Development of highly potent and selective dynorphin A analogues as new medicines.

Dynorphin A (Dyn A), a 17 amino acid peptide H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH, is a potent opioid peptide which interacts preferentially with kappa-opioid receptors. Research in the development of selective and potent opioid peptide ligands for the kappa-receptor is important in mediating analgesia. Several cyclic disulphide bridge-containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A(1-11)-NH2 cyclic analogues incorporating the sulphydryl-containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the mu- and delta-receptors) as the parent Dyn A(1-11)-NH2 peptide in the guinea-pig brain (GPB), but exhibited a much lower activity in the guinea-pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure-activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at kappa-opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand-based drug design.

Surface plasmon resonance detection of interactions between peptide fragments of N-telopeptide and its monoclonal antibodies.

As populations age, osteoporosis is becoming an important public health care problem. Urinary level of the cross-linked N-telopeptide of type I collagen has been reported to be a sensitive marker of bone resorption. Recently, we synthesized and characterized 10 overlapping peptides covering the N-telopeptide of alpha-2 type I collagen and reported their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibodies determined by a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). In this study, we design an assay based on the surface plasmon resonance (SPR) technology to detect binding interaction of each peptide fragment of NTX with the anti-NTX monoclonal antibodies. Anti-NTX monoclonal antibodies were immobilized on the surface of sensor chip by amine-coupling procedure. Serial dilutions of each peptide were prepared and injected separately onto the antibodies-immobilized sensor chip. The real-time association and dissociation interactions of each peptide were detected and reported as sensorgrams. Binding response of each peptide to the monoclonal antibodies was determined, and the SPR results were compared with the ELISA results. We demonstrate that the trends of binding potency of peptide fragments detected by SPR are in good correlation to the results obtained by ELISA, indicating that our developed SPR-based method can be further applied to detect the NTX fragments in urine and to monitor the bone loss in humans. The potent peptide fragments identified by both assays are promising for further preparation of specific monoclonal antibodies in order to develop bioassays for bone loss in humans.

Novel peptide inhibitors for Grb2 SH2 domain and their detection by surface plasmon resonance.

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.

Oligomerization of human Gadd45a protein.

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.

Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain.

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).

The central region of Gadd45 is required for its interaction with p21/WAF1.

Cell cycle arrest represents an important response to genotoxic stress and the tumor suppressor p53 has been described to act as a critical effector in this biological event. Upon stress, p53 becomes transcriptionally active and up-regulates the transcription of downstream effector genes, which contain p53 recognition sites in their regulatory regions. Among the genes activated are p21 and GADD45, each of which independently exhibits growth-suppressive activity. The Gadd45 protein has been described to form a complex with p21, and thus, work was undertaken to map the regions of Gadd45 involved in this interaction and to examine the roles of those two proteins in growth suppression. In this report, a Gadd45 overlapping peptide library and a series of Gadd45 deletion mutants were used to define the domains of Gadd45 involved in the association with p21. Results using both in vitro and in vivo methods have shown that the interaction of Gadd45 with p21 involves a central region of Gadd45. Interestingly, the p21-binding domain of Gadd45 also encodes the Cdc2-binding activity, indicating that the central region of Gadd45 may serve as an important "core," through which Gadd45 protein is able to present cross-talk with other cell cycle regulators. In addition, GADD45 inhibition of Cdc2 kinase activity was compared with Myd118 and CR6, two other members of the GADD45 family. GADD45 was shown to generate the strongest inhibitory effect on Cdc2 activity. Finally, results from short-term survival assays further demonstrated that p21 and GADD45 act upon different cellular pathways to exert their growth-suppressive function.

The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression.

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.

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

Solution structure and dynamics of G1TE, a nonphosphorylated cyclic peptide inhibitor for the Grb2 SH2 domain.

