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780

Young InvestiGATORS MINI-SYMPOSIUM LECTURES

L1

SOMATOSTATIN ANALOGUES CONTAINING SUGAR AMINO ACIDS.

Sibylle Gruner, Erich v. Roedern, Elisabeth Lohof, Marcus Born, Christian Mang, Horst Kessler from Institut für Organische Chemie und Biochemie, TU München, 85747 Garching, Germany and Gyorgy Kéri, and A. Venetianer from the Department of Medicinal Chemistry, Semmelweis Medicinal University, H-1444. Budapest 8. POB 260.

Sugar Amino Acids (SAAs) are sugar moieties containing at least one amino as well as at least one carboxyl group.[1] Their oligomers represent chimeras between the two big classes of biopolymers, the carbohydrates and proteins. They therefore have been used as both, carbohydrate and peptidomimetics. SAAs have been used in our laboratory mainly as turnmimics. We also applied SAAs to improve bioavailability and selectivity by functionalizing the carbohydrate skeleton.

In this work we present the development of new, active SAA‑containing somatostatin analogues.

[1] Lohof, E.; Burkhart, F.; Born, M. A.; Planker, E.; Kessler, H., in Sugar Amino Acids and Carbohydrates as Scaffolds and Peptidomimetics, Abell, A., Eds.; JAI Press Inc.: Stanford, Connecticut, 2, 263(1999).

L2

Macrophage chemotactic response to elastin-derived VGVAPG and VGVPG permutations: a structure-activity relationship and receptor binding assay.

Maria Portia P. Briones1 Satsuki Kamisato1, Iori Maeda1, Noboru Takami2, and Kouji Okamoto1 from 1Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan. 2RI Center, Fukuoka University School of Medicine, Fukuoka, 814-0180, Japan

VGVAPG, a hexapeptide sequence is repeated multiple times in human, bovine, and porcine elastin molecules. The most frequent pentapeptide sequence, VGVPG is present in human, bovine, porcine and chicken elastin molecules and is also repeated multiple times. Elastin digests and elastin-derived peptides interact with a variety of cell types to modulate cellular behavior and are implicated to play roles in several diseases such as atherosclerosis, emphysema, and metastasis. In our previous studies, elastin-derived polyhexapeptide (VGVAPG)n induced chemotaxis in macrophages, but elastin-derived polypentapeptide (VPGVG)n failed to stimulate such a biological activity. Hence, it is the interest of this study to investigate whether VGVAPG and its permutations, as well as VGVPG and its permutations can stimulate migration in rat peritoneal macrophages. It was observed using chemotaxis assay that four of the six permutations namely VGVAPG, GVAPGV, VAPGVG and GVGVAP were chemoattractant for macrophages at 10–9M, to 10-7M concentrations, respectively, and that they serve as signaling peptides which function to regulate macrophage migration. Such a directed cell movement is speculated to be mediated by high affinity binding of the cell receptor to these peptides. In the deactivation test wherein VGVAPG desensitized chemotactic response of macrophages to the other three potent hexapeptides (GVAPGV, VAPGVG and GVGVAP), the possibility that chemotaxis was induced by recognition of these chemotactic peptides by a single receptor was suggested. Chemotactic activity induced by these peptides was found to be profoundly sensitive to lactose. Also, we have found that these hexapeptides were recognized by a receptor distinct from that which recognizes fMLP. Macrophages stimulated by VGVAPG demonstrated an enhancement in guanosine 3’, 5’-cyclic monophosphate (cGMP) level, thus, reflecting an involvement of cGMP in the signaling mechanism of elastin hexapeptide VGVAPG-induced chemotaxis. Binding of the elastin-derived hexapeptide to macrophages was studied using a radiolabeled tyrosinated analog (Y-VGVAPG). In a parallel study using VPGVG and its permutations, only one pentapeptide permutation ie., VGVPG was found to be chemotactic with its optimal activity at 10-9M concentration. A study on the structure-function relationship of these peptides has been conducted in aqueous solution and phospholipid liposomes using CD and FTIR spectroscopy.

L3

Synthesis of Novel N-Terminal Cyclic Dynorphin A Analogues: Strategies and Side Reactions.

Balvinder S. Vig and Jane V. Aldrich, from Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, U.S.A.

