Inhibition Of Triosephosphate Isomerase Research Articles

Triosephosphate Isomerase Inhibition by Resveratrol: A New Mechanism of Anti-Glycolysis in Breast Cancer

Triosephosphate Isomerase Inhibition by Resveratrol: A New Mechanism of Anti-Glycolysis in Breast Cancer

Ligand-Based Virtual Screening and Molecular Docking of Benzimidazoles as Potential Inhibitors of Triosephosphate Isomerase Identified New Trypanocidal Agents.

Trypanosoma cruzi (T. cruzi) is a parasite that affects humans and other mammals. T. cruzi depends on glycolysis as a source of adenosine triphosphate (ATP) supply, and triosephosphate isomerase (TIM) plays a key role in this metabolic pathway. This enzyme is an attractive target for the design of new trypanocidal drugs. In this study, a ligand-based virtual screening (LBVS) from the ZINC15 database using benzimidazole as a scaffold was accomplished. Later, a molecular docking on the interface of T. cruzi TIM (TcTIM) was performed and the compounds were grouped by interaction profiles. Subsequently, a selection of compounds was made based on cost and availability for in vitro evaluation against blood trypomastigotes. Finally, the compounds were analyzed by molecular dynamics simulation, and physicochemical and pharmacokinetic properties were determined using SwissADME software. A total of 1604 molecules were obtained as potential TcTIM inhibitors. BP2 and BP5 showed trypanocidal activity with half-maximal lytic concentration (LC50) values of 155.86 and 226.30 µM, respectively. Molecular docking and molecular dynamics simulation analyzes showed a favorable docking score of BP5 compound on TcTIM. Additionally, BP5 showed a low docking score (−5.9 Kcal/mol) on human TIM compared to the control ligand (−7.2 Kcal/mol). Both compounds BP2 and BP5 showed good physicochemical and pharmacokinetic properties as new anti-T. cruzi agents.

Recent Advances in the Development of Triose Phosphate Isomerase Inhibitors as Antiprotozoal Agents.

Parasitic diseases caused by protozoa, such as Chagas disease, leishmaniasis, malaria, African trypanosomiasis, amoebiasis, trichomoniasis, and giardiasis, are considered serious public health problems in developing countries. Drug resistance among parasites justifies the search for new therapeutic drugs, and the identification of new targets becomes a valuable approach. In this scenario, the glycolysis pathway, which converts glucose into pyruvate, plays an important role in the protozoa energy supply, and it is therefore considered a promising target. In this pathway, triose phosphate isomerase (TIM) plays an essential role in efficient energy production. Furthermore, protozoa TIM shows structural differences with human enzyme counterparts, suggesting the possibility of obtaining selective inhibitors. Therefore, TIM is considered a valid approach to develop new antiprotozoal agents, inhibiting the glycolysis in the parasite. In this review, we discuss the drug design strategies, structure-activity relationship, and binding modes of outstanding TIM inhibitors against Trypanosoma cruzi, Trypanosoma brucei, Plasmodium falciparum, Giardia lamblia, Leishmania mexicana, Trichomonas vaginalis, and Entamoeba histolytica. TIM inhibitors have mainly shown aromatic systems and symmetrical structure, where the size and type of heteroatom are important for enzyme inhibition. This inhibition is mainly based on the interaction with i) the interfacial region of TIM inducing changes on the quaternary and tertiary structure or ii) with the TIM catalytic region, the main pathways that disable the catalytic activity of the enzyme. Benzothiazole, benzoxazole, benzimidazole, and sulfhydryl derivatives stand out as TIM inhibitors. In silico and in vitro studies have demonstrated that the inhibitors bind mainly at the TIM dimer interface. In this review, the development of new TIM inhibitors as antiprotozoal drugs is demonstrated as an important pharmaceutical strategy that may lead to new therapies for these ancient parasitic diseases.

Esters of quinoxaline-7-carboxylate-1,4-di-N-oxide as Trichomonas vaginalis triosephosphate isomerase inhibitors.

