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Small-molecule antivirulence agents represent a promising alternative or adjuvant to antibiotics. These compounds disarm pathogens of disease-causing toxins without killing them, thereby diminishing survival pressure to develop resistance. Here we show that the small-molecule antivirulence agents F12 and F19 block staphylococcal transcription factor AgrA from binding to its promoter. Consequently, toxin expression is inhibited, thus preventing host cell damage by Gram-positive pathogens. Broad spectrum efficacy against Gram-positive pathogens is due to the existence of AgrA homologs in many Gram-positive bacteria. F12 is more efficacious in vitro and F19 works better in vivo. In a murine MRSA bacteremia/sepsis model, F19 treatment alone resulted in 100% survival while untreated animals had 70% mortality. Furthermore, F19 enhances antibiotic efficacy in vivo. Notably, in a murine MRSA wound infection model, combination of F19 with antibiotics resulted in bacterial load reduction. Thus, F19 could be used alone or in combination with antibiotics to prevent and treat infections of Gram-positive pathogens.

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4−/−Rdh8−/− mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential., Mutations that lead to misfolding of rhodopsin can cause retinitis pigmentosa. Here, the authors carry out a high throughput screen to identify a small molecule chaperone of rod opsin, and show that it protects mouse models of retinitis pigmentosa from retinal degeneration.

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-A resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore 11-cis-retinal and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomerized product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing Food and Drug Administration (FDA)-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by MS. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that shows features of Stargardt's disease and age-related retinal degeneration.

The Amgenȁ9s Data Access Analysis Prediction Tools (ADAAPT) system is a desktop decision support tool developed to provide flexible access and analysis of chemical and biological data. The system is platform independent, adaptable, easily deployed, and scalable. It consists of four main modules: access, analysis, prediction, and tools. The access module contains numerous user interfaces designed to retrieve data easily. The analysis module provides standard computational tools to perform property calculation, QSAR/QSPR, and statistical analyses. The prediction module contains in-house models to calculate a drug-likeness score and absorption index. Finally, the tools module provides a wide array of features that are of general interest to our scientists.

Through pharmacophore models and providing quantitative analysis of structure-activity relationships (QSAR), molecular modeling techniques can be useful tools to study the interactions of ion channels and their modulators. The present review focuses on molecular modeling approaches that defined pharmacophore models of ion channel modulators in the CNS. The commonality and subtlety of the pharmacophore models of various ion channel modulators are discussed which can be used as a framework for the design of ion channel modulators.

The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66–76, 1996, and J. Virol. 71:4062–4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the α-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.

A dynamical model for the structure of two human immunodeficiency virus-1 protease (HIV-1 PR)-inhibitor complexes, H-Val-Ser-Gln-Asn-Leu Ψ [CH(OH)CH 2 ]Val-Ile-Val-OH (U85548e) and N-acetyl-Thr-Ile-Nle-Ψ [CH 2 NH]-Nle-Gln-Arg-amide (MVT-101), has been developed on the basis of molecular dynamics simulations. The study examines all possibilities of protonation state and compares the results with the corresponding crystal structures. The results indicate that only one of the four states for each inhibitor agrees well with the crystallographic data. This provides a theoretical prediction of the protonation state of the catalytic aspartic acids for each complex and may be useful for the evaluation of potential therapeutic targets

A facile synthesis of a series of potential pseudosymmetric HIV-protease inhibitors containing 1,3-diamino-2-hydroxypropane moiety is described.

A molecular dynamics simulation has been performed on the Asn88-Gln88 mutant of the aspartyl protease of the human immunodeficiency virus including explicit consideration of solvent water molecules. Studies of residue 88 protease mutants by Guenet et al. (Eur. J. Pharmacol. 1989, 777, 443-451) have previously demonstrated that the enzyme is inactivated due to dimer dissociation, even though the mutant site is removed from the dimer interface. The calculations provide leading evidence that the dimer is destabilized in the Gln88 mutant and suggest a molecular mechanism in which the mutation indirectly alters the articulation of the monomeric units

