A high-throughput screening campaign to identify inhibitors of DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD).

The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.

Structure-Activity Relationships of the MEPicides: N-Acyl and O-Linked Analogs of FR900098 as Inhibitors of Dxr from Mycobacterium tuberculosis and Yersinia pestis.

Despite continued research efforts, the threat of drug resistance from a variety of bacteria continues to plague clinical communities. Discovery and validation of novel biochemical targets will facilitate development of new drugs to combat these organisms. The methylerythritol phosphate (MEP) pathway to make isoprene units is a biosynthetic pathway essential to many bacteria. We and others have explored inhibitors of the MEP pathway as novel antibacterial agents. Mycobacterium tuberculosis, the causative agent of tuberculosis, and Yersinia pestis, resulting in the plague or "black death", both rely on the MEP pathway for isoprene production. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr) catalyzes the first committed step in the MEP pathway. We examined two series of Dxr inhibitors based on the parent structure of the retrohydroxamate natural product FR900098. The compounds contain either an extended N-acyl or O-linked alkyl/aryl group and are designed to act as bisubstrate inhibitors of the enzyme. While nearly all of the compounds inhibited both Mtb and Yp Dxr to some extent, compounds generally displayed more potent inhibition against the Yp homologue, with the best analogs displaying nanomolar IC50 values. In bacterial growth inhibition assays, the phosphonic acids generally resulted in poor antibacterial activity, likely a reflection of inadequate permeability. Accordingly, diethyl and dipivaloyloxymethyl (POM) prodrug esters of these compounds were made. While the added lipophilicity did not enhance Yersinia activity, the compounds showed significantly improved antitubercular activities. The most potent compounds have Mtb MIC values of 3-12 µg/mL. Taken together, we have uncovered two series of analogs that potently inhibit Dxr homologues from Mtb and Yp. These inhibitors of the MEP pathway, termed MEPicides, serve as leads for future analog development.

Synthesis and bioactivity of ß-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 ß-position of the hydroxamate analogue of 2. While direct addition of a ß-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 hydroxamate's 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 Yersinia pestis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase).

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.

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.

The effect of chain length and unsaturation on Mtb Dxr inhibition and antitubercular killing activity of FR900098 analogs.

Inhibition of the nonmevalonate pathway (NMP) of isoprene biosynthesis has been examined as a source of new antibiotics with novel mechanisms of action. Dxr is the best studied of the NMP enzymes and several reports have described potent Dxr inhibitors. Many of these compounds are structurally related to natural products fosmidomycin and FR900098, each bearing retrohydroxamate and phosphonate groups. We synthesized a series of compounds with two to five methylene units separating these groups to examine what linker length was optimal and tested for inhibition against Mtb Dxr. We synthesized ethyl and pivaloyl esters of these compounds to increase lipophilicity and improve inhibition of Mtb growth. Our results show that propyl or propenyl linker chains are optimal. Propenyl analog 22 has an IC50 of 1.07 µM against Mtb Dxr. The pivaloyl ester of 22, compound 26, has an MIC of 9.4 µg/mL, representing a significant improvement in antitubercular potency in this class of compounds.

Design of Potential Bisubstrate Inhibitors against Mycobacterium tuberculosis (Mtb) 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)-Evidence of a Novel Binding Mode.

In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 µM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents.