Synthetic fosmidomycin analogues with altered chelating moieties do not inhibit 1-deoxy-D-xylulose 5-phosphate reductoisomerase or Plasmodium falciparum growth in vitro

Fourteen new fosmidomycin analogues with altered metal chelating groups were prepared and evaluated for inhibition of E. coli Dxr, M. tuberculosis Dxr and the growth of P. falciparum K1 in human erythrocytes. None of the synthesized compounds showed activity against either enzyme or the Plasmodia. This study further underlines the importance of the hydroxamate functionality and illustrates that identifying effective alternative bidentate ligands for this target enzyme is challenging

Synthesis and Bioactivity of beta-Substituted Fosmidomycin Analogues Targeting 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase

Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the beta-position of the hydroxamate analogue of 2. While direct addition of a beta-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the longer compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamates methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth

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

<div><p>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 <i>Yersinia pestis</i>. Enzyme assays indicate apparent kinetic constants of K_M^DXP = 252 µM and K_M^NADPH = 13 µM, IC₅₀ values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and K_i values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the <i>Y. pestis</i> 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.</p></div

Dose-dependent inhibition of the <i>Y. pestis</i> MEP synthase.

<p>IC₅₀ values were determined using A) fosmidomycin or B) FR900098. The R² value for each plot is indicated. Assays were performed in duplicate.</p

Dose-response plot of <i>Y. pestis</i> MEP synthase with the top five rationally designed inhibitors; compounds A) 13, B) 15, C) 16, D) 42 and E) 48.

<p>Assays were performed by combining the enzyme with 150 µM NADPH, followed by addition of the inhibitor. After five minute incubation at 37°C, substrate was added to initiate the reaction. The R² value for each plot is indicated. The enzymatic activity is relative to an uninhibited control. All assays were performed in duplicate.</p

<i>Yersinia pestis</i> growth inhibition assay with extract 29.

<p><i>Y. pestis</i> A1122 was cultured in the presence of e29 at the indicated concentrations. Bacterial growth is relative to an uninhibited culture. All assays were performed in duplicate. The extract inhibits bacterial growth in a dose-dependent fashion.</p

Mechanism of inhibition by e29.

<p>A) Relative to NADPH, e29 is an uncompetitive inhibitor of the purified <i>Y. pestis</i> MEP synthase. B) Relative to DXP, e29 is a noncompetitive inhibitor. C) A model of e29 inhibition. MEP synthase (E) undergoes a conformational change (E*) upon binding of NADPH (N), exposing an allosteric site to which the inhibitor (I) binds. As the inhibitor is noncompetitive with respect to DXP (D), I may bind the E*N or E*ND complex, thereby inhibiting the enzyme.</p