Virtual Screening of potential drug-like inhibitors against Lysine/DAP pathway of Mycobacterium tuberculosis

Background: An explosive global spreading of multidrug resistant Mycobacterium tuberculosis (Mtb) is a catastrophe, which demands an urgent need to design or develop novel/potent antitubercular agents. The Lysine/DAP biosynthetic pathway is a promising target due its specific role in cell wall and amino acid biosynthesis. Here, we report identification of potential antitubercular candidates targeting Mtb dihydrodipicolinate synthase (DHDPS) enzyme of the pathway using virtual screening protocols. Results: In the present study, we generated three sets of drug-like molecules in order to screen potential inhibitors against Mtb drug target DHDPS. The first set of compounds was a combinatorial library, which comprised analogues of pyruvate (substrate of DHDPS). The second set of compounds consisted of pyruvate-like molecules i.e. structurally similar to pyruvate, obtained using 3D flexible similarity search against NCI and PubChem database. The third set constituted 3847 anti-infective molecules obtained from PubChem. These compounds were subjected to Lipinski's rule of drug-like five filters. Finally, three sets of drug-like compounds i.e. 4088 pyruvate analogues, 2640 pyruvate-like molecules and 1750 anti-infective molecules were docked at the active site of Mtb DHDPS (PDB code: 1XXX used in the molecular docking calculations) to select inhibitors establishing favorable interactions. Conclusion: The above-mentioned virtual screening procedures helped in the identification of several potent candidates that possess inhibitory activity against Mtb DHDPS. Therefore, these novel scaffolds/candidates which could have the potential to inhibit Mtb DHDPS enzyme would represent promising starting points as lead compounds and certainly aid the experimental designing of antituberculars in lesser time

Design and Synthesis of Potential Novel Antibiotic Compounds Utilising Photoredox Catalysis

The continued emergence of widespread antibiotic resistance over the prior several decades poses an increasingly severe worldwide challenge to public health. Several frontline antibiotic treatments are being rendered obsolete due to the advent of numerous bacterial resistance mechanisms, an issue further compounded by the lack of antibiotics currently residing within the antibacterial drug discovery pipeline that operate via previously unexploited mechanisms of action. There are numerous underlying issues that have propagated this unsavoury situation, some specific to antibiotic drug development and others that negatively impact the field of drug discovery as a whole. One of the latter issues centres around the implementation of high throughput target-based screening of suboptimal compound libraries for hit identification, and the narrow range of synthetic methodologies used to explore chemical space within such compound collections. Dihydrodipicolinate synthase (DHDPS) constitutes a promising biomolecular target for novel antibiotic therapies due to its key role in the biosynthesis of essential amino acid L-lysine, a process widely specific to bacteria. Despite several prior campaigns and the development of micromolar potency inhibitors of DHDPS through target–based screening approaches, so far no compounds have been developed that display in vitro antibacterial activity in the subsequent phenotypic screens. In silico screening constitutes an invaluable range of techniques used in the identification of potential hit compounds that has been implemented to great effect in numerous drug discovery campaigns, including the discovery of novel antibacterial compounds, often aiding in the design of more focused compound libraries for assessment in vitro. Photoredox catalysis has emerged as a powerful synthetic tool for enabling access to previously unexplored regions of chemical space especially within medicinal chemistry contexts, facilitating highly chemoselective activation of reagents under benign reaction conditions. Sulfonylhydrazones are well established reagents within the field of organic synthesis capable of undergoing a myriad of transformations. Recent reports concerning the photocatalytic activation of hydrazone substrates to enable radical cyclisations served as the basis for the initial interest in developing related methodologies to generate desired compounds in the search for novel antibacterial agents. In this thesis is described the design and synthesis of potential novel antibacterial compounds, initially utilising pharmacophore searches and qualitative in silico docking investigations to identify molecular scaffolds of interest as synthetic targets. The development of a novel photoredox reaction for the generation of sulfone hit structures from sulfonyl hydrazone starting materials is described, including exploration of the substrate scope and reaction mechanism studies. The synthesis of additional in silico derived hit structures is also described, as well as attempts made to expand the synthetic utility of the developed photocatalytic methodology. Initial evaluation of antibacterial activity of the compound collection is described including preliminary discussion of structure activity relationships as a foundation for the derivation of future work. The final chapter contains technical experimental details and characterisation data pertaining to the previously discussed work

