Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.

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A-1155463, a highly potent and selective BCL-X L inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-X L -dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanismbased and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-X L biology as well as a productive lead structure for further optimization.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: ␦-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or ␦-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and ␦-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or ␦-catenin complexed with ␤-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherincatenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and ␦-catenin. Finally, we showed that endogenous ␦-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that ␦-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.PDZ 1 domains are 80 -100-amino acid compact globular motifs that are usually embedded in larger multidomain scaffolding proteins (1-3). PDZ domains predominantly mediate protein/protein interactions by recognizing the C termini of various intracellular and cell-surface proteins. Type 1 PDZ domains interact with the C-terminal consensus sequence X(S/ T)X(V/I/L)-COOH, whereas type 2 domains bind to the C-terminal consensus sequence X-hydrophobe-X-hydrophobe-COOH (3-5). Structural analyses of peptides bound to PDZ domains suggest necessary interactions at both positions 0 and Ϫ2 (6-8). However, among type 1 PDZ ligands, these residues are relatively invariant, indicating that other residues within the C terminus likely contribute to the specificity of PDZ domain/ ligand interactions (3). For example, two previous studies (9 -11), as well as those presented here, demonstrated the importance of residues Ϫ1 and Ϫ3 for binding specificity and affinity for some PDZ domain/ligand interactions.Genetic evidence supports a role for several families of PDZ domain-containing proteins as scaffolding molecules that target signaling complexes to various subcellular locations. For example, the multi-PDZ domain protein INAD was found to assemble components of the Drosophila visual transduction system to allow for efficient signaling in response to light (12, 13). Another study in the nematode Caenorhabditis elegans demonstrated that the basolateral localization of the LET-23 receptor tyrosine kinase is dependent upon direct b...

The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival, which makes them attractive targets for therapeutic intervention in cancers. Herein, we describe the structure-based design of IAP antagonists with high affinities and selectivity (>2000-fold) for c-IAP1 over XIAP and their functional characterization as activators of apoptosis in tumor cells. Although capable of inducing cell death and preventing clonogenic survival, c-IAP-selective antagonists are significantly less potent in promoting apoptosis when compared to pan-selective compounds. However, both pan-IAP- and c-IAP-selective antagonists stimulate c-IAP1 and c-IAP2 degradation and activation of NF-kappaB pathways with comparable potencies. Therefore, although compounds that specifically target c-IAP1 and c-IAP2 are capable of inducing apoptosis, antagonism of the c-IAP proteins and XIAP is required for efficient induction of cancer cell death by IAP antagonists.

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with K i ‫؍‬ 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8-170 ng͞ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW ؊4 ETW ؊1 V; IC 50 ‫؍‬ ϳ0.15 M) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp ؊1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the ␤2-␤3 loop provides a binding site for the Trp ؊4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.