Inhibitors of the Herbicidal Target IspD: Allosteric Site Binding

Witschel, Matthias; Höffken, Hans Wolfgang; Seet, Michael; Parra, Liliana; Mietzner, Thomas; Thater, Frank; Niggeweg, Ricarda; Röhl, Franz; Illarionov, Boris; Rohdich, Felix; Kaiser, J.; Fischer, Markus; Bacher, Adelbert; Diederich, François

doi:10.1002/anie.201102281

Cited by 47 publications

(62 citation statements)

References 26 publications

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“…We and others have previously reported small molecule inhibitors of IspD ( 1 and 2 , Fig. 2), confirming that IspD homologs are amenable to chemical inhibition121718. These IspD inhibitors have generally shown high-micromolar activity, although some herbicidal A. thaliana species-selective nanomolar templates have also been identified ( 3a and 3b , Fig.…”

supporting

confidence: 72%

Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

Price

Armstrong

Imlay

et al. 2016

Sci Rep

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The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria.

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supporting

confidence: 72%

Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

Price

Armstrong

Imlay

et al. 2016

Sci Rep

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“…MMV-08138 also did not affect the enzyme activity of purified A. thaliana or P. vivax IspD at up to 1 mM inhibitor (see Fig. S9) (32,33).…”

Section: Resultsmentioning

confidence: 94%

A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

Herrera

Ebert

et al. 2015

Antimicrob Agents Chemother

109

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The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the "Malaria Box" library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC 50 ) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-D-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC 50 ) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action. Despite encouraging progress over the past decade, malaria caused by Plasmodium parasites continues to pose an enormous disease burden (1). New antimalarials with novel mechanisms of action are needed to circumvent existing or emerging drug resistance (2). The apicoplast is a plastid organelle unique to Plasmodium spp. (and other pathogenic Apicomplexa parasites) and is a key target for development of new antimalarials. Due to its prokaryotic origin and evolution as a secondary plastid, it contains pathways that have no counterpart in the human host (3, 4). The apicoplast in Plasmodium is essential for both intraerythrocytic and intrahepatic development in the human host (5, 6).Despite efforts to develop inhibitors of apicoplast function, to date, there have been no primary agents for treatment of acute malaria whose mechanism of action targets this unusual plastid organelle. Antibiotics that inhibit prokaryotic transcription and translation, such as doxycycline and clindamycin, block expression of the apicoplast genome and are active against Plasmodium parasites (5). Unfortunately, these drugs show a "delayed death" phenotype, in which growth inhibition occurs only after 2 replication cycles (96 h). The slow kinetics limit the use of doxycycline and clindamycin to chemoprophylaxis or as partner drugs in combination therapies with faster-acting compounds. Fosmidomycin, which inhibits the enzyme DoxR/IspC for MEP (methyl-D-erythritol-4-phosphate) isoprenoid precursor biosynthesis in the apicoplast, has immediate onset but shows high recrudescence rates clinically when used as monotherapy (7,8). The efficacy of fosmidomycin-based combination therapy is currently being evaluated, with mixe...

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“…Several groups have demonstrated an interest in developing small molecule inhibitors of the MEP pathway enzymes, including those that target DXP synthase [12], [13], MEP synthase (reviewed in [14]), CDP-ME synthase [15], [16], CDP-ME kinase [17], [18], cMEPP synthase [19], [20], HMB-PP synthase [21], [22], and HMB-PP reductase [23], [24]. While each of these MEP pathway enzymes is a viable target for drug development [25] the focus of this report is on MEP synthase.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

et al. 2014

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The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KM DXP = 252 µM and KM NADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.

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Inhibitors of the Herbicidal Target IspD: Allosteric Site Binding

Cited by 47 publications

References 26 publications

Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

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