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Purpose We have recently demonstrated that gonadotrophin-releasing hormone receptor-activating autoantibodies (GnRHR-AAb) are associated with polycystic ovary syndrome (PCOS). The aim of this study was to map the antigenic epitopes of GnRHR-AAb from PCOS patients, and develop retro-inverso peptide inhibitors that specifically target GnRHR-AAb. Methods Serum samples from ten GnRHR-AAb-positive PCOS patients and ten GnRHR-AAb-negative healthy controls were tested. Epitope mapping for GnRHR-AAb was performed using a set of 11 overlapping octapeptides spanning the second extracellular loop of GnRHR. Antibody-blocking effect of the designed retro-inverso peptide inhibitors was evaluated in a cell-based bioassay. Results Two peptide sequences, FSQCVTHC and HCSFSQWW, were found to react with all PCOS sera, but not with control sera. Two retro-inverso peptides that mimic the identified epitopes, d-CHTVCQSF and d-WWQSFSCH, significantly inhibited PCOS serum IgG-induced GnRHR activation. One of these two peptide inhibitors, d-CHTVCQSF, largely suppressed autoantibody-induced GnRHR activation, suggesting that the epitope sequence FSQCVTHC may be a major functional target of GnRHR-AAb. Conclusion We have identified a dominant functional epitope for GnRHR-AAb associated with PCOS, and demonstrated effective blocking of GnRHR-AAb activity with epitope-mimicking retro-inverso peptide inhibitors. These proteolytically stable decoy peptides may have important therapeutic implications in subjects who harbor these autoantibodies.

The application of tumor targeting ligands to the treatment of cancer holds promise for improving efficacy and reducing toxicity. LT7 (L(HAIYPRH)) peptide, a phage display-selected peptide, exhibited high binding affinity to transferrin receptor (TfR) overexpressed on tumor cells. However, its in vivo tumor targeting efficiency was impaired due to enzymatic degradation in blood circulation. To improve the stability and targeting ability, a retro-inverso analogue of LT7 peptide, named DT7 peptide (D(HRPYIAH)), was designed for targeted therapy of hepatocellular carcinoma. The result of computer simulation predicted that DT7 bound to TfR protein more efficiently than LT7, and this prediction was confirmed experimentally by surface plasmon resonance (SPR). Ex vivo stability experiment demonstrated that DT7 possessed stronger ability against proteolysis than LT7 in fresh mouse serum. We further prepared DT7-, LT7-, and transferrin (Tf)-modified liposomes (DT7-LIP, LT7-LIP, and Tf-LIP, respectively). DT7-LIP showed a significantly stronger in vitro targeting ability than LT7-LIP and Tf-LIP under normal condition and simulated biological condition. In addition, the in vitro antitumor effect of DTX-loaded DT7-LIP was markedly enhanced in comparison to DTX-loaded LT7-LIP and DTX-loaded Tf-LIP. In vivo imaging indicated that DT7-LIP had better tumor accumulation than LT7-LIP and Tf-LIP. For in vivo antitumor studies, the tumor growth rate of mice treated with DTX-loaded DT7-LIP was significantly inhibited compared to that in mice treated with DTX-loaded LT7-LIP and DTX-loaded Tf-LIP. Overall, this study verified the potential of the stable DT7 peptide in improving the efficacy of docetaxel in the treatment of hepatocellular carcinoma. STATEMENT OF SIGNIFICANCE: A phage display library-selected LT7 (L(HAIYPRH)) peptide exhibited high affinity to transferrin receptor (TfR). However, its bioactivity was impaired in vivo as L-peptides are susceptible to degradation by proteolytic enzymes. Here, we designed a retro-inverso peptide DT7(D(HRPYIAH)) and demonstrated its increased serum stability and higher binding affinity to TfR. A stabilized targeted drug delivery system was further constructed by modified DT7 peptide on the surface of liposomes. The data indicated that DT7 peptide-modified liposomes exhibited higher targeting ability in vitro and in vivo. More importantly, DT7-modified liposomes demonstrated positive preclinical significance in enhancing the therapeutic effects against hepatocellular carcinoma.

Natural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in immunology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules.

