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.

Students who demonstrate strong talent and interest in STEM are initially attracted to STEM through extracurricular experiences.

What early experiences attract students to pursue an education and career in science, technology, engineering, and mathematics (STEM)? Does hands-on research influence them to persevere and complete a major course of academic study in STEM? We evaluated survey responses from 149 high school and undergraduate students who gained hands-on research experience in the 2007-2013 Aspiring Scientists Summer Internship Programs (ASSIP) at George Mason University. Participants demonstrated their strong interest in STEM by volunteering to participate in ASSIP and completing 300 h of summer research. The survey queried extracurricular experiences, classroom factors, and hands-on projects that first cultivated students' interest in the STEM fields, and separately evaluated experiences that sustained their interest in pursuing a STEM degree. The majority of students (65.5%, p < 0.0001) reported extracurricular encounters, such as the influence of a relative or family member and childhood experiences, as the most significant factors that initially ignited their interest in STEM, while hands-on lab work was stated as sustaining their interest in STEM (92.6%). Based on these findings collected from a cohort of students who demonstrated a strong talent and interest in STEM, community-based programs that create awareness about STEM for both children and their family members may be key components for igniting long-term academic interest in STEM.

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.