Our aim was to improve techniques for drug development by facilitating the identification of small molecules that bind with high affinity to acceptor molecules (for example, cell-surface receptors, enzymes, antibodies) and so to mimic or block their interaction with the natural ligand. Previously such small molecules have been characterized individually on a serial basis. The systematic synthesis and screening of peptide libraries of defined structure represents a new approach. For relatively small libraries, predetermined sequence variations on solid-phase supports have been used, and large libraries have been produced using a bacteriophage vector into which random oligodeoxynucleotide sequences have been introduced, but these techniques have severe limitations. Here we investigate an alternative approach to synthesis and screening of peptide libraries. Our simple methodology greatly enhances the production and rapid evaluation of random libraries of millions of peptides so that acceptor-binding ligands of high affinity can be rapidly identified and sequenced, on the basis of a 'one-bead, one-peptide' approach.

No abstract

4-{[4-({(3R)-1-Butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140) is a potent noncompetitive allosteric antagonist of the CCR5 receptor (pK B ϭ 8.6 Ϯ 0.07; 95% CI, 8.5 to 8.8) with concomitantly potent antiviral effects for HIV-1. In this article, the receptor-based mechanism of action of 873140 is compared with four other noncompetitive allosteric antagonists of CCR5. Although 857), and N, with an allosteric mechanism of action. The blockade of CCR5 by 873140 was extremely persistent with a rate constant for reversal of Ͻ0.004 h Ϫ1 (t 1/2 Ͼ 136 h). Coadministration studies of 873140 with the four other allosteric antagonists yielded data that are consistent with the notion that all five of these antagonists bind to a common allosteric site on the CCR5 receptor. Although these ligands may have a common binding site, they do not exert the same allosteric effect on the receptor, as indicated by their differential effects on the binding of 125 I-RANTES. This idea is discussed in terms of using these drugs sequentially to overcome HIV viral resistance in the clinic.With the discovery that the R5 strain of HIV uses the chemokine C CCR5 receptor for cell infection (Alkhatib et al., 1996;Choe et al., 1996;Doranz et al., 1996; Dragic et al., 1996;Deng et al., 1997;Shieh et al., 1998;Zhang and Moore, 1999) has come the opportunity for a completely new approach to preventing HIV infection: blockade of CCR5 receptor interaction with the viral coat protein gp120. Subsequent reports of potent antagonists of CCR5-mediated HIV entry (Baba et al., 1999;Finke et al., 2001;Strizki et al., 2001;Kazmierski et al., 2003; Demarest et al., 2004a,b, Maeda et al., 2004 have validated this approach and have possibly opened a new era of AIDS therapy. There are data to support the notion that an allosteric mechanism is involved in the antagonism of HIV by low molecular weight antagonists of CCR5 (Kazmierski et al., 2002). The large size of the proteins involved in HIV fusion (i.e., CCR5 and gp120) and the fact that mutational studies indicate that numerous regions of both CCR5 (Atchison et al., 1996;Rucker et al., 1996;Doms and Peiper, 1997;Doranz et al., 1997;Picard et al., 1997 ABBREVIATIONS: MIP-1␣, macrophage inflammatory protein 1-alpha (standard nomenclature CCL3, also known as LD78); CHO, Chinese hamster ovary; SPA, scintillation proximity assay; DMSO, dimethyl sulfoxide; RT, room temperature; HEK, human embryonic kidney; FLIPR, fluorometric imaging plate reader; RANTES, regulated on activation, normal T cell expressed and secreted (standard nomenclature for this chemokine is CCL5); Sch-C (SCH 351125), (Z)-(4-bromophenyl

A series of cyclic, conformationally constrained peptides related to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the mu opioid receptor. The following new peptides were prepared and tested for their mu opioid receptor potency and selectively in rat brain binding assays: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (2, CTOP); D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (3, CTAP); D-Phe-Cys-Tyr-D-Trp-Nle-Thr-Pen-Thr-NH2 (4); D-Phe-Cys-Tyr-D-Trp-Lys-Val-Pen-Thr-NH2 (5); D-Phe-Cys-Tyr-D-Trp-Lys-Gly-Pen-Thr-NH2 (6); D-Phe-Cys-Tyr-Trp-Lys-Thr-Pen-Thr-NH2 (7); D-Tyr-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH (8); D-PhGly-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (9); and D-PhGly-Pen-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (10). The most selective peptide, 2 (CTOP), displayed both high affinity (IC50 = 3.5 nM) and exceptional selectivity (IC50 delta/IC50 mu = 4,000) for mu opioid receptors. Furthermore, 2 exhibited very low affinity for somatostatin receptors in the rat brain (IC50 greater than 24,000 nM), with an IC50 somatostatin/IC50 mu receptor selectivity of 8,750. These conformationally constrained cyclic peptides should provide new insight into the structural and conformational requirements for the mu opioid receptor and the physiological role of this receptor.

No abstract

Six allosteric HIV-1 entry inhibitor modulators of the chemokine (C-C motif) receptor 5 (CCR5) receptor are compared for their potency as inhibitors of HIV-1 entry [infection of human osteosarcoma (HOS) cells and peripheral blood mononuclear cells (PBMC)] and antagonists of chemokine (C-C motif) ligand 3-like 1 [CCL3L1]-mediated internalization of CCR5. This latter activity has been identified as a beneficial action of CCL3L1 in prolonging survival after HIV-1 infection (Science 307: 1434 -1440, 2005). The allosteric nature of these modulators was further confirmed with the finding of a 58-fold (HOS cells) and 282-fold (PBMC) difference in relative potency for blockade of CCL3L1-mediated internalization versus HIV-1 entry. For the CCR5 modulators, statistically significant differences in this ratio were found for maraviroc, vicriviroc, aplaviroc, Sch-C, TAK652, and TAK779. For instance, although TAK652 is 13-fold more potent as an HIV-1 inhibitor (over blockade of CCL3L1-mediated CCR5 internalization), this ratio of potency is reversed for Sch-C (22-fold more potent for CCR5-mediated internalization over HIV-1 entry). Quantitative analyses of the insurmountable antagonism of CCR5 internalization by these ligands suggest that all of them reduce the efficacy of CCL3L1 for CCR5 internalization. The relatively small magnitude of dextral displacement accompanying the depression of maximal responses for aplaviroc, maraviroc and vicriviroc suggests that these modulators have minimal effects on CCL3L1 affinity, although possible receptor reserve effects obscure complete interpretation of this effect. These data are discussed in terms of the possible benefits of sparing natural CCR5 chemokine function in HIV-1 entry inhibition treatment for AIDS involving allosteric inhibitors.HIV-1 has been shown to use the chemokine C receptor CCR5 to gain entry into cells to cause infection (Alkhatib et al., 1996;Choe et al., 1996;Deng et al., 1996;Dragic et al., 1996); therefore, efforts have centered on the development of CCR5 HIV entry inhibitors for potential treatment of AIDS and AIDS prevention. Several molecules have been described previously (Kazmierski et al., 2003(Kazmierski et al., , 2007Schols, 2006); presently, one of them, maraviroc, has been approved for therapeutic use (Fä tkenheuer et al., 2005). Another CCR5 entry inhibitor, aplaviroc (Demarest et al., 2004; Article, publication date, and citation information can be found at