Aggregation of amyloid-β peptide (Aβ) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH 2 ) on the aggregation and toxicity of Aβ. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aβ oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aβ. PINPs also bound to Aβ with high affinity ( K d =13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aβ, crossed an in vitro blood–brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APP SWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

The formation of beta-amyloid (Abeta) deposits in the brain is likely to be a seminal step in the development of Alzheimer's disease. Recent studies support the hypothesis that Abeta soluble oligomers are toxic to cells and have potent effects on memory and learning. Inhibiting the early stages of Abeta aggregation could, therefore, provide a novel approach to treating the underlying cause of AD. We have designed a retro-inverso peptide (RI-OR2, H(2)N-r

Cell penetrating peptides (CPPs), including arginine-rich peptides, are attractive tools for the intracellular delivery of various bioactive molecules with a low membrane permeability. We showed that the accelerated intracellular delivery of arginine-rich peptides was achieved by the addition of a short peptide segment (penetration accelerating sequence, Pas) to arginine-rich CPPs. The cytosolic release of the Pas-attached arginine-rich CPPs was observed within 5 min after the treatment of the cells with the peptides even in the presence of serum. Effectiveness of the Pas segment in the intracellular delivery of bioactive peptides using arginine-rich CPPs was exemplified through the enhanced growth inhibition activity of the malignant glioma cells by a retro-inverso peptide derived from the p53 C-terminal 22-amino-acid segment (positions 361-382).

The solid phase synthesis of a partially modified retro-inverso analogue of the bradykinin potentiating peptide BPP9a, [gLys6, (R,S)-mPhe7, Ala8] BPP9a is described. The analogue, which is active in vitro and in vivo, displays prolonged resistance towards cleavage by angiotensin converting enzyme (ACE).

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.

Two novel retro-inverso analogues of a conformationally restricted carbocyclic muramyl dipeptide (MDP) derivative N-(2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2carbonyl)-L-alanyl-D-isoglutamine were prepared and tested in a immunorestoration test in mice. Both retro-inverso MDP analogues did not enhance non-specific resistance to experimental fungal infection in immunosuppressed animals, suggesting that the intact amide bond is essential for the immunorestorating activity in this type of rigidified carbocyclic MDP analogues.

Complete activation of T cells requires two signals: an Ag-specific signal delivered via the TCR by the peptide-MHC complex and a second costimulatory signal largely provided by B7:CD28/CTLA-4 interactions. Previous studies have shown that B7 blockade can either ameliorate experimental autoimmune encephalomyelitis by interfering with CD28 signaling or exacerbate the disease by concomitant blockade of CTLA-4 interaction. Therefore, we developed a functional CD28 mimic to selectively block B7:CD28 interactions. The design, synthesis, and structural and functional properties of the CD28 free peptide, the end group-blocked CD28 peptide, and its retro-inverso isomer are shown. The synthetic T cell-costimulatory receptor peptides fold into a polyproline type II helical structure commonly seen in regions of globular proteins involved in transient protein-protein interactions. The binding determinants of CD28 can be transferred onto a short peptide mimic of its ligand-binding region. The CD28 peptide mimics effectively block the expansion of encephalitogenic T cells in vitro suggesting the potential usefulness of the peptides for the treatment of autoimmune disease conditions requiring down-regulation of T cell responses.

The crystallographic investigation of the retro-inverso peptide Bz-S-gAla-R-mAla-NHPh reveals an extended backbone conformation where the NH groups of the gem-diamino alkyl moiety and the CO groups of the malonyl residue face side by side. This extended conformation, presenting all carbonyls on opposite sides of the NH groups, is stabilized by interstrand H-bonds running in a single direction of the parallel beta-sheets that characterize the crystal packing. These sheets differ from the beta-sheets formed by native amino acids only. (1)H-NMR nuclear Overhauser effect spectroscopy (NOESY) experiments suggest that a conformation similar to that found in the crystal also prevails in dimethylsulfoxide solution. Previous potential energy calculations of gem-diamino alkyl (g) and malonyl (m) Ala residues predicted that extended forms were less stable than the helical ones because of strong electrostatic repulsions between the parallel polar groups. Similar arguments were invoked to give more weight to helical forms of the retro-peptide units in the proposal of packing models of some nylons in their crystalline polar regions. The present findings show that both g and m Ala residues can experience the extended conformation in the beta-sheet aggregation. The energy increase occurring in one strand, due to the parallel orientation of consecutive peptide dipoles, is more than compensated by favorable cooperative interactions among head-to-tail aligned peptide dipoles of facing strands, resulting in the formation of two C==O...H==N H-bonds per residue.

