Effects of metal ions on the structural and biochemical properties of trypanosomatid phosphoglycerate mutases

Flagellate protozoa from the order Trypanosomatida have developed a range of strategies to survive in their mammalian hosts. A consequence is that the glycolytic pathway has assumed an important role, especially in bloodstream-form Trypanosoma brucei, where it is essential as the sole producer of ATP. The seventh enzyme in the pathway, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shares no common properties with the corresponding enzyme in humans. This enzyme catalyses the conversion of 3PGA to 2PGA, with the requirement for metal ions to assist the catalytic function. In this study, two important biochemical and structural aspects of the enzyme were investigated: i) The in vitro and in vivo requirements for biologically relevant metal ions to support the activity of iPGAM, and ii) The ability of trypanosomatid iPGAM to exist in multiple conformations and oligomeric states in solution. The maximum activity of iPGAM in vitro requires Co2+, but this cannot be the case in vivo where ICP-OES analyses confirmed that Co2+ was essentially undetectable in T. brucei cytosolic fractions. The activity of iPGAM in vivo is therefore one of the lowest among the glycolytic enzymes. By contrast, Mg2+ and Zn2+ were found to be the most abundant metals in both cytosolic fractions and in purified bacterially expressed iPGAM. Our newly-developed multimode-plate reader discontinuous assay further revealed that of the biologically relevant metals, only Mg2+ can support iPGAM activity, but at less than 50% of the level of Co2+. By contrast, Zn2+ strongly inhibits iPGAM. This assay which was developed with minimal metal interference on the coupling enzymes, also showed that in solution, the ratio of the concentrations of 3PGA:2PGA (substrate:product) at equilibrium is not 1:1 as observed in the crystal structure, but is in fact 12:1, which may be due to the tighter binding of 2PGA to the enzyme. A series of biophysical analyses, notably by SEC-MALS showed that iPGAM from Leishmania mexicana, another trypanosomatid protozoan parasite exists in different forms and oligomeric states in solution, either as the closed-form monomer, openiii form monomer, or closed/open-form dimer which can be successfully separated by ion-exchange chromatography. The open-form LmiPGAM is particularly relevant for drug development, as the catalytic site in the closed-form structure is poorly inaccessible. Both virtual and high-throughput screening approaches were used to identify novel potential inhibitors. Out of a collection of 11 compounds tested at 1 mM, two showed substantial inhibition with 49% and 14% remaining activity. Taken together, the findings from this study demonstrated the potential of iPGAM to be a key modulator in controlling glycolytic flux in trypanosomes, and thus further validated it as an important drug target

Enhanced expression and purification strategy for recombinant bacterially-expressed human hexokinase II

Dengue virus hijacks the host cellular mechanism to propagate and survive during viral infection, in which the central carbon mechanism plays a crucial role to upregulate DENV infection through the increase of human hexokinase II (HKII) activity. Since the enzyme governs the glycolytic pathway, it has potentials as a target for anti-dengue (DENV) drug development. In this study, the production of human hexokinase II protein has been enhanced by using bacterial system for anti-dengue therapeutic purpose. The HKII gene was cloned into pET28b vector and transformed into the E. coli strain BL21 (DE3) for HKII expression. In order to obtain soluble recombinant HKII in an active form, we optimized protein expression under specific conditions at 18°C for 19 hours using Terrific Broth media, in the presence of 0.5 mM isopropyl-2-D-thiogalactopyranoside (IPTG). The pET28b-HKII construct expressed in BL21(DE3) system exhibited adequate protein expression, thus, this construct was subsequently proceeded to purification process. The expressed protein was purified to homogeneity by a combination of Immobilized Metal Ion Affinity Chromatography (IMAC)and size exclusion chromatography (SEC), resulting in pure, active bacterially-expressed HKII with a specific activity of 56. 67U.mg-1. The amount of HKII obtained from 2 L culture is 80 mg, with a yield percentage of 10.5%. Hence in this study, human HKII has successfully been cloned and expressed as a soluble protein that can be utilized for further therapeutic studies

Interrogating novel compounds for improved anti-dengue therapies via inhibition of human hexokinase II (HKII)

Dengue is one of the most fatal infectious diseases in the world, which is caused by dengue virus (DENV). Regrettably until now, specific treatment for the disease has not been established. It has been reported that a human glycolytic enzyme, the human hexokinase II (HKII) has a great impact in supporting viral replication in the host cell, thus the enzyme has been proposed as an anti-DENV drug target. The main aim of this research is to identify novel anti-DENV agents for the treatment of dengue disease through in silico screening and HKII enzymatic inhibition studies

