Binding Sites of Bicarbonate in Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is used in plant metabolism for fruit maturation or seed development as well as in the C4 and crassulacean acid metabolism (CAM) mechanisms in photosynthesis, where it is used for the capture of hydrated CO2 (bicarbonate). To find the yet unknown binding site of bicarbonate in this enzyme, we have first identified putative binding sites with nonequilibrium molecular dynamics simulations and then ranked these sites with alchemical free energy calculations with corrections of computational artifacts. Fourteen pockets where bicarbonate could bind were identified, with three having realistic binding free energies with differences with the experimental value below 1 kcal/mol. One of these pockets is found far from the active site at 14 Å and predicted to be an allosteric binding site. In the two other binding sites, bicarbonate is in direct interaction with the magnesium ion; neither sequence alignment nor the study of mutant K606N allowed to discriminate between these two pockets, and both are good candidates as the binding site of bicarbonate in phosphoenolpyruvate carboxylase.

Structural Optimization of Azacryptands for Targeting Three-Way DNA Junctions.

Transient melting of the duplex-DNA (B-DNA) during DNA transactions allows repeated sequences to fold into non-B DNA structures, including DNA junctions and G-quadruplexes. These noncanonical structures can act as impediments to DNA polymerase progression along the duplex, thereby triggering DNA damage and ultimately jeopardizing genomic stability. Their stabilization by ad hoc ligands is currently being explored as a putative anticancer strategy since it might represent an efficient way to inflict toxic DNA damage specifically to rapidly dividing cancer cells. The relevance of this strategy is only emerging for three-way DNA junctions (TWJs) and, to date, no molecule has been recognized as a reference TWJ ligand, featuring both high affinity and selectivity. Herein, we characterize such reference ligands through a combination of in vitro techniques comprising affinity and selectivity assays (competitive FRET-melting and TWJ Screen assays), functional tests (qPCR and Taq stop assays), and structural analyses (molecular dynamics and NMR investigations). We identify novel azacryptands TrisNP-amphi and TrisNP-ana as the most promising ligands, interacting with TWJs with high affinity and selectivity. These ligands represent new molecular tools to investigate the cellular roles of TWJs and explore how they can be exploited in innovative anticancer therapies.

Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells.

DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.

Effect of sampling on BACE-1 ligands binding free energy predictions via MM-PBSA calculations.

The BACE-1 enzyme is a prime target to find a cure to Alzheimer's disease. In this article, we used the MM-PBSA approach to compute the binding free energies of 46 reported ligands to this enzyme. After showing that the most probable protonation state of the catalytic dyad is mono-protonated (on ASP32), we performed a thorough analysis of the parameters influencing the sampling of the conformational space (in total, more than 35 µs of simulations were performed). We show that ten simulations of 2 ns gives better results than one of 50 ns. We also investigated the influence of the protein force field, the water model, the periodic boundary conditions artifacts (box size), as well as the ionic strength. Amber03 with TIP3P, a minimal distance of 1.0 nm between the protein and the box edges and a ionic strength of I = 0.2 M provides the optimal correlation with experiments. Overall, when using these parameters, a Pearson correlation coefficient of R = 0.84 (R2 = 0.71) is obtained for the 46 ligands, spanning eight orders of magnitude of Kd (from 0.017 nm to 2000 µM, i.e., from -14.7 to -3.7 kcal/mol), with a ligand size from 22 to 136 atoms (from 138 to 937 g/mol). After a two-parameter fit of the binding affinities for 12 of the ligands, an error of RMSD = 1.7 kcal/mol was obtained for the remaining ligands. © 2017 Wiley Periodicals, Inc.

A Hybrid Knowledge-Based and Empirical Scoring Function for Protein-Ligand Interaction: SMoG2016.