A novel peptide containing the lysosomally degradable sequence GlyPheLeuGly and a sequence-inverting unit has been prepared. This peptide presents the methylamino groups of sarcosine (N-methylglycine) at both termini for co-polymerisation with alpha,omega-bis(oxiranylmethoxy)poly(ethylene glycol) of mean M-w 1650. Gel permeation chromatographic analysis showed the presence of a mixture of oligomers. preliminary degradation studies showed that these oligomers are cleaved by cathepsin B, an important lysosomal enzyme. (C) 2000 Elsevier Science B.V. All rights reserved.

We determined the pharmacological profile of novel backbone-modified peptides designed as protease-resistant, selective analogues of AVP. Binding affinities of peptides were determined at both V1A and V2 subtypes of vasopressin receptor (VPR). Biological potencies of selected peptides were tested in pressor and antidiuretic bioassays. Substitution of the achiral α-aminoisobutyric acid (Aib) at position 4 or 7 of AVP produced peptides that selectively bound the V2 VPR. Both [Aib4]AVP (140 IU mg−1) and [Aib7]AVP (36 IU mg−1) are selective antidiuretic agonists with little or no activity in uterotonic and pressor assays. [Aib4] and [Aib7] derivatives of the linear V1A-selective antagonist [PhaaDTyr(Et)2Arg6Tyr(NH2)9]AVP bound selectively and with high affinity (Kd 0.51 and 4.1 nM respectively) to the V1A VPR. Bioassays confirmed that these peptides were potent antivasopressor agents (pA2 8.10 and 8.36 respectively). A total retro-inverso strategy was used to prepare protease-resistant mimetics of both AVP and linear V1A-selective antagonists. Cyclic retro-inverso mimetics of AVP did not bind either V1A or V2 VPRs. In contrast, rationally designed retro-inverso mimetics of linear V1A-selective antagonists selectively bound the V1A VPR. Our findings indicate novel methods to improve the pharmacodynamic and pharmacokinetic parameters of neurohypophysial hormone analogues which could be equally applicable to other peptide-receptor systems. British Journal of Pharmacology (1999) 128, 647–652; doi:10.1038/sj.bjp.0702857

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is alpha-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical beta-turns. This feature may explain why retro-inverso foot-and-mouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.

Peptides corresponding to the immunodominant loop located at residues 135–158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141–159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-dretro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the correspondingl-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the correspondingl-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines.

The cyclic pentapeptide cyclo(-Arg-Gly-Asp-d-Phe-Val-) is a highly potent and selective inhibitor for the αvβ3 integrin and is a prospective anticancer drug by acting to inhibit angiogenesis and by...

A novel 18 amino acid peptide PYC98 was demonstrated to inhibit JNK1 activity toward c-Jun. We observed a 5-fold increase in the potency of the retro-inverso form, D-PYC98 (a d-amino acid peptide in the reversed sequence) when compared with the inhibition achieved by L-PYC98, prompting our further evaluation of the D-PYC98 inhibitory mechanism. In vitro assays revealed that, in addition to the inhibition of c-Jun phosphorylation, D-PYC98 inhibited the JNK1-mediated phosphorylation of an EGFR-derived peptide, the ATF2 transcription factor, and the microtubule-regulatory protein DCX. JNK2 and JNK3 activities toward c-Jun were also inhibited, and surface plasmon resonance analysis confirmed the direct interaction of D-PYC98 and JNK1. Further kinetics analyses revealed the non-ATP competitive mechanism of action of D-PYC98 as a JNK1 inhibitor. The targeting of the JNK1 common docking site by D-PYC98 was confirmed by the competition of binding by TIJIP. However, as mutations of JNK1 R127 and E329 within the common docking domain did not impact on the affinity of the interaction with D-PYC98 measured by surface plasmon resonance analysis, other residues in the common docking site appear to contribute to the JNK1 interaction with D-PYC98. Furthermore, we found that D-PYC98 inhibited the related kinase p38 MAPK, suggesting a broader interest in developing D-PYC98 for possible therapeutic applications. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cells, we confirmed that the cell-permeable D-PYC98-TAT inhibited c-Jun Ser63 phosphorylation during hyperosmotic stress. Thus, D-PYC98-TAT is a novel cell-permeable JNK inhibitor.

