41 research outputs found
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 18ยฐC 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; 17ยฐC, 25ยฐC and
37ยฐC, 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 17ยฐC 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 HKIIโs substrate and product; alpha-D-glucose (GLC) and beta-D-glucose-6-phosphate (BG6), as well as a known HKIIโs inhibitor, 2-deoxyglucose (2DG), as the query molecules. The analogues of the three query molecules were subsequently docked against the HKIIโs 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. ยฉ 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 GenScriptยฎ. 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
Identifying potential inhibitors of human hexokinase II for the development of anti-dengue therapeutics via virtual screening approaches
The human hexokinase II (HK2) has been suggested as a potential therapeutic target
for the development of drugs against dengue virus (DENV) infection. In this paper,
compounds with potential HK2 inhibitory activity have been identified using ligandbased
and structure-based virtual screening approaches. Ligand-based drug design
was performed by using Ultra-Fast Shape Recognition with Atom Types (UFSRAT) and
Ultrafast Shape Recognition with CREDO Atom Types (USRCAT) programmes by
utilising 2-Deoxyglucose (2-DG) as the query molecule, which is a known HK2 inhibitor.
The molecules identified from the programmes showed great similarity to 2-DG with
scores ranged from 0.78-0.85 and 0.88-0.97 for UFSRAT and USRCAT, respectively. 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 AutoDock
Vina programme, on both A and B chains where the active sites were located. The
docking hits for molecules from UFSRAT showed binding energies ranged from -7.1 to -4.8
kcal/mol when docked on chain A, while the hits for chain B showed scores ranged
from -6.7 to -4.8 kcal/mol. On the other hand, the binding energies for molecules from
USRCAT when docked on both A and B chains were similar, which ranged from -7.0 to -
5.2 kcal/mol. The hits bind firmly at the cavities, where both GLC and BG6 were oriented
towards the active sites of HK2. Taken together, this study has successfully discovered
compounds which have potentials as potent inhibitors of HK2, thus pave the path
towards the development of dengue therapeutics
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 parasiteโs 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. ยฉ 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 Glucoseโs (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