Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor-reactive cytolytic T lymphocytes.

The recent identification of molecularly defined human tumor antigens recognized by autologous CTLs has opened new opportunities for the development of antigen-specific cancer vaccines. Despite extensive work, however, the number of CTL-defined tumor antigens that are suitable targets for generic vaccination of cancer patients is still limited, mostly because of the painstaking and lengthy nature of the procedures currently used for their identification. A novel approach is based on the combined use of combinatorial peptide libraries in positional scanning format (positional scanning synthetic combinatorial peptide libraries, PS-SCLs) and tumor-reactive CTL clones. To validate this approach, we herein analyzed in detail the recognition of PS-SCLs by Melan-A-specific CTL clones. Our results indicate that, at least for some clones, most of the amino acids composing the native antigenic peptide can be identified through the use of PS-SCLs. Interestingly, this analysis also allowed the identification of peptide analogues with increased antigenic activity as well as agonist peptides containing multiple amino-acid substitutions. In addition, biometrical analysis of the data generated by PS-SCL screening allowed the identification of the native ligand in a public database. Overall, these data demonstrate the successful use of PS-SCLs for the identification and optimization of tumor-associated CTL epitopes.

Solid phase synthesis of mixture-based acyclic and heterocyclic small molecule combinatorial libraries from resin-bound polyamides.

The development of soluble mixture-based heterocyclic combinatorial libraries derived from amino acids and peptides is described. Starting with a "toolbox" of various chemical transformations, including alkylations, reductions, acylations, and the use of a variety of bifunctional reagents, the "libraries from libraries" concept has been expanded to encompass the development of more than fifty positional scanning combinatorial libraries each composed of tens of thousands of low molecular weight acyclic and heterocyclic compounds.

Orphanin FQ/nociceptin receptor binding studies.

A review of the binding studies performed on the receptor (ORL) for Orphanin FQ/Nociceptin is presented. Binding studies have been conducted using a variety of receptor sources: cell lines expressing the cloned receptor, cell lines endogenously expressing the receptor, and brain and other tissue from several different species. Binding studies of opioids, new ligands and antagonists at the ORL receptor are briefly discussed. Saturation, competition and binding kinetic experiments, and the effects of buffer composition are reviewed. There are numerous instances of conflicting data in published reports on OFQ; the basis for these disparities is as yet undetermined. This review endeavors to compile the results and conditions employed in binding studies as an aid to current and new researchers in this field. In an attempt to explain binding disparities, we have determined that Orphanin/Nociceptin binds to glass fiber filtermats in a "specific" manner; these new data are presented.

Polyarginines are potent furin inhibitors.

The ubiquitous serine endoprotease furin has been implicated in the activation of bacterial toxins and viral glycoproteins as well as in the metastatic progression of certain tumors. Although high molecular mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomolar inhibitors have been reported to date. Here we describe the identification of such inhibitors using positional scanning amidated and acetylated synthetic l- and d-hexapeptide combinatorial libraries. The results indicated that l-Arg or l-Lys in all positions generated the most potent inhibitors. However, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-l-arginine, had a K(i) for furin of 40 nm. The K(i) values for the related convertases PACE4 and prohormone convertase-1 (PC1) were 110 nm and 2.5 microm, respectively. Although nona-l-arginine was cleaved by furin, the major products after a 6-h incubation at 37 degrees C were hexa- and hepta-l-arginines, both of which retained the great majority of their potency and specificity against furin. Hexa-d-arginine was as potent and specific a furin inhibitor as hexa-l-arginine (K(i) values of hexa-d-arginine: 106 nm, 580 nm, and 13.2 microm for furin, PACE4, and PC1, respectively). PC2 was not inhibited by any polyarginine tested; indeed, PC2 showed an increase in activity of up to 140% of the control in the presence of l-polyarginines. Data are also presented that show extended subsite recognition by furin and PC2. Whereas N-terminal acetylation was found to reduce the inhibitory potency of the l-hexapeptide LLRVKR against furin 8-fold, C-terminal amidation reduced the potency < 2-fold. Conversely, N-terminal acetylation increased the potency against PC2 nearly 3-fold, whereas C-terminal amidation of the same peptide increased the potency by a factor of 1.6. Our data indicate that non-acetylated, poly-d-arginine-derived molecules may represent excellent lead compounds for the development of therapeutically useful furin inhibitors.

