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Human African trypanosomiasis is among the World Health Organization's designated neglected tropical diseases. Repurposing strategies are often employed in academic drug discovery programs due to financial limitations, and in this instance, we used human kinase inhibitor chemotypes to identify substituted 4-aminoazaindoles, exemplified by 1 . Structure-activity and structure-property relationship analysis, informed by cheminformatics, identified 4s as a potent inhibitor of Trypanosoma brucei growth. While 4s appeared to be fast acting and cidal in the in vitro assays, it failed to cure a murine model of infection. Preliminary efforts to identify the potential mechanism of action of the series pointed to arginine kinase, though, as we demonstrate, this does not appear to be the sole target of our compounds. This comprehensive approach to drug discovery, encompassing cheminformatics, structure-potency and structure-property analysis, and pharmacophore identification, highlights our multipronged efforts to identify novel lead compounds for this deadly disease.

Most cell wall and secreted β-glucans are synthesised by the CAZy Glycosyltransferase 2 family (www.cazy.org), with different members catalysing the formation of (1,4)-β-, (1,3)-β-, or both (1,4)- and (1,3)-β-glucosidic linkages. Given the distinct physicochemical properties of each of the resultant β-glucans (cellulose, curdlan, and mixed linkage glucan, respectively) are crucial to their biological and biotechnological functions, there is a desire to understand the molecular evolution of synthesis and how linkage specificity is determined. With structural studies hamstrung by the instability of these proteins to solubilisation, we have utilised in silico techniques and the crystal structure for a bacterial cellulose synthase to further understand how these enzymes have evolved distinct functions. Sequence and phylogenetic analyses were performed to determine amino acid conservation, both family-wide and within each sub-family. Further structural analysis centred on comparison of a bacterial curdlan synthase homology model with the bacterial cellulose synthase crystal structure, with molecular dynamics simulations performed with their respective β-glucan products bound in the trans-membrane channel. Key residues that differentially interact with the different β-glucan chains and have sub-family-specific conservation were found to reside at the entrance of the trans-membrane channel. The linkage-specific catalytic activity of these enzymes and hence the type of β-glucan chain built is thus likely determined by the different interactions between the proteins and the first few glucose residues in the channel, which in turn dictates the position of the acceptor glucose. The sequence-function relationships for the bacterial β-glucan synthases pave the way for extending this understanding to other kingdoms, such as plants., Competing Interests: The authors have declared that no competing interests exist.

There is increasing experimental and computational evidence that cellulose microfibrils can exist in a stable twisted form. In this study, atomistic molecular dynamics (MD) simulations are performed to investigate the importance of intrachain hydrogen bonds on the twist in cellulose microfibrils. We systematically enforce or block the formation of these intrachain hydrogen bonds by either constraining dihedral angles or manipulating charges. For the majority of simulations a consistent right handed twist is observed. The exceptions are two sets of simulations that block the O2-O6' intrachain hydrogen bond, where no consistent twist is observed in multiple independent simulations suggesting that the O2-O6' hydrogen bond can drive twist. However, in a further simulation where exocyclic group rotation is also blocked, right-handed twist still develops suggesting that intrachain hydrogen bonds are not necessary to drive twist in cellulose microfibrils., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The question of how many chains an elementary cellulose microfibril contains is critical to understanding the molecular mechanism(s) of cellulose biosynthesis and regulation. Given the hexagonal nature of the cellulose synthase rosette, it is assumed that the number of chains must be a multiple of six. We present molecular dynamics simulations on three different models of Iβ cellulose microfibrils, 18, 24, and 36 chains, to investigate their structure and dynamics in a hydrated environment. The 36-chain model stays in a conformational space that is very similar to the initial crystalline phase, while the 18- and 24-chain models sample a conformational space different from the crystalline structure yet similar to conformations observed in recent high-temperature molecular dynamics simulations. Major differences in the conformations sampled between the different models result from changes to the tilt of chains in different layers, specifically a second stage of tilt, increased rotation about the O2-C2 dihedral, and a greater sampling of non-TG exocyclic conformations, particularly the GG conformation in center layers and GT conformation in solvent-exposed exocyclic groups. With a reinterpretation of nuclear magnetic resonance data, specifically for contributions made to the C6 peak, data from the simulations suggest that the 18- and 24-chain structures are more viable models for an elementary cellulose microfibril, which also correlates with recent scattering and diffraction experimental data. These data inform biochemical and molecular studies that must explain how a six-particle cellulose synthase complex rosette synthesizes microfibrils likely comprised of either 18 or 24 chains., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

