Search

Forster (fluorescence) resonance energy transfer (FRET) and fluores- cence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high- throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay tech- nologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual- readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed ''Dual-Readout F 2 as- say'' with F 2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F 2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.

The epithelial-derived, type II transmembrane serine protease matriptase, the mouse homologue of which is epithin, has been shown to be involved in epidermal differentiation, hair formation, and thymus function. We show in this study that epithin/matriptase (Epi/MTP) plays a significant role in mammary epithelial cell growth and morphogenesis. Epi/MTP is expressed at low level in the mouse mammary epithelium of young animals and it accumulates at the terminal end-bud of the growing ducts. The level of Epi/MTP is elevated in the mammary glands at stages when epithelial proliferation and modeling occur. It is primarily present in the luminal epithelial cells of mouse mammary ducts and lobules. Using an ex vivo three-dimensional culture system for mammary epithelial functional assays, we show that mammary epithelial growth and morphogenesis in the presence of the latent form hepatocyte growth factor (pro-HGF) are blocked either by an inhibitor of the Epi/MTP protease activity or by siRNA knockdown of the Epi/MTP expression. These studies demonstrate that Epi/MTP participates in mammary epithelial growth and modeling through activation of pro-HGF. Our findings reveal an important pathway in normal mammary epithelial morphogenesis which may participate in breast cancer progression.

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low micromolar range binding affinity, such as cyclic peptide 15 with an Kd = 0.359 μM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.

A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilyl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nin-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.

We have designed and synthesized a cyclic, bivalent Smac mimetic (compound 3) and characterized its interaction with the X-linked inhibitor of apoptosis protein (XIAP). Compound 3 binds to XIAP containing both BIR2 and BIR3 domains with a biphasic dose-response curve representing two binding sites with IC 50 values of 0.5 and 406 nM, respectively. Compound 3 binds to XIAPs containing the BIR3-only and BIR2-only domain with K i values of 4 nM and 4.4 microM, respectively. Gel filtration experiments using wild-type and mutated XIAPs showed that 3 forms a 1:2 stoichiometric complex with XIAP containing the BIR3-only domain. However, it forms a 1:1 stoichiometric complex with XIAP containing both BIR2 and BIR3 domains, and both BIR domains are involved in the binding. Compound 3 efficiently antagonizes inhibition of XIAP in a cell-free functional assay and is200 times more potent than its corresponding monovalent compound 2. Determination of the crystal structure of 3 in complex with the XIAP BIR3 domain confirms that 3 induces homodimerization of the XIAP BIR3 domain and provides a structural basis for the cooperative binding of one molecule of compound 3 to two XIAP BIR3 molecules. On the basis of this crystal structure, a binding model of XIAP containing both BIR2 and BIR3 domains and 3 was constructed, which sheds light on the ability of 3 to relieve the inhibition of XIAP with not only caspase-9 but also caspase-3/-7. Compound 3 is cell-permeable, effectively activates caspases in whole cells, and potently inhibits cancer cell growth. Compound 3 is a useful biochemical and pharmacological tool for further elucidating the role of XIAP in regulation of apoptosis and represents a promising lead compound for the design of potent, cell-permeable Smac mimetics for cancer treatment.

XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. We report the design, synthesis and characterization of a non-peptide, cell-permeable, bivalent small-molecule (SM-164) which mimics Smac protein for targeting XIAP. Our study shows that SM-164 binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000-times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. SM-164 concurrently interacts with both BIR domains in XIAP and functions as an ultra-potent antagonist of XIAP in both cell-free functional and cell-based assays. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in leukemia cancer cells, while having a minimal toxicity to normal human primary cells at 10,000 nM. The potency of bivalent SM-164 in binding, functional and cellular assays is 2–3 orders of magnitude higher than its corresponding monovalent Smac mimetics.

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.

Indolicidin is a host defense tridecapeptide that inhibits the catalytic activity of HIV-1 integrase in vitro. Here we have elucidated its mechanism of integrase inhibition. Using crosslinking and mass spectrometric footprinting approaches, we found that indolicidin interferes with formation of the catalytic integrase-DNA complex by directly binding DNA. Further characterization revealed that the peptide forms covalent links with abasic sites. Indolicidin crosslinks single- or double-stranded DNAs and various positions of the viral cDNA with comparable efficiency. Using truncated and chemically modified peptides, we show that abasic site crosslinking is independent of the PWWP motif but involves the indolicidin unique lysine residue and the N- and C- terminal NH2 groups. Because indolicidin can also inhibit topoisomerase I, we believe that multiple actions at the level of DNA might be a common property of antimicrobial peptides.

