Translational repression of thymidylate synthase by targeting its mRNA.

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.

Structure-based optimization of the terminal tripeptide in glycopeptide dendrimer inhibitors of Pseudomonas aeruginosa biofilms targeting LecA.

The galactopeptide dendrimer GalAG2 ((ß-Gal-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA-KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide-lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X-ray crystallography. Two of these tripeptides, GalA-KPY (dissociation constant (K(D))=2.7 µM) and GalA-KRL (K(D)=2.7 µM), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2, particularly G2KPY ((ß-Gal-OC6H4CO-Lys-Pro-Tyr)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine-tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions.

Clinal variation as a tool to understand climate change.

Clines are observable gradients that reflect continuous change in biological traits of species across geographical ranges. Clinal gradients could vary at geographic scales (latitude and altitude). Since clinal variations represent active genomic responses at the population level they (clines) provide an immense power to address questions related to climatic change. With the fast pace of climate change i.e. warming, populations are also likely to exhibit rapid responses; at both the phenotypic and genotypic levels. We seek to understand how clinal variation could be used to anticipate climatic responses using Drosophila, a pervasively used inter-disciplinary model system owing to its molecular repertoire. The genomic information coupled with the phenotypic variation greatly facilitates our understanding of the Drosophilidae response to climate change. We discuss traits associated with clinal variation at the phenotypic level as well as their underlying genetic regulators. Given prevailing climatic conditions and future projections for climate change, clines could emerge as monitoring tools to track the cross-talk between climatic variables and organisms.

No water, no mating: Connecting dots from behaviour to pathways.

Insects hold considerable ecological and agricultural importance making it vital to understand the factors impacting their reproductive output. Environmental stressors are examples of such factors which have a substantial and significant influence on insect reproductive fitness. Insects are also ectothermic and small in size which makes them even more susceptible to environmental stresses. The present study assesses the consequence of desiccation on the mating latency and copulations duration in tropical Drosophila melanogaster. We tested flies for these reproductive behavioral parameters at varying body water levels and with whole metabolome analysis in order to gain a further understanding of the physiological response to desiccation. Our results showed that the duration of desiccation is positively correlated with mating latency and mating failure, while having no influence on the copulation duration. The metabolomic analysis revealed three biological pathways highly affected by desiccation: starch and sucrose metabolism, galactose metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results are consistent with carbohydrate metabolism providing an energy source in desiccated flies and also suggests that the phenylalanine biosynthesis pathway plays a role in the reproductive fitness of the flies. Desiccation is a common issue with smaller insects, like Drosophila and other tropical insects, and our findings indicate that this lack of ambient water can immediately and drastically affect the insect reproductive behaviour, which becomes more crucial because of unpredictable and dynamic weather conditions.

Synthesis, biological evaluation and molecular docking of aryl hydrazines and hydrazides for anticancer activity.

Aryl hydrazine and hydrazide analogues were synthesized based on p-tolyl hydrazine, isolated as a breakdown product of a secondary metabolite from the mushroom, Agaricus bisporus, and tested to be highly active molecule than 5-fluorouracil in in vitro anticancer studies. The synthesized analogues were tested for anticancer activity using NCI protocol. Anolgues 12 and 15 emerged as molecules with significant in vitro anticancer activity. Molecular docking study revealed the binding orientations of aryl hydrazines and hydrazides analogues in the active sites of thymidylate synthase.

Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly.

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.

A small-molecule fusion inhibitor of influenza virus is orally active in mice.

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

Exploring QSTR and toxicophore of hERG K+ channel blockers using GFA and HypoGen techniques.

