Identifying analogues of 2-deoxyglucose, alpha-d-glucose and beta-d-glucose-6-phosphate as potential inhibitors of human hexokinase ii for the development of anti-dengue therapeutics

The human hexokinase isoform II (HKII) is one of the important enzymes for dengue virus (DENV) replication and thus has been suggested as a potential therapeutic target for DENV drug development. In this work, compounds were identified using Ultrafast Shape Recognition with CREDO Atom Types (USRCAT) by utilizing both HKII’s substrate and product; alpha-D-glucose (GLC) and beta-D-glucose-6-phosphate (BG6), as well as a known HKII’s inhibitor, 2-deoxyglucose (2DG), as the query molecules. The analogues of the three query molecules were subsequently docked against the HKII’s crystal structure (PDB ID: 2NZT) by using Auto Dock 4 program on Chain B, where the active sites and strong bonds were located. Among the top-ranked compounds, Compound 4 (ZINC26898487), which was the most similar to 2DG, showed the best binding energy (-7.63 kcal/mol) and contained two H bonds. Compound 9 (ZINC16930948), an analogue of GLC emerged as the best inhibitor candidate because it had six H bonds. Similarly, among the molecules similar to BG6, Compound 14 (ZINC4403351) had been suggested as a potential inhibitor because it contained four strong H bonds. All compounds were predicted to be non-toxic, based on Toxicity Estimation Software Tool (TEST) analysis. By providing these valuable findings, this study has paved the way for the discovery of compounds that should be further tested for the development of anti-dengue drugs. © 2019, Universiti Putra Malaysia Press. All rights reserved

Cloning and characterisation of Lactate Dehydrogenase Gene from Plasmodium knowlesi in bacterial system

Glycolysis is essential for Plasmodium survival during its intra-erythrocytic stage in the human host. As a consequence, enzymes in the glycolytic pathway have been proposed as ideal therapeutic targets for malaria pharmaceuticals. Specifically, lactate dehydrogenase, which is the final enzyme in glycolysis, has been validated as a good drug target. We have cloned and characterised recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was obtained from GenScript®. Pk-LDH gene was successfully amplified from the pUC57 vector and a PCR product with the size of 951bp was cloned into pEASY-Blunt E1 expression vector. The ligated product was subsequently transformed into Trans1-T1 Phage Resistant Chemically Competent Cell. A sequence alignment analysis, which was conducted to compare the sequence similarity of Pk-LDH to LDH from other human malaria parasites revealed open reading frame of 316 amino acids of Pk-LDH and showed 97.8% homology to P. vivax LDH and 90% homology to P. malariae, P. falciparum, and P. ovale LDHs, respectively. The purified recombinant Pk-LDH will later be utilised for inhibition studies in future antimalarial drug design and discovery research, specifically for P. knowlesi

Plasmodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review

Malaria continues to pose imminent threat to the world population, as the mortality rate associated with this disease remains high. Current treatment relies on antimalarial drugs such as Artemisinin Combination Therapy (ACT) are still effective throughout the world except in some places, where ACT-resistance has been reported, thus necessitating novel approaches to develop new anti-malarial therapy. In the light of emerging translational research, several plasmodial targets, mostly proteins or enzymes located in the parasite’s unique organelles, have been extensively explored as potential candidates for the development of novel antimalarial drugs. By targeting the metabolic pathways in mitochondrion, apicoplast or cytoplasm of Plasmodium, the possibility to discover new drugs is tremendous, as they have potentials as antimalarial therapeutic targets. This literature review summarizes pertinent information on plasmodial targets, especially enzymes involved in specific metabolic pathways, and the strategies used to discover new antimalarial drugs. © 2019, University of Malaya. All rights reserved

Screening potential inhibitors of lactate dehydrogenase from Plasmodium knowlesi via virtual screening approaches

Lactate dehydrogenase from Plasmodium knowlesi (Pk-LDH) has been suggested as a potential therapeutic target for the development of drugs against malaria disease. This paper reported the screening of compounds which have potentials to be developed as drugs specific for malaria caused by P. knowlesi via in silico screening. Due to the unavailability of Pk-LDH crystal structure, a protein model was built based on the crystal structure of the closest similar protein, lactate dehydrogenase from Plasmodium falciparum (Pf-LDH) with 91% sequence identity between the two enzymes. The model was developed using MODELLER program and verified in Structural Analysis and Verification Server. Primary and secondary structure features were determined and based on Globplot, two disordered regions were predicted at amino acid numbers 85-95 and 269-281. Meanwhile, results of PPCpred server predicted that Pk-LDH is crystallisable with predicted crystallisation propensity of 0.766. Verification of the model was performed with the ERRAT quality factor of 92.2% while Verify 3D gave the percentage of 85.76%. Ligand-based drug design was performed using Ultra-Fast Shape Recognition with Atom Types (USFRAT) with scores for compounds most resemble oxamate ranged from 0.832-0.914. Meanwhile, the results from structure-based screening using Autodock4 and Cygwin gave minimum binding energies ranged from -3.59 to -0.07. Taken together, this study has successfully generated a verified model structure of Pk-LDH and yielded a list of compounds that have potentials to be developed as antimalarial drugs