The design and synthesis of four nonnaturally occurring amino acid analogues of l-gamma-carboxyglutamic acid (Gla), appropriately protected for Fmoc-based solid-phase peptide synthesis (SPPS), is described. These amino acids are Bu-Mal 2, BCAH 3, Pen-Mal 4, and Cm-Gla 5. These Gla analogues have been designed to replace the glutamic acid of position 1 in the cyclic decapeptide G1TE, which is a potent inhibitor of tyrosine kinase, to further enhance binding to the Grb2-SH2 domain of signal transduction receptors. In the new amino acids, the propionic acid side chain of Glu has been replaced by a malonyl or a carboxymethylmalonyl moiety located at different distances from the alpha-carbon to optimize interactions in the phosphotyrosine-binding cavity of the Grb2-SH2 domain. Additionally, a direct and efficient synthetic route for the preparation of Fmoc-protected l-gamma-carboxyglutamic acid, which is amenable to large-scale production, has been developed to provide this important and unique amino acid(1) in 55% overall yield.

Potent, specific, non-peptide small-molecule inhibitors of the MDM2-p53 interaction were successfully designed. The most potent inhibitor (MI-63) has a K(i) value of 3 nM binding to MDM2 and greater than 10,000-fold selectivity over Bcl-2/Bcl-xL proteins. MI-63 is highly effective in activation of p53 function and in inhibition of cell growth in cancer cells with wild-type p53 status. MI-63 has excellent specificity over cancer cells with deleted p53 and shows a minimal toxicity to normal cells.

The growth factor receptor-binding protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, which involves cell proliferation and differentiation. Therefore, the Grb2-SH2 domain has been chosen as our target for development of potential antiproliferative agents. Herein, we report the study of the inhibitory effects of small nonphosphorylated peptide analogs interacting with the Grb2-SH2 domain protein by surface plasmon resonance (SPR) technology. A set of 8 related peptide analogs were synthesized, purified, and characterized. Their inhibitory effects on Grb2-SH2 were evaluated by the SPR technology developed with the BIACORE X instrument. The lead peptide, Fmoc-Glu-Tyr-Aib-Asn-NH2 (Fmoc-E-Y-Aib-N; Fmoc: 9-fluorenylmethyoxycarbonyl; Aib=alpha-amino isobutyric acid) inhibited Grb2-SH2 domain function with an IC50 value of 8.7 microM. A molecular modeling study of the lead peptide indicated that the glutamate in the Fmoc peptide is ideally positioned to form a strong salt bridge to Arg 67 in the Grb2-SH2 domain, using both its backbone carbonyl and its acidic group. Residue Glu 89 in Grb2-SH2 flips inward to fill the binding site and partially replace the phosphate group as a hydrogen-bond acceptor. Results of these studies provide important information for further development of potent nonphosphorylated peptide inhibitors of the Grb2-SH2 domain.

We found that indolicidin, a natural antimicrobial peptide, has HIV-1 integrase inhibitory activity. Subsequently, we also discovered analogs of indolicidin with substantially higher inhibitory potency. The dimers and tetramers of the most active sequence (ILPWKWPWWPWPP) were prepared by connection of the monomers' C-terminal ends, using lysine as a linker. The inhibitory potency of the dimeric peptide is higher than the monomeric peptide. The tetrameric peptide, prepared by connection of two dimers at C-ends using again lysine as the linker, is the most potent integrase inhibitor with IC(50) value of 0.6 microM for both 3'-end processing and strand transfer.