Predictive quantitative structure-toxicity and toxicophore models were developed for a diverse series of hERG K+ channel blockers, acting as anti-arrhythmic agents using QSAR+ module in Cerius2 and HypoGen module in Catalyst software, respectively. The 2D-QSTR analysis has been performed on a dataset of 68 molecules carefully selected from literature for which IC50 values measured on hERG K+ channels expressed in mammalian cells lines using the voltage patch clamp assay technique were reported. Their biological data, expressed as IC50, spanned from 7.0nM to 1.4mM, with 7 orders difference. Several types of descriptors including electrotopological, thermodynamic, ADMET, graph theoretical (topological and information content) were used to derive a quantitative relationship between the channel blockers and its physico-chemical properties. Statistically significant QSTR model was obtained using genetic function approximation methodology, having seven descriptors, with a correlation coefficient (r2) of 0.837, cross-validated correlation coefficient (q2) of 0.776 and predictive correlation coefficient (r2 pred) of 0.701, indicating the robustness of the model. Toxicophore model generated using HypoGen module in Catalyst, on these datasets, showed three important features for hERG K+ channel blockers, (i) hydrophobic group (HP), (ii) ring aromatic group (RA) and (iii) hydrogen bond acceptor lipid group (HBAl). The most predictive hypothesis (Hypo 1), consisting of these three features had a best correlation coefficient of 0.820, a low rms deviation of 1.740, and a high cost difference of 113.50, which represents a true correlation and a good predictivity. The hypothesis, Hypo 1 was validated by a test set consisting of 12 molecules and by a cross-validation of 95% confidence level. Accordingly, our 2D-QSTR and toxicophore model has strong predictivity to identify structurally diverse hERG K+ channel blockers with desired biological activity. These models provide a useful framework for understanding binding, and gave structural insight into the specific protein-ligand interactions responsible for affinity, and how one might modify any given structure to mitigate binding.

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice.

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.

hIAPP forms toxic oligomers in plasma.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in ß-sheet rich oligomers with increased ß-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.

Evaluation of proinflammatory cytokine pathway inhibitors for p38 MAPK inhibitory potential.

The target for the anti-inflammatory natural products like amentoflavone ( 2), which act by interfering with the proinflammatory cytokine pathway (e.g., TNF-alpha, IL-1beta, and NO synthase), is not yet well-defined. Data obtained from docking, electronic, and surface analyses shed some light on steric and electronic complementarity of these molecules to p38 MAPK, thereby suggesting a possible mechanism by which they might reduce the production of proinflammatory cytokines.

Computer-assisted methods in chemical toxicity prediction.

In Silico predictive ADME/Tox screening of compounds is one of the hottest areas in drug discovery. To provide predictions of compound drug-like characteristics early in modern drug-discovery decision making, computational technologies have been widely accepted to develop rapid high throughput in silico ADMET analysis. It is widely perceived that the early screening of chemical entities can significantly reduce the expensive costs associated with late stage failures of drugs due to poor ADME/Tox properties. Drug toxic effects are broadly defined to include toxicity, mutagenicity, carcinogenicity, teratogenicity, neurotoxicity and immunotoxicity. Toxicity prediction techniques and structure-activity relationships relies on the accurate estimation and representation of physico-chemical and toxicological properties. This review highlights some of the freely and commercially available softwares for toxicity predictions. The information content can be utilized as a guide for the scientists involved in the drug discovery arena.

Rational Design of Cyclic Peptide Inhibitors of U2AF Homology Motif (UHM) Domains To Modulate Pre-mRNA Splicing.

U2AF homology motifs (UHMs) are atypical RNA recognition motif domains that mediate critical protein-protein interactions during the regulation of alternative pre-mRNA splicing and other processes. The recognition of UHM domains by UHM ligand motif (ULM) peptide sequences plays important roles during early steps of spliceosome assembly. Splicing factor 45 kDa (SPF45) is an alternative splicing factor implicated in breast and lung cancers, and splicing regulation of apoptosis-linked pre-mRNAs by SPF45 was shown to depend on interactions between its UHM domain and ULM motifs in constitutive splicing factors. We have developed cyclic peptide inhibitors that target UHM domains. By screening a focused library of linear and cyclic peptides and performing structure-activity relationship analysis, we designed cyclic peptides with 4-fold improved binding affinity for the SPF45 UHM domain compared to native ULM ligands and 270-fold selectivity to discriminate UHM domains from alternative and constitutive splicing factors. These inhibitors are useful tools to modulate and dissect mechanisms of alternative splicing regulation.

