Binding determinants for substrates and inhibitors of trehalose-6-phosphate phosphatase
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Abstract
Trehalose is a sugar commonly found in archaeon, bacteria, fungi, plants, and invertebrates. It is utilized as an energy source and upregulated during stress conditions such as thermal fluctuations and oxidative stress. As mammals do not synthesize trehalose, trehalose biosynthetic pathways have become therapeutic targets for infectious diseases. The enzyme trehalose-6-phosphate phosphatase (T6PP) catalyzes the dephosphorylation of trehalose 6-phosphate to form trehalose. In its absence, the viability and virulence of bacteria, fungi, plants and nematodes are decreased. Hence T6PP is the focus of this study as a target for therapeutics of the diseases tuberculosis and lymphatic filariasis.
T6PP is a phosphohydrolase in the haloalkanoic acid dehalogenase superfamily. To identify the determinants for substrate specificity needed to guide structure-aided inhibitor design for therapeutics, atomic-resolution crystallographic information on the Michaelis complex is of great importance. Toward this goal, the structure of T6PP from Mycobacterium marinum was determined via X-ray crystallography in an unliganded form and the structure of T6PP from Salmonella typhimurium (St) was determined in the apo form, bound to the substrate analog, trehalose 6-phosphate, the product, trehalose, and the inhibitor, 4-n-octylphenyl α-D-glucopyranoside 6-sulfate. The enzyme confers specificity via hydrogen bonding to the phosphate and glucosyl group proximal to the phosphate. Specifically, the conserved residues Glu123, Lys125 and Glu167 form hydrogen bonds to the hydroxyl groups of the proximal glucose. However, the distal glucose binding sub-site can tolerate new chemotypes.
To further aid inhibitor design, the two inhibitors of Brugia malayi T6PP discovered via screening the Johns Hopkins library of FDA-approved drugs, Cephalosporin C and Closantel, were computationally docked into StT6PP. The Cephalosporin C scaffold was optimized to provide an inhibitor with a KI of 20 M that comprises a 5,6-indole scaffold to afford hydrogen bonds to the Glu/Lys/Glu motif and a computationally discovered phosphate mimic tetrazole. Closantel acts as a slow-binding inhibitor and a series of analogs were synthesized to increase potency. Two analogs show enhanced efficacy relative to Closantel with IC50 values near 60 M. Future efforts will aim to optimize these scaffolds for inhibition of T6PP to develop therapeutics for tuberculosis and lymphatic filariasis.