The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray re®nement of protein±ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force ®eld used, structures with good geometries are generated. X-ray re®nement in X-PLOR/CNS, REFMAC and SHELX using PRODRGgenerated topologies produces results comparable to re®ne-ment with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.

The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by PtdIns(3,4,5)P 3 , comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum-and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.

Chitinase B (ChiB) from Serratia marcescens is a family 18 exochitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrateassisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites ؊2 to ؉3) shows closure of the ''roof'' of the active site tunnel. It also shows that the sugar in the ؊1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the ؉1 and ؉2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.C hitinases hydrolyze chitin, a linear polymer of ␤-(1,4)-linked N-acetylglucosamine (NAG), which is an abundant biopolymer. These enzymes are essential to chitin-containing organisms (fungi, insects, crustaceans) and are used by several bacteria to exploit chitin as a source of energy. Chitinase inhibitors have generated a lot of interest given their potential as insecticides (1), fungicides (2, 3), and antimalarials (4, 5). Biotechnological exploitation of chitinases, as well as design of inhibitors with sufficiently high selectivity and affinity, requires detailed knowledge of the catalytic mechanism and enzyme-substrate interactions.Most nonplant chitinases belong to glycosidase family 18 (6) and degrade chitin with retention of the stereochemistry at the anomeric carbon (7-10). The reaction is thought to be initiated by distortion of the Ϫ1 sugar ring and protonation of the glycosidic oxygen by a protonated acidic residue. The subsequent nucleophilic attack differs from classical reaction mechanisms of retaining enzymes such as lysozyme (11) and amylases (12) in that it involves the N-acetyl group of the Ϫ1 sugar, rather than a carboxylate side chain on the protein (8,9,13,14). Thus, the first step of chitinolysis results in cleavage of the sugar chain and formation of an oxazolinium ion intermediate, and hydrolysis of this ion completes the reaction (9, 15) (Fig. 1).Although the current model for the catalytic mechanism is well established, the amount of structural evidence in its support is limited (8). Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to f...

African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.