We present the third generation of our scoring function for the prediction of protein-ligand binding free energy. This function is now a hybrid between a knowledge-based potential and an empirical function. We constructed a diversified set of ∼1000 complexes from the PDBBinding-CN database for the training of the function, and we show that this number of complexes generates enough data to build the potential. The occurrence of 420 different types of atomic pairwise interactions is computed in up to five different ranges of distances to derive the knowledge-based part. All of the parameters were optimized, and we were able to considerably improve the accuracy of the scoring function with a Pearson correlation coefficient against experimental binding free energies of up to 0.57, which ranks our new scoring function as one of the best currently available and the second-best in terms of standard deviation (SD = 1.68 kcal/mol). The function was then further improved by inclusion of different terms taking into account repulsion and loss of entropy upon binding, and we show that it is capable of recovering native binding poses up to 80% of the time. All of the programs, tools, and protein sets are released in the Supporting Information or as open-source programs.

Repurposing of rutin for the inhibition of norovirus replication.

Drug repurposing is a strategy employed to circumvent some of the bottlenecks involved in drug development, such as the cost and time needed for developing new molecular entities. Noroviruses cause recurrent epidemics and sporadic outbreaks of gastroenteritis associated with significant mortality and economic costs, but no treatment has been approved to date. Herein, a library of molecules previously used in humans was screened to find compounds with anti-noroviral activity. Antiviral testing for four selected compounds against murine norovirus infection revealed that rutin has anti-murine norovirus activity in cell-based assays.

Predicting new Ugi-smiles couplings: a combined experimental and theoretical study.

Following our previous mechanistic studies of multicomponent Ugi-type reactions, theoretical calculations have been performed to predict the efficiency of new substrates in Ugi-Smiles couplings. First, as predicted, 2,4,6-trichlorophenol experimentally gave the corresponding aryl-imidate. Theoretical predictions of nitrosophenols as good acidic partners were then successfully confirmed by experiments. In the latter case, the reaction offers a new access to benzimidazoles.

Are changes in antibiotic prophylaxis recommendations responsible for an increased risk of cefazolin allergy?

BACKGROUND: The first line of prevention of surgical site infection relies on the timely administration of antibiotic prophylaxis. First- and second-generation cephalosporins are the most recommended antibiotics in elective surgery. The incidence of cefazolin allergy has increased worldwide over the years. The sensitization mechanism of cefazolin is currently unknown, and data supporting cross-reactivity between penicillins and cephalosporins are lacking. Sensitization could occur through previous exposure either to cefazolin or to structurally related chemical agents. The objective of this study was to evaluate sensitization agents towards cefazolin. METHODS: The OpenBabel chemoinformatics toolbox was used to search for similarities between cefazolin and other molecules in an extensive drug database. Using the pholcodine-rocuronium similarity score as a threshold, we selected drugs with the most similar structure to that of cefazolin. Exposure to those drugs and cefazolin was assessed in a cohort of patients with skin test-proven cefazolin allergy at a specialized allergy centre via a self-administered anonymous questionnaire. RESULTS: Using the pholcodine-rocuronium similarity score as a threshold (score≥0.7), 42 molecules were found to be similar to cefazolin (all cephalosporins). Only 8 were marketed in France. None of the 14 cefazolin-allergic patients who answered the questionnaire (65% female, median age 56 years) reported exposure to any identified antibiotics. In contrast, 11 (78%) had at least one previous surgery requiring cefazolin before the index case. CONCLUSION: Direct previous cefazolin exposure was identified in 78% of cefazolin-allergic patients. Cefazolin started to take a central place in antibiotic prophylaxis after 2010, when cefamandole usage decreased drastically. Changes in antibiotic prophylaxis over the past 14 years in France could have been the turning point for the increased incidence of cefazolin allergy.

Substituent effects in Ugi-smiles reactions.

In a recent communication, we described the mechanism of the well-known Ugi-type reactions with a model system (J. Org. Chem. 2012, 77, 1361-1366). Herein, focusing on the Ugi-Smiles coupling, we study the effects of each of the four reactants on the energy profile to further explain the experimental results. The variations observed with different carbonyl compounds rely on their influence on the formation of the aryl-imidate, whereas the variations on the amine preferentially affect the Smiles rearrangement. The effect of substituents on the phenol derivative is seen upon both aryl-imidate formation and the rearrangement. The effect of the isocyanide substituents is less pronounced.

