Assessment of Binding Affinity via Alchemical Free-Energy Calculations.

Free-energy calculations have seen increased usage in structure-based drug design. Despite the rising interest, automation of the complex calculations and subsequent analysis of their results are still hampered by the restricted choice of available tools. In this work, an application for automated setup and processing of free-energy calculations is presented. Several sanity checks for assessing the reliability of the calculations were implemented, constituting a distinct advantage over existing open-source tools. The underlying workflow is built on top of the software Sire, SOMD, BioSimSpace, and OpenMM and uses the AMBER 14SB and GAFF2.1 force fields. It was validated on two datasets originally composed by Schrödinger, consisting of 14 protein structures and 220 ligands. Predicted binding affinities were in good agreement with experimental values. For the larger dataset, the average correlation coefficient Rp was 0.70 ± 0.05 and average Kendall's τ was 0.53 ± 0.05, which are broadly comparable to or better than previously reported results using other methods.

New inhibitor targeting human transcription factor HSF1: effects on the heat shock response and tumor cell survival.

Comparative modeling of the DNA-binding domain of human HSF1 facilitated the prediction of possible binding pockets for small molecules and definition of corresponding pharmacophores. In silico screening of a large library of lead-like compounds identified a set of compounds that satisfied the pharmacophoric criteria, a selection of which compounds was purchased to populate a biased sublibrary. A discriminating cell-based screening assay identified compound 001, which was subjected to systematic analysis of structure-activity relationships, resulting in the development of compound 115 (IHSF115). IHSF115 bound to an isolated HSF1 DNA-binding domain fragment. The compound did not affect heat-induced oligomerization, nuclear localization and specific DNA binding but inhibited the transcriptional activity of human HSF1, interfering with the assembly of ATF1-containing transcription complexes. IHSF115 was employed to probe the human heat shock response at the transcriptome level. In contrast to earlier studies of differential regulation in HSF1-naïve and -depleted cells, our results suggest that a large majority of heat-induced genes is positively regulated by HSF1. That IHSF115 effectively countermanded repression in a significant fraction of heat-repressed genes suggests that repression of these genes is mediated by transcriptionally active HSF1. IHSF115 is cytotoxic for a variety of human cancer cell lines, multiple myeloma lines consistently exhibiting high sensitivity.

Clostridium difficile has a single sortase, SrtB, that can be inhibited by small-molecule inhibitors.

BACKGROUND: Bacterial sortases are transpeptidases that covalently anchor surface proteins to the peptidoglycan of the Gram-positive cell wall. Sortase protein anchoring is mediated by a conserved cell wall sorting signal on the anchored protein, comprising of a C-terminal recognition sequence containing an "LPXTG-like" motif, followed by a hydrophobic domain and a positively charged tail. RESULTS: We report that Clostridium difficile strain 630 encodes a single sortase (SrtB). A FRET-based assay was used to confirm that recombinant SrtB catalyzes the cleavage of fluorescently labelled peptides containing (S/P)PXTG motifs. Strain 630 encodes seven predicted cell wall proteins with the (S/P)PXTG sorting motif, four of which are conserved across all five C. difficile lineages and include potential adhesins and cell wall hydrolases. Replacement of the predicted catalytic cysteine residue at position 209 with alanine abolishes SrtB activity, as does addition of the cysteine protease inhibitor MTSET to the reaction. Mass spectrometry reveals the cleavage site to be between the threonine and glycine residues of the (S/P)PXTG peptide. Small-molecule inhibitors identified through an in silico screen inhibit SrtB enzymatic activity to a greater degree than MTSET. CONCLUSIONS: These results demonstrate for the first time that C. difficile encodes a single sortase enzyme, which cleaves motifs containing (S/P)PXTG in-vitro. The activity of the sortase can be inhibited by mutation of a cysteine residue in the predicted active site and by small-molecule inhibitors.

Targeting an Initiator Allergen Provides Durable and Expansive Protection against House Dust Mite Allergy.

