A Molecular Docking Strategy Identifies Eosin B as a Non-active Site Inhibitor of Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase

Atreya, Chloé E.; Johnson, Eric F.; Irwin, John J.; Dow, Antonia; Massimine, Kristen M.; Coppens, Isabelle; Stempliuk, Valeska; Beverley, Stephen M.; Joiner, Keith A.; Shoichet, Brian K.; Anderson, Karen S.

doi:10.1074/jbc.m212690200

Cited by 23 publications

(17 citation statements)

References 37 publications

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“…It now seems probable that channeling instead occurs in conjunction with domain-domain communication or conformational changes induced by ligand binding at one active site that affect activity at the active site of the other enzyme. We have begun to address the coupling of channeling and communication through investigation of a small molecule inhibitor that binds in the channel region (3). Future research will focus on mechanistic and structural determinants of TS-DHFR domain-domain communication as it relates to substrate channeling with the ultimate goal of developing a nonactive site therapy for protozoal infection.…”

Section: Discussionmentioning

confidence: 99%

“…Targeting the channel between the TS and DHFR in bifunctional enzymes has the potential to produce more specific therapies, with fewer side effects than traditional active sitedirected medications, for protozoal diseases including malaria and toxoplasmosis. Previously reported results with eosin B signify an important step toward establishing proof of the principle that the putative channel region of TS-DHFR can serve as a molecular target that when inhibited, results in parasite death (3).…”

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confidence: 99%

“…In this report we present results of mutagenesis of solvent-exposed basic residues comprising the electrostatic highway. In an earlier paper, we reported the findings from targeted molecular docking searches to identify small molecule inhibitors that bind in this region, as a means to block the putative channel (3).…”

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confidence: 99%

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Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Atreya

Johnson

Williamson

et al. 2003

Journal of Biological Chemistry

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In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.Electrostatic channeling is a mechanism proposed based on the crystal structure of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) 1 from Leishmania major that would enable negatively charged dihydrofolate produced at the TS active site to be handed off along a series of solventexposed lysine and arginine residues to the DHFR active site, where it is converted to tetrahydrofolate (H 4 folate) (1).TS and DHFR are crucial enzymes, found in all species. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2Ј-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate H 4 folate, used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis. Only in protozoal parasites and some plants however, are TS and DHFR found on the same polypeptide chain, leading to the hypothesis that in these organisms, H 2 folate may be channeled from the TS active site to that of DHFR, never equilibrating with bulk solution (Fig. 1A). When the crystal structure of L. major TS-DHFR was solved (2.9 Å resolution), a 40 Å "electrostatic highway" across the surface of the protein was proposed as an explanation for how channeling may occur (1, 2). We sought to test the electrostatic channeling hypothesis with two parallel approaches. In this report we present results of mutagenesis of solvent-exposed basic residues comprising the electrostatic highway. In an earlier paper, we reported the findings from targeted molecular docking searches to identify small molecule inhibitors that bind in this region, as a means to block the putative channel (3).There are precedents for channeling among bifunction...

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Section: Discussionmentioning

confidence: 99%

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Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Atreya

Johnson

Williamson

et al. 2003

Journal of Biological Chemistry

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“…Whereas it is typically more difficult to achieve species specificity with inhibitors directed at the TS versus the DHFR active site, unique mechanistic features of C. hominis TS suggest the possibility for a specific inhibitor directed at this active site. Previous efforts to find a non-active site inhibitor specific for a bifunctional enzyme were targeted at the putative channeling region of L. major TS-DHFR (29). Mechanistic evidence against substrate channeling by C. hominis TS-DHFR, as well as structural differences, indicate that novel non-active site targets should be explored in this enzyme.…”

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Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