Trichomoniasis is a public health problem worldwide. Trichomoniasis treatment consists of the use of nitroimidazole derivatives; however, therapeutic ineffectiveness occurs in 5 to 20 % of the cases. Therefore, it is essential to propose new pharmacological agents against this disease. In this work, esters of quinoxaline-7-carboxylate-1,4-di-N-oxide (EQX-NO) were evaluated in in vitro assays as novel trichomonicidal agents. Additionally, an in vitro enzyme assay and molecular docking analysis against triosephosphate isomerase of Trichomonas vaginalis to confirm their mechanism of action were performed. Ethyl (compound 12) and n-propyl (compound 37) esters of quinoxaline-7-carboxy-late-1,4-di-N-oxide derivatives showed trichomonicidal activity comparable to nitazoxanide, whereas five methyl (compounds 5, 15, 19, 20 and 22), four isopropyl (compounds 28, 29, 30 and 34), three ethyl (compounds 4, 13 and 23) and one npropyl (compound 35) ester derivatives displayed activity comparable to albendazole. Compounds 6 and 20 decreased 100 % of the enzyme activity of recombinant protein triosephosphate isomerase.

Enhanced Antigiardial Effect of Omeprazole Analog Benzimidazole Compounds.

Giardiasis is a diarrheal disease that is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy are common, and have adverse effects and increased resistance of the parasite to the drug, generating the need to find new alternative treatments. In this study, we synthesized a series of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were confirmed through mass, 1H NMR, and 13C NMR techniques. The in vitro efficacy compounds against Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for 2 h at 37 °C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target compounds, H-BZM2, O2N-BZM7, and O2N-BZM9 had greater antigiardial activity (IC50: 36, 14, and 17 µM on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2, and 2.8 M−1 s−1) respectively, loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein. Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Triosephosphate Isomerase Inhibitors as Potential Drugs against Clostridium perfringens

Triosephosphate Isomerase Inhibitors as Potential Drugs against <i>Clostridium perfringens</i>

A Cytosolic Bypass and G6P Shunt in Plants Lacking Peroxisomal Hydroxypyruvate Reductase.

The oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicate that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-phosphoglycolate accumulation and cyclic electron flow in Arabidopsis (Arabidopsis thaliana). We found that 2-phosphoglycolate is a competitive inhibitor of triose phosphate isomerase, an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol, where cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as glucose-6-phosphate, forming a cytosolic bypass around the block of stromal triose phosphate isomerase. However, this also stimulates a glucose-6-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.

Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

Novel and Selective Rhipicephalus microplus Triosephosphate Isomerase Inhibitors with Acaricidal Activity.

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites causing significant economic losses for the cattle industry. The major tool of control is reducing the number of ticks, applying acaricides in cattle. However, overuse has led to selection of resistant populations of R. microplus to most of these products, some even to more than one active principle. Thus, exploration for new molecules with acaricidal activity in R. microplus has become necessary. Triosephosphate isomerase (TIM) is an essential enzyme in R. microplus metabolism and could be an interesting target for the development of new methods for tick control. In this work, we screened 227 compounds, from our in-house chemo-library, against TIM from R. microplus. Four compounds (50, 98, 14, and 161) selectively inhibited this enzyme with IC50 values between 25 and 50 μM. They were also able to diminish cellular viability of BME26 embryonic cells by more than 50% at 50 μM. A molecular docking study showed that the compounds bind in different regions of the protein; compound 14 interacts with the dimer interface. Furthermore, compound 14 affected the survival of partially engorged females, fed artificially, using the capillary technique. This molecule is simple, easy to produce, and important biological data—including toxicological information—are available for it. Our results imply a promising role for compound 14 as a prototype for development of a new acaricidal involving selective TIM inhibition.

The Photorespiratory Metabolite 2-Phosphoglycolate Regulates Photosynthesis and Starch Accumulation in Arabidopsis.