Expression and purification of soluble bacterially-expressed human hexokinase II in E.coli system

Human hexokinase II (HKII) is one of the key enzymes in the glycolytic pathway. It has been postulated that HKII is a potential target for anti-dengue (DENV) drug development, as well as involved in cancer and tumor cell growth. In this work, the human hexokinase II (HKII) gene was cloned into pETite N-His SUMO vector and transformed into the E.coli strain HI-control 10G for the propagation of clones. Two different expression hosts, E.coli HI-controlTM BL21 (DE3) and BL21 (DE3) pLysS were used to optimize HKII expression. In order to obtain the soluble recombinant HKII in a functional form, we optimized protein expression at three different temperatures; 17°C, 25°C and 37°C, at 24 hours incubation time. The soluble protein was expressed in the presence of 0.5 mM isopropyl-2-Dthiogalactopyranoside (IPTG) in TB media at 17°C for 24 hrs. The expressed protein was then purified to homogeneity by a combination of Immobilized Metal Ion Affinity Chromatography (IMAC), size exclusion chromatography (SEC) and ion-exchange chromatography (IEX), resulting in pure bacterially-expressed HK2. Taken together, this study has successfully produced soluble bacterially-expressed human HKII that can be utilized for further therapeutic studies

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

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

Effects of Daidzin and analogue of Ganoderma sinense on bacterially-expressed human hexokinase isoform 2 for anti-dengue drug design

Dengue disease, which is caused by dengue virus (DENV) has been a major worldwide concern, with increased number of cases each year. Currently, there are no specific medications to treat the disease. Hence, there is a dire need to develop novel drugs for disease treatment. Glycolysis is a metabolic pathway that serves as the main source of energy for DENV replication and targeting the pathway is one of the ideal approach to discover new anti-DENV drugs. This paper focuses on the inhibition of the human hexokinase isoform 2 (HK2) enzyme, which is one of the important enzymes in glycolysis, in the quest to disrupt DENV replication. In order to search for potential inhibitors, two methods were conducted, which are ligand-based screening and structure-based screening approaches. The docking of Daidzin, which was derived from Kudzu, a Japanese plant, has shown the nearest binding affinity score (-7.94 kcal/mol) to Glucose‘s (GLC), which is -8.15 kcal/mol. Meanwhile, from the ligand-based screening, Ethyl (2R)-2-[[3-[2-[(4-methylbenzoyl)amino]ethyl]-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]sulfanyl]butanoate, a compound which is the analogue of Ganoderma sinense with a binding score of -8.43 kcal/mol was chosen for the subsequent inhibition studies. These compounds were further analysed in an inhibition assay to determine the effects of of the potential naturally-derived inhibitors on the activity of HK2. The outcome from the inhibition study shows that both compounds exhibited substantial inhibition on HK2 enzyme, where Daidzin, at 0.5 mM, resulted in HK2 remaining activity of 88.98%, while Ethyl (2R)-2-[[3-[2-[(4-methylbenzoyl)amino]ethyl]-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]sulfanyl]butanoate (Ethyl (2R)) resulted in 69.58% of HK2 remaining activity, also at 0.5 mM concentration. In conclusion, this study has served as a platform for the development of anti-dengue drugs based on naturally-derived compounds, which is anticipated to be a safer option for dengue treatmen

Virtual screening for potential inhibitors of human hexoki-nase ii for the development of anti-dengue therapeutics

