PHOSPHOGLYCERATE KINASE FROM TRYPANOSOMA BRUCEI: SELECTIVE INHIBITORS, HOMOLOGY MODELING AND CRYSTALLIZATION. Bradley E. Bernstein, Christophe L. M. J. Verlinde, Wim G. J. Hol^*, Biomolecular Structure Center, University of Washington, Box 357742, ^*Howard Hughes Medical Institute, Seattle, WA 98195-7420

Trypanosoma brucei, the causative agent of African sleeping sickness is completely reliant on glycolysis for its energy needs while infecting its human host. Trypansomal glycolytic enzymes are, therefore, excellent targets for "selective" structure-based drug design. We found that an empirically identified inhibitor of phosphoglycerate kinase, "SPADNS," has an IC₅₀ of 20uM towards the trypanosomal version of this enzyme but does not inhibit a mammalian PGK in the measurable range. SPADNS has a rigid, multi-ringed structure and several functional groups which can be modified and is therefore an excellent lead compound for drug design. A topology based similarity search identified 89 commercially available derivatives of SPADNS. Two of these compounds were found to be even more potent inhibitors of trypanosomal PGK than SPADNS; with IC₅₀s of 1.2uM and 2.5uM and two orders of magnitude of selectivity. To investigate binding modes for SPADNS and its derivatives a homology model of T.brucei PGK has been built on the basis of the crystal structures of B.stearothermophilus and porcine PGK (49% and 46% identical in sequence to the T.brucei enzyme). Sequence conservation, as well as structure validation algorithms, suggest that this model is particularily accurate in the active site cleft region where these inhibitors are expected to bind. Based on this model a binding mode has been identified which explains: (i) the relative affinities of SPADNS and its derivatives for trypanosomal PGK and (ii) the selectivity which these compounds exhibit with respect to mammalian PGK. We are interested in obtaining crystallographic data for critical analysis of the model and binding hypothesis. Towards this end, a large scale expression and purification of T.brucei PGK has been carried out and conditions which yield small protein crystals are currently being optimized.