Identification of Leishmania major UDP-Sugar Pyrophosphorylase Inhibitors Using Biosensor-Based Small Molecule Fragment Library Screening.

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

In silico Predicted Glucose-1-phosphate Uridylyltransferase (GalU) Inhibitors Block a Key Pathway Required for Listeria Virulence.

Peptidoglycan walls of gram positive bacteria are functionalized by glycopolymers called wall teichoic acid (WTA). In Listeria monocytogenes, multiple enzymes including the glucose-1-phosphate uridylyltransferase (GalU) were identified as mandatory for WTA galactosylation, so that the inhibition of GalU is associated with a significant attenuation of Listeria virulence. Herein, we report on a series of in silico predicted GalU inhibitors identified using structure-based virtual screening and experimentally validated to be effective in blocking the WTA galactosylation pathway in vitro. Several hits such as C04, a pyrimidinyl benzamide, afforded promising experimental potencies. This proof-of-concept study opens new perspectives for the development of potent and selective GalU inhibitors of high interest to attenuate Listeria virulence. It also underscores the high relevance of using molecular modeling for facilitating the identification of bacterial virulence attenuators and more generally antibacterials.

Screening assay for inhibitors of a recombinant Streptococcus pneumoniae UDP-glucose pyrophosphorylase.

The UDP-glucose pyrophosphorylase of Streptococcus pneumoniae (GalUSpn) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Since the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts, we propose that the GalU enzyme is a critical target to fight the pneumococcal disease. A recombinant GalUSpn was overexpressed and purified. An enzymatic assay that is rapid, sensitive and easy to perform was developed. This assay was appropriate for screening chemical libraries for searching GalU inhibitors. This work represents a fundamental step in the exploration of novel antipneumococcal drugs.

Structure and Function of Prokaryotic UDP-Glucose Pyrophosphorylase, A Drug Target Candidate.

UDP-glucose is an essential metabolite for a variety of processes in the cell physiology in all organisms. In prokaryotes, it is involved in the synthesis of trehalose, an osmoprotectant, in galactose utilization via the Leloir pathway and it plays a key role in the synthesis of the components of the bacterial envelope, particularly the lipopolysaccharide and the capsule, which represent necessary virulence factors of many bacterial pathogens. UDP-glucose is synthesized in bacteria by the prokaryotic UDP-glucose pyrophosphorylase (UGP, EC 2.7.7.9), an enzyme belonging to the family of sugar:nucleotidyl transferases. Despite the ubiquitous distribution of UGP activity in all domains of life, prokaryotic UGPs are evolutionarily unrelated to their eukaryotic counterparts. Taken together, these features make of bacterial UGP an attractive target candidate for the discovery and development of new generation antibiotics. This review summarizes the current knowledge on structure and function of bacterial UGPs, underlying their potential as drug target candidates.

Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase.

The pyrophosphorylase that condenses UTP and GlcNAc-1-P was purified 9500-fold to near homogeneity from the soluble fraction of pig liver extracts. At the final stage of purification, the enzyme was quite stable and could be kept for at least 4 months in the freezer with only slight loss of activity. On native gels, the purified enzyme showed a single protein band, and this band was estimated to have a molecular mass of approximately125 kDa on Sephacryl S-300. SDS-polyacrylamide gel electrophoresis analysis of the enzyme gave three protein bands of 64, 57, and 49 kDa, but these polypeptides are all closely related based on the following. 1) All three polypeptides show strong cross-reactivity with antibody prepared against the 64-kDa band. 2) All three proteins become labeled with either the UDP-GlcNAc photoaffinity probe azido-125I-salicylate-allylamine-UDP-GlcNAc or a similar UDP-GalNAc photoaffinity probe, and either labeling was inhibited in a specific and concentration-dependent manner by unlabeled UDP-GlcNAc or UDP-GalNAc. Thus, the enzyme is probably a homodimer composed of two 64-kDa subunits. The purified enzyme had an unusual specificity in that, at higher substrate concentrations, it utilized UDP-GalNAc as a substrate as well as UDP-GlcNAc in the reverse direction and GalNAc-1-P as well as GlcNAc-1-P in the forward direction. However, the Km for the GalNAc substrates was considerably higher than that for GlcNAc derivatives. This activity for synthesizing UDP-GalNAc was not due to epimerase activity since no UDP-GalNAc could be detected when the enzyme was incubated with UDP-GlcNAc for various periods of time. The pyrophosphorylase required a divalent cation, with Mn2+ being best at 0.5-1 mM, and the pH optimum was between 8.5 and 8.9.

Molecular cloning, nucleotide sequencing, and affinity labeling of bovine liver UDP-glucose pyrophosphorylase.

A bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2.7.7.9], which catalyzes the reversible uridylyl transfer between glucose 1-phosphate and MgUTP, has been cloned by the use of oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant sequence similarities with the enzymes from potato tuber and a slime mold, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the purified enzyme was incubated with an affinity labeling reagent, uridine triphosphopyridoxal, and then reduced with sodium borohydride. The enzyme was inactivated rapidly and irreversibly by the reagent at low concentrations. The inactivation was almost completely retarded by UDP-glucose and MgUTP. Structural analysis of the labeled enzyme revealed that three lysyl residues, Lys291, Lys357, and Lys396, were modified by the reagent. The three lysyl residues are conserved at the corresponding positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the active-site structure of bovine liver UDP-glucose pyrophosphorylase is very similar to that of the potato tuber enzyme.

Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.

Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA.

The expression of the enzyme UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) from potato (Solanum tuberosum L.) was analysed with respect to sink-source interactions and potato tuber storage. The highest level of expression was found in developing tubers, the strongest sink tissue. Storage of mature tubers at low temperatures led to an increase of the steady-state level of UGPase mRNA, implicating a role of this enzyme in the process of "cold-sweetening". Transgenic plants were created expressing UGPase antisensee RNA under the control of the 35S promoter of the Cauliflower Mosaic Virus with the polyadenylation signal of the octopine-synthase gene. Regenerated plants were tested for reduction of UGPase at the RNA, protein and activity levels. Plants with a 95%-96% reduction of UGPase activity in growing tubers showed no change in growth and development. Also, carbohydrate metabolism in tubers of these plants was not substantially affected, indicating that only 4% of the wild-type UGPase activity is sufficient for the enzyme to function in plant growth and development.

Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals.

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.

Inactivation of skeletal-muscle UDP-glucose pyrophosphorylase by reaction with carboxylate-directed reagents.

Skeletal-muscle UDP-glucose pyrophosphorylase is inactivated by reaction with 2-ethoxy-N-(ethoxy-carbonyl)-1,2-dihydroquinoline (EEDQ) and 1-(3-dimethylaminopropyl-3-ethylcarbodi-imide (EDAC), two reagents specific for carboxylate groups. The former reagent is a more effective inactivator than EDAC. Although no evidence of reversible enzyme-reagent complexes of the affinity-labelling type was obtained by kinetic analysis of the inactivation, the selective protection of UDP-glucose pyrophosphorylase activity against inactivation by EEDQ in the presence of uridine substrates is indicative of an active-site-directed effect. The results are consistent with the hypothesis that EEDQ modifies a single carboxylate group located in a hydrophobic domain close to the substrate-binding site, leading to enzyme inactivation. In contrast, the reaction between UDP-glucose pyrophosphorylase and EDAC appears to involve a different region of the enzyme.

Non-Michaelian kinetics of rabbit muscle uridine diphosphoglucose pyrophosphorylase.

The kinetic properties of rabbit muscle uridine diphosphoglucose (UDP-Glc) pyrophosphorylase have been studied, in both directions, with respect to substrate saturation, product inhibition, and cation requirement for activity. The results demonstrate that UDP-Glc pyrophosphorylase is a non-Michaelian enzyme: the synthetic reaction is characterized by a marked inhibition by glucose-1-phosphate (at concentrations higher than 0.3 mM) and by an hyperbolic saturation for UTP. In the reverse reaction, instead, the saturation function for UDP-Glc is hyperbolic and that for inorganic pyrophosphate is sigmoid, with a high Hill coefficient of (nH) 2.5. The study of the metal requirement indicates a distinctive ability of cations to stimulate the reactions of synthesis and degradation of the sugar nucleotide and a different stoichiometry of the metal chelates involved. The reaction mechanism is of the ordered-sequential type and the data of product inhibition allowed the identification of glucose-1-phosphate as the first substrate bound and UDP-Glc as the last product released. The inhibition pattern by UDP-Glc gives evidence for cooperativity also in the binding of this molecule.

Regulation of UDPG pyrophosphorylase in Acetabularia mediterranea.

The kinetic properties of UDPG pyrophosphorylase (glucosyl-1-phosphate uridyl transferase, EC 2.7.7.9) suggest that it may play a key role in the regulation of metabolism in Acetabularia mediterranea. The enzyme-catalyzed reaction is readily reversible in vitro, and has been assayed in both directions. The enzyme shows substrate inhibition by UDPG and UTP at substrate concentrations in excess of 2 mM. The kinetic behavior of the enzyme is consistent with the hypothesis that it catalyzes an ordered bisubstrate biproduct reaction in which G-1-P is the leading substrate, and UTP is the leading product. A plot of initial velocity vs. PPi concentration is sigmoid, indicating a cooperative homotropic effect. PGAL inhibits the reaction in the direction: UTP + G-1-P leads to UDPG + PPi It has no effect on the reverse reaction. The responses of the enzyme may serve to regulate the allocation of G-1-P between anabolic and catabolic pathways.