Inhibition of beta-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the beta-amyloid peptide (Abeta). Evidence of a direct correlation between the ability of Abeta to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Abeta have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Abeta25-35. In an effort to generate novel inhibitors of Abeta neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Abeta25-35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Abeta25-35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Abeta1-42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Abeta25-35 and Abeta1-42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to beta-sheet transition and self-aggregation of Abeta25-35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Abeta aggregation process and neurotoxicity.

Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4+ T cells and induce T cell responses to native peptide ligands.

Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.

Discovery of novel peptidic dopamine transporter ligands by screening a positional scanning combinatorial hexapeptide library.

The acute reinforcing effects of cocaine are thought by some to result from cocaine binding to the dopamine (DA) transporter, which inhibits DA uptake and increases synaptic DA levels in the mesolimbic system. Other data suggest that neurotransmitters other than DA contribute to cocaine reinforcement and addiction. These considerations illustrate the need to have additional research tools with which to test the "DA hypothesis." One strategy is to identify drugs which bind to the DA transporter (DAT ligands) but which do not inhibit DA uptake as effectively as cocaine. The purpose of the present study was to identify members of a novel structural class of DAT ligands and to characterize their interactions at the DA transporter. A positional scanning hexapeptide D-amino acid library was screened for inhibition of [(125)I]RTI-55 binding to rat caudate DA transporters. Based on the results, 12 peptides were synthesized. All 12 peptides inhibited [(125)I]RTI-55 binding to DA transporters with IC(50) values, which ranged from 1.8 microM to 12 microM. The two most potent peptides (TPI-669-1 and TPI-669-4) were prepared in larger quantities and were characterized further for activity at the DAT and 5-HT transporter. Both peptides inhibited DA and 5-HT uptake and transporter binding with IC(50)/K(i) values in the low micromolar range. In vivo microdialysis studies demonstrated that both peptides increase extracellular DA and 5-HT in the nucleus accumbens of rats. These data demonstrate that peptides can function as inhibitors of biogenic amine transport. Future work will focus on developing more potent and selective peptides. Published 1999 Wiley-Liss, Inc.

Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library.

A positional scanning synthetic peptide combinatorial library containing approximately 52 million hexapeptides was used to identify potential inhibitory peptides for recombinant mouse prohormone convertase 1 (PC1) and PC2 and to provide information on the specificity of these enzymes. The library surveys revealed that a P6 Leu, a P4 Arg, a P2 Lys, and a P1 Arg were most inhibitory against PC1, and a P6 Ile and a P4 Arg were most inhibitory against PC2. Using information derived from the library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2. The data obtained revealed the preference of both enzymes for a P3 Val. At P5, many substitutions were well tolerated. PC1 and PC2 proved to differ mainly in the selectivity of their S6 subsites. In PC1, this subsite displayed a strong preference toward occupation by Leu; the Ki value for peptide Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 28 times lower than that for peptide Ac-Ile-Ile-Arg-Val-Lys-Arg-NH2. In contrast, PC2 discriminated little between Leu and Ile at P6, as evidenced by the small (1.5-fold) difference in Ki values for these two peptides. Several hexapeptides synthesized as a result of the screen were found to represent potent inhibitors of PC2 (with Ki values in the submicromolar range) and, particularly, of PC1 (with Ki values in the low nanomolar range). The most potent inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, proved to be the same peptide for both enzymes and inhibited PC1 and PC2 in a competitive, fast-binding manner with Ki values of 3.2 and 360 nM, respectively. The four most potent peptide inhibitors of PC1 and PC2 were also tested against soluble human furin and found to exhibit a different rank order of inhibition; for example, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 440-fold less potent against furin than against PC1, with a Ki of 1400 nM.

Selective ligands for the mu, delta, and kappa opioid receptors identified from a single mixture based tetrapeptide positional scanning combinatorial library.

A combinatorial library of 6,250,000 tetrapeptides in the mixture based positional scanning format was screened in binding assays for the three opioid receptors, mu, delta, and kappa. Three different binding profiles were found. Individual peptides were synthesized representing all possible combinations of the active amino acids identified from the screening data. New, highly active peptides selective for each of the three receptors were chosen. This study demonstrates the power of mixture-based combinatorial libraries to identify distinctly different ligands for closely related receptors.

Comparison of the binding of alpha-helical and beta-sheet peptides to a hydrophobic surface.

The induction and stabilisation of secondary structure for a series of amphipathic alpha-helical and beta-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from beta-sheet to alpha-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85 degrees C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.

Selected peptides targeted to the NMDA receptor channel protect neurons from excitotoxic death.