The Calvin-Benson cycle and its photorespiratory repair shunt are in charge of nearly all biological CO2 fixation on Earth. They interact functionally and via shared carbon flow on several levels including common metabolites, transcriptional regulation, and response to environmental changes. 2-Phosphoglycolate (2PG) is one of the shared metabolites and produced in large amounts by oxidative damage of the CO2 acceptor molecule ribulose 1,5-bisphosphate. It was anticipated early on, although never proven, that 2PG could also be a regulatory metabolite that modulates central carbon metabolism by inhibition of triose-phosphate isomerase. Here, we examined this hypothesis using transgenic Arabidopsis thaliana lines with varying activities of the 2PG-degrading enzyme, 2PG phosphatase, and analyzing the impact of this intervention on operation of the Calvin-Benson cycle and other central pathways, leaf carbohydrate metabolism, photosynthetic gas exchange, and growth. Our results demonstrate that 2PG feeds back on the Calvin-Benson cycle. It also alters the allocation of photosynthates between ribulose 1,5-bisphosphate regeneration and starch synthesis. 2PG mechanistically achieves this by inhibiting the Calvin-Benson cycle enzymes triose-phosphate isomerase and sedoheptulose 1,7-bisphosphate phosphatase. We suggest this may represent one of the control loops that sense the ratio of photorespiratory to photosynthetic carbon flux and in turn adjusts stomatal conductance, photosynthetic CO2 and photorespiratory O2 fixation, and starch synthesis in response to changes in the environment.

In silico investigation of morpholines as novel class of trypanosomal triosephosphate isomerase inhibitors

Human African trypanosomiasis, also known as sleeping sickness, is caused by the single-celled kinetoplastid parasite Trypanosoma brucei transmitted to humans by infected tsetse flies. The disease threatens millions of people. Currently available treatment options are faced with some important challenges. In this work, a total of eighty-seven (87) morpholine derivatives were evaluated for drug-likeness based on Lipinski’s rule of five and their ability to inhibit the activities of trypanosomal triosephosphate isomerase was assessed by molecular docking and calculation of free energy of binding. Analysis of the results revealed that 97.7 % of the dataset complied with Lipinski’s criteria for a molecule to be orally bioavailable. Also, 50.0 % of the studied compounds had a good total polar surface area profile, a parameter which is of great importance for the treatment of stage two Trypanosoma infections. Docking studies showed that all the dataset demonstrated affinity for triosephosphate isomerase. Moreover, six morpholines scored higher than the co-crystallized inhibitor of triosephosphate isomerase (2-phosphoglycerate). These derivatives inhibited the activity of triosephosphate isomerase by making significant interactions with Glu 167, Val 214, 233, Asn 11, Lys 13, Ser 213, Leu 232, Ile 172, Gly 211, 212, 234, 235 and His 92 in the active site of the protein. Furthermore, besides the acceptable pharmacokinetic profiles of the six morpholines, they also showed inhibitory potencies toward four other validated antitrypanosomal drug targets. In view of the foregoing findings, we propose that the six morpholine derivatives be given worthwhile attention to develop them into novel trypanocides.

Development of bis-thiazoles as inhibitors of triosephosphate isomerase from Trypanosoma cruzi. Identification of new non-mutagenic agents that are active in vivo

The neglected disease American trypanosomiasis is one of the major health problems in Latin America. Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the etiologic agent of this disease, has been proposed as a druggable target. Some bis-benzothiazoles have been described as irreversible inhibitors of this enzyme. On the other hand, new bioactive furane-containing thiazoles have been described as excellent in vivo anti-T. cruzi agents. This encouraged us to design and develop new bis-thiazoles with potential use as drugs for American trypanosomiasis. The bis-thiazol 5, 3,3′-allyl-2,2′-bis[3-(2-furyl)-2-propenylidenehydrazono]-2,2′,3,3′-tetrahydro-4,4′-bisthiazole, showed the best in vitro anti-T. cruzi profile with a higher selectivity index than the reference drugs Nifurtimox and Benznidazole against amastigote form of the parasite. This derivative displayed marginal activity against TcTIM however the bis-thiazol 14, 3-allyl-2-[3-(2-furyl)-2-propenylidenehydrazono]-3′-phenyl-2′-(3-phenyl-2-propenylidenehydrazono]-2,2′,3,3’-tetrahydro-4,4′-bisthiazole, was an excellent inhibitor of the enzyme of the parasite. The absence of both in vitro mutagenic and in vivo toxicity effects, together with the activity of bis-thiazol 5in vivo, suggests that this compound is a promising anti-T. cruzi agent surpassing the “hit-to-lead” stage in the drug development process. ©2015 Elsevier Science Ltd. All rights reserved.