Analogues of Oxamate, Pyruvate, and Lactate as potential inhibitors of Plasmodium Knowlesi lactate Dehydrogenase identified using virtual screening and verified via Inhibition Assays

Malaria management remains a challenge, due to the resistance of malaria parasites to current antimalarial agents. This resistance consequently delays the global elimination of malaria throughout the world. Hence, the demand is increasing for new and effective antimalarial drugs. The identification of potential drugs that target Pk-LDH can be obtained through virtual screening analyses, as this has been previously applied to discover Pf-LDH inhibitors. In this study, the selected candidates from our virtual screening analyses were subsequently tested against purified Pk-LDH, and verified through an inhibition of Pk-LDH via enzymatic activity assays. Virtual screening analysis from this study showed that 3,3-Difluoropyrrolidine hydrochloride and 3-hydroxytetrahydrofuran exhibited binding affinity values of −3.25 kcal/mol and −3.74, respectively. These compounds were selected for evaluation towards inhibitory activity against Pk-LDH assays, including two compounds from a previous study which are oxalic acid and glycolamide. The earlier compounds were structurally similar to lactate and pyruvate, and the latter two compounds were structurally similar to a known LDH inhibitor, oxamate. Among all of the compounds tested, oxalic acid showed the highest inhibition activity at 54.12%; interestingly, this correlated well with the virtual screening analyses, which showed that this compound was the best among the Oxamate analogues, with a binding affinity value of −2.59 kcal/mol. Hence, further exploration and development of this compound may result in a promising antimalarial drug for malaria treatment, especially for infection involving P. Knowlesi

The glycolytic pathway is a target for drug discovery against tropical diseases

Glycolysis is the heart of various metabolic processes, ranging from energy generation to biosynthesis of molecules, indicating its importance as the most primeval metabolic pathway in nearly all living organisms. The pathway has also been perceived as an energy provider for viral replication, which has been previously shown to be altered upon virus infection. Here, we discuss the potential role of glycolysis as a target for the discovery of new drugs against infectious tropical diseases, such as malaria and dengue. Compounds that may possess the ability to inhibit some of the enzymes in the glycolytic pathway, such as lactate dehydrogenase (LDH) and hexokinase isoform 2 (HK2) were initially screened through virtual means, prior to the production of these enzymes in the bacterial system. The effects of these potential inhibitors on the activity of the enzymes were subsequently evaluated, by benchmarking with known inhibitors of these enzymes. Some of the virtually screened compounds, notably Chitin, an analogue of a known inhibitor of human HK2, 2- deoxyglucose (2-DG), exhibited substantial inhibition on the enzyme’s activity, which indicates its potential to disrupt dengue virus (DENV) replication. Meanwhile, oxalic acid, which is an analogue of a known inhibitor of LDH, oxamate, has shown promising inhibition on the activity of Plasmodium knowlesi LDH (Pk-LDH), also indicating the possibility of the virtually screened compound to be developed into a potent drug that specifically targets the enzyme. These initial works may provide future explorative potential and have paved the path towards the discovery of new drugs for the treatment of tropical infectious diseases

Analogues of 2-Deoxyglucose as inhibitor candidates for hexokinase II identified via virtual screening analyses