Although dynorphin A (Dyn A) is thought to be an endogenous ligand for -opioid receptors, it also exhibits high affinity for - and -opioid binding sites. It has been known for several years that the N-terminal amine and the aromatic group of Tyr1 are key pharmacophoric groups in Dyn A. To date, no cyclic analogues of opioid peptides have restricted the backbone conformation of the N-terminal Tyr residue of this opioid peptide. A cyclization involving the N-terminus will restrict the conformation of the N-terminal amine and Tyr1. Here we describe the synthetic strategies for two series of novel N-terminal cyclic analogues of Dyn A. Various synthetic schemes involving the use of allyl (allyl; Alloc, allyloxycarbonyl) and hyperacid labile (Pip, phenylisopropyl; Mtt, 4-methyltrityl) protecting groups for the side chains of amino acids involved in the cyclization were explored. Pyroglutamate formation, which was observed when the side chain of Glu was protected by allyl, was avoided by the use of the bulkier protecting group Pip. In the case where the  amine of Lys was deprotected, separate neutralization of the amine had to be avoided to prevent premature loss of the N-Fmoc protecting group from the peptide. The successful cyclization schemes and various side reactions during the synthesis of novel N-terminal cyclic Dyn A analogues will be presented.

[Research supported by NIDA grant DA05195.]

L4

VINYL SULFIDE CYCLIZED ANALOGUES OF ANGIOTENSIN II WITH HIGH AFFINITY TO THE AT1-RECEPTOR.

Petra Johannesson1, Gunnar Lindeberg1, Gregory V. Nikiforovich2, Anja Johansson1, Adolf Gogoll3, Anders Karlén1 and Anders Hallberg1 from 1Organic Pharmaceutical Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden; 2Department of Biochemistry and Molecular Biophysics, Washington University, St Louis, MO 63110, U.S.A.; 3Organic Chemistry, Uppsala University, Box 531, SE-751 21 Uppsala, Sweden

We herein present synthesis and studies of the two conformationally constrained analogues of angiotensin II (Ang II), 2 and 3, shown below.

An aldehyde precursor with the chain length of four carbons was synthesized from 2-aminoadipic acid (Aad) and incorporated into precursor peptide 1. Cyclization of 1 was achieved by treatment with 95% TFA.

Analogues 2 and 3 exhibit nanomolar affinity to the AT1-receptor. Both of them possess low-energy conformers compatible to 3D model(s) of Ang II suggested earlier.

[Supported by the Swedish Research Council for Engineering Sciences and the Swedish Foundation for Strategic Research.]

L5

SYNTHESIS OF BACKBONE CYCLIC ANALOGUES OF -CONOTOXIN ImI BY CHEMOSELECTIVE LIGATION OF UNPROTECTED LINEAR PRECURSORS.

Christopher J. Armishaw, Julie Dutton, David J. Craik and Paul F. Alewood, from, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, AUSTRALIA.

A chemoselective backbone cyclisation strategy [1],[2] was successfully applied to unprotected precursors for the preparation of cyclic analogues of -conotoxin ImI, cImI, cImIA and cImIAG. The linear thioester peptide precursors were assembled using manual Boc solid phase peptide synthesis on a Boc-Gly-SCH2CH2CO-Phe-OCH2 PAM resin and cleaved from the resin with HF/p-cresol. The cyclic amide backbone and disulfide bonds were conveniently prepared in a one pot backbone cyclisation/oxidation by stirring the linear thioester precursor in aqueous base (2M GnHCl with 0.1M Tris, pH 8.3). Cyclisation was shown to be very rapid (<5 minutes) and formation of disulfide bonds was complete within 24 hours. cImIA displayed only one disulfide bonded isomer corresponding to the native disulfide bond connectivity and cImI and cImIAG displayed a mixture of different disulfide bonded isomers determined by directed disulfide bond formation and partial reduction/alkylation. The 3D structure of the native disulfide bond isomers of each of cImI, cImIA and cImIAG was resolved by high field n.m.r spectroscopy and compared to the native structure [3] and their binding affinity to the 7 subunit of the nicotinic acetylcholine receptor was determined.

[1] Camarero, J.A., Muir, T.W. mun., 1369-1370 (1997).

[2] Daly, N.L., Love, S., Alewood, P.F., Craik, D.J. Biochemistry 38, 10606-10614 (1999).

[3] Gehrmann, J., Daly, N.L., Alewood, P.F., Craik, D.J. J.Med.Chem. 42 2364-2372 (1999)

L6

VERIFICATION OF A  OPIOID RECEPTOR MODEL BY Zn2+-BINDING SITE ENGINEERING AND SITE-DIRECTED MUTAGENESIS.

Carol Fowler1, Irina Pogozheva1,Harry LeVine,III.2 and Henry I. Mosberg1 from 1Division of Medicinal Chemistry, University of Michigan and 2Dept. of Neuroscience, Pfizer, Ann Arbor, MI, U.S.A.

