Inhibition of the Escherichia coli 6‑Oxopurine Phosphoribosyltransferases by Nucleoside Phosphonates: Potential for New Antibacterial Agents Dianne T. Keough, † Dana Hockova ́ , ‡ Dominik Rejman, ‡ Petr S ̌ pac ̌ ek, ‡ Silvie Vrbkova ́ , ‡ Marcela Krec ̌ merova ́ , ‡ Wai Soon Eng, † Harmen Jans, § Nicholas P. West, † Lieve M. J. Naesens, § John de Jersey, † and Luke W. Guddat* ,† † The School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072 QLD, Australia ‡ Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic § Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium ABSTRACT: Escherichia coli (Ec) cells possess two purine salvage enzymes: xanthine-guanine phosphoribosyltransferase (XGPRT) and hypoxanthine phosphoribosyltransferase (HPRT). EcXGPRT shares a common structural feature with other members of this family, a flexible loop that closes over the active site during catalysis. The replacement of six of these amino acids by alanine has no effect on the K m for the two substrates. However, the K i for the nucleoside monophosphate increases by 27-fold, and the k cat is reduced by ∼200-fold. Nucleoside phosphonates (NP) are good inhibitors of EcXGPRT and EcHPRT, with K i values as low as 10 nM. In the absence of the flexible loop, these values increase by 5- to 30-fold, indicating the importance of the loop for high-affinity inhibition. Crystal structures of two NPs in complex with EcXGPRT explain the tight binding. Prodrugs of NPs with low K i values for EcXGPRT or EcHPRT exhibit IC 50 values between 5 and 23 μM against Mycobacterium tuberculosis in cell-based assays, suggesting that these compounds are therapeutic leads against pathogenic bacteria. ■ INTRODUCTION Escherichia coli cells possess two 6-oxopurine phosphoribosyl- transferases (PRTases): xanthine-guanine phosphoribosyltrans- ferase (EcXGPRT) and hypoxanthine PRTase (EcHPRT). EcXGPRT and EcHPRT catalyze the Mg 2+ -dependent transfer of the ribosyl-5-phosphate group from 5-phospho-α-D-ribosyl- 1-pyrophosphate (PRib-PP) to the N 9 position of guanine, hypoxanthine, or xanthine to yield the corresponding nucleo- side monophosphate (GMP, IMP, or XMP, respectively) and pyrophosphate (PP i ) (Figure 1). As signified by their names, EcXGPRT and EcHPRT have different specificities for the naturally occurring purine bases. EcXGPRT has a strong preference for guanine, with a k cat /K m of 6.5 μM −1 s −1 , whereas for xanthine this value is 1.2 μM −1 s −1 . Hypoxanthine is a weak substrate, having a k cat /K m value of 0.2 μM −1 s −1 . 1 In comparison to EcXGPRT, EcHPRT prefers hypoxanthine. The k cat /K m for this substrate is 4.9 μM −1 s −1 , and both guanine and xanthine are weak substrates with k cat /K m values of 0.03 and 0.0003 μM −1 s −1 , respectively. 1 This enzyme is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both guanine and xanthine. A number of structures of EcXGPRT have been determined in the absence or presence of substrates or products. In the crystal structure of the free enzyme, sulfate and magnesium ions are observed in the active site. 2 In this case, the amino acid residues between 61-SSYDHDNQRELK-72 have poor electron density, suggesting that this area of the enzyme is flexible. In complex with the inactive analog of PRib-PP (1-α-pyrophos- phoryl-2-α,3-α-dihydroxy-4-β-cyclopentane-methanol-5-phos- phate, cPRPP)·guanine·Mg 2+ , or with IMP or GMP, the loop Received: May 26, 2013 Published: August 8, 2013 Figure 1. Reactions catalyzed by the 6-oxopurine PRTases. The naturally occurring purine bases are guanine (R is −NH 2 ), hypoxanthine (R is −H), and xanthine (R is −OH). Article pubs.acs.org/jmc © 2013 American Chemical Society 6967 dx.doi.org/10.1021/jm400779n | J. Med. Chem. 2013, 56, 6967−6984