Whereas treatment of allergic diseases such as asthma relies largely on the targeting of dysregulated effector pathways, the conceptually attractive alternative of preventing them by a pharmaceutical, at-source intervention has been stymied until now by uncertainties about suitable targets and the challenges facing drug design. House dust mites (HDMs) are globally significant triggers of allergy. Group 1 HDM allergens, exemplified by Der p 1, are cysteine proteases. Their degradome has a strong disease linkage that underlies their status as risk and initiator allergens acting directly and through bystander effects on other allergens. Our objective was to test whether target-selective inhibitors of group 1 HDM allergens might provide a viable route to novel therapies. Using structure-directed design to optimize a series of pyruvamides, we undertook the first examination of whether pharmaceutically developable inhibitors of group 1 allergens might offer protection against HDM exposure. Developability criteria included durable inhibition of clinically relevant signals after a single aerosolized dose of the drug. The compounds suppressed acute airway responses of rats and mice when challenged with an HDM extract representing the HDM allergome. Inhibitory effects operated through a miscellany of downstream pathways involving, among others, IL-33, thymic stromal lymphopoietin, chemokines, and dendritic cells. IL-13 and eosinophil recruitment, indices of Th2 pathway activation, were strongly attenuated. The surprisingly expansive benefits arising from a unique at-source intervention suggest a novel approach to multiple allergic diseases in which HDMs play prominent roles and encourage exploration of these pharmaceutically developable molecules in a clinical setting.

The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma.

De novo ligand design to partially flexible active sites: application of the ReFlex algorithm to carboxypeptidase A, acetylcholinesterase, and the estrogen receptor.

Reflex is a recent algorithm in the de novo ligand design software, SkelGen, that allows the flexibility of amino acid side chains in a protein to be taken into account during the drug-design process. In this paper the impact of flexibility on the solutions generated by the de novo design algorithm, when applied to carboxypeptidase A, acetylcholinesterase, and the estrogen receptor (ER), is investigated. The results for each of the targets indicate that when allowing side-chain movement in the active site, solutions are generated that were not accessible from the multiple static protein conformations available for these targets. Furthermore, an analysis of structures generated in a flexible versus a static ER active site suggests that these additional solutions are not merely noise but contain many interesting chemotypes.

SkelGen: a general tool for structure-based de novo ligand design.

The recent lapse in productivity in the pharmaceutical industry has facilitated the emergence of experimental and in silico structure-based design methodologies, based on identification of biologically active low molecular weight fragments that can be exploited to produce potential drug candidates with diverse chemistries. SkelGen, an in silico example of this methodology, is reviewed. The ability of this algorithm to identify chemically diverse low molecular weight fragments that would potentially bind to DNA gyrase is recounted, as is the first purely de novo structure-based design of five compounds that show at least micromolar activity against the estrogen receptor. The ability of the algorithm to incorporate partial protein flexibility during its design of compounds to the estrogen receptor is discussed, and an opinion as to the near and long-term futures for de novo design algorithms is expressed.

Exhaustive de novo design of low-molecular-weight fragments against the ATP-binding site of DNA-gyrase.

We present a de novo design approach to generating small fragments in the DNA-gyrase ATP-binding site using the computational drug design platform SkelGen. We have generated an exhaustive number of structural possibilities, which were subsequently filtered for site complementarity and synthetic tractability. A number of known active fragments are found, but most of the species created are potentially novel and could be valuable for further elaboration and development into lead-like structures.

Generation and selection of novel estrogen receptor ligands using the de novo structure-based design tool, SkelGen.

A de novo design approach to generating novel estrogen receptor (ER) ligands is described. The SkelGen program was used to generate ligands in the active sites of seven crystal structures of ERalpha. Seventeen high-scoring, diverse structures were selected from the SkelGen output and synthesized without introducing any modifications to the structures. Five ligands, four of which are novel, showed < or = 25 microM affinity, with the best compound displaying an IC50 of 340 nM. SkelGen can, therefore, be a powerful tool for designing active molecules.

Including tightly-bound water molecules in de novo drug design. Exemplification through the in silico generation of poly(ADP-ribose)polymerase ligands.

Different strategies for the in silico generation of ligand molecules in the binding site of poly(ADP-ribose)polymerase (PARP) were studied in order to observe the effect of the targeting and displacement of tightly bound water molecules. Several molecular scaffolds were identified as having better interactions in the binding site when targeting one or two tightly bound water molecules in the NAD binding site. Energy calculations were conducted in order to assess the ligand-protein and ligand-water-protein interactions of different functional groups of the generated ligands. These calculations were used to evaluate the energetic consequences of the presence of tightly bound water molecules and to identify those that contribute favorably to the binding of ligands.