We have constructed three-dimensional computational models of the , , and  opioid receptor transmembrane domains by distance geometry with H-bonding constraints [1]. Comparison of our model to the recent X-ray crystal structure of Rhodopsin [2], showed that there are differences in the alignment of various transmembrane helices, and differences in the shape of the binding pocket. To clarify the vertical alignment of these helices, we constructed potential Zn2+-binding centers by mutating residues located near the proposed binding pocket of the  opioid receptor to Cys or His, making use of native histidines as some of the Zn2+ coordination sites. The demonstration of Zn2+ binding, as evidenced by reduced ligand binding in the presence of Zn2+,indicates that those residues forming the Zn2+-binding center are in close proximity. By comparing the Zn2+ binding ability of engineered binding centers composed of residues located at varying positions along the helices, the relative alignment of these helices can be determined. Single and double point mutations of residues around the binding pocket, together with various analogues of our opiate peptide, JOM-6, also test our proposed model of ligand docking inside the opioid receptor.

JOM-6 Tyr[DCys-Phe-DPen]NH2 (S-Et-S)

[1] Pogozheva, I., Lomize, A., Mosberg, H. I. Biophys J.75, 612 (1998).

[2] Palczewski, K., et. Al. Science 289, 739 (2000).

L7

EXTENDING SYNTHETIC ACCESS TO PROTEINS.

John Offer and Philip E. Dawson, fromThe Scripps Research Institute, La Jolla, CA 92037, U.S.A.

The ligation of peptides under mild aqueous conditions has given rapid synthetic access to many small proteins. In particular for native chemical ligation, the exquisite chemoselective reaction between an unprotected peptide thioester and an N-terminal cysteine peptide, both of which may be of either synthetic or biological origin, gives unprecedented versatility for protein engineering. Furthermore, uniquely of existing ligation reactions it leaves the natural amide bond at the ligation site. It is apparent that the scope of this reaction would be increased by the addition of alternative ligation sites. A temporary handle that could confer the ligation properties of cysteine and yet be removable after ligation is an appealing avenue of investigation. This approach would be able to draw on the robust methodology that has been developed for native chemical ligation. The availability, ease of introduction, ligation properties and removal of such a handle are of primary concern for any extension to be useful. The 2-mercaptobenzylamine skeleton combined the familiar rearrangement properties of 2-hydroxylbenzylamine (from backbone amide protection) with the favorable properties of thiophenol for thioester exchange. Derivatives of the 2-mercaptobenzylamine have been developed for optimum generality of ligation and ease of removal.

L8

Evaluation of synergistic interaction between cytokines and peptide epitope vaccines in protection against HER-2 expressing lung metastases.

1,3Naveen K. Dakappagari, 4Parihar, R, Pyles, J, 4,5Carson, W. E, and 1,2,,3,5Kaumaya, P. T. P from1Departments of Obstetrics and Gynecology, 2Molecular and Cellular Biochemistry, 3Microbiology, 4Molecular Virology, Immunology and MedicalGenetics, and the 5Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210 USA.

HER-2, a member of the epidermal growth factor receptor family is overexpressed in 25-30% of breast, ovarian and colon cancers. HER-2 overexpression is associated with poor prognosis and resistance to chemo- and hormonal therapies. Treatment of metastatic breast cancer patients with monoclonal antibody (HerceptinTM) produced objective responses. Active specific immunotherapy offers the possibility of generating sustained anti-HER-2 immune responses and is potentially more applicable than passive approaches. Synthetic peptide vaccines targeting B-cell epitopes of the extracellular domain of the HER-2 oncoprotein were evaluated in transgenic mice destined to develop mammary tumors due to HER-2/neu overexpression. One of the B-cell epitope peptide vaccine elicited high titered antibodies and prevented tumor development in 83% of the transgenic mice. Cytokines such as interlekin-12 (IL-12) and interferon-α (IFNα) are known to enhance anti-tumor activity of cancer vaccines by skewing the immune response towards IgG2a via interferon-γ release. Mouse IgG2a subtype is known to have superior capacity to fix complement and mediate tumor cell lysis due to its high affinity for the activation Fc receptors while interferon-γ by itself can inhibit tumor growth by activating the release of reactive super-oxide ions. To enhance the quality of immune response and the anti-tumor activity, Balb/c mice were immunized with a combination of peptide epitope vaccines and IL-12 or IFNα . High titered vaccine specific immune response of the IgG2a subtype were observed when supplemented with IL-12 but not IFNα. We are currently evaluating the effectiveness of these differential immune responses in preventing the formation of HER-2 overexpressing lung metastasis following syngeneic tumor cell challenge. The results of these studies will be discussed. This work was supported by NCI grant CA 84356 to PTPK.

