Escherichia coli 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH).These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9 -91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues. 5Ј-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN)1 functions at two steps in bacterial pathways related to polyamine biosynthesis, quorum sensing, methylation, and purine and methionine salvage reactions ( Fig. 1; see Refs. 1-4). It catalyzes the physiologically irreversible hydrolysis of 5Ј-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose (MTR). The product adenine is subsequently recycled into the adenine nucleotide pool by the widely distributed adenine phosphoribosyltransferase (5), and the 5-methylthio-D-ribose is subsequently phosphorylated to 5-methylthio-␣-D-ribose 1-phosphate and converted into methionine (6). MTA is a by-product of the reactions involving sequential transfers of the aminopropyl group from decarboxylated S-adenosylmethionine to form spermidine and spermine (although spermine is absent in Escherichia coli). Polyamine synthesis is sensitive to product inhibition by MTA, with inhibition constants reported to be 0.3 M for bovine spermine synthase (7) and 50 M for rat spermidine synthase (8). Inhibition of MTAN is therefore expected to inhibit polyamine biosynthesis and the salvage pathways for adenine and methionine. Another function of MTAN in bacteria is generation of S-ribosylhomocysteine (SRH) from SAH. SRH is a precursor for synthesis of tetrahydrofurans, quorum-sensing molecules involved in expression of the enzymes for biofilm formation, exotoxin synthesis, and antibiotic resistance factors (9 -14). Previously characterized nucleoside and nucleotide N-ribosyl hydrolases proceed through transition state structures in which the N-ribosidic

Summary Plasmodium falciparum, the primary cause of deaths from malaria, is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5′-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. Here we present a new class of HGXPRT inhibitors, the acyclic Immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K Ã i ¼ 58 pM, firstgeneration) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K Ã i ¼ 9 pM, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (K Ã i ¼ 9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K Ã i ¼ 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5′-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second-to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transitionstate analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.uman PNP catalyzes the phosphorolysis of 6-oxypurine nucleosides and deoxynucleosides to generate α-D-(deoxy) ribose 1-phosphate and the purine base. The purine is recycled or oxidized to uric acid for excretion. A rare genetic deficiency of PNP reveals that the enzyme is essential for recycling d-guanosine and formation of free purines leading to uric acid synthesis. PNP deficiency causes the presence of elevated concentrations of d-guanosine in the blood resulting in apoptosis of dividing T-cells due to the metabolic accumulation of dGTP, an inhibitor of ribonucleotide reductase (1, 2). Inhibitors of PNP have been used for the treatment of T-cell cancers and autoimmune disorders where T-cell clones are misdirected against self-antigens causing disorders, including psoriasis, rheumatoid arthritis, and multiple sclerosis (2, 3). PNP inhibitors are also in clinical trials for gout because formation of purine base precursors for uric acid formation requires PNP in humans.Knowledge of enzymatic transition-state structure is obtained from the experimental approach of kinetic isotope effects combined with quantum-chemical models (4). This analysis provides an atomic view of the difference in bond-vibrational environment between the reactants and th...

Helicobacter pylori is a Gram-negative bacterium that colonizes the gut of over 50% of the world's population. It is responsible for most peptic ulcers and is an important risk factor for gastric cancer. Antibiotic treatment for H. pylori infections is challenging as drug resistance has developed to antibiotics with traditional mechanisms of action. H. pylori uses an unusual pathway for menaquinone biosynthesis with 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzing an essential step. We validated MTAN as a target with a transition-state analogue of the enzyme [Wang, S.; Haapalainen, A. M.; Yan, F.; et al. Biochemistry 2012, 51, 6892-6894]. MTAN inhibitors will only be useful drug candidates if they can both include tight binding to the MTAN target and have the ability to penetrate the complex cell membrane found in Gram-negative H. pylori. Here we explore structural scaffolds for MTAN inhibition and for growth inhibition of cultured H. pylori. Sixteen analogues reported here are transition-state analogues of H. pylori MTAN with dissociation constants of 50 pM or below. Ten of these prevent growth of the H. pylori with IC90 values below 0.01 μg/mL. These remarkable compounds meet the criteria for potent inhibition and cell penetration. As a consequence, 10 new H. pylori antibiotic candidates are identified, all of which prevent H. pylori growth at concentrations 16-2000-fold lower than the five antibiotics, amoxicillin, metronidazole, levofloxacin, tetracyclin, and clarithromycin, commonly used to treat H. pylori infections. X-ray crystal structures of MTAN cocrystallized with several inhibitors show them to bind in the active site making interactions consistent with transition-state analogues.

We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to "fold" to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time.