Dengue fever, which is a disease caused by the dengue virus (DENV), is a major unsolved issue in many tropical and sub-tropical regions of the world. The absence of treatment that effectively prevent further viral propagation inside the human’s body resulted in a high number of deaths globally each year. Thus, novel anti-dengue therapies are required for effective treatment. Hu-man hexokinase II (HKII), which is the first enzyme in the glycolytic pathway, is an important drug target due to its significant impact on viral replication and survival in host cells. In this study, 23.1 million compounds were computationally-screened against HKII using the Ultrafast Shape Recognition with a CREDO Atom Types (USRCAT) algorithm. In total, 300 compounds with the highest similarity scores relative to three reference molecules, known as Al-pha-D-glucose (GLC), Beta-D-glucose-6-phosphate (BG6), and 2-deoxyglucose (2DG), were aligned. Of these 300 compounds, 165 were chosen for further structure-based screening, based on their similarity scores, ADME analysis, the Lipinski’s Rule of Five, and virtual toxicity test results. The selected analogues were subsequently docked against each domain of the HKII structure (PDB ID: 2NZT) using AutoDock Vina programme. The three top-ranked compounds for each query were then selected from the docking results based on their binding energy, the number of hydrogen bonds formed, and the specific catalytic residues. The best docking results for each analogue were observed for the C-terminus of Chain B. The top-ranked analogues of GLC, compound 10, compound 26, and compound 58, showed predicted binding energies of −7.2, −7.0, and −6.10 kcal/mol and 7, 5, and 2 hydrogen bonds, respectively. The analogues of BG6, compound 30, compound 36, and compound 38, showed predicted binding energies of −7.8, −7.4, and −7.0 kcal/mol and 11, 9, and 5 hydrogen bonds, while the top three analogues of 2DG, known as compound 1, compound 4, and compound 31, showed predicted binding energies of −6.8, −6.3, and −6.3 kcal/mol and 4, 3, and 1 hydrogen bonds, sequentially. The highest-ranked compounds in the docking analysis were then selected for molecular dynamics simulation, where compound 10, compound 30, and compound 1, which are the analogues of GLC, BG6, and 2DG, have shown strong protein-ligand stability with an RMSD value of ±5.0 A° with a 5 H bond, ±4.0 A° with an 8 H bond, and ±0.5 A° with a 2 H bond, respectively, compared to the reference molecules throughout the 20 ns simulation time. Therefore, by using the computational studies, we pro-posed novel compounds, which may act as potential drugs against DENV by inhibiting HKII’s activity

Virtual screening for potential inhibitors of human hexokinase II for the development of anti-dengue therapeutics

Dengue fever, which is a disease caused by the dengue virus (DENV), is a major unsolved issue in many tropical and sub-tropical regions of the world. The absence of treatment that effectively prevent further viral propagation inside the human’s body resulted in a high number of deaths globally each year. Thus, novel anti-dengue therapies are required for effective treatment. Hu-man hexokinase II (HKII), which is the first enzyme in the glycolytic pathway, is an important drug target due to its significant impact on viral replication and survival in host cells. In this study, 23.1 million compounds were computationally-screened against HKII using the Ultrafast Shape Recognition with a CREDO Atom Types (USRCAT) algorithm. In total, 300 compounds with the highest similarity scores relative to three reference molecules, known as Al-pha-D-glucose (GLC), Beta-D-glucose-6-phosphate (BG6), and 2-deoxyglucose (2DG), were aligned. Of these 300 compounds, 165 were chosen for further structure-based screening, based on their similarity scores, ADME analysis, the Lipinski’s Rule of Five, and virtual toxicity test results. The selected analogues were subsequently docked against each domain of the HKII structure (PDB ID: 2NZT) using AutoDock Vina programme. The three top-ranked compounds for each query were then selected from the docking results based on their binding energy, the number of hydrogen bonds formed, and the specific catalytic residues. The best docking results for each analogue were observed for the C-terminus of Chain B. The top-ranked analogues of GLC, compound 10, compound 26, and compound 58, showed predicted binding energies of −7.2, −7.0, and −6.10 kcal/mol and 7, 5, and 2 hydrogen bonds, respectively. The analogues of BG6, compound 30, compound 36, and compound 38, showed predicted binding energies of −7.8, −7.4, and −7.0 kcal/mol and 11, 9, and 5 hydrogen bonds, while the top three analogues of 2DG, known as compound 1, compound 4, and compound 31, showed predicted binding energies of −6.8, −6.3, and −6.3 kcal/mol and 4, 3, and 1 hydrogen bonds, sequentially. The highest-ranked compounds in the docking analysis were then selected for molecular dynamics simulation, where compound 10, compound 30, and compound 1, which are the analogues of GLC, BG6, and 2DG, have shown strong protein-ligand stability with an RMSD value of ±5.0 A° with a 5 H bond, ±4.0 A° with an 8 H bond, and ±0.5 A° with a 2 H bond, respectively, compared to the reference molecules throughout the 20 ns simulation time. Therefore, by using the computational studies, we pro-posed novel compounds, which may act as potential drugs against DENV by inhibiting HKII’s activity