L9

ANALYSIS OF THE ACTIVE SITE SPECIFICITY OF PLASMEPSINS FROM THE FOUR SPECIES OF PLASMODIUM USING TARGETED CHROMOGENIC COMBINATORIAL PEPTIDE LIBRARIES.

Bret B. Beyer, Alfred Y. Chung, Jodie V. Johnson, and Ben M. Dunn, from the Department of Biochemistry, University of Florida, Gainesville, FL 34610, U.S.A.

Plasmepsins (PMs) are aspartic proteinases (APs) found in the four species of Plasmodium that produce malaria. It has been shown that addition of AP inhibitors to cells in culture results in malarial parasite death 1, 2. Thus, it is necessary to understand the specificity of the four PMs, as these enzymes are possible drug targets. Two different targeted chromogenic octapeptide combinatorial libraries were used to analyze the enzyme/substrate interactions of the four PMs. The first library (P1 Library), comprised of 19 pools of 361 different peptides each, was based on the sequence Lys-Pro-Xaa-Glu-P1*Nph-Xaa-Leu, where P1 is a fixed amino acid for a given pool and Xaa is a mixture of 19 different amino acids. Results from studies with the first library revealed that the optimal P1 residues for the four PMs are the hydrophobic Phe, nLeu, and Leu. The second library (P2’ Library) is similar to the P1 library, but was based on the sequence Lys-Pro-Xaa-Xaa -P1(mix)*Nph-P2’-Leu, where P2’ is a fixed amino acid for a given pool and P1(mix) is a mixture of Phe, Leu, and nLeu at equimolar concentration. Kinetic assays monitoring the rates of cleavage of these substrates were performed on a diode array spectrophotometer and cleavage products were analyzed by utilizing Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS analysis of cleavage products produced by Plasmodium falciparum PMII catalyzed digestion of the P1-Phe containing pool revealed the favored amino acids at the P3 and P2' positions. These data were compared to previous studies with single peptide substrates, which have shown Alanine to be preferred at the P2’ position. From the combinatorial approach, it has been observed that basic residue Lysine was the most preferred residue at this site. Future experiments will probe different combinatorial libraries with substitutions at other positions. [Supported by NIH grants DK18865 & AI39211]

[1] Francis, S., Gluzman, I., Oksman, A., et. al. EMBO 13, 306 (1994)

[2] Moon, R., Tyas, L., Certa, U., et. al. Eur J Biochem, 244, 552 (1997)

L10

SYNTHETIC GLYCOPEPTIDE MODELS OF MUC1 CORE PROTEIN.

Mare Cudic, David Craik, and Laszlo Otvos, Jr., from The Wistar Institute, Philadelphia, PA 19104, U.S.A., and Centre for Drug Design and Development, University of Queensland, Brisbane, QLD 4072, Australia

MUC1 is a heavily glycosylated protein expressed on normal epithelial cells in humans, mainly consisting of multiple peptide repeats of a highly conserved sequence of 20 amino acids (PDTRPAPGSTAPPAHGVTSA). In cancer cells, MUC1 is overexpressed and the oligosaccharides pattern is changed compared to normal mucin. In order to understand how glycosylation may modulate or regulate the recognition process of mucin protein core epitopes, we have synthesized a series of peptide and glycopeptide models that contain no sugar thus mimicking the core sequence, peptides with the short carbohydrate side chains (Glc-Glc-GalNAc-1) that will mimic the carcinoma-originated mucin protein fragments, and finally peptides with extended carbohydrate chains (Glc-Glc-Glc-Glc-Glc-Glc-GalNAc-1) that will mimic the mucin secreted by normal cells. N-acetyl galactosamine (GalNAc) was the primary sugar unit linked to either a serine or threonine residue, since it represents the only consistent feature of MUC1 glycosylation. O-glycosylated Thr/Ser building blocks, were prepared from acetylated tri- and heptasaccharide bromides as a glycosyl donors. The azido functionality at C-2 of the GalNAc facilitated stereoselective glycosylation, and the -anomer was formed in great excess, even for long chain glycosyl donors. The non-glycosylated MUC1 peptide and the glycopeptides carrying one or two sugar units were assembled on solid-phase employing standard Fmoc-strategy. Azido group on the sugar ring was transformed into an N-acetyl group by reaction with thioacetic acid on the polymer-bound glycopeptide. After cleveage and deacetylation of the sugar hydroxyls, peptides were purified by RP-HPLC and characterized by MALDI-MS. The conformational effects, as well as, antibody and T-cell recognition, as regulated by O-glycosylation were studied and compared with the corresponding non-glycosylated counterpart.

L11

COVALENT AND NON-COVALENT OLIGOMERIZATION OF THE TRANSMEMBRANE -HELIX 4 FROM THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR.