Allosteric inhibitors of dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is an enzyme which catalyzes the first step of the lysine biosynthesis pathway in bacteria and plants. Deletion of the gene encoding DHDPS results in non-viable bacteria, therefore DHDPS is considered a validated drug target. The enzyme is feedback-regulated by lysine, and structural studies have shown that the tetrameric enzyme contains two allosteric sites, each of which bind two lysine molecules. The Palmer laboratory has previously developed a potent inhibitor "bislysine" that mimics the structure of two bound lysine molecules. Previous work showed that S-aminoethylcysteine ("thialysine") was a much poorer inhibitor than lysine, despite the structural similarity of the two compounds. This thesis describes the synthesis of new allosteric inhibitors of DHDPS, with the goal of defining their structural and chemical properties, such as inhibitor side chain length and pKa, that lead to inhibition. Racemic analogs of lysine were generated using the amidomalonic ester synthesis. Analogs of bislysine were generated from dimethyl 2,5-bis([(tertbutoxy)carbonyl]amino) hexanedioate by treatment with lithium diisopropylamide followed by alkylation using various electrophiles. This alkylation step hampers the overall process because it proceeds in low yield (typically near 10%). Studies were undertaken in an attempt to understand the factors influencing this reaction; however, variations in the reaction times, solvent composition, and additives did not improve the yield appreciably. All the inhibitors were tested using the established DHDPS-DHDPR coupled assay to estimate the IC50 values. The lysine analogue (±)-(E)-2,6-diaminohex-4-enoic acid, which has a double bond in the side chain as the only modification, showed weaker inhibition (IC50 = 3.7 mM) compared to racemic lysine (IC50 = 0.2 mM). The altered pKa of the ε-amino group, which makes a hydrogen bond with His59 when bound to the allosteric site, is proposed to account for the loss of activity. Triazolylmethylglycine, which is predicted to have a pKa value closer to lysine, but contains a shorter side chain, was an even weaker inhibitor. Bis-amino acid versions of these compounds were much stronger inhibitors. A bis-analog of para-aminobenzylglycine showed weak inhibition as well, suggesting this bulkier compound, with a much lower side chain pKa, could still bind to the allosteric cavity

A dual-target herbicidal inhibitor of lysine biosynthesis

Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of- concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.Emily RR Mackie, Andrew S Barrow, Rebecca M Christoff, Belinda M Abbott, Anthony R Gendall, Tatiana P Soares da Cost

KiDoQ: using docking based energy scores to develop ligand based model for predicting antibacterials

