Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate that AG1343 is well absorbed orally in a variety of species and possesses favorable pharmacokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing for the treatment of AIDS.

IpaH proteins are E3 ubiquitin ligases delivered by the type III secretion apparatus into host cells upon infection of humans by the Gram-negative pathogen Shigella flexneri. These proteins comprise a variable leucine-rich repeat-containing N-terminal domain and a conserved C-terminal domain harboring an invariant cysteine residue that is crucial for activity. IpaH homologs are encoded by diverse animal and plant pathogens. Here we demonstrate that the IpaH C-terminal domain carries the catalytic activity for ubiquitin transfer and that the N-terminal domain carries the substrate specificity. The structure of the IpaH C-terminal domain, determined to 2.65-Å resolution, represents an all-helical fold bearing no resemblance to previously defined E3 ubiquitin ligases. The conserved and essential cysteine residue lies on a flexible, surface-exposed loop surrounded by conserved acidic residues, two of which are crucial for IpaH activity. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptType III secretion (T3S) systems are used by numerous Gram-negative pathogenic bacteria to deliver effector proteins into the cells of their human, animal or plant hosts. T3S systems comprise the T3S apparatus (T3SA) that form syringe-like structures that span the bacterial envelope and extend like a needle from the bacterial surface, translocators that transit through the T3SA and form a pore within the target cell membrane, effectors that transit through the T3SA and the pore into the cytosol of host cells, and specific chaperones and transcription regulators for secretion and transcription of these effectors 1,2 . Some effectors target the actin cytoskeleton to promote entry or inhibit phagocytosis of the bacterium, whereas other effectors interfere with the host's innate immune responses 1 .The ubiquitin pathway is a common target of bacterial effectors. This pathway involves one ubiquitin-activating enzyme (E1), a limited number of ubiquitin-conjugating enzymes (E2s) and many ubiquitin-ligating enzymes (E3s). The C-terminal glycine residue of 76-residue ubiquitin is first charged via a thioester linkage onto a cysteine residue of E1 and then transferred to a cysteine residue of an E2. E3s recruit an E2 or a subset of E2s for ubiquitin transfer to specific substrates. Two classes of E3s are differentiated on the basis of their mechanism of action and on sequence or structural similarities. RING (really interesting new gene) and U-box (a modified RING motif) domain-containing E3s act as adaptor-like molecules by bringing a ubiquitinated E2 and the substrate into sufficiently close proximity to promote the ubiquitination of the substrate. In contrast, HECT (homologous to E6-associated protein C terminus) domain-containing E3s possess an essential cysteine residue that acts as an acceptor for the ubiquitin carried by the E2 before its transfer to the substrate. The N-and C-terminal domains of HECT E3s are usually involved in substrate binding and catalytic activity, respectively 3 .Several T3S effector...

A lead compound obtained from a high volume human non-pancreatic secretory phospholipase A2 (hnps-PLA2) screen has been developed into a potent inhibitor using detailed structural knowledge of inhibitor binding to the enzyme active site. Four crystal structures of hnps-PLA2 complexed with a series of increasingly potent indole inhibitors were determined and used as the structural basis for both understanding this binding and providing valuable insights for further development. The application of structure-based drug design has made possible improvements in the binding of this screening lead to the enzyme by nearly three orders of magnitude. Furthermore, the optimized structure (LY311727) displayed 1,500-fold selectivity when assayed against porcine pancreatic s-PLA2.