We have utilized a computational structure-based approach to identify nonpeptidic small organic compounds that bind to a human leukocyte antigen (HLA) DR1301 molecule (HLA-DR1301 or DR1301) and block the presentation of myelin basic protein peptide 152-165 (MBP 152-165) to T cells. A three-dimensional (3D) structure of DR1301 was derived by homology modeling followed by extensive molecular dynamics simulation for structural refinement. Computational structure-based database searching was performed to identify nonpeptidic small-molecule candidates from the National Cancer Institute (NCI) database containing over 150 000 compounds that can effectively interact with the peptide-binding groove of the HLA molecule. By in vitro testing of 106 candidate small molecules, two lead compounds were confirmed to specifically block IL-2 secretion by DR1301-restricted T cells in a dose-dependent and reversible manner. The specificity of blocking DR1301-restricted MBP presentation was further validated in a binding assay using an analogue of the most potent lead compound. Computational docking was performed to predict the three-dimensional binding model of these confirmed small molecule blockers to the DR1301 molecule and to gain structural insight into their interactions. Our results suggest that computational structure-based searching is an effective approach to discover nonpeptidic small organic compounds to block the interaction between DR1301 and T cells. The nonpeptidic small organic compounds identified in this study are useful pharmacological tools to study the interactions between HLA molecules and T cells and a starting point for the development of a novel therapeutic strategy for the treatment of multiple sclerosis (MS) or other immune-related disorders.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor bound protein 2 (Grb2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferative agents due to its potential to shut down the Ras activation pathway. Generally, the pTyr-X-Asn minimal binding motif is required for a high-affinity ligand binding to the Grb2-SH2 domain. Using phage-display techniques, we discovered a non-pTyr-containing cyclic peptide G1 with moderate binding affinity from 107 different sequences. To understand the structural basis for the high-affinity binding of these novel non-phosphorus-containing inhibitors to the Grb2-SH2 domain, we extensively studied herein the unique functional requirements of the acidic side chain in Tyr-2 position due to the absence of the phosphate group in these non-phosphorylated peptides. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low nanomolar range binding affinity, such as cyclic peptide 20 with an IC 50 =0.026 μ M, which is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. Whole cell assays indicate that peptide 20 can penetrate the cell membranes and inhibit the association of Grb2 with p185erbB2 in erbB2-overexpressing MDA-MA-453 cancer cells at low micromolar concentrations.

[reaction: see text] L-Phosphonomethylphenylalanine (L-Pmp) is an important phosphatase-resistant pTyr analogue. A most concise and stereoselective approach to the synthesis of the suitably protected Fmoc-Pmp(Bu(t))(2)-OH was developed in order to incorporate the functionally significant L-Pmp residue into peptides and peptidomimetics efficiently using standard Fmoc protocol. With this key building block, we are able to efficiently synthesize a series of potent Pmp-containing Grb2-SH2 domain antagonists, which can be used as chemotherapeutic leads for the treatment of erbB2-overexpressed breast cancer.

Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative and antimetastatic effects on many kinds of cancer cells in vitro. HT-29 human carcinoma cell line is one of the most susceptible cell lines to gossypol-induced cell death. Here, it is shown that treatment of HT-29 cells with gossypol not only induces cell cycle arrest on the G0/G1 phase, but also induces apoptosis. With a serial of Western blot analysis, it is revealed that gossypol-induced cell cycle arrest is involved in P21 up-regulation and cyclin D1 down-regulation; gossypol-induced apoptosis triggers down-regulation of anti-apoptosis Bcl-2 members: Bcl-X(L), Bag-1 and Mcl-1, up-regulation of pro-apoptosis Bcl-2 member Bak, activation of caspase-3, -6, -7, -8, and -9, up-regulation of Apaf-1, release of cytochrome c (cyto-c) from mitochondria, and activation of both DFF45 and PARP. Taken together, gossypol-induced cell death initiates extensive alterations of cell cycle and apoptosis proteins. Gossypol-induced apoptosis of HT-29 cells is through first the mitochondrial pathway, then the death receptor pathway, and the mitochondria pathway is, at least in part, involved in cyto-c release.

A series of phosphotyrosine containing cyclic peptides was designed and synthesized based upon the phage library derived cyclopeptide, G1TE . Considering the type-I β-turn feature of peptidic ligand binding to Grb2 SH2 domain, we introduce α,α-disubstituted cyclic amino acid, Ach, into the 4th position of the cyclic peptide to induce a local right handed 3 10 helical conformation. In order to stabilize the favorable binding conformation, the bulky and hydrophobic amino acids, neopentylglycine (NPG) and phenylalanine, were introduced into the 8th and 2nd positions of the peptide ligand, respectively. To facilitate the sidechain of pTyr3 reaching into the phosphotyrosine binding pocket, a less bulky alanine was preferred in position 1. Based upon these global modifications, a highly potent peptide ligand 12 was discovered with an IC 50 =1.68 nM, evaluated by ELISA binding essay. Ligand 12 is at least 10 5 more potent than the lead peptide, termed G1TE .

