Chemistry and Chemical Biology ETDs

Chunliang LIU

Publication Date

9-1-2015

Abstract

The D-glucose disaccharide α, α-D-trehalose is synthesized by a variety of bacteria, fungi, plants and invertebrates to support cell survival by functioning as a fuel, a metabolic regulator or a protectant against environmental stress. Five different trehalose biosynthetic pathways are known to exist, one of which, the OtsA/OtsB pathway is common among pathogenic bacteria and fungi and is also found in parasitic nematodes. Previously reported otsA and otsB gene knockout (or knockdown) experiments have shown that both pathway enzymes are essential for M. tuberculosis cell growth and host colonization. RNAi gene silencing carried out in the nematode model system Caenohabditis elegans revealed that the T6P phosphatase is essential. Based on these findings we concluded that trehalose-synthesizing pathogens are likely to be vulnerable to the action of small molecule inhibitors of T6P phosphatase. We designed a bi-module inhibitor prototype. Accordingly, the phosphate group of the trehalose 6-phosphate moiety was replaced by a phosphate mimetic for targeting the active site of the catalytic domain, and the glucose unit was modified for targeting the cap domain for induced cap closure over the active site. Sulfate proved to be the most effective warhead for the substrate-binding site. T6S binding was shown by using Single Angle X-ray Scattering (SAXS) analysis to induce cap closure. Using glucose-6-sulfate as the platform, a series of synthetic derivatives possessing 'drug-like' functions tethered to the glucose ring were evaluated. Of these first generation inhibitors 4-n-octylphenyl-α-D-glucopyranoside 6-sulfate proved to be the tightest binding T6PP competitive inhibitor. In parallel, we have used the glucose-6-sulfate as the scaffold in the design of active site-directed, irreversible inhibitors. From the adducts which were synthesized and tested for T6PP inactivation, 4-n-octylphenyl-2-(3-(flurosulfonyl) benzoylamino)-2-deoxy-α-D-glucopyranoside-6-sulfate was selected for further characterization, and subsequently shown to inactivate the T6PPs with stoichiometric covalent modification and reasonable efficiency. Bioinformatic analysis and site-directed mutagenesis methods were used to identify the modified residue, a stringently conserved lysine residue was identified. Based on this result, the design and synthesis of the second generation of irreversible inhibitors that are optimized this target site is planned for future work.

Project Sponsors

National Institutes of Health (NIH)

Language

English

Keywords

T6PP, bivalent inhibitors, phosphatase, covalent inhibitors, trehalose-6-phosphate phosphatase

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Dunaway-Mariano, Debra

Second Committee Member

Mariano, Patrick

Third Committee Member

Liang, Fu-Sen

Fourth Committee Member

Allen, Karen

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