Novel PNA oligomers containing an N-(aminomethyl) β-alanine backbone (‘retroinverso’ PNA) were prepared via blocksynthesis or via a combination of solution and solid-phase chemistry. The half-life time of the unprotected N terminus is 38 h at pH 7.0 (20 °C) and is pH dependent.

Novel monomeric building blocks 4a and 4b of peptide nucleic acids with an N-(aminomethyl) β-alanine backbone (‘retro-inverso’ PNA) are conveniently synthesized via Hofmann rearrangement. In the major rotamer the side chain carbonyl points toward the C terminus.

A retro-inverso peptide is made up of d-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Despite their limited success as antigenic mimicry, retro-inverso isomers generally fail to emulate the protein-binding activities of their parent peptides of an alpha-helical nature. In studying the interaction between the tumor suppressor protein p53 and its negative regulator MDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D. (2004) J. Am. Chem. Soc. 126, 16288-16289) made a surprising finding that the retro-inverso isomer of p53(15-29) retained the same binding activity as the wild type peptide as determined by inhibition enzyme-linked immunosorbent assay. The authors attributed the unusual outcome to the ability of the D-peptide to adopt a right-handed helical conformation upon MDM2 binding. Using a battery of biochemical and biophysical tools, we found that retro-inverso isomerization diminished p53 (15-29) binding to MDM2 or MDMX by 3.2-3.3 kcal/mol. Similar results were replicated with the C-terminal domain of HIV-1 capsid protein (3.0 kcal/mol) and the Src homology 3 domain of Abl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopic as well as x-ray crystallographic studies showed that D-peptide ligands of MDM2 invariably adopted left-handed helical conformations in both free and bound states. Our findings reinforce that the retro-inverso strategy works poorly in molecular mimicry of biologically active helical peptides, due to inherent differences at the secondary and tertiary structure levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structure level.

The end group modified retro-inverso bombesin C-terminal nonapeptide 3 and its diastereomer 4 have been synthesized. The absolute configurations at the substituted malonamic acid residues in 3 and 4 were determined by chemical correlation with D- and L-2-aminobutyric acids of known absolute configuration. Neither 3 nor 4 had any bombesin antagonist activity

The Gly 4 and/or Tyr 5 residues in dermorphin hexapeptide (H-Tyr- d -Ala-Phe-Gly-Tyr-Pro-OH) were replaced by Nα-methyl- or d -amino acids in order to examine the effect on opioid activity. Two pseudopeptides (H-Tyr- d -Ala-Phe-Gly-ψ (NHCO)-Xaa-Pro-OH, Xaa — Tyr or Phe) in which the Gly 4 Xaa bond is reversed, were also prepared. Metabolic stability, analgesia and selectivity of these compounds for different receptor populations have been investigated. Results suggest that the 12 new analogues showed a negligible affinity for the K binding site and some selectivity for μ- or δ receptors. In some cases the analgesic potencies seems to be related to enzymatic stability of the peptides.

Alzheimer's disease (AD) is a progressive neurodegenerative condition that results in severe cognitive and functional decline in sufferers and for which there are currently no effective treatments to halt or reverse disease progression. AD is the most common form of dementia and age is the major risk factor for this disease. With worldwide population structures changing as increasing number of individuals survive into old age, there is urgent need for novel disease modifying treatments for this condition, which has profound effects upon sufferers in addition to those around them. Some of us have previously developed a peptide inhibitor of Aβ1-42 aggregation (RI-OR2-TAT) that has been shown to reduce Aβ1-42 pathology in vivo in mouse models of AD. ~1690 copies of RI-OR2-TAT have been covalently attached to nanoliposome carrier particles forming Peptide Inhibitor NanoParticles (PINPs), and this study investigated the effect of PINPs upon Aβ1-42 aggregation at the molecular level. Our results show that PINPs are able to reduce Aβ1-42 aggregation and do so by binding early (oligomers) and late (fibrillar) stage aggregates. These results highlight the ability of PINPs to disrupt the formation of multiple Aβ1-42 aggregates capable of causing neurotoxicity and thus provide a strong case for PINPs to be carried forward into early stage clinical trials as a novel therapeutic option for the treatment of AD.

