant human and rat enzyme to find out the mode of kinetic interaction with this target. Its pattern of enzyme inhibition was different from that of cinchoninic, isoxazol and naphthoquinone derivatives and was of a non-competitive type for the human (K(ic)=402 nM; K(iu)=506 nM) and the rat enzyme (K(ic)=116 nM; K(iu)=208 nM). The characteristic species-related inhibition of DHOdehase found with other compounds was less expressed with redoxal. In human and rat mitochondria, redoxal did not inhibit NADH-induced respiration, its effect on succinate-induced respiration was marginal. This was in contrast to the sound effect of atovaquone and dichloroallyl-lawsone, studied here for comparison. In human mitochondria, the IC(50) value for the inhibition of succinate-induced respiration by atovaquone was 6.1 microM and 27.4 microM for the DHO-induced respiration; for dichlorallyl-lawsone, the IC(50) values were 14.1 microM and 0.23 microM.     

Novelties in Adenia (Passifloraceae): Four new species, a new combination, a vegetative key, and diagnostic characters for known Madagascan species

Four new species and one new combination ofAdenia are presented, along with a vegetative key and diagnostic characters of the Madagascan species. Additional notes are provided about unusual specimens and field observations.Adenia kigogoensis from Tanzania is shown to be distinct fromA. stenodactyla (its putative closest relative) andA. digitata based on anther connation and other floral traits. The remaining new taxa are from Madagascar.Adenia litoralis has been observed from one coastal locality in northern Madagascar. It is distinctive in fruit size and leaf form.Adenia metamorpha is the only Madagascan taxon with a narrow cylindrical trunk and large napiform tuber; it is also known from only one locality in Madagascar in the Ankarana Reserve.Adenia mcdadiana is a robust liana with highly reduced glands and leaves that appear to be neotenic compared to its closest putative relative,A. sphaerocarpa. Finally, the position ofA. stylosa has been clarified. This species was once treated asA. firingalavensis var.stylosa, and prior to that asA. epigea var.stylosa, but molecular and morphological data suggest it is separate from these species.     

Gadolinium-based contrast agent toxicity: a review of known and proposed mechanisms

Gadolinium chelates are widely used as contrast media for magnetic resonance imaging. The approved gadolinium-based contrast agents (GBCAs) have historically been considered safe and well tolerated when used at recommended dosing levels. However, for nearly a decade, an association between GBCA administration and the development of nephrogenic systemic fibrosis (NSF) has been recognized in patients with severe renal impairment. This has led to modifications in clinical practices aimed at reducing the potential and incidence of NSF development. Newer reports have emerged regarding the accumulation of gadolinium in various tissues of patients who do not have renal impairment, including bone, brain, and kidneys. Despite the observations of gadolinium accumulation in tissues regardless of renal function, very limited clinical data regarding the potential for and mechanisms of toxicity is available. This significant gap in knowledge warrants retrospective cohort study efforts, as well as prospective studies that involve gadolinium ion (Gd³⁺) testing in patients exposed to GBCA. This review examines the potential biochemical and molecular basis of gadolinium toxicity, possible clinical significance of gadolinium tissue retention and accumulation, and methods that can limit gadolinium body burden.     

Carbazole and hydrazone derivatives as new competitive inhibitors of tyrosinase: Experimental clues to binuclear copper active site binding

In this work a total of 12 carbazoles and hydrazone-bridged thiazole-pyrrole derivatives have been identified as new competitive inhibitors of tyrosinase. Carbazole derivative with 2-benzoimidazole substitution showed most potent inhibition in the series. Other carbazole derivatives containing benzothiazole and benzoxazole substitutions showed comparable levels of tyrosinase inhibition. The hydrazone derivatives also showed potent tyrosinase inhibitory activity with comparable K_i values except one with fluoride at its terminal position. Kinetic studies showed competitive inhibition of tyrosinase by all compounds that increased the substrate K_m without changing the V_max value. Moreover, experimental evidence suggests that the target compounds specifically bind to the binuclear copper center of the tyrosinase active site in an apparent mono-dentate fashion. Carbazoles and hydrazones are new and emerging classes of compounds as tyrosinase inhibitors that may provide new structural avenues to discovery of drugs targeting the treatment of hyperpigmentation and related dermatological disorders.     

Outer-membrane phospholipase A: known structure, unknown biological function

Outer-membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria. The enzymatic activity of OMPLA is strictly regulated to prevent uncontrolled breakdown of the surrounding phospholipids. The activity of OMPLA can be induced by membrane perturbation and concurs with dimerization of the enzyme. The recently elucidated crystal structures of the inactive, monomeric and an inhibited dimeric form of the enzyme provide detailed structural insight into the functional properties of the enzyme. OMPLA is a serine hydrolase with a unique Asn-156-His-142-Ser-144 catalytic triad. Only in the dimeric state, complete substrate binding pockets and functional oxyanion holes are formed. A model is proposed for the activation of OMPLA in which membrane perturbation causes the formation of non-bilayer structures, resulting in the presentation of phospholipids to the active site of OMPLA and leading to the formation of the active dimeric species. Possible roles for OMPLA in maintaining the cell envelope integrity and in pathogenicity are discussed.     

The genus Dietzia: a new home for some known and emerging opportunist pathogens

The genus Dietzia has only been established fairly recently. The Gram morphology and colony appearance of the species of this genus is remarkably similar to Rhodococcus equi . In the absence of simple, accurate methods for their identification, Dietzia spp. might have been misidentified as a Rhodococcus spp. and/or considered to be contaminants only. This MiniReview is designed to summarize current evidence on the clinical significance of Dietzia species, to consider their potential role as human pathogens, and to outline approaches that can be used to accurately classify and identify members of the genus, with the overall aim of alerting the medical microbiological community to a little known genus that contains clinically significant organisms.     

Phylogeny of ion channels: clues to structure and function

Voltage-gated ion channels are responsible for the electrical activity in a variety of cell types in modern-day animals. However, they represent the result of many millions of years of evolution of a family of ion channel proteins that are also found in prokaryotes and diverse eukaryotes, and probably exist in all life forms. This review traces the evolution of ion channels, with particular emphasis on the factors and evolutionary pathways that may have given rise to voltage-gated potassium (K+), calcium (Ca2+), and sodium (Na+) channels. The review also highlights the utility of comparing phylogenetically distinct versions of the same protein as a means to better understand the structure and function of proteins.     

5-Substituted-2,3-diphenyltetrahydrofurans: a new class of moderately selective COX-2 inhibitors

The nature of C-5 substituent and the configuration at C-5 carbon of 2,3-diphenyltetrahydrofurans, with chiral centres at C-2, C-3 and C-5, show a remarkable influence on their COX-2 inhibition and selectivity. Out of the eight compounds investigated here, 1b with COOH group and R* configuration at C-5, and 2d with CH2SCH2CH3 group and S* configuration at C-5 have been identified as lead molecules for further studies on COX-2 inhibition.