2275explore the effects of thiols on the partitioning between 4'-and S'-hydrogen abstraction and on the reduction of peroxide intermediates are in progress. and extends the findings of Saito et aLs and suggests that this is a general property of all G T steps. Recent observations have demonstrated that the cleavage resulting from 4'-hydrogen abstraction at GT steps occurs most frequently as part of a staggered double-strand break.23 Additional studies using oligomers to (23) Dedon, P. C.; Goldberg, 1. H. Abstract:We have proposed that development of methods for controlling the side-chain topography of amino acid residues in peptides and proteins provides a new approach to the topographical design of biologically active peptides. An example of this approach is the use of the 1,2,3,4-tetrahydroisoquinolinecarboxylic acid (Tic) residue, which favors a gauche (-) side-chain conformation when in the N-terminal position, whereas in its acylated form (internal position), the most stable side-chain conformation is gauche (+). This approach has been tested by incorporating D-Tic or Tic at different positions of 1. 1 opioid receptor specific octapeptides such as D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH, (CTP, l), examination of the biological consequences of these modifications, and detailed 'H NMR based conformational analysis. The compounds prepared and their biological activities were as follows: ~-Tic-Cys-Tyr-~-Trp-Lys-Thr-Pen-Thr-NH~ (2; gauche (-), 6/1.1 = 7800, ICso 1. 1 = 1.2 nM); Gly-~-Tic-Cys-Tyr-~-Trp-Orn-Thr-Pen-Thr-NH~ (3; gauche (+), 6/1.1 = 19, ICso p = 278.7 nM); and D-PheCys-Tic-~-Trp-Orn-Thr-Pen-Thr-NH~ (4; gauche (+), 6 / p = -7 , ICso p = 1439.0 nM). In the absence of a geminal pair of protons suitable for distance calibration, a new technique (Davis, D. G. J. Am. Chem. SOC. 1987, 109, 3471-3472) of transverse and longitudinal cross-relaxation rate measurements has been utilized in conjunction with other 2D NMR methods in order to determine the three-dimensional solution conformations for the peptides 1-4, with subsequent application of restrained molecular dynamics (GROMOS). The average backbone conformations in peptides 1-4 were very similar, but the side-chain conformational preferences in the analogues differed, suggesting that the different affinities and selectivities for 1.1 opioid receptors were primarily due to differences in the side-chain conformations of Tic (D-Tic), and thus due to differences in the topographies of these peptides, and not the backbone conformations. A detailed analysis of these relationships is presented.

SUMMARYHepatitis C virus (HCV) infects more than 3% of the world's population, leading to an increased risk of cirrhosis and hepatocellular carcinoma. The current standard of care, a combination of pegylated interferon alfa and ribavirin, is poorly tolerated and often ineffective against the most prevalent genotype of the virus, genotype 1. The very recent approval of boceprevir and telaprevir, two HCV protease inhibitors, promises to significantly improve treatment options and outcomes. In addition to the viral protease NS3 and the viral polymerase NS5B, direct-acting antivirals are now in development against NS5A. A multifunctional phosphoprotein, NS5A is essential to HCV genome replication, but has no known enzymatic function. Here we report how the design of small-molecule inhibitors against NS5A has evolved from promising monomers to highly potent dimeric compounds effective against many HCV genotypes. We also highlight recent clinical data and how the inhibitors may bind to NS5A, itself capable of forming dimers. HEPATITIS C VIRUS (HCV): AN INTERNATIONAL HEALTH CONCERNHepatitis C virus (HCV) infects approximately 170 million individuals, with an estimated 2.3-4.7 million new infections each year (1). The primary mode of transmission of HCV is via exposure to infected blood, including transfusions from infected donors, and through injection drug use. It is estimated that 15-30% of all HCV infections will spontaneously clear, but the remaining 70-85% of infections will develop into chronic hepatitis (2, 3). Chronic infections can subsequently lead to steatosis, cirrhosis and hepatocellular carcinoma (4). Among all recognized positive-strand RNA viruses, the ability to establish a chronic infection is exclusive to HCV (5), although how the virus mediates persistence remains unknown.Current treatment options for HCV are relatively poor. The standard of care is often a grueling 48-week combination of pegylated interferon alfa (IFN-α) and the nucleoside analogue ribavirin. Effective clearance of the virus is achieved in less than 50% of genotype