Conservation and Role of Electrostatics in Thymidylate Synthase.

Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

Advances in targeting nucleocapsid-nucleic acid interactions in HIV-1 therapy.

The continuing challenge of HIV-1 treatment resistance in patients creates a need for the development of new antiretroviral inhibitors. The HIV nucleocapsid (NC) protein is a potential therapeutic target. NC is necessary for viral RNA packaging and in the early stages of viral infection. The high level of NC amino acid conservation among all HIV-1 clades suggests a low tolerance for mutations. Thus, NC mutations that could arise during inhibitor treatment to provide resistance may render the virus less fit. Disruption of NC function provides a unique opportunity to strongly dampen replication at multiple points during the viral life cycle with a single inhibitor. Although NC exhibits desirable features for a potential antiviral target, the structural flexibility, size, and the presence of two zinc fingers makes small molecule targeting of NC a challenging task. In this review, we discuss the recent advances in strategies to develop inhibitors of NC function and present a perspective on potential novel approaches that may help to overcome some of the current challenges in the field.

CH-π "T-shape" interaction with histidine explains binding of aromatic galactosides to Pseudomonas aeruginosa lectin LecA.

The galactose specific lectin LecA mediates biofilm formation in the opportunistic pathogen P. aeruginosa . The interaction between LecA and aromatic ß-galactoside biofilm inhibitors involves an intermolecular CH-π T-shape interaction between C(ε1)-H of residue His50 in LecA and the aromatic ring of the galactoside aglycone. The generality of this interaction was tested in a diverse family of ß-galactosides. LecA binding to aromatic ß-galactosides (KD ∼ 8 µM) was consistently stronger than to aliphatic ß-galactosides (KD ∼ 36 µM). The CH-π interaction was observed in the X-ray crystal structures of six different LecA complexes, with shorter than the van der Waals distances indicating productive binding. Related XH/cation/π-π interactions involving other residues were identified in complexes of aromatic glycosides with a variety of carbohydrate binding proteins such as concanavalin A. Exploiting such interactions might be generally useful in drug design against these targets.

Selective mapping of chemical space for Pseudomonas aeruginosa deacetylase LpxC inhibitory potential.

UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase enzyme of Pseudomonas aeruginosa is an interesting target for development of anti-infective drugs against this gram-negative bacterium. Many segregated studies analyzing the P. aeruginosa UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase and its inhibitors have been reported in the recent past. In the present study, an attempt has been made to integrate this knowledge for the development of an effective multilayer screening approach. Eventually, an extensive chemical space was screened to filter out three potential P. aeruginosa UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase inhibitors.

Evaluation of Pseudomonas aeruginosa deacetylase LpxC inhibitory activity of dual PDE4-TNFalpha inhibitors: a multiscreening approach.

In this study, we have focused on the implication of a multiscreening approach in the evaluation of Pseudomonas aeruginosa deacetylase LpxC inhibitory activity of dual PDE4-TNFalpha inhibitors. A genetic function approximation (GFA) directed quantitative structure-activity relationship (QSAR) model was developed for LpxC inhibition on the basis of reported biological activity (Kline and Andersen, J. Med. Chem. 2002, 45, 3112-3129). Subsequently, reported PDE4-TNFalpha inhibitors (Klienman and Campbell, J. Med. Chem. 1998, 41, 266-270) were screened using the QSAR model. Whereby, the compounds were predicted to have equipotent activity with the most potent compound in reported LpxC inhibitor series. A docking analysis of these compounds carried out on the LpxC homology model corroborated the initial results. The compounds were then validated using surface electronic properties analysis and subjected to an adsorption, distribution, metabolism, excretion, and toxicity filter. Taken together, a multiscreening strategy was used to validate potential leads for LpxC inhibition.