Validating targets antiparasitic chemotherapy

Guanine and the tetrasodium salt of PRPP were purchased from Sigma Chemical Co. Louis, Mo.) and are of the highest purity available., and concentrations were determined by integration of nuclear magnetic resonance peaks with methylene chloride as an internal standard. UC_Select (25) in combination with the Daylight version of the Available Chemicals Directory was used to identify original reagent sets, as well as for the elimination of reagents that had unattractive chemical or pharmaceutical properties.

The concentration of dimethyl sulfoxide in the assays was kept at 10%. Similarity and superstructure searches of the Available Chemicals Directory were performed with Daylight's Merlin system (version 4.61; Daylight Chemical Information Systems, Inc., Santa Fe, N.

M.), using a Tanimoto similarity metric and Daylight's hashed connectivity fingerprints.

Sybyl's CONCORD module was used to build the virtual reagent library.

It could be expected to form unfavorable interactions with the backbone carbonyls of Asp181 and Asp187, which directly interact with the exocyclic N PRTs, we decided that screening of our in-house phthalimidocarboxanilide database for inhibitory activity against GPRT could potentially result in identification of a starting point for our lead discovery efforts.

This approach resulted in identification of two active molecules (Fig. Both phthalimide derivatives contained bulky fused ring systems that most likely derived much of their binding energy from burying the large hydrophobic substituents.

Parasitic protozoa lack the ability to synthesize purine nucleotides de novo, relying instead on purine salvage enzymes for their survival.

Guanine phosphoribosyltransferase (GPRT) from the protozoan parasite Giardia lamblia is a potential target for rational antiparasitic drug design, based on the experimental evidence, which indicates the lack of interconversion between adenine and guanine nucleotide pools.

A number of groups have reported on the successful design of inhibitors directed against trypanosomal (2, 4, 15–16), leishmanial (6), malarial (19), and tritrichomonal (3, 27) targets active in the 10 n M to 50 μM range. GPRT shows little homology with the known sequences of other purine PRTs (26).Lys152 of GPRT positions its -NH group 6.3 Å away from the O6 of guanine, in sharp contrast to the typically observed distance of 3 Å, with two ordered water molecules spanning the distance.Despite the noted structural differences, the active site preserves the bifocal character observed in other purine PRTs, formed by the stacking interaction at the purine binding site and the hydrogen bonding interactions at the 5′-phosphate binding site of PRTs.In every case, the previously described (3) three-step docking procedure was used involving (i) library preorientation, followed by (ii) rigid docking and (iii) flexible scoring.Spheres were chosen by superimposing phthalimide on the purine or pseudopurine atom positions derived from the respective crystal structures as reported previously (3).

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