One of the critical intracellular signal transduction pathways involves the binding of the Grb2 SH2 domain to the phosphotyrosine (pTyr) motifs on growth factor receptors, such as epidermal growth factor receptor (EGFR) and erbB2, leading to downstream activation of the oncogenic Ras signaling pathway. Therefore, the Grb2 SH2 domain has been chosen as our target for the development of potential anticancer agents. As a continuation of our earlier work, herein we report the design and synthesis of new peptide analogs, and their inhibitory effect on the Grb2 SH2 domain using surface plasmon resonance (SPR) technology. These novel agents do not contain phosphotyrosine or phosphotyrosine mimics. Binding interactions between these peptides and the Grb2 SH2 domain were measured and analyzed using a BIAcore X instrument, which provides detailed information on the real-time detection of the binding interaction. The results of this study should provide important information for the further development of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain.

Gadd45a is an 18-kDa acidic protein that is induced by genotoxic and certain other cellular stresses. The exact function of this protein is not known. However, there is evidence for its involvement in growth control, maintenance of genomic stability, DNA repair, cell cycle control, and apoptosis. Consistently, Gadd45a has previously been shown to interact in vitro and/or in vivo with a number of proteins playing central roles in these cellular processes: proliferating cell nuclear antigen, p21(Cip1/Waf1), Cdc2-CyclinB complex, MTK1, and histones. Adding to this complexity, we have found that Gadd45a self-associates in solution, both in vitro and when expressed in the cell. Moreover, Gadd45a can complex with the two other members of the Gadd45 family of stress-induced proteins, human Gadd45b (MyD118) and Gadd45g (CR6). Gel-exclusion chromatography, native gel electrophoretic analysis, enzyme-linked immunosorbent assay, and chemical cross-linking showed that recombinant Gadd45a forms dimeric, trimeric, and tetrameric species in vitro, the dimers being the predominant form. Deletion mutant and peptide scanning analyses suggest that Gadd45a has two self-association sites: within N-terminal amino acids 33-61 and within 40 C-terminal amino acids. Despite the low abundance of Gadd45a in the cell, oligomer-forming concentrations can probably be achieved in the foci-like nuclear structures formed by the protein upon overexpression. Evidence for a potential role of Gadd45a self-association in altering DNA accessibility on damaged nucleosomes is presented.

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 microM) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a beta-turn-like conformation. Replacement of Tyr3 or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).

Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45-mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth.

Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin β family, ASAP1 (also known as centaurin β4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin β family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin β4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P2binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P2 binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.

Systematic modification of amino acid at position Y-2 of a library-derived non-phosporylated thioether-cyclized peptide, cyclo(CH 2 CO-Glu −2 -Leu-Tyr 0 -Glu-Asn-Val-Gly-Met-Tyr-Cys)-amide, aided by molecular modeling, demonstrates that the Glu −2 sidechain compensates for the absence of Tyr 0 phosphorylation in retaining effective binding to Grb2-SH2 domain. Replacement of Glu −2 with γ-carboxy-glutamic acid produced a high affinity inhibitor, the first example with submicromolar affinity (IC 50 = 640 nM).

Michael reaction of the methoxymethyl-protected monodiazeniumdiolate of piperazine (MOM-PIPERAZI/NO) with 4-maleimidobutyric acid followed by its conversion to the N-hydroxy-succinimido ester produces a reagent capable of transferring the nitric oxide (NO)-donating diazeniumdiolate group to the terminal amines of the lysine residues contained in proteins. The reagent has been used to produce diazeniumdiolated bovine serum albumin (D-BSA) and diazeniumdiolated human serum albumin (D-HSA) containing 22 and 19 modified lysyl groups, respectively. Upon dissolution in pH 7.4 phosphate buffer at 37 degrees C, these albumin derivatives gradually released all of their contained NO (approximately 40 mol/mol of protein) with initial rates of about 30-40 pmol/min/mg and half-lives on the order of 3 weeks. This methodology is now available for use in exploiting the unique specific metabolic interactions of proteins to target NO therapy to specific physiological processes in vivo.

