New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Condensing enzymes are essential in type II fatty acid synthesis and are promising targets for antibacterial drug discovery. Recently, a new approach using a xylose-inducible plasmid to express antisense RNA in Staphylococcus aureus has been described; however, the actual mechanism was not delineated. In this paper, the mechanism of decreased target protein production by expression of antisense RNA was investigated using Northern blotting. This revealed that the antisense RNA acts posttranscriptionally by targeting mRNA, leading to 5 mRNA degradation. Using this technology, a two-plate assay was developed in order to identify FabF/ FabH target-specific cell-permeable inhibitors by screening of natural product extracts. Over 250,000 natural product fermentation broths were screened and then confirmed in biochemical assays, yielding a hit rate of 0.1%. All known natural product FabH and FabF inhibitors, including cerulenin, thiolactomycin, thiotetromycin, and Tü3010, were discovered using this whole-cell mechanism-based screening approach. Phomallenic acids, which are new inhibitors of FabF, were also discovered. These new inhibitors exhibited target selectivity in the gel elongation assay and in the whole-cell-based two-plate assay. Phomallenic acid C showed good antibacterial activity, about 20-fold better than that of thiolactomycin and cerulenin, against S. aureus. It exhibited a spectrum of antibacterial activity against clinically important pathogens including methicillinresistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae.Hundreds of essential proteins have been identified in bacteria as potential drug targets (1,16,18,23). Of these, only a few are targets of therapeutically useful drugs. These include penicillin binding proteins, D-Ala-D-Ala ligase, MurA, undecaprenyl pyrophosphate, and alanine racemase for cell wall; 30S and 50S ribosomal subunits, elongation factor G, and IletRNA synthetase for protein synthesis; RNA polymerase for RNA synthesis; InhA (FabI) for fatty acid synthesis; dihydrofolate reductase (FolA) and p-aminobenzoic acid synthase (FolP) for metabolism; and DNA gyrase and topoisomerase IV for DNA synthesis. In past decades, extensive chemical modification of existing antibiotics has afforded improved activity against their targets. This strategy served well to develop new and effective antibiotics; however, such modification is becoming increasingly difficult and identification of new classes of compounds with different modes of action is critical to combat emerging resistance and meet clinical needs.

We found that N- {4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca 2ϩ assays 7-to 8-fold with EC 50 values in the 400 to 800 nM range, and at 10 M shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4-to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3Ј-difluorobenzaldazine (DFB) affected [ 3 H]quisqualate binding to mGluR5, but although DFB partially competed for [ 3 H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 M) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 M CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors (GPCRs) that bind glutamate to modulate neurotransmitter release or postsynaptic excitatory neurotransmission, and hence they modulate the strength of synaptic transmission. The mGluRs are members of GPCR family C and possess a large extracellular agonist binding domain in the amino-terminal portion of the receptor. This agonist binding domain distinguishes family C from the other GPCR families in which the agonist binding sites are associated with the seven-strand transmembrane spanning region or with the extracellular loops that connect the strands of this region. Thus, in the mGluRs, interaction of the agonist with the transmembrane domains is thought to be indirect (O'Hara et al., 1993; for reviews, see Conn and Pin, 1997;Bockaert and Pin, 1999).Article, publication date, and citation information can be found at

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We have identified a family of highly selective allosteric modulators of the group I metabotropic glutamate receptor subtype 5 (mGluR5). This family of closely related analogs exerts a spectrum of effects, ranging from positive to negative allosteric modulation, and includes compounds that do not themselves modulate mGluR5 agonist activity but rather prevent other family members from exerting their modulatory effects. 3,3Ј-Difluorobenzaldazine (DFB) has no agonist activity, but it acts as a selective positive allosteric modulator of human and rat mGluR5. DFB potentiates threshold responses to glutamate, quisqualate, and 3,5-dihydroxyphenylglycine in fluorometric Ca 2ϩ assays 3-to 6-fold, with EC 50 values in the 2 to 5 M range, and at 10 to 100 M, it shifts mGluR5 agonist concentration-response curves approximately 2-fold to the left. The analog 3,3Ј-dimethoxybenzaldazine (DMeOB) acts as a negative modulator of mGluR5 agonist activity, with an IC 50 of 3 M in fluorometric Ca 2ϩ assays, whereas the analog 3,3Ј-dichlorobenzaldazine (DCB) does not exert any apparent modulatory effect on mGluR5 activity. However, DCB seems to act as an allosteric ligand with neutral cooperativity, preventing the positive allosteric modulation of mGluRs by DFB as well as the negative modulatory effect of DMeOB. None of these analogs affects binding of [ 3 H]quisqualate to the orthosteric (glutamate) site, but they do inhibit [ 3 H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding to the site for 2-methyl-6-(phenylethynyl)-pyridine, a previously identified negative allosteric modulator. With the use of these compounds, we provide evidence that allosteric sites on GPCRs can respond to closely related ligands with a range of pharmacological activities from positive to negative modulation as well as to neutral competition of this modulation.Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors that bind glutamate to exert a modulatory influence on neuronal excitability and synaptic transmission in the central nervous system. The eight known members of the mGluR subfamily have been divided into three groups on the basis of their sequence identity, pharmacology, and preferred signal transduction mechanism. Group I mGluRs (mGluRs 1 and 5) are primarily localized postsynaptically where they modulate ion channel activity and neuronal excitability. The group I mGluRs are coupled to G ␣q and its associated effectors, such as phospholipase C. Groups II (mGluRs 2 and 3) and III (mGluRs 4, 6, 7, and 8) are primarily located presynaptically and regulate the release of neurotransmitters, including glutamate. Group II and III mGluRs are coupled to G ␣i and its associated effectors, such as adenylate cyclase. These latter two groups are distinguished from each other by their pharmacology; selective agonists and antagonists have been identified for each group (Conn and Pin, 1997). All mGluR subtypes possess a large (ϳ560 amino acids) extracellular amino-terminal domain that contains the glutamate agonist binding site. Thus, in...

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

Two birds with one stone: Platencin (1) is a novel and potent broad‐spectrum Gram‐positive antibiotic. Whereas platensimycin is a selective inhibitor of FabF, platencin exerts its activity by a novel mode of action by dual inhibition of FabH and FabF.