Design of beta-turn based therapeutic agents

Peptides and proteins are essential to many biological processes. The interaction between the peptide ligands and their receptor targets commonly involves beta-turn structures. Yet poor bioavailability and unfavorable pharmacokinetics significantly compromise the use of peptides as drugs. Thus, there has been a great deal of interest in the design of peptidomimetics (modified peptides) as therapeutic agents by mimicking beta-turn structures. This review highlights the importance of beta-turn in the design of various peptidomimetics for many diseases. This review also outlines several beta-turn mimicking strategies and its application in the design of potent peptide analogues. beta-turn mimetics often tend to be more rigid in positioning the critically important amino acid residues and thus optimize the surface conformation for productive interaction with the receptors.     

Structure-activity studies of peptides from the "hot-spot" region of human CD2 protein: development of peptides for immunomodulation

CD2 is a cell surface protein belonging to the immunoglobulin superfamily (IgSF) that plays a key role in mediating adhesion between human T-lymphocytes and target cells. The interaction between cell-adhesion molecules CD2 and CD58 is critical for immune response. Modulation or inhibition of these interactions has been shown to be therapeutically useful. Synthetic 12-mer linear and cyclic peptides and cyclic hexapeptides from the beta-turn and beta-strand region (hot spot) of human CD2 protein were designed to modulate CD2-CD58 interaction. The 12-amino acid synthetic cyclic peptides effectively blocked the interaction between CD2 and CD58 proteins as demonstrated by E-rosetting and heterotypic adhesion assays. NMR and molecular modeling studies indicated that these cyclic peptides exhibit beta-turn structure in solution and closely mimic the beta-turn structure of the surface epitopes of CD2 protein. The designed cyclic peptides with beta-turn structure have the ability to modulate CD2-CD58 interaction.     

Probing opioid receptor interactions with azacycloalkane amino acids. Synthesis of a potent and selective ORL1 antagonist

Azacycloalkane turn mimics 6-9 were used to explore the relationship between conformation and biological activity of peptide ligands to the opioid receptor-like (ORL1) receptor. Three azabicyclo[x.y.0]alkane amino acids and a 5-tBuPro type VI beta-turn mimic were introduced into peptides 10-13 by solid-phase synthesis on MBHA resin. Biological examination of peptides 10-13 showed two new antagonists (10 and 12) exhibiting increased selectivity for the ORL1 receptor.     

Deamidation of model beta-turn cyclic peptides in the solid state

To investigate the importance of secondary structure on peptide deamidation in the solid state, two cyclic beta-turn peptides and their linear analogs were used as models of Asn residues in structured and unstructured domains, and incorporated into poly(vinyl pyrrolidone) (PVP)-based lyophilized solids. The secondary structure of the model peptides was determined in solution and the solid state using a combination of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. The model beta-turn cyclic peptides were found to be type II beta-turns while the linear analogs were determined to be predominantly unstructured. Quantitatively, the cyclic peptides consisted of approximately 80% beta-turn while the linear analogs contained only 30%-35% beta-turn. To characterize the solid environment, T(g), and moisture content of the solid-state formulations were determined. Accelerated stability studies were conducted in the solid state at 37 degrees C using formulations lyophilized from solutions at pH 8.8 (0.1 M borate buffer). The effect of matrix mobility on solid-state deamidation was investigated by altering the moisture content through variation of relative humidity or the addition of a plasticizer. Cyclic peptides degraded 1.2-8 times slower than the linear analogs under all of the conditions studied. The observed rate constants, however, for all of the peptides decreased dramatically (four orders of magnitude) in the glassy solids. This suggests the greater importance of matrix mobility in solid-state degradation. Molecular dynamics (MD) simulations were also performed to explore the low energy, preferred state of the peptides, and determine the structure around the beta-turn.     

Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb2(1)

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.     

Design and evaluation of small peptides mapping the exposed surface of IL-8

In an effort to determine which regions of IL-8 are involved in interactions with its receptors, eight peptides were designed to correspond to distinct exposed regions of the IL-8 monomer, using the proton NMR-derived structure of the dimer as a basis. The peptides were evaluated singularly, and as equimolar mixtures of two to six peptides, in an IL-8 receptor binding assay and found to have no binding interaction with either α or β IL-8 receptor as single peptides or mixtures of two peptides. In contrast, one of these peptides having the sequence AVLPRSAKEL, which corresponds to the N-terminal 10 amino acid residues of the 77 amino acid form of IL-8, exhibited potent chemotactic activity in human neutrophils. These results indicate that there is no contiguous ligand that can be designed based on the NMR and X-ray determined structure of IL-8 and that there may be multiple receptors responsible for neutrophil activation and chemotaxis.     

Preparation of ‘side‐chain‐to‐side‐chain’ cyclic peptides by Allyl and Alloc strategy: potential for library synthesis

Abstract: Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side‐chain‐to‐side‐chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibé method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh₃)₄/PhSiH₃)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium‐mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.     

