BcL-xL Conformational Changes Upon Fragment Binding Revealed by NMR.

BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR.

PLoS One. 2013; 8(5): e64400
Aguirre C, Ten Brink T, Walker O, Guillière F, Davesne D, Krimm I

Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices [Formula: see text]2, [Formula: see text]3 and the very beginning of [Formula: see text]5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach. HubMed – drug


Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer.

PLoS One. 2013; 8(5): e64338
Yadav S, Sehrawat A, Eroglu Z, Somlo G, Hickey R, Yadav S, Liu X, Awasthi YC, Awasthi S

Triple-negative breast cancer (TNBC) is one of the hardest subtypes of breast cancer to treat due to the heterogeneity of the disease and absence of well-defined molecular targets. Emerging evidence has shown the role of cohesin in the formation and progression of various cancers including colon and lung cancer but the role of cohesin in breast cancer remains elusive. Our data showed that structural maintenance of chromosome 1 (SMC1), a subunit of the cohesin protein complex, is differentially overexpressed both at RNA and protein level in a panel of TNBC cell lines as compared to normal epithelial or luminal breast cancer cells, suggesting that the amplified product of this normal gene may play role in tumorigenesis in TNBC. In addition, our results show that induced overexpression of SMC1 through transient transfection enhanced cell migration and anchorage independent growth while its suppression with targeted small interfering RNA (siRNA) reduced the migration ability of TNBC cells. Increased expression of SMC1 also lead to increase in the mesenchymal marker vimentin and decrease in the normal epithelial marker, E-cadherin. Immunocytochemical studies along with flow cytometry and cell fractionation showed the localization of SMC1 in the nucleus, cytoplasm and also in the plasma membrane. The knockdown of SMC1 by siRNA sensitized the TNBC cells towards a PARP inhibitor (ABT-888) and IC50 was approximately three fold less than ABT-888 alone. The cytotoxic effect of combination of SMC1 suppression and ABT-888 was also confirmed by the colony propagation assay. Taken together, these studies report for the first time that SMC1 is overexpressed in TNBC cells where it plays a role in cell migration and drug sensitivity, and thus provides a potential therapeutic target for this highly invasive breast cancer subtype. HubMed – drug


NeuroChip: A Microfluidic Electrophysiological Device for Genetic and Chemical Biology Screening of Caenorhabditis elegans Adult and Larvae.

PLoS One. 2013; 8(5): e64297
Hu C, Dillon J, Kearn J, Murray C, O’Connor V, Holden-Dye L, Morgan H

Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. Here we make a significant addition to this repertoire through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. We demonstrate this device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. We also combine NeuroChip with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and has applications in neurogenetics, drug discovery and neurotoxicology. HubMed – drug


Description of HIV-1 Group M Molecular Epidemiology and Drug Resistance Prevalence in Equatorial Guinea from Migrants in Spain.

PLoS One. 2013; 8(5): e64293
Yebra G, de Mulder M, Holguín A

The HIV epidemic is increasing in Equatorial Guinea (GQ), West Central Africa, but few studies have reported its HIV molecular epidemiology. We aimed to describe the HIV-1 group M (HIV-1M) variants and drug-resistance mutations in GQ using sequences sampled in this country and in Spain, a frequent destination of Equatoguinean migrants.We collected 195 HIV-1M pol sequences from Equatoguinean subjects attending Spanish clinics during 1997-2011, and 83 additional sequences sampled in GQ in 1997 and 2008 from GenBank. All (n?=?278) were re-classified using phylogeny and tested for drug-resistance mutations. To evaluate the origin of CRF02_AG in GQ, we analyzed 2,562 CRF02_AG sequences and applied Bayesian MCMC inference (BEAST program).Most Equatoguinean patients recruited in Spain were women (61.1%) or heterosexuals (87.7%). In the 278 sequences, the variants found were CRF02_AG (47.8%), A (13.7%), B (7.2%), C (5.8%), G (5.4%) and others (20.1%). We found 6 CRF02_AG clusters emerged from 1983.9 to 2002.5 with origin in GQ (5.5 sequences/cluster). Transmitted drug-resistance (TDR) rate among naïve patients attended in Spain (n?=?144) was 4.7%: 3.4% for PI (all with M46IL), 1.8% for NRTI (all with M184V) and 0.9% for NNRTI (Y188L). Among pre-treated patients, 9/31 (29%) presented any resistance, mainly affecting NNRTI (27.8%).We report a low (<5%) TDR rate among naïve, with PI as the most affected class. Pre-treated patients also showed a low drug-resistance prevalence (29%) maybe related to the insufficient treatment coverage in GQ. CRF02_AG was the prevalent HIV-1M variant and entered GQ through independent introductions at least since the early 1980s. HubMed – drug