Atreya¹,

Anderson

2004

Journal of Biological Chemistry

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This study presents a kinetic characterization of the recently crystallized bifunctional thymidylate synthasedihydrofolate reductase (TS-DHFR) enzyme from the apicomplexa parasite, Cryptosporidium hominis. Our study focuses on determination of the C. hominis TS-DHFR kinetic mechanism, substrate channeling behavior, and domain-domain communication. Unexpectedly, the unique mechanistic features of C. hominis TS-DHFR involve the highly conserved TS domain. At 45 s ؊1 , C. hominis TS activity is 10 -40-fold faster than other TS enzymes studied and a new kinetic mechanism was required to simulate C. hominis TS behavior. A large accumulation of dihydrofolate produced at TS and a lag in product formation at DHFR were observed. These observations make C. hominis TS-DHFR the first bifunctional TS-DHFR enzyme studied for which there is clear evidence against dihydrofolate substrate channeling. Furthermore, whereas with Leishmania major TS-DHFR there are multiple lines of evidence for domain-domain communication (ligand binding at one active site affecting activity of the other enzyme), no such effects were observed with C. hominis TS-DHFR.Protozoal parasites such as Cryptosporidium hominis, Plasmodium falciparum, Toxoplasma gondii, and Leishmania major are unusual in that their thymidylate synthase (TS) 1 and dihydrofolate reductase (DHFR) enzymes exist on the same polypeptide as part of a single bifunctional TS-DHFR enzyme.2 TS and DHFR are critical enzymes and established drug targets. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate tetrahydrofolate (H 4 folate), used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis.Almost a decade of research on bifunctional TS-DHFR from protozoal parasites has relied on the only available TS-DHFR crystal structure, that from the kinetoplastid protozoa L. major (1). The recent solution of the crystal structures of TS-DHFR enzymes from two clinically relevant apicomplexan protozoa, P. falciparum and C. hominis, however, has revealed unanticipated deviations from the kinetoplastid model (2, 3). An obvious next question is whether these significant structural differences translate as significant mechanistic differences between parasite classes. We sought to address this question through a detailed kinetic characterization of TS-DHFR from C. hominis, again yielding surprising results.A major conclusion of the recent solution of the apicomplexan TS-DHFR structures is that there exist two families of bifunctional TS-DHFR structures: a short linker family with an Nterminal tail, as in the kinetoplastids (a class that includes Leishmania and the trypanosomes); and a long linker family with a donated or crossover helix, as in the apicomplexan paras...

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“…This compound, eosin B (4Ј,5Ј-dibromo-2Ј,7Ј-dinitrofluorescein), targets a unique non-active-site region on the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme (2). Inhibitors specific to this area have the potential to be powerful antiparasitics since they target folate metabolism, an essential feature of the parasitic life cycle, through a different mode of action than the currently utilized antifolate therapies.…”

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Eosin B as a Novel Antimalarial Agent for Drug-Resistant Plasmodium falciparum

Massimine

McIntosh

Doan³

et al. 2006

Antimicrob Agents Chemother

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4,5-Dibromo-2,7-dinitrofluorescein, a red dye commonly referred to as eosin B, inhibits Toxoplasma gondii in both enzymatic and cell culture studies with a 50% inhibitory concentration (IC 50 ) of 180 M. As a non-active-site inhibitor of the bifunctional T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS), eosin B offers a novel mechanism for inhibition of the parasitic folate biosynthesis pathway. In the present study, eosin B was further evaluated as a potential antiparasitic compound through in vitro and cell culture testing of its effects on Plasmodium falciparum. Our data revealed that eosin B is a highly selective, potent inhibitor of a variety of drug-resistant malarial strains, with an average IC 50 of 124 nM. Furthermore, there is no indication of cross-resistance with other clinically utilized compounds, suggesting that eosin B is acting via a novel mechanism. The antimalarial mode of action appears to be multifaceted and includes extensive damage to membranes, the alteration of intracellular organelles, and enzymatic inhibition not only of DHFR-TS but also of glutathione reductase and thioredoxin reductase. In addition, preliminary studies suggest that eosin B is also acting as a redox cycling compound. Overall, our data suggest that eosin B is an effective lead compound for the development of new, more effective antimalarial drugs.Plasmodium falciparum, the causative agent of malaria, is a protozoan parasite that has a significant impact on health worldwide. Due to the emergence and spread of resistance to antimalarial drugs, novel chemotherapeutics for the treatment of malaria are urgently needed. To achieve this goal, antiparasitic research needs not only to identify new, chemically diverse molecules for the circumvention of cross-resistance but also to discover novel targets (44). Ideal putative targets will have a variety of characteristics, including involvement in an essential feature of the parasite life cycle, parasitic selectivity over the host, and low potential for resistance development (44).Along these lines, we recently identified a compound that acts as an inhibitor for a novel chemotherapeutic target in the related apicomplexan organism Toxoplasma gondii. This compound, eosin B (4Ј,5Ј-dibromo-2Ј,7Ј-dinitrofluorescein), targets a unique non-active-site region on the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme (2). Inhibitors specific to this area have the potential to be powerful antiparasitics since they target folate metabolism, an essential feature of the parasitic life cycle, through a different mode of action than the currently utilized antifolate therapies. Furthermore, the nonactive target site is unique to the bifunctional parasitic enzyme-it is not contained within mammalian TS and DHFR counterparts-and therefore provides for a high level of selectivity.Cell culture and biochemical assays agree that the average 50% inhibitory concentration (IC 50 ) of eosin B for T. gondii is 180 M (2). Importantly, the relative safety of eosin has b...

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A Molecular Docking Strategy Identifies Eosin B as a Non-active Site Inhibitor of Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase

Cited by 23 publications

References 37 publications

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

Eosin B as a Novel Antimalarial Agent for Drug-Resistant Plasmodium falciparum

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