Excitotoxic neuronal death, associated with neurodegeneration and stroke, is triggered primarily by massive Ca2+ influx arising from overactivation of glutamate receptor channels of the N-methyl-D-aspartate (NMDA) subtype. To search for channel blockers, synthetic combinatorial libraries were assayed for block of agonist-evoked currents by the human NR1-NR2A NMDA receptor subunits expressed in amphibian oocytes. A set of arginine-rich hexapeptides selectively blocked the NMDA receptor channel with IC50 approximately 100 nM, a potency similar to clinically tolerated blockers such as memantine, and only marginally blocked on non-NMDA glutamate receptors. These peptides prevent neuronal cell death elicited by an excitotoxic insult on hippocampal cultures.

Characterization of antigen-antibody interactions using single substitution analogs and mixture-based synthetic combinatorial libraries.

In an effort to use monoclonal antibodies (mAbs) as selective probes for early detection of breast cancer, the specificities of a number of antipeptide mAbs have been studied at the individual amino acid level using single substitution peptide analogs and peptide combinatorial libraries. In this study, the mapping results are presented for mAb172-12A4, which was raised against the haptenic peptide LGSGAFGTIYKG(C), corresponding to residues 138-149 of the oncogene v-erbB. This peptide is homologous with a region in epidermal growth factor receptor (EGFR) and human oncogene c-erbB-2, and contains the ATP binding motif that is common among protein kinases. The substitution profile of this interaction correlated well with the results from the screening of hexa- and decapeptide positional scanning libraries. Based on the results of this mAb's specificity for the antigenic determinant (-AFGTIYK-), proteins that have sequence homology were found from a database search of human sequences. Thirty-two unique peptide sequences, a majority of which was from protein kinases, were synthesized and tested for recognition by mAb 172-12A4. Eleven peptides had activities that differed from the original peptide by less than an order of magnitude, and the activities for 29 of the 32 (90%) could be accurately predicted based on the individual substitution analog results. While both epitope mapping approaches address the amino acid level of mAb specificity, positional scanning libraries offer an advantage of identifying the positional importance of each antigenic determinant residue without any prior knowledge of the mAb's specificity. The fine specificity mapping of peptide-specific mAbs using the synthetic tools illustrated here will be useful for the development of immunodiagnostics that detect cancer-related proteins in clinical samples.

Identification of novel antitumor agents from mixture-based synthetic combinatorial libraries using cell-based assays.

A new strategy is presented here which integrates combinatorial library technology with the antitumor in vitro screening system at the National Cancer Institute in the search for novel antitumor agents. Mixture-based synthetic combinatorial libraries (SCLs) representing hundreds of thousands to millions of individual compounds were screened against the cell-based assay, which evaluates compounds for their ability to inhibit the growth of 60 different human tumor cell lines. Five different SCLs, composed of peptides, peptidomimetics, polyamines or small molecules were first tested against three cell lines to identify the most active SCLs. Two SCLs, namely the N-perbenzylated pentamine and the N-acylated permethylated triamine, were deconvoluted to yield individual compounds having significant activities against the 60 tumor cell lines. Active compounds were tested in mice to determine the maximum tolerated dose, followed by in vivo testing in a hollow fiber assay. Using this strategy, three different compounds identified directly from SCLs are currently being evaluated in human tumor xenografts. This study demonstrates for the first time the use of in vitro cell-based assays to identify antitumor lead compounds from mixture-based combinatorial libraries.

Combinatorial chemistry: from peptides and peptidomimetics to small organic and heterocyclic compounds.

Modified dipeptides have been used successfully for the generation of a variety of small organic and heterocyclic combinatorial libraries, including linear urea, polyamine, hydantoin, thiohydantoin, cyclic urea, cyclic thiourea and bicyclic guanidine. The synthesis and screening results for a number of these libraries are described. The solid phase synthesis of heterocyclic compounds such as diazepine and thiomorpholinone are also described.

Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1.

Fifteen hexapeptides having high affinity for the opioid-like receptor ORL1 were identified from a combinatorial library containing more than 52 million different hexapeptides. The five compounds with the highest affinity were characterized further by use of a variety of in vitro models. Binding studies indicated that these five peptides have affinity for ORL1 in the nanomolar range, similar to the recently discovered endogenous ligand called nociceptin and orphanin FQ (N/OFQ). The activity of these compounds was investigated in three different assays: stimulation of [35S]GTPgammaS binding and inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells transfected with ORL1, and inhibition of electrically induced contractions in the mouse vas deferens. In each assay, the five hexapeptides acted as partial agonists. The EC50 values for stimulation of [35S]GTPgammaS binding and inhibition of cAMP accumulation were in the range of that for N/OFQ, but maximal effects ranged from 70 to 90% of N/OFQ in the cAMP assay, and 30 to 60% of N/OFQ in the GTPgammaS assay. The positive hexapeptides identified were found to have minimal structural similarity to N/OFQ. The peptides are positively charged, which could enable them to bind to the negatively charged second extracellular loop thought to be a likely binding site for N/OFQ.