Novel PCU cage diol peptides as potential targets against wild-type CSA HIV-1 protease: synthesis, biological screening and molecular modelling studies

We have synthesized a series of novel pentacycloundecane cage diol diacid (PCU diol diacid) incorporated C ₂-symmetric peptides. Their activity against the resistance-prone wild-type C-South African (C-SA) HIV protease (HIV PR) is reported. These compounds were obtained in moderate yields of 42–72 %. Amongst the nine compounds reported herein only compound 6, 7, 10 and 11 showed moderate IC₅₀ values of 5–10 μM. Peptides 7 and 10 contain two phenylglycine and two tryptophan amino acids, respectively as side arms to the cage diol. Phenylglycine is a non-natural amino acid. Docking and molecular dynamics (MD) studies were carried out to understand the binding mode of the PCU moiety at the active site of the HIV PR enzyme. The computational results show that the cage diol peptide fits quite comfortably inside the active site of the enzyme. Not much movement is observed during the MD simulation and the hydrogen bonds that develop between the inhibitor and the enzyme pocket suggest that the inhibitor/HIV PR complex is stable at room temperature.     

Synthesis and physico‐chemical properties of peptides in soil humic substances

Abstract: Soil humic substances (HS) are heterologous, polydispersive, and multi‐functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with ¹³C‐glucose or ¹⁵N‐nitrate for 8.5 months. Evidence for random‐coil peptidic structures in the labeled HS was obtained from 2‐D nuclear magnetic resonance (NMR), pyrolysis gas chromatography‐mass spectrometry (pyro‐GC‐MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid‐state NMR data. Detailed ¹³C and ¹⁵N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC‐MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre‐existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.     

Antimicrobial activity of arginine‐ and tryptophan‐rich hexapeptides: the effects of aromatic clusters,d‐amino acid substitution and cyclization

Abstract: Many antimicrobial peptides bear arginine (R)‐ and tryptophan (W)‐rich sequence motifs. Based on the sequence Ac‐RRWWRF‐NH₂, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, l ‐d ‐amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW‐peptides displayed moderate activity towards Gram‐positive Bacillus subtilis (15 < MIC < 31 μm ) and were inactive against Gram‐negative Escherichia coli at peptide concentrations <100 μm . Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d ‐amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW‐peptides at 100 μm concentration was low (<7% lysis for linear R/W‐rich peptides and <28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high‐performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.     

Structure determination of neoefrapeptins A to N: peptides with insecticidal activity produced by the fungus Geotrichum candidum

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in alpha,alpha-dialkylated amino acids. CD spectra suggested a 3(10) helical structure for neoefrapeptins A and F.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

Influence of the hydrophilic face on the folding ability and stability of α‐helix bundles: relevance to the peptide catalytic activity

Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self‐aggregating 18‐mer peptides designed previously to adopt a high level of α‐helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four α‐helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far‐ and near‐UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well‐defined tertiary structure acting as true catalysts.     

The isolation and characterization of a peptide that alters sodium channels from Buthus martensii Karsch.

The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography and found to greatly prolong action potentials at nanomolar concentrations when applied to frog and mouse nerves. The N-terminal primary amino acid sequence of one of the peptides, BMK 16(5), was determined. The first 23 amino acids of BMK 16(5) were found to be: VKDGYIADDRNCPYFCGRNAYYD. The two cysteine residues in the sequence appeared as Edman sequence cycle blanks; however, they were assigned to be cysteines due to sequence similarity to other peptide toxins that bind to sodium channels and identification of the presence of cysteines obtained from single time point amino acid analysis. The MW of BMK 16(5) was determined by a Perkin Elmer API 300 LC/MS/MS to be 3,695. The amino acid residues of BMK 16(5) show strong similarity with the first 23 amino acid residues of a number of scorpion alpha neurotoxins. Unlike these neurotoxins, BMK 16(5) possesses a proline residue at position 13 which will likely make it fold in a unique way so as to bind to and alter sodium channels.     

Guanidyl-carboxylate interactions: crystal structures of arginine dipeptides*

The crystal structures of the hydrated dipeptides l -arginyl-l -aspartic acid and l -arginyl-l -glutamic acid have been determined from three-dimensional X-ray diffraction data. Each peptide crystallizes as a double-zwitterion with both the main and side-chain carboxyl groups ionized and the amino and guanidyl termini protonated. The peptide backbone conformation in both peptides is remarkably similar. Both peptides adopt a trans conformation for the peptide linkage with the guanidyl and acidic side-chains extended on opposite sides of the peptide backbone. The arginyl side-chain conformations differ between peptides; the conformation observed for arginyl aspartic acid is unique. Extensive intermolecular hydrogen bonding networks are observed in both structures; however, in neither structure is there evidence of intramolecular hydrogen bonding. The intermolecular guanidyl-carboxylate interactions are detailed. These interactions include a modified Type A interaction which models the possible bridging of adjacent peptide carbonyl oxygens in an α-helix by the guanidinium moiety.     