In this study, a novel approach for the development of a peptide-based vaccine has been tested. We investigated the possibility of replacing an all- l amino acid peptide sequence corresponding to the protective B-cell epitope hemagglutinin (HA) 91–108 from influenza HA with a retro-inverso analogue encompassing this sequence. Retro-inverso peptides are composed of d -amino acids assembled in a reverse order from that of the parent l -sequence, thus maintaining the overall topology of the native sequence. This explains the observed antigenic cross-reactivity with anti-influenza virus antibodies. Mice immunized intranasally with the ovalbumin-conjugated retro-inverso analogue and cholera toxin as an adjuvant, produced strong systemic (serum IgG) and mucosal (lung IgA) antibody responses, and were protected against intranasal challenge with a lethal dose of influenza virus. The weight loss pattern in the protected group indicated that the vaccinated animals developed a disease of low severity resulting in a quick recovery. Furthermore, splenocytes of the immunized mice cultured in the presence of inactivated influenza virus, secreted high levels of IFN-γ. The half-life of the retro-inverso analogue in the presence of lung homogenate proteases was at least 700 times greater than that of the parent l -peptide. These results demonstrate that peptidomimetic analogues with high resistance to proteolytic degradation are very effective immunogens when administered via the intranasal route, inducing protective immunity against a viral infection. This approach might be advantageous for vaccine development.

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi , a protozoan parasite that is the causative antigen of Chagas’ disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi -infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10–50 μM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.

In 1998 we reported that an L-peptide derived from H1 of c-Myc (Int-H1-S6A,F8A), linked to an internalization sequence from the third a-helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro-inverso (RI-) peptides can assume a 3D conformation very close to their corresponding L-forms and can be recognized by the same monoclonal antibody. We synthesized a RI-peptide form of our original L-peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI-form of the original Antennapedia internalization sequence was perfectly capable of carrying a D-peptide into human cells. We have studied three different potentially active peptides. L-peptides: Int-H1wt, Int-H1-S6A,F8A. D-peptides: RI-Int -H1-S6A,F8A. We have also studied three presumed control peptides: Int and RI-Int (no H1 motif), H1-S6A,F8A (no internalization sequence). Both 'active' and 'control' peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 mM) of the control peptide RI-Int, non-Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c-Myc (and molecules of the family), we chose an iso-amphipathic modified peptide of the H1 motif, with a proximity coefficient50% and where the major change was at position 7 (F--A). From a family of 73 H1 motifs belonging to (H1-Loop-H2) hu man sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N-Myc, L-Myc, c-Myc, H1-S6A,F8A of c-Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int-H1isoamph and the corresponding RI-Int-H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF-7 cells) a colon cancer line (HCT-116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso-amphipathic-modified H1 sequence were always very clearly (3-10 times) less active than the corresponding peptides carrying a conserved "H1 of Myc" motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF-7 cells treated with L-peptides; MCF-7 cells treated with RI-peptides; HCT-116 cells treated with L-peptides; PBLs treated with L-peptides; PBLs treated with RI-peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF-7 cells. On a molar basis, RI-peptides were about 5-10 times more potent and 30-35 times more stable in complete culture medium, than their corresponding L-forms. RI-Int can probably internalize longer peptido-mimetic molecules (for instance molecules mimetic of (H1-Loop-H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors-two steps closer to a potential drug.

An HIV-1-specific miniantibody, a peptide representing the third heavy chain complementarity-determining region (CDR) of an HIV-specific mouse antibody, was characterized and modified with unnatural D-isomeric amino acids. The CDR peptide and its parent antibody bound to a similar epitope, located in the V3 region of HIV-1 gp120. A shortened CDR sequence was modified with D-amino acids to create an all-D-amino acid retro-inverso (RI) peptide with a reversed sequence order. The RI CDR was less susceptible to proteolytic degradation than its L-counterpart and had a higher affinity for HIV-1 peptides. The miniantibody and its parent antibody showed neutralization of both primary and laboratory strains of HIV-1. In accordance with the binding studies, the RI CDR showed a stronger HIV-inhibiting capacity than its L-counterpart. We conclude that the anti-HIV retro-inverso CDR identified in this study has the potential to become a future anti-HIV drug. It has a virus-neutralizing capacity in vitro and appears to be stable. Future research should focus on characterizing its antiviral activity in vivo.

