Renaud Hardré

The crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho-d-arabinonohydroxamic acid

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the second step in glycolysis, the reversible isomerization of d-glucose 6-phosphate to d-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a cis-enediol(ate) intermediate. 5-Phospho-d-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a Ki of 2 × 10−7 M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 Å resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2.

Synthesis and evaluation of a new inhibitor of phosphoglucose isomerases: the enediolate analogue 5-phospho-D-arabinohydroxamate

Designed as a high energy intermediate analogue inhibitor of the potent chemotherapeutic target phosphoglucose isomerases, 5-phospho-D-arabinohydroxamate was efficiently synthesized in a two steps procedure. To date, it proved to be the strongest competitive inhibitor with respect to substrate D-fructose-6-phosphate (Ki down to 98 nM and Km/Ki values up to 513). A comparative inhibition study of this compound and other known strong inhibitors on phosphoglucose isomerases from three different sources is also reported.

Competitive inhibitors of yeast phosphoglucose isomerase: synthesis and evaluation of new types of phosphorylated sugars from the synthon d-arabinonolactone-5-phosphate

Designed as competitive inhibitors of the isomerization reaction catalyzed by the potential chemotherapeutic target phosphoglucose isomerases (PGI), D-arabinonamide-5-phosphate and D-arabinohydrazine-5-phosphate were synthesized and fully characterized. These new types of phosphorylated sugar derivatives were easily and efficiently obtained in a one-step procedure from the promising synthon D-arabinono-1,4-lactone 5-phosphate. These two compounds proved to be new good competitive inhibitors of yeast PGI with the substrate D-fructose-6-phosphate, though not as strong as D-arabinohydroxamic acid-5-phosphate. Overall, our results are in accord with the postulated 1,2-cis-enediolate species as a probable high-energy intermediate of the PGI-catalyzed reaction.

Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.

Competitive inhibition of Trypanosoma brucei phosphoglucose isomerase by D-arabinose-5-phosphate derivatives.

Abstract We report four new strong high energy intermediate analog competitive inhibitors of fructose-6-phosphate isomerization catalyzed by purified Trypanosoma brucei phosphoglucose isomerase: D-arabinonhydroxamic acid-5-phosphate, D-arabinonate-5-phosphate, D-arabino-namide-5-phosphate and D-arabinonhydrazide-5-phosphate. For comparison, the inhibitory properties of the corresponding non-phosphorylated analogues D-arabinonhydroxamic acid, D-arabinonate, D-arabinonamide and D-arabinonhydrazide were aslo evaluated. D-Arabi-nonhydroxamic acid-5-phosphate appears as the most potent competitive inhibitor ever evaluated on a phosphoglucose isomerase with an inhibition constant value of 50 nM and a Michaelis constant over inhibition constant ratio of about 2000. Our results show that anionic high energy intermediate analogues, and more particularly D-arabinonhydroxamic acid-5-phosphate, display a weak but significant specificity for Trypanosoma brucei phosphoglucose isomerase versus yeast phosphoglucose isomerase, while neutral high energy intermediate analogues are not selective at all. This would indicate the presence of more positively charged residues in the active site for Trypanosoma brucei phosphoglucose isomerase as compared to that of yeast phosphoglucose isomerase.

A convenient preparation of aldonohydroxamic acids in water and crystal structure of L-erythronohydroxamic acid.

Hydroxamic acids derived from aldonic acids, namely aldonohydroxamic acids, have become an increasingly important class of inhibitors of enzymes involved in the metabolism of carbohydrates. We now report the straightforward preparation of various types of aldonohydroxamic acids by a new methodology involving the use of commercial 50% aqueous hydroxylamine as the source of the free base hydroxylamine that reacts directly with the corresponding aldonolactone dissolved in water. The reaction proceeds almost instantaneously in water at room temperature, yielding generally pure products in quantitative yield. To date, this methodology is probably the most facile and efficient way to synthesize aldonohydroxamic acids. We also determined by X-ray diffraction analysis the first crystal structure of a free aldonohydroxamic acid reported to date. Crystals of L-erythronohydroxamic acid belonged to the monoclinic system, space group P2(1), a=5.511(3), b=7.556(1), c=8.071(3) A, beta=109.10 degrees, and Z=2.

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

Glucose-6-phosphate isomerase (PGI; EC 5.3.1.9; also often called by its old nomenclature phosphoglucose isomerase) is an intracellular enzyme that catalyses the reversible conversion of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). The native Leishmania PGI is a homodimeric molecule of 60 kDa per monomer with 47% sequence identity to human PGI. It has been shown to be present in both the cytosol and the glycosome of Leishmania promastigotes and represents a potential target for rational drug design. The present work describes the crystallization of two bacterially expressed Leishmania PGI constructs, one corresponding to the natural protein and the other to an N-terminally deleted form. Crystals of both forms are identical and present a large c unit-cell parameter. A complete data set was collected from the N-terminally deleted PGI to a resolution of 3.3 A in space group P6(1), with unit-cell parameters a = b = 87.0, c = 354.7 A, alpha = beta = 90, gamma = 120 degrees. A preliminary study of the first inhibitors to be evaluated on the Leishmania enzyme is also reported.