One of the critical intracellular signaling pathways involves specific interactions between growth factor receptors and the adaptor protein Grb2. These interactions normally involve specific tyrosine phosphorylated regions in receptors and other cognate proteins. Following the lead of our recent findings that a phage library based non-phosphorylated disulfide linked 11-mer peptide inhibited such interactions, we report here the synthesis of novel redox-stable cyclic peptide analogs. These include thioether cyclized and backbone cyclized structures. The thioether analog was prepared under mild conditions from an N-terminally chloroacetylated and C-terminally cysteine extended peptide precursor. The thioether peptide showed equipotent binding affinity for the Grb2-SH2 domain (IC50=10–15 μM) when compared to the disulfide cyclized lead-peptide. The bioactive thioether linked peptide was demonstrated to offer advantages to the disulfide cyclized peptides under physiological conditions.

We have developed a new synthetic route for the preparation of Fmoc protected l -γ-carboxyglutamic acid in 60% overall yield (>99% ee) via a six-step synthesis from d -Garner’s aldehyde. An aldol condensation and the selective cleavage of the acetonide protective group are key steps.

We have prepared glycosylated analogues of the principal neutralizing determinant of gp120 and studied their conformations by NMR and circular dichroism spectroscopies. The 24-residue peptide from the HIV-1IIIB isolate (residues 308-331) designated RP135, which contains the immunodominant tip of the V3 loop, was glycosylated with both N- and O-linked sugars. The structures of two glycopeptides, one with an N-linked beta-glucosamine (RP135NG) and the other with two O-linked alpha-galactosamine units (RP135digal), were studied by NMR and circular dichroism spectroscopies. Molecular dynamics calculations based on the NMR data obtained in water solutions were performed to explore the conformational substates sampled by the glycopeptides. The data showed that covalently linking a carbohydrate to the peptide has a major effect on the local conformation and imparts additional minor changes at more distant sites of partially defined secondary structure. In particular, the transient beta-type turn comprised of the -Gly-Pro-Gly-Arg- segment at the "tip" of the V3 loop is more highly populated in RP135digal that in the native peptide and N-linked analogue. Binding data for the glycopeptides with 0.5beta, a monoclonal antibody mapped to the RP135 sequence, revealed a significant enhancement in binding for RP135digal as compared with the native peptide, whereas binding was reduced for the N-linked glycopeptide. These data show that glycosylation of V3 loop peptides can affect their conformations as well as their interactions with antibodies. The design of more ordered and biologically relevant conformations of immunogenic regions from gp120 may aid in the design of more effective immunogens for HIV-1 vaccine development.

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.

Development of phosphotyrosyl (pTyr) mimetics which are stable to protein-tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug development. Since a majority of pTyr mimetics derive their "phosphofunctionality" from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, L-O-(2-malonyl)tyrosine (OMT,5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp,2) introduction of fluorines alpha to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4'-O-[2-(2-fluoromalonyl)]-L-tyrosine (FOMT,6) a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC(50) = 10 microM) relative to the unfluorinated peptide, X = OMT (18) (IC(50) = 10 microM. Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pKa values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC(50) = 18 microM) relative to the unfluorinated peptide, X = OMT (21) (IC(50) = 14 microM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.

Synthesis of N-Boc-L-2-amino-4-(diethylphosphono)-4,4-difluorobutanoic acid is reported. This analogue was utilized for the solid-phase synthesis of a peptide containing a nonhydrolyzable phosphoserine mimetic.

Signaling by the T cell antigen receptor (TCR) is mediated by 17-residue tyrosine-based activation motifs (TAM) present in the cytoplasmic tails of the TCR zeta and CD3 chains. TAMs become tyrosine-phosphorylated upon TCR stimulation, creating a high affinity binding site for the tandem SH2 domains of ZAP-70. In permeabilized T cells, the association of TCR and ZAP-70 was inhibited by a protein tyrosine phosphatase (PTPase)-resistant TAM peptide analog, in which difluorophosphonomethyl phenylalanyl (F2Pmp) residues replaced phosphotyrosine. Inhibition of this association prevented TCR-stimulated tyrosine phosphorylation of ZAP-70 and reduced ZAP-70 kinase activity to basal levels. The reduction in ZAP-70 activity coincided with reduced tyrosine phosphorylation of a number of substrates. Such PTPase-resistant peptides, capable of disrupting SH2 domain-mediated protein-protein interactions, should prove useful in further dissection of multiple signaling pathways and may serve as models for rationally designed chemotherapeutic agents for the treatment of autoimmune and neoplastic disorders.