Anthony W Partridge, Roman A Melnyk, and Charles M Deber, from Department of Structural Biology and Biochemistry, The Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8; Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these mutations occur within the transmembrane (TM) helices and are thought to manifest in the disease through a disruption of the structure/function of the TM domain. Specifically, mutations may disrupt the native helix-helix interactions within the TM domain. Although these interactions are generally thought to be important for the folding and association of membrane proteins, the detailed rules for how TM helices pack together are currently unknown. To address this issue, we have employed mutagenesis of a self-associating TM peptide named CFTR-TM4(V232D) with the aim of uncovering the specific residues that mediate its oligomerization. CFTR-TM4(V232D) has the sequence (KKK-L219QASAFCGLGFLIDLALFQAGLGR242M-KKK) and corresponds to TM helix 4 from CFTR protein with the CF phenotypic mutation V232D. Under reducing conditions, this peptide is monomeric on an SDS-PAGE gel. However, once a disulfide homodimer is formed, the peptide then is able to further associate through non-covalent interactions to form a “ladder” of tetramers, hexamers, and octamers. Ala-scanning mutagenesis has shown that D232 and Q237 are vital for the formation of the non-covalent oligomers. On the contrary, all other positions, with the exception of the necessary residue C225, can be individually replaced with Ala without the loss of the non-covalent oligomers. These results suggest that the peptide oligomerizes through interhelical hydrogen bonds between the Asp and the Gln residues. In addition, these results may relate to the dimerization of the full-length CFTR molecule and the manner in which the V232D mutation results in CF disease.

L12

DESIGN, SYNTHESIS AND CHARACTERIZATION OF SCAFFOLD-ASSEMBLED COLLAGEN MIMETICS.

Garth A. Kinberger and Murray Goodman, Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, U.S.A.

A collagen mimetic program has been a major effort in our laboratories.1 We have recently utilized a scaffold (template) derived from TRIS (tris(hydroxymethyl)aminomethane) which posses three carboxylic acid moieties for N-terminal peptide chain attachment and with a free amine for further attachment to surfaces or core structures to result in novel biomaterials. Collagen-like peptides composed of the sequence (Gly-Pro-Nleu)n have been assembled on the scaffold where n equals 1, 3, 5, 7 and 10 (Figure 1). When n is 5 the structure forms a triple helical array in an ethylene glycol water solvent (2:1) with a melting transition at 21C. Furthermore, when n is 7 and 10 the melting transitions are 25 and 37C, respectively in water. The purity and identity of these molecules are established by HPLC and mass spectrometry MALDI-TOF. The structure of these molecules are elucidated by temperature dependant optical rotation measurements and circular dichroism studies. The potential biological activity of these triple helical collagen mimetic structures will be discussed. The family of molecules shown in Figure 1 represent building blocks for dendrimer and biomedical materials.

Figure 1. A Z-protected TRIS scaffold-assembled collagen mimetic where n = 1, 3, 5, 7 and 10.

[1]. Goodman, M., Melacini, G., Feng, Y. J. Am. Chem. Soc., 1996, 118, 10928.

L13

POSITIONAL CYCLIZATION SCANNING: A NEW METHOD TO SEARCH FOR THE BIOACTIVE CONFORMATION OF GLUCAGON.

Jung-Mo Ahn, Dev Trivedi, Peter M. Gitu, Matthew Medeiros, Jennifer R. Swift and Victor J. Hruby, from Department of Chemistry, University of Arizona, Tucson, Arizona, 85721, U.S.A.

"Positional cyclization scanning" was used to determine secondary structures of glucagon required for maximal interaction with the glucagon receptor. A series of glucagon analogues, conformationally constrained via disulfide bridges between cysteine i and cysteine i+5 or lactam bridges between lysine i and glutamic acid i+4 were designed to induce and stabilize -turns and -helical structures, respectively.

Binding assays showed that all of the cyclic analogues with disulfide bridges bound to the receptor with significantly reduced binding affinities compared to the analogues containing lactam bridges. Interestingly, c[Lys5,Glu9]glucagon amide and c[Lys17,Glu21]glucagon amide demonstrated more than 7 fold increased receptor binding affinities relative to native glucagon. These results suggest that the bioactive conformation of glucagon may adopt a helical conformation at the N-terminal region as well as the C-terminal region, which was not evident from previous biophysical studies of glucagon [1,2]. [Supported by a grant from the U.S. Public Health Service]

[1] Sasaki, K., Dockerill, S., Adamiak, D.A., Tickle, I.J., Blundell, T. Nature 257, 751-757 (1975).