Structure-activity relationship study of collagen-derived anti-angiogenic biomimetic peptides

Structure-activity relationship (SAR) studies are essential in the generation of peptides with enhanced activity and efficacy as therapeutic agents. In this study, we report a Structure-activity relationship study for a family of mimetic peptides derived from type IV collagen with potent anti-angiogenic properties. The Structure-activity relationship study was conducted using a number of validated in vitro assays including cell proliferation, adhesion, migration, and tubule formation. We report a critical sequence (NINNV) within this peptide series, which is required for the potent anti-angiogenic activity. Detailed amino acid substitutions resulted in peptides with superior efficacy. Specifically, substitutions with isoleucine at positions 12 and 18 along with the substitution of the methionine at position 10 with the non-natural amino acid D-alanine led to an increase in potency by two orders of magnitude over the parent peptide. Several mimetic peptides in this series exhibit a significant improvement of activity over the parent peptide. This improved in vitro activity is expected to correlate with an increase in in vivo activity leading to effective peptides for anti-angiogenic therapy for different disease applications including cancer and age-related macular degeneration.     

Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis australis)

Two basic phospholipases A2 (Pa-11 and Pa-13) have been isolated from the venom of an Australian elapid snake, Pseudechis australis (king brown snake). The reduced and S-carboxymethylated phospholipases A2 were digested with trypsin and the resulting peptides were purified by a combination of chromatography on a DEAE-cellulose DE-52 column and gel filtration procedures. Eleven main peptides from Pa-11 and 9 peptides from Pa-13 could account for the amino acid compositions of the respective enzyme molecules. The alignment of the tryptic peptides and unelucidated regions of the amino acid sequences of tryptic peptides were established by the analysis of the peptides obtained by chymotryptic and/or Staphylococcal protease digestions. Each phospholipase A2 consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. Although Pa-11 is enzymatically 30-times as active as Pa-13 and highly toxic as compared to Pa-13, they are highly homologous in their amino acid sequences. They are also homologous to the enzymes from mammalian pancreas and the other snake venom phospholipases A2, especially to those from snakes belonging to the subfamilies Acanthophiinae and Laticaudinae.     

Unraveling the active conformation of urotensin II

Urotensin II (U-II) is a disulfide-bridged undecapeptide recently identified as the ligand of an orphan G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date.With the aim of elucidating the active conformation of hU-II, we have performed a spectroscopic analysis of hU-II minimal active fragment hU-II(4-11) in different environmental conditions. The analysis indicated that hU-II(4-11) was highly structured in the anisotropic membrane mimetic SDS solution, showing a type II' beta-turn structure, which is almost unprecedented for L-amino acid peptides. Micelle bound structure of hU-II(4-11) was then compared with those of four synthetic analogues recently synthesized in our lab, bearing modified Cys residues at position 5 and/or position 10 and characterized by different levels of agonist activity. The structures of the active compounds were found to be very similar to that of hU-II(4-11), while a barely active compound does not show any propensity to beta-turn formation. Furthermore, distances among putative pharmacophoric points in the structures of the active compounds obtained in SDS solution are in good agreement with those found in a recently described non-peptide agonist of the hU-II receptor. A type II' beta-turn structure was already found for the somatostatin analogue octreotide. On the basis of the similarity of the primary and 3D structures of U-II and somatostatin analogues and on the basis of the sequence homology between the GPR14/UT-II receptor and members of the somatostatin receptor family, a common evolutionary pathway for the signal transmission system activated by these peptide can be hypothesized.     

Studies on analogs of a peptide derived from alpha‐fetoprotein having antigrowth properties

Abstract: A 34‐amino acid portion of the third domain of alpha‐fetoprotein possesses antigrowth and anticancer activities. Three analogs of this sequence were chemically synthesized, in which the two cysteines of the original sequence were replaced by alanines, glycines or serines. The original cysteine and alanine peptides formed trimers at 0.20 g/L in pH 7.4 phosphate buffer, and the glycine and serine peptides formed dimers. Trimer preparations were more potent in inhibiting estrogen‐induced growth in the mouse uterine assays than the two dimeric oligomers. Of salient importance is that the alanine peptide retained its trimeric form in solution much longer than the cysteine peptide. Antigrowth assays were performed starting with stock solutions at a peptide concentration of 0.20 g/L, because at very high peptide concentration (8.0 g/L) the peptides aggregated extensively. All the peptides, although differing in biological activity, had almost identical secondary structures. Unlike alpha‐fetoprotein, the three peptides have low amounts of α‐helix. Trifluoroethanol has the ability to convert peptides into a helical conformation when they have a propensity for that structure. At trifluoroethanol concentrations of 20% and higher, the alanine and glycine peptides were changed into highly helical structures.