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Structures of 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase/Lipophilic Phosphonate Complexes.

ABSTRACT: Fosmidomycin, a potent inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), has antibacterial and antimalaria activity. Due to its poor pharmacokinetics, more lipophilic DXR inhibitors are needed. However, the hydrophobic binding site(s) in DXR remains elusive. Here, pyridine/quinoline containing phosphonates are identified to be DXR inhibitors with IC(50) values as low as 840 nM. We also report three DXR:inhibitor structures, revealing a novel binding mode. The indole group of Trp211 is found to move ~4.6 Å to open up a mainly hydrophobic pocket, where the pyridine/quinoline rings of the inhibitors are located and have strong π-π stacking/charge-transfer interactions with the indole. Docking studies demonstrate our structures could be used to predict the binding modes of other lipophilic DXR inhibitors. Overall, this work shows an important role of Trp211 in inhibitor recognition and provides a structural basis for future drug design and development.

SUBMITTER: Deng L

PROVIDER: S-EPMC3046873 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase by lipophilic phosphonates: SAR, QSAR, and crystallographic studies.

Project description:1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a novel target for developing new antibacterial (including antituberculosis) and antimalaria drugs. Forty-one lipophilic phosphonates, representing a new class of DXR inhibitors, were synthesized, among which 5-phenylpyridin-2-ylmethylphosphonic acid possesses the most activity against E. coli DXR (EcDXR) with a K(i) of 420 nM. Structure-activity relationships (SAR) are discussed, which can be rationalized using our EcDXR:inhibitor structures, and a predictive quantitative SAR (QSAR) model is also developed. Since inhibition studies of DXR from Mycobacterium tuberculosis (MtDXR) have not been performed well, 48 EcDXR inhibitors with a broad chemical diversity were found, however, to generally exhibit considerably reduced activity against MtDXR. The crystal structure of a MtDXR:inhibitor complex reveals the flexible loop containing the residues 198-208 has no strong interactions with the 3,4-dichlorophenyl group of the inhibitor, representing a structural basis for the reduced activity. Overall, these results provide implications in the future design and development of potent DXR inhibitors.

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Thermodynamic Investigation of Inhibitor Binding to 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase.

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| S-EPMC3462030 | biostudies-literature

Expression, characterization and inhibition of Toxoplasma gondii 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

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Enamide Prodrugs of Acetyl Phosphonate Deoxy-d-xylulose-5-phosphate Synthase Inhibitors as Potent Antibacterial Agents.

Project description:To fight the growing threat of antibiotic resistance, new antibiotics are required that target essential bacterial processes other than protein, DNA/RNA, and cell wall synthesis, which constitute the majority of currently used antibiotics. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase is a vital enzyme in bacterial central metabolism, feeding into the de novo synthesis of thiamine diphosphate, pyridoxal phosphate, and essential isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. While potent and selective inhibitors of DXP synthase in vitro activity have been discovered, their antibacterial activity is modest. To improve the antibacterial activity of selective alkyl acetylphosphonate (alkylAP) inhibitors of DXP synthase, we synthesized peptidic enamide prodrugs of alkylAPs inspired by the natural product dehydrophos, a prodrug of methyl acetylphosphonate. This prodrug strategy achieves dramatic increases in activity against Gram-negative pathogens for two alkylAPs, butyl acetylphosphonate and homopropargyl acetylphosphonate, decreasing minimum inhibitory concentrations against Escherichia coli by 33- and nearly 2000-fold, respectively. Antimicrobial studies and LC-MS/MS analysis of alkylAP-treated E. coli establish that the increased potency of prodrugs is due to increased accumulation of alkylAP inhibitors of DXP synthase via transport of the prodrug through the OppA peptide permease and subsequent amide hydrolysis. This work demonstrates the promise of targeting DXP synthase for the development of novel antibacterial agents.

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Project description:The nonmevalonate pathway of isoprenoid biosynthesis present in Plasmodium falciparum is known to be an effective target for antimalarial drugs. The second enzyme of the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), catalyzes the transformation of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP). For crystallographic studies, DXR from the human malaria parasite P. falciparum (PfDXR) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method in the presence of NADPH. X-ray diffraction data to 1.85 A resolution were collected from a monoclinic crystal form belonging to space group C2 with unit-cell parameters a = 168.89, b = 59.65, c = 86.58 A, beta = 117.8 degrees. Structural analysis by molecular replacement is in progress.

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Molecular Cloning and Functional Analysis of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase from <i>Santalum album</i>.

Project description:Sandalwood (Santalum album L.) heartwood-derived essential oil contains a high content of sesquiterpenoids that are economically highly valued and widely used in the fragrance industry. Sesquiterpenoids are biosynthesized via the mevalonate acid and methylerythritol phosphate (MEP) pathways, which are also the sources of precursors for photosynthetic pigments. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a secondary rate-limiting enzyme in the MEP pathway. In this paper, the 1416-bp open reading frame of SaDXR and its 897-bp promoter region, which contains putative conserved cis-elements involved in stress responsiveness (HSE and TC-rich repeats), hormone signaling (abscisic acid, gibberellin and salicylic acid) and light responsiveness, were cloned from 7-year-old S. album trees. A bioinformatics analysis suggested that SaDXR encodes a functional and conserved DXR protein. SaDXR was widely expressed in multiple tissues, including roots, twigs, stem sapwood, leaves, flowers, fruit and stem heartwood, displaying significantly higher levels in tissues with photosynthetic pigments, like twigs, leaves and flowers. SaDXR mRNA expression increased in etiolated seedlings exposed to light, and the content of chlorophylls and carotenoids was enhanced in all 35S::SaDXR transgenic Arabidopsis thaliana lines, consistent with the SaDXR expression level. SaDXR was also stimulated by MeJA and H2O2 in seedling roots. α-Santalol content decreased in response to fosmidomycin, a DXR inhibitor. These results suggest that SaDXR plays an important role in the biosynthesis of photosynthetic pigments, shifting the flux to sandalwood-specific sesquiterpenoids.

| S-EPMC8143465 | biostudies-literature

Kinetic characterization and allosteric inhibition of the Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).

Project description: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 KMDXP = 252 µM and KMNADPH = 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.

| S-EPMC4149570 | biostudies-literature

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