Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

Smith, Jessica M.; Warrington, Nicole V.; Vierling, Ryan J.; Kuhn, Misty L.; Anderson, W.F.; Koppisch, Andrew T.; Meyers, Caren L. Freel

doi:10.1038/ja.2013.105

Cited by 39 publications

(82 citation statements)

References 50 publications

(68 reference statements)

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“…MMV-08138 showed no enzyme inhibition activity against E. coli DXS at up to a 100 M concentration of the inhibitor, DXR/IspC at up 50 M, or IspD, IspE, and IspF at 10 M (see Fig. S8 in the supplemental material) (28)(29)(30)(31). 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.…”

Section: Resultsmentioning

confidence: 97%

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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Section: Resultsmentioning

confidence: 97%

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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“…However, basal expression of DXP synthase in BL21 cells harboring the dxs -pET37b plasmid is sufficient to confer resistance to BAP ( 4 ). 23 Thus, we evaluated 1 − 7 against E. coli BL21 cells harboring the dxs -pET37b plasmid grown in LB medium where basal DXP synthase expression is evident relative to the pET37b control (Figure S9). Increasing intracellular levels of DXP synthase confers resistance to alkylAPs 3 − 6 relative to the pET37b control (Figure 3), despite the potentially opposing effects on cell growth caused by aggregation and toxicity in this system.…”

Section: Resultsmentioning

confidence: 99%

“…Our previous work has established that sterically demanding alkylacetylphosphonates (alkylAPs) can selectively inhibit DXP synthase 15 (Figure 1b) and butylacetylphosphonate (BAP) possesses antimicrobial activity by a mechanism likely involving reversible inhibition of DXP synthase. 23 …”

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confidence: 99%

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

et al. 2017

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1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate. DXP is at a metabolic branch point in bacteria, feeding into the methylerythritol phosphate pathway to indispensable isoprenoids and acting as a precursor for biosynthesis of essential cofactors in central metabolism, pyridoxal phosphate and ThDP, the latter of which is also required for DXP synthase catalysis. DXP synthase follows a unique random sequential mechanism and possesses an unusually large active site. These features have guided the design of sterically demanding alkylacetylphosphonates (alkylAPs) toward the development of selective DXP synthase inhibitors. alkylAPs studied here display selective, low μM inhibitory activity against DXP synthase. They are weak inhibitors of bacterial growth in standard nutrient rich conditions. However, bacteria are significantly sensitized to most alkylAPs in defined minimal growth medium, with minimal inhibitory concentrations (MICs) ranging from low μM to low mM and influenced by alkyl-chain length. The longest analog (C8) displays the weakest antimicrobial activity and is a substrate for efflux via AcrAB-TolC. The dependence of inhibitor potency on growth environment emphasizes the need for antimicrobial screening conditions that are relevant to the in vivo microbial microenvironment during infection. DXP synthase expression and thiamin supplementation studies offer support for DXP synthase as an intracellular target for some alkylAPs and reveal both the challenges and intriguing aspects of these approaches to study target engagement.

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“…Contrary to our expectations, unnatural bisubstrate analog inhibitors were not identified; rather, trihydroxybenzaldoximes emerged as new d -GAP competitive inhibitors with the 2,4,5-trihydroxybenzaldoxime scaffold exhibiting the most potent inhibitory activity. To date, the few inhibitors of DXP synthase described 15,16,18–20,34,35 exhibit micromolar inhibitory activities with K i values ranging from ~4 µ m (acetylphosphonates) to 75 µ m (ketoclomazone). Here, we have shown that oxime-based inhibitors display inhibitory potencies with K i values as low as 1 µ m , placing them amongst the most potent inhibitors of DXP synthase described to date (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Hydroxybenzaldoximes Are D‐GAP‐Competitive Inhibitors of E. coli 1‐Deoxy‐D‐Xylulose‐5‐Phosphate Synthase

et al. 2015

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1-Deoxy-d-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branchpoint, serving also as a precursor in the biosynthesis of vitamins B1 and B6 which are critical for central metabolism. Toward identifying novel bisubstrate analog inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low micromolar inhibitors, competitive against d-glyceraldehyde 3-phosphate (d-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of d-GAP-competitive DXP synthase inhibitors offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.

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Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

Cited by 39 publications

References 50 publications

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

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

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

Hydroxybenzaldoximes Are D‐GAP‐Competitive Inhibitors of E. coli 1‐Deoxy‐D‐Xylulose‐5‐Phosphate Synthase

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