Dimers of known HIV-1 integrase inhibitory hexapeptide H-His-Cys-Lys-Phe-Trp-Trp-NH(2) containing different lengths of cross linkers in the place of cysteine residue, were designed, and synthesized. The inhibitory potency of these dimeric peptides is consistently higher than the lead hexapeptide. The dimeric peptide with djenkolic acid linker exhibited IC(50) values of 5.3 and 6.5 microM, for 3'-end processing and strand transfer, respectively.

Laminin, a large multidomain glycoprotein specific to basement membranes, is a heterotrimer with alpha, beta, and gamma chains held together in an alpha-helical coiled-coil structure. Synthetic peptides comprising two 51-mers (B1 and B2) from the beta 1 and gamma 1 subunits and a 55-mer (M) from alpha 2 were used to study the molecular mechanisms in laminin chain assembly. Using the synthetic peptides in various mixing experiments, the heterotrimer (B1-B2-M) was preferentially produced. The thermal stability of the heterotrimer increased dramatically (by approximately 20 degrees C) over that of the B1-B2 heterodimer as measured by circular dichroism (CD) spectroscopy. The B1-B1 homodimer (Tm = 60 degrees C) showed higher thermal stability when compared to B1-B2 and B2-B2 dimers. However, the B1 + B2 mixture produced principally the B1-B2 heterodimer. These results suggested that the preferential formations of heterodimer was regulated by kinetic interactions between each chain. The B2 and M peptides have many hydrophobic isoleucine residues which were replaced by leucines. These substitutions were predicted to favor an alpha-helical conformation and a higher propensity for zipper formation. B2L and ML, in which all isoleucine residues were replaced by leucine, showed significantly increased alpha-helicities. While B2L was able to form heterodimers and heterotrimers similar to B2, ML was not able to participate in heterotrimer formation as efficiently as the M peptide. The thermal stability of B1-B2L was comparable to that of B1-B2, but B2L and/or ML containing trimers showed lower thermal stability than B1-B2-M. These results suggest that the isoleucine residues in the alpha 2 and gamma 1 chains are critical for stabilizing the heteromeric triple-stranded coiled-coil structure.

Endothelin converting enzyme (ECE) is essential for generation of the biological effects of endothelin-1 (ET-1) from a precursor, big endothelin-1 (Big ET-1). We synthesized four analogs of human Big ET-1[16–38], substituted with single d-amino acids at P1, P2, P1′and P2′ positions. ECE activity was determined using an ET-1 specific radioimmunoassay system. None of the d-amino acid containing Big ET-1 analogs were apparently cleaved by ECE, however, one of the synthetic peptides, [d-Val22]Big ET-1[16–38], strongly inhibited the ECE activity. Furthermore, when this d-Val22 containing peptide was preadministrated to rat striatum, it was found to inhibit the dopamine release induced by Big ET-1. This result suggests that the d-Val22 containing peptide inhibits the ECE activity in vivo. The d-Val22 containing inhibitor offers hope of developing more potent and highly specific ECE inhibitors of therapeutic significance.

Phosphonomethyl phenylalanine (Pmp) is a non-hydrolyzable phosphotyrosyl (pTyr) mimetic, which has been incorporated into eleven-mer Pmp-containing peptides that have previously been reported to competitively inhibit the protein-tyrosine phosphatases PTP1 and PTP1B. We have recently shown that phosphonodifluoromethyl phenylalanine (F 2 Pmp) is superior to Pmp as a pTyr mimetic in SH2 domain-binding peptides. Herein we find using the hexameric peptide sequence Ac-D-A-D-E- X -L-amide, where X = (D/L)-Pmp or L-F 2 Pmp, that the half maximal inhibition values of these two peptides against PTP 1B-mediated dephosphorylation of autophosphorylated insulin receptor to be 200 μM and 100 nM, respectively. These data indicate that F 2 Pmp induces a three orders of magnitude enhancement in affinity relative to Pmp, resulting in an exceptionally potent peptide-based PTP inhibitor. We conclude that F 2 Pmp may be a generally useful tool in the preparation of selective, high affinity PTP inhibitors.

A Met residue in the pTyr+3 position has previously been shown to be an important determinant for high affinity binding of peptides to PI 3-kinase p85 SH2 domains. In the present work, a series of linear and cyclic peptides based on the sequence “Gly-pTyr-Val-Pro-Met-Leu” as well as analogues having pTyr replaced by the phosphatase-resistant pTyr mimetics, phosphonomethyl phenylalanine (Pmp) or difluorophosphonomethyl phenylalanine (F 2 Pmp), were synthesized and their binding potency in p85 SH2 domain preparations compared with corresponding peptides in which the Met has been substituted by Nle. Nle is a chemically more stable, isosteric Met homologue in which the sulfur has been replaced by a methylene. Significant binding potency was retained by the Nle-containing peptides, indicating that Met is not absolutely essential for high affinity binding to this SH2 domain.

