Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics.

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.

Webina: an open-source library and web app that runs AutoDock Vina entirely in the web browser.

MOTIVATION: Molecular docking is a computational technique for predicting how a small molecule might bind a macromolecular target. Among docking programs, AutoDock Vina is particularly popular. Like many docking programs, Vina requires users to download/install an executable file and to run that file from a command-line interface. Choosing proper configuration parameters and analyzing Vina output is also sometimes challenging. These issues are particularly problematic for students and novice researchers. RESULTS: We created Webina, a new version of Vina, to address these challenges. Webina runs Vina entirely in a web browser, so users need only visit a Webina-enabled webpage. The docking calculations take place on the user's own computer rather than a remote server. AVAILABILITY AND IMPLEMENTATION: A working version of the open-source Webina app can be accessed free of charge from http://durrantlab.com/webina. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

New Stretching Method for Aligning Gels: Its Application to the Measurement Residual Chemical Shift Anisotropies (RCSAs) without the Need for Isotropic Shift Correction.

An existing gel stretching device is modified, permitting the use of organic solvents for the study of small molecules. Different from the original device, gels are stretched into 4 mm open-ended NMR tubes and then inserted into regular 5 mm NMR tubes. No open-ended tubes are inserted in the NMR probe avoiding the risk of sample leaking. It is also shown that residual chemical shift anisotropies (RCSAs) measured with the device are free of isotropic shift interferences and corrections for them are not needed during the post-acquisition data analysis. Three internal references for chemical shift were evaluated (CCl4 , CBr4 and TMS), being CCl4 the most convenient one to measure RCSAs in CDCl3 . RCSAs measured with the modified stretching device using CCl4 as the internal chemical shift reference were enough to determine the relative configuration of three small molecules with an excellent degree of configuration discrimination.

Measurement of residual chemical shift anisotropies in compressed polymethylmethacrylate gels. Automatic compensation of gel isotropic shift contribution.

Mechanical compression of polymer gels provides a simple way for the measurement of residual chemical shift anisotropies, which then can be employed, on its own, or in combination with residual dipolar couplings, for structural elucidation purposes. Residual chemical shift anisotropies measured using compression devices needed a posteriori correction to account for the increase of the polymer to solvent ratio inside the swollen gel. This correction has been cast before in terms of a single-free parameter which, as shown here, can be simultaneously optimized along with the components of the alignment tensor while still retaining discriminating power of the different relative configurations as illustrated in the stereochemical analysis of α-santonin and 10-epi-8-deoxycumambrin B.

Application of Residual Dipolar Couplings and Selective Quantitative NOE to Establish the Structures of Tetranortriterpenoids from Xylocarpus rumphii.

Nine triterpenoid derivatives were isolated from the heartwood of Xylocarpus rumphii and were identified as xylorumphiins E (1), C (2), L (3), and M-R (4-9). Compounds 4-9 have a hemiacetal group in the triterpenoid side chain, making them impossible to purify. Purification was achieved after acetylation and subsequent separation of the epimeric mixtures of acetates; however differentiaition of the R and S epimers was not possible using standard NMR techniques. In one case, the relative configuration of a remotely located stereocenter with respect to the stereocenters in the main skeleton was unambiguously determined using residual dipolar couplings. Dipolar couplings were collected from the sample oriented in compressed poly(methyl methacrylate) gels swollen in CDCl3. In another case, the relative configuration was determined using 1D selective quantitative NOE experiments. Xylorumphiin K (10), xyloccensin E, taraxer-14-en-3ß-ol, (22S)-hydroxytirucalla-7,24-diene-3,23-dione, and 25-hydroxy-(20S,24S)-epoxydammaran-3-one were isolated from the bark of the same plant. Compounds 3-10 are new compounds. Compounds 1-6 and xyloccensin E were tested at one concentration, 1 × 10-5 M, in the NCI59 cell one-dose screen but did not show significant activity.

Di(ethylene glycol) methyl ether methacrylate (DEGMEMA)-derived gels align small organic molecules in methanol.

Residual dipolar couplings (RDCs) constitute an important NMR parameter for structural elucidation in all areas of chemistry. In this study, di(ethylene glycol) methyl ether methacrylate (DEGMEMA)-based gels are introduced as alignment media for the measurement of RDCs of small organic molecules in polar solvents such as methanol. The low viscosity of methanol permits the execution of J-scaled BIRD HSQC experiments that yield very sharp lines in anisotropic conditions. The gels have excellent mechanical properties, and their compression and expansion in the swollen state can be reversed and performed multiple times. This process enables the easy loading and release of analytes. The excellent performance of these new aligning gels is demonstrated by analyzing the structure of the alkaloid retrorsine. Copyright © 2016 John Wiley & Sons, Ltd.

