Highly specific inactivation of triosephosphate isomerase from Trypanosoma cruzi

doi:10.1016/S0006-291X(02)00796-9

Biochemical and Biophysical Research Communications

Volume 295, Issue 4, 26 July 2002, Pages 958-963

https://doi.org/10.1016/S0006-291X(02)00796-9 Get rights and content

Abstract

We searched for molecules that selectively inactivate homodimeric triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the parasite that causes Chagas' disease. We found that some benzothiazoles inactivate the enzyme. The most potent were 3-(2-benzothiazolylthio)-propanesulfonic acid, 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid, and 2-(2-4(4-aminophenyl)benzothiazole-6-methylbenzothiazole-7-sulfonic acid. Half-maximal inactivation by these compounds was attained with 33, 56, and $8 μM$ , respectively; in human TIM, half-maximal inactivation required $422 μM$ , 3.3 mM, and 1.6 mM. In TcTIM, the effect of the benzothiazoles decreased as the concentration of the enzyme was increased. TcTIM has a cysteine (Cys 15) at the dimer interface, whereas human TIM has methionine in that position. In M15C human TIM, the benzothiazole concentrations that caused half-maximal inactivation were much lower than in the wild type. The overall findings suggest that the benzothiazoles perturb the interactions between the two subunits of TcTIM through a process in which the interface cysteine is central in their deleterious action.

Section snippets

Materials and methods

Chemicals. The benzothiazoles that were studied are given in Fig. 1. Except for 10 which was obtained from ICN, the rest were obtained from Aldrich. Compounds 1–8 were more than 95% pure; compound 9 was of unknown purity (Sigma–Aldrich Library of Rare Chemicals); and 10 was 75% pure. The two latter compounds were purified. For 9, 500 mg commercial product was stirred in 40 ml chloroform for 30 min; the residue was collected and taken to dryness. For the purification of 10, 250 mg product was mixed

Results

The benzothiazoles that were assessed are given in Fig. 1. At a concentration of $250 μM$ , only some benzothiazoles inactivated TcTIM. For example, compound 3 had no effect, 1 brought about moderate inactivation, whereas 8, 9, and 10 caused strong inactivation. The effect of the latter three benzothiazoles on TcTIM was studied in more detail. Some experiments were also carried out with 1.

The inactivating effect of 1, 8, 9, and 10 was concentration dependent (Fig. 2). However, it is noted that the

Discussion

Other groups have searched for agents that perturb the interactions between the two monomers of TIM from parasites. Singh et al. [10] found that a peptide that mimics loop 3 of TIM from P. falciparum inactivated the enzyme. Likewise, Kunz et al. [25] tested several peptides that mimicked the β turns of loop 3 on TIM from T. brucei; they found that the peptides did not affect the activity of that TIM, albeit they found that other peptides exerted inhibitory activity at low concentrations. Here,

Acknowledgements

The authors are indebted to Dr. Rosario Muñoz Clares for helpful discussions. The technical assistance of Nallely Cabrera, Maria Isabel Chávez Uribe, and Maria Elena Orduña is greatly appreciated. This work was supported by Grant No. G27551M from Consejo Nacional de Ciencia y Tecnologı́a, México (to A.G.P.) and Grant No. IN200600 from Dirección General de Apoyo a Personal Académico, UNAM (to R.P.M.).

