Abstract
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.
Highlights
Referred to as ‘‘The Great Mortality’’ by contemporaries, Black Death irrevocably changed the social and economic structure of 14th century Europe, killing one-third of the Western European population [1]
Isoprenoids are a crucial family of molecules that includes compounds such as quinones and cholesterol and are involved in a number of cellular processes, from electron transport to signal transduction to the regulation of membrane fluidity. Each member of this diverse family of molecules is derived from two common building blocks; isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), synthesized via the mevalonic acid (MVA) or methyl erythritol phosphate (MEP) pathways (Figure 1)
Because the methylerythritol phosphate (MEP) pathway is exclusively utilized by many human pathogens, and knockout of MEP pathway genes has proven lethal in bacteria such as Mycobacterium tuberculosis [3], Francisella tularensis [4], Escherichia coli [5], and Vibrio cholerae [6], the MEP pathway enzymes have received considerable attention as promising targets for the development of novel antibiotics
Summary
Referred to as ‘‘The Great Mortality’’ by contemporaries, Black Death irrevocably changed the social and economic structure of 14th century Europe, killing one-third of the Western European population [1]. In light of its high morbidity/mortality rate, ease of dissemination, associated emergency response procedures, and significant social impact, Y. pestis is categorized by the US Centers for Disease Control and Prevention (CDC) as a Category A biological threat agent (i.e. an agent of greatest concern). The ease by which antibiotic resistance can be deliberately engineered into bacteria, and the increasing prevalence of antibiotic resistant strains, emphasizes the need for continued development of novel antibiotics against new bacterial targets. Because the MEP pathway is exclusively utilized by many human pathogens, and knockout of MEP pathway genes has proven lethal in bacteria such as Mycobacterium tuberculosis [3], Francisella tularensis [4], Escherichia coli [5], and Vibrio cholerae [6], the MEP pathway enzymes have received considerable attention as promising targets for the development of novel antibiotics (reviewed in [7] [8] [9])
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