Molecular docking and molecular dynamics simulation studies of Trypanosoma cruzi triosephosphate isomerase inhibitors. Insights into the inhibition mechanism and selectivity

Trypanosoma cruzi (T. cruzi) triosephosphate isomerase (TcTIM) is a glycolytic enzyme essential for parasite survival and has been considered an interesting target for the development of new antichagasic compounds. The homodimeric enzyme is catalytically active only as a dimer. Interestingly, significant differences exist between the human and parasite TIMs interfaces with a sequence identity of 52%. Therefore, compounds able to specifically disrupt TcTIM but not Homo sapiens TIM (hTIM) dimer interface could become selective antichagasic drugs. In the present work, the binding modes of 1,2,4-thiadiazol, phenazine and 1,2,6-thiadiazine derivatives to TcTIM were investigated using molecular docking combined with molecular dynamics (MD) simulations. The results show that phenazine and 1,2,6-thiadiazine derivatives, 2 and 3, act as dimer-disrupting inhibitors of TcTIM having also allosteric effects in the conformation of the active site. On the other hand, the 1,2,4-thiadiazol derivative 1 binds into the active site causing a significant decrease in enzyme mobility in both monomers. The loss of conformational flexibility upon compound 1 binding suggests that this inhibitor could be preventing essential motions of the enzyme required for optimal activity. The lack of inhibitory activity of 1 against hTIM was also investigated and seems to be related with the high mobility of hTIM which would hinder the formation of a stable ligand–enzyme complex. This work has contributed to understand the mechanism of action of this kind of inhibitors and could result of great help for future rational novel drug design.

A novel inhibitor of Mammalian triosephosphate isomerase found by an in silico approach.

Triosephosphate isomerase (TIM) is an essential, highly conserved component of glycolysis. Tumors are often dependent on glycolysis for energy and metabolite production (the Warburg effect). Glycolysis inhibitors thus show promise as cancer treatments. TIM inhibition, unlike inhibition of other glycolysis enzymes, also produces toxic methylglyoxal targeted to regions of high glycolysis, an effect that might also be therapeutically useful. Thus TIM is an attractive drug target. A total of 338,562 lead-like molecules were analyzed computationally to find TIM inhibitors by an efficient “double screen” approach. The first fragment-sized compounds were studied using structure-based virtual screening to identify binding motifs for mammalian TIM. Subsequently, larger compounds, filtered to meet the binding criteria developed in the first analysis, were ranked using a second round of structure-based virtual screening. A compound was found that inhibited mammalian TIM in vitro in the micromolar range. Docking and molecular dynamics (MD) suggested that the inhibitor made hydrogen bond contacts with TIM catalytic residues. In addition, hydrophobic contacts were made throughout the binding site. All predicted inhibitor-TIM interactions involved TIM residues that were highly conserved. The discovered compound may provide a scaffold for elaboration of other inhibitors.

Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK–TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.

A Phosphoglycolate Phosphatase from Saccharomyces cerevisiae

The nitrophenyl phosphatase family of the Haloacid Dehalogenase superfamily consists of a number of phosphatases. The homolog in S. cerevisiae was only known to hydrolyze p‐nitrophenyl phosphate. BLAST searches and alignments indicated that PHO13 from S. cerevisiae was most similar to an algae PGPase. We cloned and overexpressed PHO13 and determined that PHO13 is a PGPase. In photosynthetic organisms, phosphoglycolate is generated in the Calvin cycle; how it is produced in nonphotosynthetic organisms is less clear. Since it a strong inhibitor of triose phosphate isomerase, it must be degraded, regardless of its origin. The PGPase from S. aureus is a virulence factor, indicating its importance. To better understand the role of PGPase in the cell, we are using yeast as a model organism, studying a pho13 knockout mutant and doing complementation studies. Enzymatic characterization, including substrate specificity, pH optimum, divalent metal ion requirements, and kinetics, has been completed, and crystal growth for structural determination is in progress. This research has been supported by an NIH AREA grant, an ASM summer undergraduate research fellowship, and a RIT honors summer research fellowship.