Background: Identification of novel drug targets and their inhibitors is a major challenge in the field of drug designing and development. Diaminopimelic acid (DAP) pathway is a unique lysine biosynthetic pathway present in bacteria, however absent in mammals. This pathway is vital for bacteria due to its critical role in cell wall biosynthesis. One of the essential enzymes of this pathway is dihydrodipicolinate synthase (DHDPS), considered to be crucial for the bacterial survival. In view of its importance, the development and prediction of potent inhibitors against DHDPS may be valuable to design effective drugs against bacteria, in general. Results: This paper describes a methodology for predicting novel/potent inhibitors against DHDPS. Here, quantitative structure activity relationship (QSAR) models were trained and tested on experimentally verified 23 enzyme's inhibitors having inhibitory value (Ki) in the range of 0.005-22(mM). These inhibitors were docked at the active site of DHDPS (1YXD) using AutoDock software, which resulted in 11 energy-based descriptors. For QSAR modeling, Multiple Linear Regression (MLR) model was engendered using best four energy-based descriptors yielding correlation values R/q2 of 0.82/0.67 and MAE of 2.43. Additionally, Support Vector Machine (SVM) based model was developed with three crucial descriptors selected using F-stepping remove-one approach, which enhanced the performance by attaining R/q2 values of 0.93/0.80 and MAE of 1.89. To validate the performance of QSAR models, external cross-validation procedure was adopted which accomplished high training/testing correlation values (q2/r2) in the range of 0.78-0.83/0.93-0.95. Conclusions: Our results suggests that ligand-receptor binding interactions for DHDPS employing QSAR modeling seems to be a promising approach for prediction of antibacterial agents. To serve the experimentalist to develop novel/potent inhibitors, a webserver "KiDoQ" has been developed http://crdd.osdd.net/raghava/kidoq webcite, which allows the prediction of Ki value of a new ligand molecule against DHDPS

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from streptococcus pneumoniae

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1 , KM PYR = 2.55 ± 0.05 mM and KM ASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TM app) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TM app = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD 4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD 4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2 ) compared to its E. coli counterpart (500 Å2 ). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS

Studies on the protein biology of the plant pathogen, Candidatus Liberibacter solanacearum.

Characterisation determined the protein was folded and not functional. Solution structural analysis found that CLso adenyltransferase/IMP cyclohydrolase exists as a monomer in solution, which contrasts to the dimer observed in other bacterial homologues. Dimerisation is required for activity of the enzyme and the oligomeric state of the enzyme was determined to account for the lack of activity in CLso adenyltransferase/IMP cyclohydrolase. Chaperone co-expression resulted in the isolation of an additional five CLso proteins: serralysin (virulence factor), dehydrorhamnose epimerase, dehydrorhamnose reductase (L- rhamnose biosynthesis), pyruvate kinase (glycolysis) and dihydrodipicolinate synthase (lysine biosynthesis), which are hypothesised to be required for the organisms’ survival since gene expression studies show high bacterial expression when in infected psyllids and in planta. Characterisation of purified dihydrodipicolinate synthase and pyruvate kinase showed that both enzymes were active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial homologues. The establishment of a robust method for the expression of CLso proteins allows for the structural and kinetic characterisation of dihydrodipicolinate synthase, a key enzyme as it catalyses the committed step and is a potential antimicrobial target. The structure of the CLso dihydrodipicolinate synthase was solved in complex with the substrates: pyruvate, pyruvate and succinic semi-aldehyde (an analogue of (S)-aspartate semialdehyde) and with the allosteric inhibitor, lysine. Structural analysis showed that there was little difference in the CLso dihydrodipicolinate synthase compared with the Agrobacterium tumefaciens orthologue. Kinetic analysis of the enzyme reports constants that suggest a high binding affinity for substrates. It was proposed that CLso dihydrodipicolinate synthase has evolved a high binding affinity for its substrates in response to nutrient limitation, which could occur as a result of the intracellular lifestyle of the organism. Overall, this work provides new data that enriches our understanding of CLso biology and the relationship of the organism with the psyllid host and provides a method for future characterisation of CLso proteins, enabling new research and drug discovery programmes to study and manage the pathogenicity of the organism