The scrapie amyloid (prion) protein (PrP27-30) is a host-derived component of the infectious scrapie agent; the potential to replicate, propagate, and form amyloid is a result of the posttranslational event or conformational abnormality. In low concentrations of guanidine hydrochloride (Gdn.HCl), PrP27-30 dissociates into a compact equilibrium intermediate with a substantial portion of secondary structure, partially denatured tertiary structure, and tryptophan residues in an apolar environment [Safar, J., Roller, P. P., Gajdusek, D. C., & Gibbs, C. J., Jr. (1993) J. Biol. Chem. 27, 20276-20284]. Here we describe the characteristics of this metastable form as monitored by 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectroscopy and circular dichroism (CD) spectroscopy, and we propose a mechanism for scrapie amyloid association. The Gdn.HCl-induced equilibrium intermediate of PrP27-30 had multiple high-affinity hydrophobic binding sites for ANS, some close to the Trp residues. The amide CD spectrum of an acid-induced intermediate (A-form), in equilibrium at pH < 2.0, was similar to the Gdn.HCl-induced intermediate and suggested the presence of a significant portion of an alpha-helical or beta-turn secondary structure. In contrast, the PrP27-30 associated into aggregates in an all beta-sheet conformation with less ordered and more exposed hydrophobic side chains. The noncooperative unfolding of the Gdn.HCl-induced intermediate at high temperature was irreversible and correlated with the loss of infectivity. The results demonstrate that PrP27-30 associates through a compact, metastable hydrophobic intermediate with an nonnative, nondenatured secondary structure and a tertiary structure close to the unfolded form.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic hexameric peptides based on the amino acid sequence "Gly-Xxx-Val-Pro-Met-Leu", where Xxx is either phosphotyrosyl (pTyr) residue or a hydrolytically stable pTyr mimetic, were examined for their ability to bind to the C-terminal SH2 domain of the p85 phosphoinositol 3-kinase (PI 3-kinase). The cyclic peptides retained significant binding affinity relative to their linear counterparts. Potency varied depending on Xxx in the order: phosphonomethyl phenylalanine (Pmp, ID50 = 5.2 microM)phosphonodifluoromethyl phenylalanine (F2Pmp, ID50 = 2.2 microM)pTyr (ID50 = 1.0 microM), with Xxx = Tyr being inactive (ID50500 M). Greatly reduced potency was observed when Xxx was of the unnatural D-configuration. The cyclic peptides represent conformationally constrained ligands which should be useful in the development of p85 SH2 domain-directed inhibitors.

Src homology 2 (SH2) domains participate in protein tyrosine kinase (PTK)-mediated cellular signal transduction through their ability to bind with high affinity to phosphotyrosyl (pTyr)-bearing protein sequences. Although peptides containing pTyr competitively inhibit the binding between phosphoproteins and cognate SH2 proteins in a sequence-specific manner, such peptides are rapidly dephosphorylated by cellular phosphatases. We now describe our efforts to develop SH2 inhibitory peptides containing phosphatase-resistant pTyr surrogates. The parent compound, (phosphonomethyl)phenylalanine (Pmp), is a phosphonate-based mimetic of pTyr in which the phosphate ester oxygen (> COPO3H2) has been replaced by a methylene unit (> CCX2PO3H2, X2 = H2). Pmp analogues bearing fluorine (X2 = H, F or X2 = F2) or hydroxyl (X2 = H, OH) substituents on the phosphonate alpha-methylene carbon have been prepared and incorporated into peptides for use as SH2 domain inhibitors. In an assay using the C-terminal SH2 domain of phosphatidylinositol (PI) 3-kinase, peptides having a GXVPML sequence [where X = pTyr, Pmp, hydroxy-Pmp (HPmp), monofluoro-Pmp (FPmp), and difluoro-Pmp (F2Pmp)] exhibited binding potency in the order HPmp < Pmp < FPmp < F2Pmp = pTyr. Distinct peptide sequences which bind selectively with Src and Grb2 SH2 domains were also prepared with pTyr and F2Pmp. The F2Pmp peptides bound with high (0.2- to 5-fold) relative affinity, compared to analogous pTyr peptides. We conclude that peptides containing F2Pmp bind to SH2 domains with high affinity and specificity and, being resistant to cellular phosphatases, should provide a generally useful tool for disrupting SH2 domain-mediated signaling pathways in intact cells.