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Examination of multiple Trypanosoma cruzi targets in a new drug discovery approach for Chagas disease
2022, Bioorganic and Medicinal Chemistry
Chagas disease (CD) is a centenarian neglected parasitosis caused by the protozoan Trypanosoma cruzi (T. cruzi). Despite the continuous efforts of many organizations and institutions, CD is still an important human health problem worldwide. A lack of a safe and affordable treatment has led drug discovery programmes to focus, for years, on the search for molecules enabling interference with enzymes that are essential for T. cruzi survival. In this work, the authors want to offer a brief overview of the different validated targets that are involved in diverse parasite pathways: glycolysis, sterol synthesis, the de novo biosynthesis of pyrimidine nucleotides, the degradative processing of peptides and proteins, oxidative stress damage and purine salvage and nucleotide synthesis and metabolism. Their structural aspects, function, active sites, etc. were studied and considered with the aim of defining molecular bases in the search for new effective treatments for CD. This review also compiles, as much as possible, all the inhibitors reported to date against these T. cruzi targets, serving as a reference for future research in this field.
Developing a new drug against trichomoniasis, new inhibitory compounds of the protein triosephosphate isomerase
2020, Parasitology International
Citation Excerpt :
All assays were done in independent triplicates, and the average error for each measurement did not exceed 10%. To test the effect of potential ligands on the enzymatic activity of TvTIM, each protein was preincubated for 2 h at 25 °C with each of the potential ligands, at 30 μgmL−1 in 1% DMSO [29–34] before activity assays. Fluorescence spectra were obtained using a LS-55 Spectrofluorometer (Perkin-Elmer), equipped with a water-jacketed cell holder for temperature control at 25 °C.
Trichomonas vaginalis is the protozoan parasite responsible for the most prevalent, non-viral, sexually transmitted disease, which affects millions of people around the world. The main treatment against this disease is metronidazole and some other nitroimidazole derivatives. However, between five and 20% of clinical cases of trichomoniasis are caused by parasites resistant to these drugs. Here we present three compounds that were selected using an innovative strategy, to propose them as possible drugs to combat trichomoniasis, using the glycolytic enzyme triose phosphate isomerase (TvTIM) as the drug target. In the genome of Trichomonas vaginalis there are two genes that encode for two isoforms of TvTIM, known as TvTIM1 and TvTIM2, varying by four out of 254 aminoacid residues. In this study, we used high-throughput virtual screening to search molecules that bind specifically to TvTIM isoforms, in which 34 compounds were selected from a library of nearly 450,000 compounds. The effects of the 34 compounds on the conformation and enzymatic activity of both TvTIM isoforms and their human homolog (HsTIM) were evaluated. We found three compounds that bind specifically, modify the conformation and inhibit TvTIM2 only; although the sequence of both isoforms of TvTIM is almost identical. The selectivity of these compounds towards TvTIM2 is explained by the lower conformational stability of this isoform and that these interactions can inhibit the activity of this enzyme and have an effect against this parasite. These compounds represent promising alternatives for the development of new therapeutic strategies against trichomoniasis.
Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei
2016, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
The TIM dimer interface is a starting point towards rational drug design against diseases originated by protozoan parasites that heavily depend on glycolysis for energy [15]. Molecules that interact with exposed cysteines like methylmethane thiosulfonate (MMTS) selectively inhibit TIMs from Leishmania donovani [18], T. cruzi [19], T. brucei [20] and Giardia lamblia [21]. The strength of the inhibition is dependent on the degree of conjugation of a reactive cysteine (Cys15 in the case of T. cruzi) and the perturbation of the interactions at the homodimeric interface.
Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166–176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a “ball and socket” interaction. LvTIM presents a Cys47 at the “ball” inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.
Synthesis and trypanocidal activity of novel benzimidazole derivatives
2016, Bioorganic and Medicinal Chemistry Letters
The present work reports the synthesis and biological activity of a series of 14 benzimidazole derivatives designed to act on the enzyme triosephosphate isomerase of Trypanosoma cruzi (TcTIM). This enzyme is involved in the metabolism of glucose, the only source of energy for the parasite. In this study, we found four compounds that inhibit TcTIM moderately and lack inhibitory activity against human TIM (HsTIM). In vitro studies against T. cruzi epimastigotes showed two compounds that were more active than the reference drug nifurtimox, and these presented a low cytotoxic effect in mouse macrophages (J744 cell line).
How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics
2015, Biophysical Journal
Citation Excerpt :
Subunit interfaces of oligomeric enzymes, including TIM, may serve as species-specific drug target sites that are especially important for parasitic diseases (4) since they are generally less conserved than the active site (4,18,19). Several benzothiazole derivatives have been reported as candidate drugs against Chagas disease because they inhibit TcTIM’s activity (5,19,20). One such inhibitor, referred to as bt10 (Fig. 1b), binds to the specific tunnel region at TcTIM’s interface (5,19).
The tunnel region at triosephosphate isomerase (TIM)’s dimer interface, distant from its catalytic site, is a target site for certain benzothiazole derivatives that inhibit TIM’s catalytic activity in Trypanosoma cruzi, the parasite that causes Chagas disease. We performed multiple 100-ns molecular-dynamics (MD) simulations and elastic network modeling (ENM) on both apo and complex structures to shed light on the still unclear inhibitory mechanism of one such inhibitor, named bt10. Within the time frame of our MD simulations, we observed stabilization of aromatic clusters at the dimer interface and enhancement of intersubunit hydrogen bonds in the presence of bt10, which point to an allosteric effect rather than destabilization of the dimeric structure. The collective dynamics dictated by the topology of TIM is known to facilitate the closure of its catalytic loop over the active site that is critical for substrate entrance and product release. We incorporated the ligand’s effect on vibrational dynamics by applying mixed coarse-grained ENM to each one of 54,000 MD snapshots. Using this computationally efficient technique, we observed altered collective modes and positive shifts in eigenvalues due to the constraining effect of bt10 binding. Accordingly, we observed allosteric changes in the catalytic loop’s dynamics, flexibility, and correlations, as well as the solvent exposure of catalytic residues. A newly (to our knowledge) introduced technique that performs residue-based ENM scanning of TIM revealed the tunnel region as a key binding site that can alter global dynamics of the enzyme.
Development of bis-thiazoles as inhibitors of triosephosphate isomerase from Trypanosoma cruzi. Identification of new non-mutagenic agents that are active in vivo
2015, European Journal of Medicinal Chemistry
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 5 in 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.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

References (25)

Using evolutionary changes to achieve species-specific inhibition of enzyme action studies with triosephosphate isomerase

Chem. Biol.

Triosephosphate isomerase from Plasmodium falciparum: the crystal structure provides insights into antimalarial drug design

Structure

Three hTIM mutants that provide new insights on why TIM is a dimer

J. Mol. Biol.

Synthetic peptides as inactivators of multimeric enzymes: inhibition of Plasmodium falciparum triosephosphate isomerase by interface peptides

FEBS Lett.

Inhibition of Plasmodium falciparum triosephosphate isomerase by chemical modification of an interface cysteine: electrospray ionization mass spectrometric analysis of differential cysteine reactivities

J. Biol. Chem.

The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

Proteins Struct. Funct. Genet.

A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability

Biochemistry

Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues. The cysteine residues of triosephosphate isomerase

Eur. J. Biochem.

Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme

Biochemistry

Refolding of triosephosphate isomerase

Biochem. J.

Cited by (61)

Examination of multiple Trypanosoma cruzi targets in a new drug discovery approach for Chagas disease

Developing a new drug against trichomoniasis, new inhibitory compounds of the protein triosephosphate isomerase

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

Synthesis and trypanocidal activity of novel benzimidazole derivatives

How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics

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