The solution structure and dynamics of G1TE, a nonphosphorylated cyclic peptide inhibitor for the Grb2 SH2 domain, was determined using two-dimensional NMR and simulated annealing methods. G1TE consists of 10 amino acids and a C-terminal Cys cyclized through its side-chain sulfur atom by a thioether linkage to its N terminus. The results indicate that G1TE assumes a circle-like shape in solution in which all the side chains are protruding outside, and none of the residues are involved in intramolecular hydrogen bonding. The average root-mean-square deviations were found to be 0.41 +/- 0.11 A for the backbone heavy atoms C, Calpha, and N, and 1.03 +/- 0.14 A for all heavy atoms in a family of 10 structures. (15)N relaxation measurements indicate that G1TE has rather restricted dynamics in the fast time scale within its backbone. However, residues Tyr3, Val6, and Gly7 may be involved in a possible conformational exchange. The structural comparison between G1TE in solution and the BCR-Abl phosphopeptide bound to Grb2 SH2 domain revealed that G1TE may form a larger circle-like binding surface than the BCR-Abl phosphopeptide in the bound form. Also, the restricted backbone dynamics of G1TE may result in a reduced loss of entropy and can compensate for the absence of a phosphate group at the Tyr3 position. These structural and dynamic properties of G1TE may provide a molecular basis for understanding its interactions with the Grb2 SH2 domain.

Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain.

The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.

Significant compensatory role of position Y-2 conferring high affinity to non-phosphorylated inhibitors of Grb2-SH2 domain.

Systematic modification of amino acid at position Y-2 of a library-derived non-phosporylated thioether-cyclized peptide, cyclo(CH2CO-Glu2-Leu-Tyr0-Glu-Asn-Val-Gly-Met-Tyr-Cys) -amide, aided by molecular modeling, demonstrates that the Glu(-2) sidechain compensates for the absence of Tyr0 phosphorylation in retaining effective binding to Grb2-SH2 domain. Replacement of Glu(-2) with gamma-carboxyglutamic acid produced a high affinity inhibitor, the first example with submicromolar affinity (IC50 = 640 nM).

A p21(Waf1/Cip1)carboxyl-terminal peptide exhibited cyclin-dependent kinase-inhibitory activity and cytotoxicity when introduced into human cells.

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps.

Translational reading gaps occur when genetic information encoded in mRNA is not translated during the normal course of protein synthesis. This phenomenon has been observed thus far only in prokaryotes and is a mechanism for extending the reading frame by circumventing the normal stop codon. Reading frames of proteins may also be extended by suppression of the stop codon mediated by a suppressor tRNA. The rabbit beta-globin read-through protein, the only known, naturally occurring read-through protein in eukaryotes, was sequenced by ion trap mass spectrometry to determine how the reading frame is extended. Seven different proteolytic peptide fragments decoded by the same sequence that spans the UGA stop codon of rabbit beta-globin mRNA were detected. Three of these peptides contain translational reading gaps of one to three amino acids that correspond to the UGA stop codon site and/or one or two of the immediate downstream codons. To our knowledge, this is the first reported example of the occurrence of reading gaps in protein synthesis in eukaryotes. This event is unique in that it is associated with bypasses involving staggered lengths of untranslated information. Four of the seven peptides contain serine, tryptophan, cysteine, and arginine decoded by UGA and thus arise by suppression. Serine is donated by selenocysteine tRNA, and it, like the other tRNAs, has previously been shown to suppress UGA in vitro in mammals, but not in vivo.

Nonphosphorylated peptide ligands for the Grb2 Src homology 2 domain.

Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC50 10-25 microM) with a 9-amino acid pTyr-containing peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.

Glycosylation affects both the three-dimensional structure and antibody binding properties of the HIV-1IIIB GP120 peptide RP135.

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.

Effects of modifications of residues in position 3 of dynorphin A(1-11)-NH2 on kappa receptor selectivity and potency.

Tyrosine1 and phenylalanine4 in dynorphin A (Dyn A) have been reported to be important residues for opioid agonist activity and for potency at kappa receptors. The glycine residues in the 2 and 3 positions of dynorphin A may affect the relative orientation of the aromatic rings in positions 1 and 4, but their flexibility precludes careful analysis. To examine these effects on dynorphin A, we previously have synthesized the linear analogues [D-Ala3]Dyn A(1-11)-NH2 (2) and [Ala3]Dyn A(1-11)-NH2 (3) and reported their biological activities. Analogues 2 and 3 displayed affinities for the central kappa opioid receptor (IC50 = 0.76 and 1.1 nM, respectively) similar to that of Dyn A(1-11)-NH2 (1) (IC50 = 0.58 nM) and greatly enhanced selectivities for kappa vs mu and kappa vs delta receptors (IC50 ratios of 350 and 1300 for 2, and 190 and 660 for 3, respectively). These results suggest that the structure and lipophilicity of the amino acid present in position 3 of Dyn A(1-11)-NH2 as well as the conformational changes they induce in the message sequence of dynorphin have important effects on potency and selectivity for kappa opioid receptors. To further investigate structure-activity relationships involving the residue at the 3 position of Dyn A(1-11)-NH2, a series of Dyn A analogues with aromatic, charged, and aliphatic side chain substitutions at the 3 position was designed, synthesized, and evaluated for their affinities for kappa, mu, and delta opioid receptors. It was found that analogues with lipophilic amino acids at the 3 position of Dyn A(1-11)-NH2 generally displayed higher affinity but similar selectivities for the kappa receptor than analogues with charged residues at the same position. It is suggested that the structural, configurational, and steric/lipophilic effects of amino acids at position 3 of Dyn A(1-11)-NH2 may play an important role in potency and selectivity for the kappa receptor.