Mechanical Behavior of Polymer Gels for RDCs and RCSAs Collection: NMR Imaging Study of Buckling Phenomena.

Anisotropic NMR parameters, such as residual dipolar couplings (RDCs), residual chemical shift anisotropies (RCSAs) and residual quadrupolar couplings (RQCs or ΔνQ ), appear in solution-state NMR when the molecules under study are subjected to a degree of order. The tunable alignment by reversible compression/relaxation of gels (PMMA and p-HEMA) is an easy, user-friendly, and very affordable method to measure them. When using this method, a fraction of isotropic NMR signals is observed in the NMR spectra, even at a maximum degree of compression. To explain the origin of these isotropic signals we decided to investigate their physical location inside the NMR tube using deuterium 1D imaging and MRI micro-imaging experiments. It was observed that after a certain degree of compression the gels start to buckle and they generate pockets of isotropic solvent, which are never eliminated. The amount of buckling depends on the amount of cross-linker and the length of the gel.

One-Shot Determination of Residual Dipolar Couplings: Application to the Structural Discrimination of Small Molecules Containing Multiple Stereocenters.

A novel approach for the fast and efficient structural discrimination of molecules containing multiple stereochemical centers is described. A robust J-resolved HSQC experiment affording highly resolved 1JCH/1TCH splittings along the indirect dimension and homodecoupled 1H signals in the detected dimension is proposed. The experiment enables in situ distinction of both isotropic and anisotropic components of molecules dissolved in compressed PMMA gels, allowing a rapid and direct one-shot determination of accurate residual dipolar coupling constants from a single NMR spectrum.

Worth the Weight: Sub-Pocket EXplorer (SubPEx), a Weighted Ensemble Method to Enhance Binding-Pocket Conformational Sampling.

Structure-based virtual screening (VS) is an effective method for identifying potential small-molecule ligands, but traditional VS approaches consider only a single binding-pocket conformation. Consequently, they struggle to identify ligands that bind to alternate conformations. Ensemble docking helps address this issue by incorporating multiple conformations into the docking process, but it depends on methods that can thoroughly explore pocket flexibility. We here introduce Sub-Pocket EXplorer (SubPEx), an approach that uses weighted ensemble (WE) path sampling to accelerate binding-pocket sampling. As proof of principle, we apply SubPEx to three proteins relevant to drug discovery: heat shock protein 90, influenza neuraminidase, and yeast hexokinase 2. SubPEx is available free of charge without registration under the terms of the open-source MIT license: http://durrantlab.com/subpex/.

Worth the weight: Sub-Pocket EXplorer (SubPEx), a weighted-ensemble method to enhance binding-pocket conformational sampling.

Structure-based virtual screening (VS) is an effective method for identifying potential small-molecule ligands, but traditional VS approaches consider only a single binding-pocket conformation. Consequently, they struggle to identify ligands that bind to alternate conformations. Ensemble docking helps address this issue by incorporating multiple conformations into the docking process, but it depends on methods that can thoroughly explore pocket flexibility. We here introduce Sub-Pocket EXplorer (SubPEx), an approach that uses weighted ensemble (WE) path sampling to accelerate binding-pocket sampling. As proof of principle, we apply SubPEx to three proteins relevant to drug discovery: heat shock protein 90, influenza neuraminidase, and yeast hexokinase 2. SubPEx is available free of charge without registration under the terms of the open-source MIT license: http://durrantlab.com/subpex/.

Allosteric inhibition of TEM-1 ß lactamase: Microsecond molecular dynamics simulations provide mechanistic insights.

ß-lactam antibiotics target DD-transpeptidases, enzymes that perform the last step of bacterial cell-wall synthesis. To block the antimicrobial activity of these antibiotics, bacteria have evolved lactamases that render them inert. Among these, TEM-1, a class A lactamase, has been extensively studied. In 2004, Horn et al. described a novel allosteric TEM-1 inhibitor, FTA, that binds distant from the TEM-1 orthosteric (penicillin-binding) pocket. TEM-1 has subsequently become a model for the study of allostery. In the present work, we perform molecular dynamics simulations of FTA-bound and FTA-absent TEM-1, totaling ~3 µS, that provide new insight into TEM-1 inhibition. In one of the simulations, bound FTA assumed a conformation different than that observed crystallographically. We provide evidence that the alternate pose is physiologically plausible and describe how it impacts our understanding of TEM-1 allostery.

Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn2C6 transcription factors YRR1 and YRM1 (p < 1 × 10-100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.