[2] Braun, W., Wider, G., Lee, K.H., Wüthrich, K. J. Mol. Biol. 169, 921-948 (1983).

L14

Design and synthesis of pseudopeptide antagonists intended to block coactivator binding to steroid nuclear receptors.

Anne-Marie Leduc, James L. Wittliff, and Arno F. Spatola from Department of Chemistry and of Biochemistry and Molecular Biology and the Institute for Molecular Diversity and Drug Design, University of Louisville, Louisville, KY 40292, U.S.A.

Estrogen receptors  and  are members of the nuclear receptor superfamily. Analysis of crystal structures of the ER binding domain has provided insight into the mechanism of action of these transcriptional regulators [1]. Upon binding to an agonist the ER undergoes a conformational change that facilitates the binding of coactivators such as SRC-1, RIP-140, and CBP. Coactivators consistently contain an -helix fragment with an LXXLL motif known as the NR Box. Furthermore, peptides containing that motif, such as a 13 residue truncation of SRC-1, have been shown to bind to the ER [2]. We have used this work as a model for the design of shorter peptides, pseudopeptides, and peptidomimetics that may mimic this interaction. To induce the required helicity, Aib residues, lactams or disulfide bridges have been introduced within these structures. Molecular dynamics and superposition with the coactivator helped to refine the final targets. Using MacroModel molecular modeling software, structures were designed to emulate the -helical character and preserve the hydrophobic leucine side chain orientations seen in the crystal structure [2]. Cysteine residues were introduced at positions i and i+3 while amide side chain to side chain bonds were formed with on-resin cyclization between Lys and Asp residues at i and i+4. In the case of the disulfide, the solid phase precursor was deprotected and cleaved from the MBHA resin and the ring was formed via DMSO oxidation [3]. The -helical contents of the resulting peptides, along with linear controls, were evaluated by NMR and CD. These compounds could function as novel regulators of downstream transcription events.

[1] Shiau et al. Cell, 95, 927 (1998).

[2] Heery et al. Nature, 387, 733 (1997).

[3] Tam, J.P. et al. J.Am.Chem.Soc., 86, 2018(1964).

L15

DESIGN OF NOVEL CELL PENETRATING PEPTIDES.

Kristen Sadler, Khee Dong Eom and James P. Tam, from Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN, U.S.A.

Recent advances in genomics and proteomics have increased the demand for transport peptides that deliver drugs, peptides and proteins into cells. Our laboratory is interested in exploiting transport peptides to study intracellular protein-protein interactions, gene expression and immune responses. Different transport peptides involving lipids, signal sequences, polycations and amphipathic regions have proven to be successful although there are disadvantages associated with each ranging from insolubility to low efficiency and cytotoxicity. We therefore intend to minimise these limitations by designing novel transport peptides that are efficient without causing cell lysis and cell death. The peptides we have designed contain duplicated proline or proline mimetics. Consequently these peptides are soluble in aqueous solutions, a property that facilitates ligation to cargo peptides or proteins. Furthermore, these novel proline-rich peptides are capable of penetrating cells without causing cell lysis. Our results may also provide a rational explanation for the translocating activity of many naturally occurring proline-rich peptides and proteins including peptide antibiotics, e.g. Bac 5 and Bac 7, and the CS surface protein of malaria Plasmodium sporozoites. A detailed structure-activity study of the ability of proline-rich peptides to penetrate cells without fatally disrupting the cell membrane will be presented.

SYMPOSIUM LECTURES

L17

ONE-DIMENSIONAL SPATIAL ENCODING: SPLIT/MIX SYNTHETIC PARALLELISM WITH TAG-FREE IDENTIFICATION AND ASSAYS AT THE SPEED OF LIGHT.

Alan Schwabacher Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, U.S.A.

A linear organization confers several benefits to a combinatorial library. We have recently demonstrated that linear spatially encoded synthesis can be carried out in a fully parallel manner with complete retention of compound identity by position. Fully parallel synthesis, as in the split/mix technique, is characterized by simultaneous reaction of each library member at each reaction sequence step, with the additional requirement that each reagent used in a given step need be handled only once.

The linear sequence also simplifies instrumental requirements for assays, and facilitates sample selection for representative chemical analysis. Treatment as a single time-varying signal, of the sequence of assay values for the entire library, allows application of the powerful tools of digital signal processing to the description of activity variation among library members.

In work carried out in collaboration with the group of Peter Geissinger, optical fibers were investigated as linear supports along which libraries can be distributed as sensor arrays. A single laser pulse down the fiber allows probing of all library members, with time resolved identification of the output of each. Apparent limitations set by the fluorescence lifetimes on the spacing of sensor regions have been overcome.