Molecular dynamics simulations have been carried out based on the GROMOS force field on the aspartyl protease (PR) of the human immunodeficiency virus HIV-1. The principal simulation treats the HIV-1 PR dimer and 6990 water molecules in a hexagonal prism cell under periodic bundary conditions and was carried out for a trajectory of 100 psec. Corresponding in vacuo simulations, i.e., treating the isolated protein without solovent, were carried out to study the influence of solvent on the simulation. The results indicate that including waters explicitly in the simlation results in a model considerably closer to the crystal structure than when solvent is neglected. Detailed conformational and helicoidal analysis was perfomed on the solvated form to determine the exact nature of the dynamical model and the exact points of agreement and disagreement with the crystal structure. The calculated dynamical model was furthr elucidated by means of studies of the time evolution of the cross-correlation coefficients for atomic displacements of the atoms comprising the protein backbone. The cross-correlation analysis revealed significant aspects of structure originating uniquely in the dynamical motions of the molecule. In particular, an unanticipated troughspace, domain-domain correlation was found between the mobile flap region covering the active site and a remote regions of the structure, which collectively act somewhat like a molecular cantilever. The significance of these results is discussed with respect to the inactivation of the protease by site-specific mutagenesis, andin the design of inhibitors. © 1992 Wiley-Liss, Inc.

4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB. Selected 4-thiazolidinones displayed activity against the enzyme in vitro. This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.

An x-ray crystallographic analysis of the potent anti-HIV agent D4T revealed two independent conformations (conformers a and b) with different glycosyl bonds and furanose geometries. Conformer a exhibits the unusual O4' exo configuration and chi (C2, N1, C1', O4') of -118 degrees. Conformer b exhibits a nearly planar furanose geometry and chi of -174 degrees. The reduced form of D4T, ddT, is poorly active against HIV and also exists in two independent conformations. Chi of forms a and b (-129 and -170.9 degrees) are similar to that found with D4T. However, the furanoses exhibit the classical C2' endo and C3' endo geometries, respectively. These observed differences are not sufficient to account for the differing potencies of D4T versus ddT.

The inhibition of HMG-CoA reductase is an efficient way to lower plasma chlosterol levels in humans. The 9,9-bis (4-fluorophenyl)-3,5-dihydroxy-8-(substituted)-6,8-nonadienoic acids analogues represent a novel class of HMG-CoA reductase inhibitors developed at Bristol-Myers. The goal of this study was to delineate from inhibitory potency values the main topographical and physico-chemical features of the binding site probed by the substituent attached to the C"8 position of the analogues. Using a combination of receptor mapping and 3D-QSAR it was possible to obtain a quantitative map of the binding site which relates the HMG-CoA reductase inhibitory potency to the shape and size of both the binding site and C"8-substituent of the inhibitor. We prove that the 3D-QSAR derived here is reliable, robust, and affords predictive utility.

Native sulfatides, as well as many sulfated glycolipids, have been shown to avidly bind to the selectin receptors. In vivo, native sulfatides significantly block activity in selectin-dependent inflammatory responses. The fact that nonsulfated galactocerebrosides did not inhibit selectin-mediated adhesion identified a critical role for the anionic sulfate residue. We therefore initiated a program to evaluate the activity of position isomers. This study showed a binding selectivity for the positions 2 and 3 of the sulfate group on the carbohydrate ring as well as enhanced activity for the disulfated analogs. Furthermore, it was discovered that the attachment of lipophilic substituents on the carbohydrate ring was tolerated, consistent with the presence of a lipophilic pocket in the binding activity. This resulted in compounds with a 6-fold increased potency.

In the initial stages of influenza virus infection, the hemagglutinin (HA) protein of influenza virus mediates both adsorption and penetration of the virus into the host cell. Recently, we identified and characterized BMY-27709 as an inhibitor of the H1 and H2 subtypes of influenza A virus that specifically inhibits the HA function necessary for virus-cell membrane fusion (G.-X. Luo, R. Colonno, and M. Krystal, Virology 226:66-76, 1996). Studies presented herein show that the inhibition is mediated through specific interaction with the HA protein. This binding represses the low-pH-induced conformational change of the HA protein which is a prerequisite for membrane fusion. In an attempt to define the binding pocket within the HA molecule, a number of drug-resistant viruses have been isolated and characterized. Sequence analyses of the HA gene of these drug-resistant viruses mapped amino acid changes responsible for drug resistance to a region located near the amino terminus of HA2. In addition, we have identified inactive analogs of BMY-27709 which are able to compete out the inhibitory activity of BMY-27709. This finding suggests that inhibition of the HA-mediated membrane fusion by this class of compounds is not solely the result of binding within the HA molecule but requires specific interactions.

