The emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics demand the discovery of new therapeutic approaches. Targeting bacterial virulence is an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium. Certain virulence factors have been shown to be potential targets for drug design and therapeutic intervention, whereas new insights are crucial for exploiting others. Targeting virulence represents a new paradigm to empower the clinician to prevent and treat infectious diseases.

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTPbinding proteins. How these receptors recognize and promote nucleotide exchange on G protein ␣ subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) ␣ subunit C-terminal undecapeptide Gt␣(340-350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gt␣(340-350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an ␣ L type. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325-346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor-G protein interface is demonstrated.

VALIDATE is a hybrid approach to predict the binding affinity of novel ligands for receptors of known three-dimensional structure. This approach calculates physicochemical properties of the ligand and the receptor−ligand complex to estimate the free energy of binding. The enthalpy of binding is calculated by molecular mechanics while properties such as complementary hydrophobic surface area are used to estimate the entropy of binding through heuristics. A diverse training set of 51 crystalline complexes was assembled, and their relevant physicochemical properties were computed. These properties were analyzed by partial least squares (PLS) statistics, or neural network analysis (SONNIC), to generate models for the general prediction of the affinity of ligands with receptors of known three-dimensional structure. The ability of the model to predict the affinity of novel complexes not included in the training set was demonstrated with three independent test sets:  14 complexes of known three-dimensional structure including 3 DNA complexes, a class of compound not included in the training set, 13 HIV protease inhibitors fit to HIV-1 protease, and 11 thermolysin inhibitors fit to thermolysin.

The presence of multiple a,a-dialkyl amino acids such as a-methylalanine (a-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with a-helical or 310-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-AibVal-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure a-helical character, whereas other similar compounds show predominantly 310-helical structures. The factors that govern helical preference include the inherent relative stability of the a-helix compared with the 310-helix, the extra hydrogen bond seen with 310-helices, and the enhanced electrostatic dipolar interaction of the 310-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions. The presence of a,a-dialkyl amino acids in microbial natural products, such as the peptaibol antibiotics, requires novel biosynthetic pathways to produce and incorporate these unusual amino acids in the face of the usual ribosomal mechanisms available for normal amino acids. This argues strongly for a special role related to function, one aspect of which may be their increased resistance to proteolytic degradation. Another aspect is the conformational restrictions imposed by these amino acids as first pointed out by Marshall and Bosshard (1, 2) and verified by others (3-7). While most work has focused on a-methylalanine (a-aminoisobutyric acid, Aib), a-ethylalanine (isovaline) has also been found to be a natural component of several peptaibol antibiotics (8,9). In addition, chiral a,a-dialkyl amino acids, such as amethylphenylalanine, have been incorporated into naturally occurring peptides in an effort to restrict their conformational freedom (10, 11).From the Ramachandran plots published by Marshall and Bosshard in 1972 (1), the presence of an additional alkyl substituent on the a-carbon severely restricted the values of the torsional variables 4 and qk as compared with those available to normal amino acids. While the two major allowed conformational areas were associated with either right-or left-handed helical conformations (both a and 310), the calculation also revealed other sets of energetically feasible values for 4 and q, adjacent to the a,a-dialkyl residue associated with extended structures as well as turns. The effect on conformation of alkyl groups larger than methyl as substituents in a,a-dialkyl amino acids has also been investigated (11,12). Despite the variety of conformations theoretically available to a,a-dialkyl amino acids, the impact of multiple substitutions ofthis type of amino acid on the overall conformation of a peptide is dramatic. The crystal structure of alamethicin (13), which contains 8 Aib residues out of 20, is predominantly a-helical, with NMR data (14) supporting a similar solution conformation in methanol. A review (15) of crystal structures of tr...

Between 2004 and 2008, the NIH molecular libraries and imaging initiative (MLI) pilot phase funded ten high-throughput Screening Centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the MLI output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 A) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

Nnve! flunrngenic substrates for human immunodeficiency viril pmteise hnve been dcvslopod based on the principle of fluorescence energy transfer. Starting from a p24/p 15 cleavage site-derived hexapeptide substrate, Ac-Thr-Ile-Nle-Nle-Gln-Arg-NH2, incorporation of 2-aminobenzoic acid in place of the acetyl group as the donor and p-NO,-Phe at the P1' position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N-terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96=wcll microtitcr plotcs and a fluoromctric platc reader have hccn devclopcd, which allow high throughput of compounds in the search for HIV protease inhibitors.

Several non-peptide systems have been designed to mimic different types of reverse turns. The incorporation of some of these mimetics into biologically active peptides has led to peptidomimetics with enhanced activity or metabolic stability. This paper reports the conformational analysis of tetrapeptides containing several bicyclic mimetics, sequences containing proline, other N-methyl and N-hydroxy amino acids, and pipecolic acid at residue i + 2 of the turn, and control peptide sequences using the Monte Carlo/stochastic dynamics simulation with the new set of AMBER* parameters for proline-containing peptides in water as implicitly represented by the GB/SA solvation model. Simple N-methylation (Pro-d-NMeAA and d-Pro-NMeAA) and N-hydroxylation of the amide bond between residues i + 1 and i + 2 or inclusion of the larger ring homolog pipecolic acid (d-Pro-Pip) in the third position (i + 2) causes significant nucleation of reverse-turn structures. Spirotricycle analogs restrict three of the four torsion angles that characterize the type II β-turn. Spirolactam analogs also restrict two of the four torsion angles as effective β-turn constraints. However, the geometry of a turn induced by indolizidinone and BTD differs significantly from that of an ideal β-turn and (S)-indolizidinone is more effective as a reverse turn than as a β-turn mimetic. These systems provide useful conformational constraints when incorporated into the structure of selected bioactive peptides. Such analogs can scan receptors for biological recognition of β-turn scaffolds with oriented side chains through combinatorial libraries to efficiently develop three-dimensional structure−activity relationships.