Secondary structure induction in aqueous vs membrane-like environments.

The conformational propensity of the 20 naturally occurring amino acids was determined in aqueous 3-[N-morpholino]propane-sulfonic acid (MOPS) buffer, protein interior-like [nonmicellar sodium dodecylsulfate (SDS)] and membrane-like environments (micellar SDS and lysophosphatidylglycerol/lysophosphatidylcholine micelles) using a single "guest" position in a polyalanine-based model host peptide (Ac-KYA13K-NH2). This model system allows the intrinsic alpha-helical or beta-sheet propensity of the amino acids to be determined without intra- and interchain side chain interactions. The overall environment dependence observed for the conformational propensity for the amino acids studied confirms the importance of determining propensity in lipidic environments to better elucidate the biological functions of proteins. The hydrophobic interactions between peptide side chains and lipids appeared to be the primary forces driving the conformational induction in lipidic environments of the model peptides studied. Finally, when comparing the results of these studies with those reported in the literature, the local environment was found to highly influence 65% of the 20 naturally occurring amino acids.

Immune activation in cervical neoplasia: cross-sectional association between plasma soluble interleukin 2 receptor levels and disease.

In a previous study (Tsukui et al., Cancer Res., 56: 3967-3974, 1996), we observed an inverse association between degree of cervical neoplasia and interleukin (IL) 2 production by peripheral blood mononuclear cells in response to human papillomavirus (HPV) 16 E6 and E7 peptides in vitro. This suggested that a Th1-mediated cellular immune response might be important in host immunological control of HPV infection and that a lack of such a response might predispose to progression of cervical disease. To follow up on these findings, we have conducted a cross-sectional study of women with various degrees of cervical neoplasia to investigate the association between overall immune activation and cervical disease. A total of 235 women were recruited into our study; 120 of these women were participants in our previous study in which IL-2 production in response to HPV-16-specific peptides was measured. The study population included 34 women with invasive cancer, 62 women with high-grade squamous intraepithelial lesions (HSILs), and 105 women with low-grade squamous intraepithelial lesions (LSILs). In addition, 34 cytologically normal women with no past history of squamous intraepithelial lesions despite confirmed HPV-16 infection in the 5 years preceding the study were selected as controls. As our measure of overall immune activation, serum samples obtained from study participants were tested for soluble IL-2 receptor (sIL-2R) level using an ELISA method. The mean sIL-2R levels were found to increase with increasing disease severity (Ptrend = 0.0002). Among cytologically normal, HPV-exposed women, the mean receptor level in serum was 465.8 units/ml compared to 467.6 units/ml among LSIL subjects, 514.9 units/ml among HSIL subjects, and 695.5 units/ml among women with invasive cervical cancer. Similarly, the proportion of women with elevated sIL-2R levels (defined as > or = 450 units/ml) increased with increasing disease severity from 35.2% among normal study subjects to 70.6% among cancer patients (Ptrend = 0.003). Among the subgroup of subjects for whom in vitro IL-2 production in response to HPV-16-specific peptides was measured, we examined the association between in vitro IL-2 production and serum levels of sIL-2R. sIL-2R levels were higher, on average, among those women who were positive in our IL-2 production assay compared to those who were negative, but the differences did not reach statistical significance (P > 0.05). We also observed a trend of increasing sIL-2R level with increasing disease severity both in women who were positive and in women who were negative for our IL-2 production assay, but the trend was only significant among those who were negative for IL-2 production (Ptrend = 0.01). Results from our studies suggest that although the immune system of women with cervical neoplasia is nonspecifically activated as disease severity increases, the ability of those women with HSILs or cancer to mount a Th1-mediated immune response to HPV peptides appears to decrease compared to women with LSILs or normal women infected with HPV. Increased overall activation along with decreased Th1 immune response among women with increasing cervical disease severity might be explained by an increased Th2-mediated immune response, a response that we hypothesize is ineffective in controlling the viral infection and its early cytological manifestations. Future studies should directly assess Th2-mediated responses to confirm this hypothesis. Also, future efforts should be aimed at determining whether the associations observed are causally related to disease progression or an effect of the disease.

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.