Synthesis and kinetic evaluation of phosphomimetic inhibitors targeting type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis.

Because tuberculosis is still a major health threat worldwide, identification of new drug targets is urgently needed. In this study, we considered type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis as a potential target, and addressed known problems of previous inhibitors in terms of their sensitivity to hydrolysis catalyzed by phosphatase enzymes, which impaired their potential use as drugs. To this end, we synthesized six novel phosphomimetic compounds designed to be hydrolytically stable analogs of the substrate ribose 5-phosphate and the best known inhibitor 5-phospho-d-ribonate.

Novel N-substituted 5-phosphate-d-arabinonamide derivatives as strong inhibitors of phosphoglucose isomerases: Synthesis, structure-activity relationship and crystallographic studies.

Phosphoglucose isomerase (PGI) is a cytosolic enzyme that catalyzes the reversible interconversion of d-glucose 6-phosphate and d-fructose 6-phosphate in glycolysis. Outside the cell, PGI is also known as autocrine motility factor (AMF), a cytokine secreted by a large variety of tumor cells that stimulates motility of cancer cells in vitro and metastases development in vivo. Human PGI and AMF are strictly identical proteins both in terms of sequence and 3D structure, and AMF activity is known to involve, at least in part, the enzymatic active site.

Calibration of the dianionic phosphate group: Validation on the recognition site of the homodimeric enzyme phosphoglucose isomerase.

We calibrate and validate the parameters necessary to represent the dianionic phosphate group (DPG) in molecular mechanics. DPG is an essential fragment of signaling biological molecules and protein-binding ligands. It is a constitutive fragment of biosensors, which bind to the dimer interface of phosphoglucose isomerase (PGI), an intracellular enzyme involved in sugar metabolism, as well as an extracellular protein known as autocrine motility factor (AMF) closely related to metastasis formation.

Bisphosphonate Inhibitors of Mammalian Glycolytic Aldolase.

The glycolytic enzyme aldolase is an emerging drug target in diseases such as cancer and protozoan infections which are dependent on a hyperglycolytic phenotype to synthesize adenosine 5'-triphosphate and metabolic precursors for biomass production. To date, structural information for the enzyme in complex with phosphate-derived inhibitors has been lacking. Thus, we determined the crystal structure of mammalian aldolase in complex with naphthalene 2,6-bisphosphate (1) that served as a template for the design of bisphosphonate-based inhibitors, namely, 2-phosphate-naphthalene 6-bisphosphonate (2), 2-naphthol 6-bisphosphonate (3), and 1-phosphate-benzene 4-bisphosphonate (4).

Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-d-arabinonhydrazide.

Type I phosphomannose isomerases (PMIs) are zinc-dependent monofunctional metalloenzymes catalysing the reversible isomerization of d-mannose 6-phosphate to d-fructose 6-phosphate. 5-Phospho-d-arabinonhydrazide (5PAHz), designed as an analogue of the enediolate high-energy intermediate, strongly inhibits PMI from Candida albicans (CaPMI). In this study, we report the 3D crystal structure of CaPMI complexed with 5PAHz at 1.

Carbohydrate-based electrochemical biosensor for detection of a cancer biomarker in human plasma.

Autocrine motility factor (AMF) is a tumor-secreted cytokine that stimulates tumor cell motility in vitro and metastasis in vivo. AMF could be detected in serum or urine of cancer patients with worse prognosis. Reported as a cancer biomarker, AMF secretion into body fluids might be closely related to metastases formation.

Complexes of a Zn-metalloenzyme binding site with hydroxamate-containing ligands. A case for detailed benchmarkings of polarizable molecular mechanics/dynamics potentials when the experimental binding structure is unknown.

Zn-metalloproteins are a major class of targets for drug design. They constitute a demanding testing ground for polarizable molecular mechanics/dynamics aimed at extending the realm of quantum chemistry (QC) to very long-duration molecular dynamics (MD). The reliability of such procedures needs to be demonstrated upon comparing the relative stabilities of competing candidate complexes of inhibitors with the recognition site stabilized in the course of MD.

Structural basis for competitive inhibition of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Vibrio cholerae.

The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate.

Exploring inhibition of Pdx1, a component of the PLP synthase complex of the human malaria parasite Plasmodium falciparum.