Dengue fever is one of the major global health issues, yet specific treatment remains vague. The human hexokinase isoform II (HK2) is one of the crucial enzyme for dengue virus (DENV) replication and thus has been suggested as a potential therapeutic target for DENV drug development. In this paper, compounds with potential HK2 inhibitory activity have been identified by using a combination of ligand-based and structurebased virtual screening approaches. In the initial ligand-based approach, a known HK2 inhibitor, 2- Deoxyglucose (2-DG) was used as the query molecule, which resulted in the identification of analogues of 2-DG with scores ranging from 0.739-0.763. The analogues were docked against the crystal structure of HK2 (PDB ID: 2NZT) in complex with alpha-D-glucose (GLC) and beta-D-glucose-6-phosphate (BG6) by using Auto Dock 4 programme, on chain B where the active sites were located. The docking hits exhibited binding energy ranging from -7.63 to -4.98kcal/mol. Six top-ranked compounds which are most similar to 2-DG were subsequently analysed based on their predicted binding affinity with the catalytic residues (Thr620, Lys621, Asn656, Lys618, Asp657, Phe604, Asn735, Glu629, Gly710, Gly622 and Glu708), H bond analysis and toxicity effect. Among the selected top six compounds, only compound 4 (ZINC26898487) is non-toxicant and has shown good binding energy (-7.63 kcal/mol) relative to 2-DG and contains four H bonds; two with Lys621, one with Glu629 and another one with is Thr620. Meanwhile, the binding energy of 2-DG itself is -7.40kcal/mol when in complex with HK2, with two H bonds; one with Lys621 and another one with Glu708. In this drug design computational studies, compound 4 (ZINC26898487) is suggested to have potentials to inhibit HK2 activity relative to the known inhibitor (2-DG), thus pave the way towards the discovery of new dengue therapeutic

Potential inhibitors of the glycolytic enzymes from Plasmodium knowlesi identified by virtual screening analysis

Malaria remains pandemic globally and continues to threaten half of the current world’s population. In Malaysia, most malaria-related cases are now caused by Plasmodium knowlesi, an emerging zoonotic monkeyborne parasite, which is potentially fatal if remains untreated. Improved strategy in drug discovery is extremely important due to the development of parasite’s resistance towards the current antimalarial drugs. Since the glycolytic pathway is essential for parasite’s survival, the enzymes involved in this pathway, including hexokinase (Pk-HK) and lactate dehydrogenase (Pk-LDH), are regarded as attractive drug targets. This study aims to screen for small molecule compounds which may potentially be developed into potent drugs that specifically target these two enzymes. Virtual screening analyses comprising of structure and ligand-based screens were conducted to search for potential novel inhibitors of both enzymes. By using MODELLER, a comparative protein structure modeling programme, the model of Pk-HK and Pk-LDH have been generated based on hexokinase from Schistosoma mansoni (Sm-HK) and lactate dehydrogenase from Plasmodium falciparum (Pf-LDH), with 33% and 91% sequence similarity respectively. By using this model, docking and scoring functions through Ligand Discovery at Edinburgh UniverSity (LIDAEUS), a structurebased approach, were conducted and yielded more than 200 binding candidates for Pk-HK. The binding affinity scores for the best ten ligands docked to Pk-HK are in the range of -83 kcal/mol to -93 kcal/mol. Through ligand-based approach using Ultra-Fast Shape Recognition with Atom Types (UFSRAT), another 200 compound similar to glucose and oxamate were discovered, where the majority displays great similarity to glucose with similarity score (Sqc) of more than 0.5. This study has successfully discovered potential inhibitors of these two glycolytic enzymes from P.knowlesi, however further validation through suitable inhibition assay system has yet to be conducted

Cloning and expression of lactate dehydrogenase from plasmodium knowlesi for anti-malarial drug development

Malaria remains a global burden, where drug resistance issue has triggered a major concern in the affected regions. Plasmodium lactate dehydrogenase, which is the key enzyme in the parasite‘s glycolytic pathway has shown to be a potential novel therapeutic target. The aim of the study is to clone and express the recombinant lactate dehydrogenase from Plasmodium knowlesi in bacterial system. Methods: The synthetic Pk-LDH gene was amplified and the PCR product with the size of 951bp was cloned into pET21a expression vector. The ligated product was transformed into BL21 (DE3) strain to induce Pk-LDH expression. Soluble expression was obtained at 20°C, incubated for 18 hours in Terrific Broth media in the presence of 0.5 mM isopropyl β-d-thiogalactoside (IPTG). The expressed Pk-LDH protein was later purified by using a combination of Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC) methods. Sequencing and BLAST analysis revealed an open reading frame of 316 amino acids of Pk-LDH, which shows 91.8% sequence similarity with Plasmodium falciparum‘s LDH. The SDS–PAGE analysis exhibits that Pk-LDH protein of 34kDa in size was present in the soluble fraction. A sharp protein peak corresponding to the size of Pk-LDH was also observed upon gel filtration elution, indicating that the protein has successfully been purified to homogeneity. MALDI-TOF analysis gave a peptide score of 282, which is significant with L-lactate dehydrogenase from P. knowlesi, as revealed from the Mascot analysis. Pure and active Pk-LDH was obtained. Conclusions: The successful expression and purification system developed of Pk-LDH in this study offer a reliable method to produce soluble Pk-LDH that is biologically active, which can be used for future antimalarial drug development study