X-ray crystallography studies have identified that most cyclic inhibitors of HIV protease (including cyclic ureas) bind in a symmetric manner, however some cyclic inhibitors, such as cyclic sulfamides, bind in a non-symmetric manner. This raises the question as to whether it is possible for cyclic sulfamides to bind symmetrically and conversely for cyclic ureas to bind non-symmetrically. Herein we report an analysis of the conformational preference of cyclic ureas and sulfamides both free in solution and bound to HIV protease, including an investigation of the effect of branching. Quantum chemical calculations (B3LYP, M06-2X, MP2, CCSD(T)) predict the cyclic urea to prefer a symmetric conformation in solution, with a large activation barrier towards inter-conversion to the non-symmetric conformation. This differs from the cyclic sulfamides, which marginally prefer a non-symmetric conformation with a much smaller barrier to inter-conversion making it more likely for a non-preferred conformation to be observed. It is predicted that the cyclic scaffold itself favours a symmetric form, while branching induces a preference for a non-symmetric form. MD simulations on the free inhibitors identified inter-conversion with the cyclic sulfamides but not the cyclic ureas, in support of the quantum chemical results. MM-PB(GB)SA calculations on the cyclic inhibitors bound to HIV protease corroborate the X-ray crystallography studies, identifying the cyclic ureas to bind symmetrically and the cyclic sulfamides in a non-symmetrical manner. While the non-preferred form of the sulfamide may well be present as a free molecule in solution, our results suggest that it is unlikely to bind to HIV protease in a symmetric manner.

The molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) and MM-generalized-Born surface area (MM-GBSA) approaches are commonly used in molecular modeling and drug design. Four critical aspects of these approaches have been investigated for their effect on calculated binding energies: (1) the atomic partial charge method used to parameterize the ligand force field, (2) the method used to calculate the solvation free energy, (3) inclusion of entropy estimates, and (4) the protonation state of the ligand. HIV protease has been used as a test case with six structurally different inhibitors covering a broad range of binding strength to assess the effect of these four parameters. Atomic charge methods are demonstrated to effect both the molecular dynamics (MD) simulation and MM-PB(GB)SA binding energy calculation, with a greater effect on the MD simulation. Coefficients of determination and Spearman rank coefficients were used to quantify the performance of the MM-PB(GB)SA methods relative to the experimental data. In general, better performance was achieved using (i) atomic charge models that produced smaller mean absolute atomic charges (Gasteiger, HF/STO-3G and B3LYP/cc-pVTZ), (ii) the MM-GBSA approach over MM-PBSA, while (iii) inclusion of entropy had a slightly positive effect on correlations with experiment. Accurate representation of the ligand protonation state was found to be important. It is demonstrated that these approaches can distinguish ligands according to binding strength, underlining the usefulness of these approaches in computer-aided drug design., (Copyright © 2012 Wiley Periodicals, Inc.)

Resistance remains a major issue with regards to HIV-1 protease, despite the availability of numerous HIV-1 protease inhibitors and copious amounts of structural and binding data. In an effort to improve our understanding of how HIV-1 protease is able to "outsmart" new drugs, we have investigated the flexibility of HIV-1 protease and in particular how it adapts to different structural stresses. Our analysis has highlighted the effects of space group on the variability between structures of HIV-1 protease and suggests that consideration of multiple structures and appropriate consideration of different conformations of the Ile50 residue is necessary in any structural analysis. Calculation of the root-mean-square deviation on a per-residue basis has been used to identify 'natural variation', while mutational and ligand analyses have been carried out to identify the effect on structure as a result of specific stresses. It was observed that mutations readily cause changes to occur at sites both close to and distant from a mutation site, with changes more likely to occur at residues that are sites of other major mutations. It is also revealed that HIV-1 protease adaption is dependent on the type and the structure of any bound ligand. Identification of the specific changes that occur due to these stresses will aid in the understanding of resistance and also aid in the design of new drugs.

A novel docking protocol using a long, all atom molecular dynamics (MD) simulation, in an explicit solvent medium, without using any distance constraints is presented. This MD docking protocol is able to dock ligands, based on the C-terminal domain (CTD) of RNA polymerase II, into the tryptophan-tryptophan (WW) domain of Pin1. In this docking process, a significant loop-bending event occurs in order to encircle the ligand into its solvent exposed binding site, which cannot be simulated using current protocols. The simulations were validated structurally and energetically against an X-ray structure to confirm correct sampling of conformational space. Based on these simulations, and justification of the starting structure as a valid intermediate structure, a potential molecular basis for binding was predicted as well as confirming the key residues involved in the formation of the final strong and stable Pin1 WW domain-ligand complex.