DrugPred_RNA—A Tool for Structure-Based Druggability Predictions for RNA Binding Sites

RNA is an emerging target for drug discovery. However, like for proteins, not all RNA binding sites are equally suited to be addressed with conventional drug-like ligands. To this end, we have developed the structure-based druggability predictor DrugPred_RNA to identify druggable RNA binding sites. Due to the paucity of annotated RNA binding sites, the predictor was trained on protein pockets, albeit using only descriptors that can be calculated for both RNA and protein binding sites. DrugPred_RNA performed well in discriminating druggable from less druggable binding sites for the protein set and delivered predictions for selected RNA binding sites that agreed with manual assignment. In addition, most drug-like ligands contained in an RNA test set were found in pockets predicted to be druggable, further adding confidence to the performance of DrugPred_RNA. The method is robust against conformational and sequence changes in the binding sites and can contribute to direct drug discovery efforts for RNA targets.publishedVersio

Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl-enzyme intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB (PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E. coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Binding site comparison between PaFabB and PaFabF revealed a conserved malonyl binding site but differences in the fatty acid binding channel. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.publishedVersio

How To Design Selective Ligands for Highly Conserved Binding Sites: A Case Study Using N-Myristoyltransferases as a Model System

Under embargo until: 2020-08-19A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side-chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds more strongly to the off-target than to the target. On the basis of this finding, a virtual screen for selective compounds was conducted, resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.acceptedVersio

In silico identification and experimental validation of hits active against KPC-2 \u3b2-lactamase

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type \u3b2-lactamases are often reported as resistant to available \u3b2-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non \u3b2-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-\u3b2-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the \u3b2-lactam antibiotic meropenem by four fold

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening.publishedVersio

An Experimental Toolbox for Structure-Based Hit Discovery for P. aeruginosa FabF, a Promising Target for Antibiotics

FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target for new antibiotics. Here, we focus on FabF from P. aeruginosa (PaFabF) as antibiotics against this pathogen are urgently needed. To facilitate exploration of this target we have set up an experimental toolbox consisting of binding assays using bio-layer interferometry (BLI) as well as saturation transfer difference (STD) and WaterLOGSY NMR in addition to robust conditions for structure determination. The suitability of the toolbox to support structure-based design of FabF inhibitors was demonstrated through the validation of hits obtained from virtual screening. Screening a library of almost 5 million compounds resulted in 6 compounds for which binding into the malonyl-binding site of FabF was shown. For one of the hits, the crystal structure in complex with PaFabF was determined. Based on the obtained binding mode, analogues were designed and synthesised, but affinity could not be improved. This work has laid the foundation for structure-based exploration of PaFabF.publishedVersio

Design and Synthesis of Brain Penetrant Trypanocidal N-Myristoyltransferase Inhibitors

<i>N</i>-Myristoyltransferase (NMT) represents a promising drug target within the parasitic protozoa <i>Trypanosoma brucei</i> (<i>T. brucei</i>), the causative agent for human African trypanosomiasis (HAT) or sleeping sickness. We have previously validated <i>T. brucei</i> NMT as a promising druggable target for the treatment of HAT in both stages 1 and 2 of the disease. We report on the use of the previously reported DDD85646 (<b>1</b>) as a starting point for the design of a class of potent, brain penetrant inhibitors of <i>T. brucei</i> NMT

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening

Evaluation of two-year recall of self-reported pesticide exposure among Ugandan smallholder farmers

OBJECTIVES: To evaluate smallholder farmers' recall of pesticide use and exposure determinants over a two-year period in a low-income country context. METHODS: The Pesticide Use in Tropical Settings (PESTROP) study in Uganda consists of 302 smallholder farmers who were interviewed in 2017. In the same season in 2019, these farmers were re-questioned concerning pesticide use (e.g., use of active ingredients) and exposure information (e.g., crops, personal protective equipment [PPE], hygienic behaviours) they had previously provided. The extent of recall bias was assessed by comparing responses at follow-up in 2019 with practices and behaviours reported from the baseline interview in 2017. RESULTS: An 84% (n = 255) follow-up response rate was attained. We found instances of better recall (e.g., overall agreement >70% and Area Under the Curve (AUC) values > 0.7) for the use of some active ingredients, commonly used PPE items, and washing clothes after application, whereas only 13.3% could correctly recall their three major crops. We observed a trend where more individuals reported the use of active ingredients, while fewer reported the use of PPE items, two years later. In general, we found better agreement in the recall of years working with pesticides compared to hours per day or days per week in the field, with no apparent systematic over or under reporting by demographic characteristics. CONCLUSIONS: While some of these findings provide consistency with those from high-income countries, more research is needed on recall in poorly educated agriculture communities in low- and middle-income settings to confirm these results