New chemotypes as Trypanosoma cruzi triosephosphate isomerase inhibitors: a deeper insight into the mechanism of inhibition

Context: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of new small molecular weight compounds used against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified phenazine and 1,2,6-thiadiazine chemotypes as novel inhibitors of TIM from T. cruzi (TcTIM).Objective: Study the mechanism of TcTIM inhibition by a phenazine derivative and by a 1,2,6-thiadiazine derivative.Methods: We performed biochemical and theoretical molecular docking studies to characterize the interaction of the derivatives with wild-type and mutant TcTIM.Results and conclusion: At low micromolar concentrations, the compounds induce highly selective irreversible inactivation of parasitic TIM. The molecular docking simulations indicate that the phenazine derivative likely interferes with the association of the two monomers of the dimeric enzyme by locating at the dimer interface, while 1,2,6-thiadiazine could act as an inhibitor binding to a region surrounding Cys-118.

Synthesis and theoretic calculations of benzoxazoles and docking studies of their interactions with triosephosphate isomerase

One-pot synthesis was carried out for Z or E stereoisomer derivates of 3-(benzoxazoyl)-2-propenoic acid following kinetic or thermodynamic control. All compounds were characterized by 1H and 13C NMR, and the single crystal X-ray structure of (2Z)-3-(6-methyl-1,3-benzoxazol-2-yl)prop-2-enoic acid (3) was obtained. Furthermore, a theoretic study was done for all the synthesized compounds at the B3LYP/6-31G(d,p) level. The target compounds were docked on triosephosphate isomerase and trypanocidal activity was explored for the 4 and 6 compounds. The Z isomers showed an intramolecular hydrogen bond O–H···N according to the X-ray structure of 3. The docking studies indicate that the test compounds insert themselves between the monomers of triosephosphate isomerase, reaching the known binding site located at interdimeric shapes of triosephosphate isomerase by means of π–π interactions and electrostatic interactions, and in this way interrupt interactions between these monomers. Thus, could explain the biologic effects of the E isomer on triosephosphate isomerase. Finally, compounds 4 and 6 showed trypanocidal activity, which could be mediated by triosephosphate isomerase inhibition.

Blind Dockings of Benzothiazoles to Multiple Receptor Conformations of Triosephosphate Isomerase from Trypanosoma cruzi and Human.

We aim to uncover the binding modes of benzothiazoles, which have been reported as specific inhibitors of triosephosphate isomerase from the parasite Trypanosoma cruzi (TcTIM), by performing blind dockings on both TcTIM and human TIM (hTIM). Detailed analysis of binding sites and specific interactions are carried out based on ensemble dockings to multiple receptor conformers obtained from molecular dynamics simulations. In TcTIM dimer dockings, the inhibitors preferentially bind to the tunnel-shaped cavity formed at the interface of the subunits, whereas non-inhibitors mostly choose other sites. In contrast, TcTIM monomer binding interface and hTIM dimer interface do not present a specific binding site for the inhibitors. These findings point to the importance of the tunnel and of the dimeric form for inhibition of TcTIM. Specific interactions of the inhibitors and their sulfonate-free derivatives with the receptor residues indicate the significance of sulfonate group for binding affinity and positioning on the TcTIM dimer interface. One of the inhibitors also binds to the active site, which may explain its relatively higher inhibition effect on hTIM.

On the Self-Assembly of a Highly Selective Benzothiazole-Based TIM Inhibitor in Aqueous Solution

Benzothiazole is a common scaffold on which many bioactive structures, including protein inhibitors and biosensors, are based. The potential self-aggregation of such molecules to form nanoparticles is relevant for a number of practical applications. 3-(2-Benzothiazolylthio)-propanesulfonic acid (BTS) has been reported as a powerful and selective inhibitor of triosephosphate isomerase from Trypanosoma cruzi, the parasite that causes the Chagas' disease. Electrical conductivity, sound velocity, density, and nuclear magnetic resonance experiments as a function of temperature and of NaCl concentration have been performed in the present work to provide a comprehensive physicochemical description of this compound in aqueous solution. Molecular dynamics simulations of the same system were also performed to characterize the structure and dynamic behavior of the corresponding aggregates at several concentrations of BTS.