Antigenic peptides are thought to bind to class I major histocompatibility complex (MHC) molecules through three modes of interaction: van der Waals interaction and, to a lesser extent, hydrogen bonding of anchor side chain atoms to residues comprising the binding pockets of the MHC molecule; hydrogen bonding of N- and C-termini to residues at the ends of the binding groove; and hydrogen bonding of peptide backbone atoms to residues lining the binding groove. To dissect the relative contribution of each of these interactions to class I MHC-peptide stability, a retro-inverso (RI) analog of VSV-8, an H-2K b restricted cytotoxic T lymphocyte (CTL) epitope and terminally modified variants of both VSV-8 and RI VSV-8 were synthesized and their ability to target H-2K b bearing cells for CTL mediated lysis was compared. None of RI VSV-8 analogs elicited lysis of target cells by CTL specific for VSV-8 nor did they appear to compete with the native peptide for binding to H-2K b . In contrast, terminally modified VSV-8 peptides elicited target lysis. These findings suggest that side chain topochemistry of the peptide is insufficient for stable peptide binding to H-2K b ; rather, hydrogen bonding of the peptide backbone atoms to H-2K b side chain atoms to appears to play a major role in the stability of the complex. Computer modeling confirmed that none of the RI analogs participate in the extensive hydrogen bonding network between the peptide backbone and the MHC molecule seen in the native structure.

There is pharmacological evidence indicating that, in addition to the inhibition of angiotensin converting enzyme (ACE; EC 3.4.15.1), the potentiation of bradykinin (BK) responses may also involve the BK receptor or some binding site in the structures involved in the contractile response to this peptide. Dipeptides such as Val-Trp and some of its analogues as well as tripeptide homologues, including total and partial retroi-nverso peptides, were synthesized and assayed for their ability to inhibit purified guinea pig plasma ACE and to potentiate the action of BK on the isolated ileum of the same species. The peptides containing the P 2 -P 1 , P 1 -P′ 1 , and P′ 1 -P′ 2 inverted amide bonds inhibited ACE, were resistant to hydrolysis, and, depending on the amino acid composition, some of them potentiated the contractile response to BK while others did not. Des-[Arg 1 ]-BK, which has an intrinsic activity at concentrations higher than 10 −5 M, and the very dissimilar angiotensin I (AI) analogue [Cys 5 -Cys 10 ]-angiotensin-I-(5–10)-amide, which has no detectable contractile activity, were able to inhibit ACE and potentiate BK. In contrast to these peptides, BPP 5a and BPP 9a from Bothrops jararaca venom, and Potentiators B and C from Agkistrodon halys blomhoffii venom were more effective as BK potentiators than as ACE inhibitors. In conclusion, we have synthesized and assayed compounds that preferentially inhibit ACE, e.g. retro-inverso tripeptides, or potentiate the response of smooth muscle to BK, e.g. snake venom peptides.

The syntheses of a series of analogues of the C-tenninal hepta- and octa-peptide of cholecystokinin are described in detail. These peptide and pseudopeptide analogues were obtained by modifying the NIe28-Gly29 region, either by replacing the Gly29 residue by D-Alanine, Sarcosine, or by replacing the peptide bond between Nle28 and Gly29 by a “ketomethylene bond” (COCH2), by a’ “carba bond” (CH2CH2) or by a “retro-inverso modification” (NHCO).

The complete solid phase synthesis of a partially modified retro-inverso analogue of Substance P, [Glp6, gPhe8, mGly9]SP 6–11, [gPhe =–NHCH(CH2Ph)NH–, mGly =–OCCH2CO–] has been performed using a polydimethylacrylamide-based solid support and [bis(trifluoroacetoxy)iodo]benzene.

New human renin inhibitors were designed from transition-state analogues of angiotensinogen, synthesized and evaluated. The peptide derovative, which contained 1-naphthylmethylsuccinylamide residue (P3) with a retro-inverso amide bond and a norstatine isoamylamide residue (P1-P1'), was stable to proteases and had potent human renin inhibitory activity. This compact inhibitor exhibited hypotension when administered orally to a monkey.