Malaria tropica is a devastating infectious disease caused by Plasmodium falciparum. This parasite synthesizes vitamin B6 de novo via the PLP (pyridoxal 5'-phosphate) synthase enzymatic complex consisting of PfPdx1 and PfPdx2 proteins. Biosynthesis of PLP is largely performed by PfPdx1, ammonia provided by PfPdx2 subunits is condensed together with R5P (D-ribose 5-phosphate) and G3P (DL-glyceraldehyde 3-phosphate).

Synthesis and evaluation of malonate-based inhibitors of phosphosugar-metabolizing enzymes: class II fructose-1,6-bis-phosphate aldolases, type I phosphomannose isomerase, and phosphoglucose isomerase.

In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g.

Polarizable water networks in ligand-metalloprotein recognition. Impact on the relative complexation energies of Zn-dependent phosphomannose isomerase with D-mannose 6-phosphate surrogates.

Using polarizable molecular mechanics, a recent study [de Courcy et al. J. Am.

The reaction mechanism of type I phosphomannose isomerases: new information from inhibition and polarizable molecular mechanics studies.

Type I phosphomannose isomerases (PMIs) are zinc-dependent metalloenzymes involved in the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). 5-Phospho-D-arabinonohydroxamic acid (5PAH), an inhibitor endowed with nanomolar affinity for yeast (Type I) and Pseudomonas aeruginosa (Type II) PMIs (Roux et al., Biochemistry 2004; 43:2926-2934), strongly inhibits human (Type I) PMI (for which we report an improved expression and purification procedure), as well as Escherichia coli (Type I) PMI.

Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase.

Crystallographic binding studies have been carried out to probe the active-site binding properties of a monomeric variant (A-TIM) of triosephosphate isomerase (TIM). These binding studies are part of a structure-based directed-evolution project aimed towards changing the substrate specificity of monomeric TIM and are therefore aimed at finding binders which are substrate-like molecules. A-TIM has a modified more extended binding pocket between loop-7 and loop-8 compared with wild-type TIM.

Synthesis and evaluation of non-hydrolyzable D-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-D-mannose as a new strong inhibitor of phosphomannose isomerases.

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs) from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-d-mannose and 6-deoxy-6-carboxymethyl-D-mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxymethyl-D-mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E.

Competitive inhibitors of type B ribose 5-phosphate isomerases: design, synthesis and kinetic evaluation of new D-allose and D-allulose 6-phosphate derivatives.

This study reports syntheses of d-allose 6-phosphate (All6P), D-allulose (or D-psicose) 6-phosphate (Allu6P), and seven D-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichiacoli (EcRpiB). 5-Phospho-D-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho-D-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step.

D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not.

Interconversion of D-ribose-5-phosphate (R5P) and D-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-phosphate (All6P) and D-allulose-6-phosphate.

Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily.

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.

Speciation of europium (III) surface species on monocrystalline alumina using time-resolved laser-induced fluorescence-scanning near-field optical microscopy.

The aim of this work was to perform highly localized spectroscopic surface measurements by combining time-resolved laser spectroscopy and scanning near-field optical microscopy. The final purpose of that was to study surface sorption at the molecular level of trivalent ions in the framework of nuclear waste disposal assessment. Time-resolved laser spectroscopy presents the advantages of being selective, sensitive, and noninvasive and scanning near-field optical microscopy is a promising technique for high resolution surface speciation.

Binding of 5-phospho-D-arabinonohydroxamate and 5-phospho-D-arabinonate inhibitors to zinc phosphomannose isomerase from Candida albicans studied by polarizable molecular mechanics and quantum mechanics.

Type I phosphomannose isomerase (PMI) is a Zn-dependent metalloenzyme involved in the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate. One of our laboratories has recently designed and synthesized 5-phospho-D-arabinonohydroxamate (5PAH), an inhibitor endowed with a nanomolar affinity for PMI (Roux et al., Biochemistry 2004, 43, 2926).

Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism.

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km values of 4 mM (R5P) and 1.

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose.

Preliminary studies on the inhibition of D-sorbitol-6-phosphate 2-dehydrogenase from Escherichia coli with substrate analogues.

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 +/- 1) microM and (48 +/- 3) microM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E.

Competitive inhibitors of Mycobacterium tuberculosis ribose-5-phosphate isomerase B reveal new information about the reaction mechanism.

Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases.

The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis.

Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site. This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea.

Leishmania mexicana mexicana glucose-6-phosphate isomerase: crystallization, molecular-replacement solution and inhibition.

Glucose-6-phosphate isomerase (PGI; EC 5.3.1.