L18

UNICHEMO PROTECTION (UCP): A NEW CONCEPT FOR CHEMICAL SYNTHESIS.

Les P. Miranda and Morten Meldal, from Department of Chemistry, Carlsberg Laboratory, DK-2500, Copenhagen, Denmark.

The applicability of chemical synthesis to peptides, oligosaccharides and other organic molecules is limited and inherently complicated by existing functional group protecting strategies. To circumvent these restrictions, here we introduce UniChemo Protection (UCP), a novel protection strategy to allow the preparation of complex polyfunctional molecules in an intrinsically simple manner. The UCP concept is based on the controlled step-wise removal of oligomeric protecting group units with a single chemical process, and importantly avoids the problematic interplay of different chemistries inherent to conventional orthogonal strategies. UCP facilitates an orthogonal process that is not dependent on a multitude of finely tuned and different orthogonal chemistries. Functional site selectivity is easily engineered by varying the degree of oligomerization at each site. After each deprotection cycle, only the newly liberated functional site is available for derivatization. The effectiveness and utility of this powerful concept was demonstrated by the efficient controlled derivatization of a pentalysine-based amino functionalized scaffold on the solid-support. With efficient and practical UCP chemistry, the number of a given functional group that can be successfully protected with UCP with modular assembled scaffolds may well exceed ten or twenty without much difficulty. This new approach should prove valuable for the extension and automation of chemical synthesis.

L19

ORIGINAL AND GENERAL STRATEGY OF DIMERIZATION OF BIOACTIVE MOLECULES.

Florine Cavelier, Matthieu Giraud, Nicole Bernad, and Jean Martinez, from Laboratoire des Aminoacides, Peptides et Protéines, UMR-CNRS 5810, Université Montpellier I et II, 34095 Montpellier cedex 05 – France.

Dimerization of an active compound often results in enhanced binding and improved pharmacological properties. One of the cause of this potency could be higher concentration of pharmacophores in proximity to recognition sites. Usually, this bivalent ligant approach supposes to use a symmetrical bifunctionnal linker X to anchor two substrates P, giving rise to the general structure P-X-P.

In this study, we describe a new way of obtaining dimeric structures based on intermolecular diketopiperazine formation. The bioactive substance to dimerize was first linked to a glycine moiety. Then a coupling step using a coupling reagent and DMAP in stoichiometric quantity resulted in the cyclization involving both glycine C‑terminal carboxylic functions and the amide nitrogen.

As first exemples, we have dimerized the minimal active CCK sequence (Ac-Trp-Leu-Asp-Phe-NH2) and serotonin as non-peptide active molecule. Biological activity of resulting dimers was evaluated.

This original strategy does not require a bifunctional linker and the reaction is easy to carry out. It is worth pointing out that this dimerization reaction can be a concurrent reaction during the coupling reagent/DMAP activation of peptide fragments on their C‑terminal extremity in peptide syntheses, which is common when glycine is at the C‑terminus.

L20

SOLID-PHASE TOTAL SYNTHESES OF TRUNKAMIDE A AND KAHALALIDE F, CYCLIC PEPTIDES FROM MARINE ORIGIN.

Fernando Albericio,1 Josep M. Caba,1 Àngel López,1 Jose C. Jiménez,1 Marta Carrascal,1 Laia Solé,1 Ignacio Rodriguez,2 Ignacio Manzanares,2 Miriam Royo,1 and Ernest Giralt,1 from 1Department of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain; and 2Phama Mar Inc., c/ de la Calera 3, 28760-Tres Cantos, Spain.

Marine organisms are a rich source of novel, biologically active compounds. Herein, the solid-phase total synthesis of Trunkamide A and Kahalalide F, currently in preclinical and clinical phase I trials respectively, are presented. Trunkamide A contains a thiazoline heterocycle and two residues of Ser and Thr with the hydroxy function modified as reverse prenyl (rPr). Kahalalide F contains: (i) an ester bond between two sterically hindered amino acids (Val and D-alloThr); (ii) the didehydroaminobutyric acid (Dhb); and (iii) a rather hydrophobic sequence with two fragments containing several -branched amino acids in a row, one of them terminated with a fatty acid. Common features of both syntheses are: (i) solid-phase peptide chain elongation using a chlorotrityl resin and a quasi-orthogonal protecting scheme with allyl, t-butyl, and fluorenyl based groups; (ii) use of HOAt based coupling reagents; and (iii) cyclizations in solution. Furthermore, the following synthetic steps will be discussed: (i) preparation of the reverse prenyl derivatives of Ser and Thr (TA); (ii) introduction of the thiazoline precursor as a protected amino thionoacid derivative (TA); (iii) formation of the thiazoline ring with DAST (TA); (iv) solid-phase formation of the Dhb residue (KF). Finally, large-scale synthesis, as well as structure-activity relationship of Kahalalide F will be also presented.