Design, synthesis, and biological activities of cyclic lactam peptide analogues of dynorphine A(1-11)-NH2.

We previously have reported four possible binding conformation of dynorphin A (Dyn A) for the central kappa opioid receptors, induced by the address sequence, using a molecular mechanics energy minimization approach. The lowest energy conformation was found to exhibit an alpha-helical conformation in the cyclized address sequence. It was suggested that an alpha-helical conformation in the cyclized address sequence or a helical conformation induced by the conformational characteristics of the message sequence may be important for binding potency and kappa opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i + 4 positions have been shown to stabilize alpha-helical conformation. Thus, a series of cyclic lactam analogues of dynorphin A(1-11)-NH2 have been designed, synthesized and evaluated by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate the conformational analysis prediction and, further, to investigate the conformational requirements for high potency and selectivity for kappa opioid receptors. Positions 2-6, 3-7, and 5-9 were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between D-Asp(2) and Lys(6) in c[D-Asp(2),Lys(6)]Dyn A(1-11)-NH2 led to an analogue with pronounced potency and selectivity enhancement for the mu opioid receptor, whereas cyclization between D-Asp(3) and Lys(7) in c[D-Asp(3),Lys(7)]Dyn A(1-11)-NH2 led to a potent ligand (IC(50) 4.9 nM) with kappa receptor selectivity. The other analogues in the series proved to be less selective. The biological results led to the suggestion that the binding conformation for the kappa receptor may have structural requirements that are distinct from those of mu and delta receptors. Interestingly, analogues with a D-Asp at position 2, 3, or 9 were found to be more potent for the kappa receptor than analogues with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position 2, 3, or 9 of Dyn A(1-11)-NH2 may have stereochemical and conformational effects on the nearby amino acids which can help discriminate the preference between kappa, mu, and delta receptors.

Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21-activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment.

Design, synthesis, and biological properties of highly potent cyclic dynorphin A analogues. Analogues cyclized between positions 5 and 11.

We have recently reported the synthesis of several cyclic disulfide bridge-containing peptide analogues of dynorphin A (Dyn A), which were conformationally constrained in the putative address segment of the opioid ligand. Several of these analogues, bridged between positions 5 and 11 of Dyn A1-11-NH2, exhibited unexpected selectivities for the kappa and mu receptors of the central over the peripheral nervous systems. In order to further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, we have synthesized a systematic series of Dyn A1-11-NH2 analogues incorporating the sulfydryl containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11, thus producing 16 cyclic peptides. In addition, Dyn A1-11-NH2, [D-Leu5]Dyn A1-11-NH2, and [D-Lys11]Dyn A1-11-NH2 were synthesized as standards. Several of these cyclic analogues, especially c[Cys5,D-Cys11] Dyn A1-11-NH2, c[Cys5, L- or D-Pen11]Dyn A1-11-NH2, c[Pen5, L-Pen11]Dyn A1-11-NH2 and c[Pen5, L- or D-Cys11]Dyn A1-11-NH2, retained the same affinity and selectivity (vs the mu and delta receptors) as the parent compound Dyn A1-11-NH2 in the guinea pig brain (GPB). These same analogues and most others exhibited a much lower activity in the guinea pig ileum (GPI), thus leading to centrally vs peripherally selective peptides, but showed a different structure-activity relationship than found previously. In a wider scope, this series of analogues also provided new insights into which amino acids (and their configurations) may be used in positions 5 and 11 of Dyn A analogues for high potency and good selectivity at kappa opioid receptors. The results obtained in the GPB suggest that requirements for binding are not the same for the kappa, mu, or delta central receptors.