Ethyl groups on 4-[(diethylphosphono)difluoromethyl]-D,L-phenylalanine are efficiently removed using S N 2-type acidic reagents. These methods were utilized for the solid-phase synthesis of SH2-binding peptides containing nonhydrolyzable phosphotyrosyl mimetics.

The infectious form of the scrapie amyloid (prion) precursor, PrPSc, is a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the carboxyl-terminal proteinase K resistant core, PrP27-30, have the potential to form amyloid as a result of a post-translational event or conformational abnormality. We have studied the conformational transitions of both proteins reconstituted into liposomes, associated in solid state in thin films, and dissociated by guanidine HCl. The secondary structure of PrPSc in liposomes deduced from analysis of circular dichroism spectra contained approximately 34% beta-sheets, approximately 20% alpha-helix, and approximately 46% beta-turns and random coil. Cleavage of the amino-terminal region of PrPSc resulted in all-beta PrP27-30, with an estimated approximately 43% beta-sheet, no alpha-helix, and approximately 57% beta-turns and random coil. The PrPSC associated in thin films with a tertiary structure perturbation corresponding to unfolding, while the secondary structure was preserved. The PrP27-30 assembled into the solid state with a similar perturbation of tertiary structure but with a large increase in the beta-sheet content, probably due to an intermolecular alignment of the external beta-sheets, or to a secondary structure transition, or both. The various conformational states had little or no impact on infectivity. Equilibrium dissociation and unfolding demonstrated a greater resistance of PrP27-30 to denaturation. The dissociated monomers unfolded through intermediate(s), suggesting the presence of protein domains with distinct secondary structure stabilities. The results provide experimental evidence for the beta-sheet type assembly of scrapie amyloid PrP27-30 in the solid state and demonstrate the importance of amino-terminal cleavage in the stability and alignment of the amyloid-forming monomers.

The P-32-postlabeling method was used to examine the adducts in DNA, polynucleotides, and mononucleotides reacted in vitro with the N-hydroxy and N-acetoxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Adduct profiles were compared to those found in vivo in liver of cynomolgus monkeys fed IQ, MeIQx or PhIP. The N-acetoxy derivatives of IQ, MeIQx and PhIP (generated in situ from the corresponding N-hydroxylamine in the presence of acetic anhydride) each formed three principal adducts in DNA. Adduct 1 of IQ, MeIQx and PhIP was chromatographically identical to the P-32-labeled bis(phosphate) derivative of N-(deoxyguanosin-8-yl)-IQ, N-(deoxyguanosin-8-yl)-MeIQx, and N-(deoxyguanosin-8-yl)-PhIP respectively, and this adduct comprised approximately 65% of total adduct levels found in DNA in vitro. The C8-guanine adduct and the two minor adducts were also found in poly(dG-dC).poly(dG-dC), suggesting that the two minor adducts of IQ, MeIQx and PhIP are also formed on the guanine base. The N-acetoxy derivatives of IQ, MeIQx, and to a much lesser extent PhIP, also formed adducts with adenine-containing polynucleotides including poly(dA), poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT), but these adenine adducts were chromatographically different from those found in DNA. The three guanine adducts of N-acetoxy-IQ, -MeIQx and -PhIP found in vitro in DNA and in guanine-containing polynucleotides were also found in the liver of monkeys fed IQ, MeIQx or PhIP respectively, indicating that metabolic activation via N-hydroxylation and esterification occurred in vivo in monkeys. With each compound, the C8-guanine adduct was the predominant adduct found in vivo. The results indicate similarities among IQ, MeIQx and PhIP in the DNA adducts formed in vitro and in vivo and substantiate the use of the P-32-postlabeling method for comparative adduct studies.

Synthesis of 4-phosphono(difluoromethyl)-D,L-phenylalanine as well as its diethyl phosphonate analogues bearing either Boc or Fmoc-amino protection are reported. The latter two derivatives were utilized for the solid-phase synthesis of SH2-related peptides containing nonhydrolyzable phosphotyrosyl mimetics.