Dynamic Modelling Reveals 'Hotspots' on the Pathway to Enzyme-Substrate Complex Formation.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the diaminopimelate pathway of bacteria, yielding amino acids required for cell wall and protein biosyntheses. The essentiality of the enzyme to bacteria, coupled with its absence in humans, validates DHDPS as an antibacterial drug target. Conventional drug design efforts have thus far been unsuccessful in identifying potent DHDPS inhibitors. Here, we make use of contemporary molecular dynamics simulation and Markov state models to explore the interactions between DHDPS from the human pathogen Staphylococcus aureus and its cognate substrate, pyruvate. Our simulations recover the crystallographic DHDPS-pyruvate complex without a priori knowledge of the final bound structure. The highly conserved residue Arg140 was found to have a pivotal role in coordinating the entry of pyruvate into the active site from bulk solvent, consistent with previous kinetic reports, indicating an indirect role for the residue in DHDPS catalysis. A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways. By means of umbrella sampling we show that these binding intermediates are thermodynamically metastable, consistent with both the available experimental data and the substrate binding model presented in this study. Our results provide insight into an important enzyme-substrate interaction in atomistic detail that offers the potential to be exploited for the discovery of more effective DHDPS inhibitors and, in a broader sense, dynamic protein-drug interactions

Investigations of dihydrodipicolinate synthase and dihydrodipicolinate reductase

The L-lysine biosynthetic pathway provides a unique and exciting target for the design of novel antibacterial agents through inhibition of the synthesis of meso—DAP or L—lysine, crucial components of the bacterial peptidoglycan cell wall. The first two enzymes in this pathway—dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR)—were targeted for inhibition. DHDPS catalyses the first committed step to L—lysine biosynthesis, the condensation of pyruvate and (S)-aspartate B-semialdehyde to give DHDP. DHDPR, the next enzyme in the pathway, then reduces DHDP in an NAD(P)H dependent reaction to give THDP. A suite of inhibitors were designed and synthesised to act as product- or substrate-based analogues of DHDPS and DHDPR, respectively. Heterocyclic compounds based on chelidamic acid or thiazane-3,5-dicarboxylates were synthesised as potential inhibitors of DHDPS. Stereoselective procedures for the oxidation of thiazane-3,5-dicarboxylates were developed. It was found that when direct oxidants were used (such as sodium periodate, peroxides and peracids) the axial sulfur lone-pair reacts preferentially, providing the axial S—oxide. Oxidation via a two step mechanism using bromine/water gives the epimeric equatorial S—oxide. Acyclic analogues of the heterocyclic compounds were also synthesised as potential inhibitors of the intermediate in the DHDPS reaction pathway. Two methods of synthesising the DHDPS substrate aspartate B-semialdehyde (ASA) were investigated. The first four step procedure beginning from racemic allylglycine, installed the aldehyde moiety through an osmium tetroxide/sodium periodate reaction. The ASA produced by this procedure was of variable yield and purity. The second method of synthesising ASA was achieved by reduction of a Weinreb amide derivative of aspartic acid. This procedure gave ASA in excellent yield and purity and was the method of choice for preparing ASA for use in kinetic studies. Additionally the required enzymes, DHDPS and DHDPR were purified to homogeneity as judged by SDS-PAGE visualised by Coomassie brilliant blue staining and specific activity tables. The inhibitors synthesised were tested for inhibition of DHDPS and DHDPR. The heterocyclic compounds were not found to be potent inhibitors of DHDPS. Furthermore, chelidamic acid and its dimethyl ester were shown NOT to be competitive inhibitors of DHDPS. Unfortunately, none of the analogous acyclic compounds proved to be potent DHDPS inhibitors either. Two potent acyclic irreversible inhibitors synthesised en route to other target inhibitors of DHDPS were discovered, possessing micromolar—millimolar activity. These compounds provide a new lead in the design of more potent DHDPS inhibitors. From the DHDPS inhibitory results obtained in this study the mechanism of DHDPS was revised. It was postulated that the DHDPS-catalysed reaction proceeds via a protonated aldehyde intermediate that is consistent with the inhibitory data obtained herein. None of the synthesised inhibitors evaluated against DHDPR were found to be more potent then the known inhibitor dipicolinic acid. Consistent with the literature results dipicolinic acid was found to be a competitive inhibitor of DHDPR, however dimethyl chelidamate was found to be an uncompetitive inhibitor of DHDPR