Challenging 50 years of established views on Ugi reaction: a theoretical approach.

The Ugi reaction is one of the most famous multicomponent couplings, and its efficiency is still explained by the original mechanism suggested by Ugi in the 60s. This article aims to present a thorough theoretical study of this reaction. It describes how the imine is activated and how the new stereogenic center is formed. Our calculations strongly suggest alternatives to some commonly accepted features, such as the reversibility of the intermediate steps, and temper the nature of the driving force of the reaction.

A qualitative failure of B3LYP for textbook organic reactions.

Depending on the selected DFT functional, two different mechanisms are found for two organic reactions (an intramolecular nucleophilic aromatic substitution and a nucleophilic addition on a carbonyl moiety). Indeed, B3LYP predicts a concerted mechanism whereas M06-2X foresees a multistep one. Calculations at the MP4(SDQ) level proved the mechanisms to be stepwise. We studied these reactions with a large panel of exchange-correlation functionals and demonstrated that the amount of exact exchange is of first importance. For some borderline cases, the form of the functional has also an impact, e.g. the Meisenheimer σ-adduct of the intramolecular nucleophilic aromatic substitution can be located with B3PW91 but not with B3LYP. These results stress the need to use recently proposed functionals to investigate chemical reactivity.

Click-Chemistry-Based Biomimetic Ligands Efficiently Capture G-Quadruplexes In Vitro and Help Localize Them at DNA Damage Sites in Human Cells.

Interrogating G-quadruplex (G4) biology at its deepest roots in human cells relies on the design, synthesis, and use of ever smarter molecular tools. Here, we demonstrate the versatility of biomimetic G4 ligands referred to as TASQ (template assembled synthetic G-quartet) in which a biotin handle was incorporated for G4-focused chemical biology investigations. We have rethought the biotinylated TASQ design to make it readily chemically accessible via an efficient click-chemistry-based strategy. The resulting biotinylated, triazole-assembled TASQ, or BioTriazoTASQ, was thus shown to efficiently isolate both DNA and RNA G4s from solution by affinity purification protocols, for identification purposes. Its versatility was then further demonstrated by optical imaging that provided unique mechanistic insights into the actual strategic relevance of G4-targeting strategies, showing that ligand-stabilized G4 sites colocalize with and, thus, are responsible for DNA damage foci in human cells.

Evidences for the key role of hydrogen bonds in nucleophilic aromatic substitution reactions.

The effect of hydrogen bonds on the fate of nucleophilic aromatic substitutions (S(N)Ar) has been studied in silico using a density functional theory approach in the condensed phase. The importance of these hydrogen bonds can explain the "built-in solvation" model of Bunnett concerning intermolecular processes between halogenonitrobenzenes and amines. It is also demonstrated that it can explain experimental results for a multicomponent reaction (the Ugi-Smiles coupling), involving an intramolecular S(N)Ar (the Smiles rearrangement) as the key step of the process. Modeling reveals that when an intramolecular hydrogen bond is present, it lowers the activation barrier of this step and enables the multicomponent reaction to proceed.

A density functional theory study of the Nef-isocyanide reaction: mechanism, influence of parameters and scope.

The Nef reaction between isocyanides and acyl chlorides is studied at the M06-2X/6-311+G(d,p) level of theory in toluene. After proving that the reaction follows a concerted mechanism instead of an addition-elimination path, we study the influences of the solvent, the isocyanide, the acyl moiety and the leaving group on the energy profile of the reaction. The calculated data can be rationalized with the pK(a) of the leaving group, or more generally with the population of the oxygen lone pairs of the acyl moiety.

Protein Preferential Solvation in Water:Glycerol Mixtures.