L21

TRIPLE-HELICAL PEPTIDE ANALYSIS OF COLLAGENOLYTIC PROTEASE ACTIVITY.

Gregg B. Fields1, Janelle L. Lauer-Fields1, Thilaka Sritharan1, and Hideaki Nagase2, from 1Dept. Chem. & Biochem., Florida Atlantic University, Boca Raton, FL 33431, U.S.A. and 2Kennedy Institute of Rheumatology, London W6 8LH, U.K.

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we constructed fluorogenic triple-helical peptide (fTHP) models of the MMP-1 cleavage site in type II collagen. Substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5’ positions, respectively. Investigation of MMP family hydrolysis of fTHP showed kcat/KM values in the order of MMP-13 > MMP-1 ~ a C-terminal domain-deleted MMP-1 [MMP-1(243-450)] ~ MMP-2 >> MMP-3 ~ a C-terminal domain-deleted MMP-3 [MMP-3(248-460)]. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that "triple-helical peptidase" activity can be distinguished from "collagenolytic" activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.

L22

Design and synthesis of a Selective PSA Cleavable Peptide-Doxorubicin Prodrug Which Targets PSA Positive Tumor Cells.

Victor M. Garsky1, Patricia K. Lumma1, Dong-Mei Feng1, Jenny Wai1, Allen Oliff3, Raymond E. Jones2, Deborah DeFeo-Jones2,Roger M. Freidinger1, Departments of Medicinal Chemistry1 and Cancer Research2, Merck Research Laboratories, West Point, PA 19486 and Dupont Pharmaceuticals Company3, Wilmington, DE 19880, U.S.A.

Doxorubicin (Dox) has limited utility in prostate cancer due to systemic toxicities consisting primarily of cardiotoxicity and myelosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index.

Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug.

Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA secreting tumor cells. Based on these studies, we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, L-377,202, for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

L23

A HIGHLY SELECTIVE CELL PERMEABLE PEPTIDE INHIBITOR OF PROPROTEIN CONVERTASE 1: DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION IN CELLULAR PC1-MEDIATED PROTEOLYSIS.

Ajoy Basak1, Peter Koch1, Francine Sirois1, Michel Chrétien1, Nabil G. Seidah2 and Majambu Mbikay1. 1Molecular Medicine & Diseases of Ageing, Loeb Health Research Institute, Ottawa University, 725 Parkdale Ave, Ottawa, ON K1Y 4K9, Canada, and 2Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, QC, H3W 1R7, Canada.

Prohormone Convertase 1 (PC1) mediating the proteolytic processing of neural and endocrine precursors, is regulated by 260amino acid long neuroendocrine protein proSAAS. Studies have revealed that this unique PC1 inhibitory property is mostly confined within a decapeptide segment near the C-terminus of the conserved human proSAAS that contains the critical dibasic KR244 motif. This decapeptide, h (human)proSAAS235-244 VLGALLRVKRis the most potent slow but tight binding reversible competitive PC1-inhibitor (Ki ~9.1 nM) with a very high degree of selectivity as compared to other PCs. In an effort to enhance the cell permeability of the inhibitor necessary for cellular transport, a number of arginine rich peptides derived from HIV-I Tat48-60 as well as DNA and RNA binding proteins were coupled via a 15-atom linear spacer to the N-terminus of hproSAAS235-244. In vitro enzyme kinetics towards PC1 showed that the resulting peptide conjugates retained selective PC1 inhibitory property (Ki in low nM range). Their ability to block PC1-induced ex vivo cleavage of POMC into ACTH and -LPH in ATt-20 cell lines was investigated and compared with that of nonconjugated hproSAAS235-244. Using synthetic model substrates containing gp160 processing site of HIV-1, the cell permeable peptides such as RRRRRR-Ahx-Ahx-VLGALLRVKR (Ahx=amino hexanoic acid) was examined in vitro as selective antiproteolytic agent towards PC1. Additionally a fluorescent tagged cell permeable PC1 inhibitor RRRRRRKFluo-Ahx-Ahx-VLGALLRVKR (KFlu=Lysine with a side chain fluorescent function) was also synthesised as specific PC1-marker for application in cell culture experiments. The biochemical applications of this and other PC1-inhibitors with high degree of cell permeable and internalization property will be presented. Moreover circular dichroism analysis was performed to assess the significance of metal ions and pH in secondary structure and PC1 inhibition. (funding from CIHR and NSERC).



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