P-Selectin (CD62/GMP140/PADGEM) is an inducible cell-surface glycoprotein expressed by endothelial cells and platelets following stimulation by inflammatory mediators such as thrombin, histamine, or peroxides. P-Selectin mediates the binding of leukocytes to activated vascular endothelium at sites of inflammation and plays a role in mediating the binding of activated platelets to leukocytes and the vascular cell wall. The adhesive function of P-selectin is mediated by its calcium-dependent (or C-type) lectin domain, which is known to bind to carbohydrate ligands including fucosyl-N-acetyllactosamine (Lex, CD15), sialyl-Lex, and 3-sulfated galactosylceramides (sulfatides). Sulfatides can efficiently block P-selectin/myeloid cell binding in vitro and are excreted at high levels by activated granulocytes. These observations led to the hypothesis that sulfatide may play a role in facilitating the disengagement of CD62, allowing the efficient exit of granulocytes from the blood stream at sites of inflammation. In this report, we extend our previous mutagenesis analysis of the P-selectin binding site [Hollenbaugh, D., Bajorath, J., Stenkamp, R., & Aruffo, A. (1993) Biochemistry 32, 2960] and show that replacement of Tyr48 with Ser or Lys113 with Arg results in P-selectin mutants that, although correctly folded, do not bind to HL60 cells. These results suggest that the conservation of charged and hydrogen-bonding site chains is not sufficient to maintain the P-selectin function and that the exact stereochemistry provided by the side chains of residues lining the P-selectin binding pocket is critical for P-selectin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

This chapter has focused on the application of molecular dynamics computer simulations and related molecular modeling techniques to the study of HIV protease structure and structure-function relationships. The abundance of crystallographic data provides ample experimental quantities (average structures, temperature factors, and hydrogen bond topography) to validate the computational techniques employed. Furthermore, these studies provide insight into the structure and functional energetics of HIV-1 protease that would be difficult or impossible to study experimentally. This chapter covers studies that investigate correlated motion between and within subunits of the protease, mutants of the protease that disrupt the tertiary structure and dimer formation, and studies of HIV-1 protease-inhibitor complexes that rationalize both the protonation state of the active site and the observed binding strength of these complexes. These studies demonstrate that MD is capable of contributing to our understanding of structure-function relationships and may aid in the design of potential therapeutics.

A series of 9,9-bis(4-fluorophenyl)-3,5-dihydroxy-8-(alkyltetrazol-5-yl)- 6,8-nonadienoic acid derivatives 1 were synthesized and found to inhibit competitively the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The analogues having 1N-methyltetrazol-5-yl attached to the C8-position (3a, 4a, R1 = R2 = F) are the most active in suppressing cholesterol biosynthesis in both in vitro and in vivo models: the IC50 for the chiral form of 3a is 19 nM, Ki = 4.3 x 10(-9)M when Km for HMG-CoA is 28 x 10(-6) M;1 the ED50 (oral) value corresponding to the lactone derivative (4a, BMY 22089) is approximately 0.1 mg/kg. Further, BMY 21950 is nearly 2 orders of magnitude more active in parenchymal heptaocytes, from which most of the serum cholesterol originates, than in other cell preparations (such as spleen, testes, ileum, adrenal, and ocular lens epithelial cells; Table III). This apparent tissue specificity may be highly beneficial since the blocking of cholesterol biosynthesis in other vital organs could eventually lead to undesirable side effects. In addition to the chemical synthesis and biological evaluation, a theoretical study aimed at relating the HMG-CoA reductase inhibitory potency to the three-dimensional structure of the inhibitors was undertaken. With a combination of molecular mapping and 3D-QSAR techniques, it was possible to determine a logical candidate for the conformation of the bound inhibitor and to quantitatively relate inhibitory potency to the shape and size of both the binding site and the C8-substituent.