For proteins in solvent mixtures, the relative abundances of each solvent in their solvation shell have a critical impact on their properties. Preferential solvation of a series of proteins in water-glycerol mixtures is studied here over a broad range of solvent compositions via classical molecular dynamics simulations. Our simulation results reveal that the differences between shell and bulk compositions exhibit dramatic changes with solvent composition, temperature, and protein nature. In contrast with the simple and widely used picture where glycerol is completely excluded from the protein interface, we show that for aqueous solutions with less than 50% glycerol in volume, protein solvation shells have approximately the same composition as the bulk solvent and proteins are in direct contact with glycerol. We further demonstrate that at high glycerol concentration, glycerol depletion from the solvation shell is due to an entropic factor arising from the reduced accessibility of bulky glycerol molecules in protein cavities. The resulting molecular picture is important to understand protein activity and cryopreservation in mixed aqueous solvents.

Coupled Valence-Bond State Molecular Dynamics Description of an Enzyme-Catalyzed Reaction in a Non-Aqueous Organic Solvent.

Enzymes are widely used in nonaqueous solvents to catalyze non-natural reactions. While experimental measurements showed that the solvent nature has a strong effect on the reaction kinetics, the molecular details of the catalytic mechanism in nonaqueous solvents have remained largely elusive. Here we study the transesterification reaction catalyzed by the paradigm subtilisin Carlsberg serine protease in an organic apolar solvent. The rate-limiting acylation step involves a proton transfer between active-site residues and the nucleophilic attack of the substrate to form a tetrahedral intermediate. We design the first coupled valence-bond state model that simultaneously describes both reactions in the enzymatic active site. We develop a new systematic procedure to parametrize this model on high-level ab initio QM/MM free energy calculations that account for the molecular details of the active site and for both substrate and protein conformational fluctuations. Our calculations show that the reaction energy barrier changes dramatically with the solvent and protein conformational fluctuations. We find that the mechanism of the tetrahedral intermediate formation during the acylation step is similar to that determined under aqueous conditions, and that the proton transfer and nucleophilic attack reactions occur concertedly. We identify the reaction coordinate to be mostly due to the rearrangement of some residual water molecules close to the active site.

OpenGrowth: An Automated and Rational Algorithm for Finding New Protein Ligands.

We present a new open-source software, called OpenGrowth, which aims to create de novo ligands by connecting small organic fragments in the active site of proteins. Molecule growth is biased to produce structures that statistically resemble drugs in an input training database. Consequently, the produced molecules have superior synthetic accessibility and pharmacokinetic properties compared with randomly grown molecules. The growth process can take into account the flexibility of the target protein and can be started from a seed to mimic R-group strategy or fragment-based drug discovery. Primary applications of the software on the HIV-1 protease allowed us to quickly identify new inhibitors with a predicted Kd as low as 18 nM. We also present a graphical user interface that allows a user to select easily the fragments to include in the growth process. OpenGrowth is released under the GNU GPL license and is available free of charge on the authors' website and at http://opengrowth.sourceforge.net/ .

Evolutionary dynamics of viral escape under antibodies stress: A biophysical model.

Viruses constantly face the selection pressure of antibodies, either from innate immune response of the host or from administered antibodies for treatment. We explore the interplay between the biophysical properties of viral proteins and the population and demographic variables in the viral escape. The demographic and population genetics aspect of the viral escape have been explored before; however one important assumption was the a priori distribution of fitness effects (DFE). Here, we relax this assumption by instead considering a realistic biophysics-based genotype-phenotype relationship for RNA viruses escaping antibodies stress. In this model the DFE is itself an evolvable property that depends on the genetic background (epistasis) and the distribution of biophysical effects of mutations, which is informed by biochemical experiments and theoretical calculations in protein engineering. We quantitatively explore in silico the viability of viral populations under antibodies pressure and derive the phase diagram that defines the fate of the virus population (extinction or escape from stress) in a range of viral mutation rates and antibodies concentrations. We find that viruses are most resistant to stress at an optimal mutation rate (OMR) determined by the competition between supply of beneficial mutation to facilitate escape from stressors and lethal mutagenesis caused by excess of destabilizing mutations. We then show the quantitative dependence of the OMR on genome length and viral burst size. We also recapitulate the experimental observation that viruses with longer genomes have smaller mutation rate per nucleotide.