A computer-based method was developed for rapid and automatic identification of potential "frequent hitters". These compounds show up as hits in many different biological assays covering a wide range of targets. A scoring scheme was elaborated from substructure analysis, multivariate linear and nonlinear statistical methods applied to several sets of one and two-dimensional molecular descriptors. The final model is based on a three-layered neural network, yielding a predictive Matthews correlation coefficient of 0.81. This system was able to correctly classify 90% of the test set molecules in a 10-times cross-validation study. The method was applied to database filtering, yielding between 8% (compilation of trade drugs) and 35% (Available Chemicals Directory) potential frequent hitters. This filter will be a valuable tool for the prioritization of compounds from large databases, for compound purchase and biological testing, and for building new virtual libraries.

The 3.0-Å resolution x-ray structure of human des-Glacoagulation factor Xa (fXa) has been determined in complex with the synthetic inhibitor DX-9065a. The binding geometry is characterized primarily by two interaction sites: the naphthamidine group is fixed in the S1 pocket by a typical salt bridge to Asp-189, while the pyrrolidine ring binds in the unique aryl-binding site (S4) of fXa. Unlike the large majority of inhibitor complexes with serine proteinases, Gly-216 (S3) does not contribute to hydrogen bond formation. In contrast to typical thrombin binding modes, the S2 site of fXa cannot be used by DX-9065a since it is blocked by Tyr-99, and the arylbinding site (S4) of fXa is lined by carbonyl oxygen atoms that can accommodate positive charges. This has implications for natural substrate recognition as well as for drug design.Hemostasis is the blood clotting process that, when functioning properly, occurs when an injury to the vasculature leads to a series of vasculomotor and cellular reactions and the activation of the blood coagulation cascade. The latter process is initiated via the extrinsic pathway, leading first to thrombin activation and then massively amplified thrombin activation due to the positive feedback of the intrinsic pathway (1). Both extrinsic and intrinsic pathways merge at the factor X activation step. An imbalance between these clotting processes, clotting inactivation processes (protein C inactivation of hemostasis cofactors), and thrombolytic processes (tissue plasminogen activator, plasminogen) can lead to thrombotic or bleeding disorders. Antithrombotics include inhibitors of thrombin, factor Xa and factor IXa, factors involved in both the extrinsic and intrinsic pathways (2). As evidence accumulates that thrombin has other important functions in cellular (3, 4) and neurological (5-10) processes, new synthetic anticoagulants increasingly target factor Xa. Daiichi published the first tight binding (K i ϭ 41 nM), specific inhibitor of fXa, 1 DX-9065a (11-13). We present here the crystal structure of the factor Xa⅐DX-9065a complex. The inhibitor binds in the active site in an extended conformation, which was expected from earlier studies (14, 15). Both hydrophobic and electrostatic interactions characterize the complex formation, which is also accompanied by local rearrangements in the active site of fXa. Considering these subtle interactions and the unpredictable ligand-induced motions involved in binding, there is clearly a need for a series of fXa-ligand complex structures to provide adequate information for structure-based drug design and an understanding of how fXa recognizes physiological substrates.EXPERIMENTAL PROCEDURES fX was isolated from human plasma; des-Gla-fX was produced via chymotryptic cleavage (removing amino acids L1-L44; chymotrypsinogen numbering is used for the catalytic domain; the sequential fX numbering, which is used for the light chain, will be indicated by the prefix "L"). Subsequently, fX was activated with the purified factor X activator from Russell's vip...

The structural features of a new class of non-nucleoside HIV-1 reverse transcriptase inhibitors (3) are presented. Comparison of the structural and electronic properties with those of TIBO (1) and Nevirapine (2) yields a common three-dimensional model. This model permits the improvement of the lead compound 3 by chemical modification (5,6). Additionally, two new types of inhibitors (4, 7) with similar biological activity can be derived from this model. The structure of the new compounds, including their absolute configuration, are determined by X-ray crystallography.

Protein flexibility and variable water structures are essential elements in protein-ligand interactions. Ligand design strategies that fail to take this into account may overlook or underestimate the potential of lead structures. Further, the significance of 'weak' interactions must be considered both in crystallographic refinement and in analysis of binding mechanisms.

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)