Inhibition of the activation of the first component of complement, C1, by various amino acids or peptides

The effects of various amino acids (or their analogues) and peptides on the activation or consumption of human complement by erythrocytes bound with hemolysin or heat-aggregated immunoglobulin G (aggIgG) were studied by using the hemolysis of hemolysin-bound erythrocytes, the consumption of complement by aggIgG in the serum, the hydrolysis of acetyl tyrosine ethyl ester by activated Cl (Cls), Cl hemolysis and a newly developed enzyme immunoassay, which directly measures interaction between Clq and aggIgG. Amino acids or peptides which were proposed to comprise Clq binding sites of the C2 region of IgG or their analogues were used. CH50 was inhibited by lysine or arginine to the largest extent, but other amino acids, including tranexamic acid and epsilon-amino caproic acid were not inhibitory up to 60 mM. The consumption of serum complement by aggIgG was prevented by arginine or lysine (about 60% inhibition at 60 mM) and by histidine to a lesser extent. The activation of Cls in the Cl complex by aggIgG precipitated at pH 5.5 was most inhibited by lysine, and to a lesser extent by tranexamic acid, arginine and epsilon-aminocaproic acid, but not glutamic acid or glycine. The results of Cl hemolysis indicated that, of all amino acids soluble at neutral pH, lysine and arginine were most effective in the inhibition of Cl hemolysis. Tranexamic acid and epsilon-aminocaproic acid were less effective, and glycine and norleucine were hardly effective. Among the dipeptides used, those that are composed of aromatic amino acids were very effective in the inhibition of Cl hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

4-Toluenesulfonylureido derivatives of amines, amino acids and dipeptides: a novel class of potential antitumor agents

The screening of a series of arylsulfonylureido derivatives of amines (such as histamine, or dopamine), aliphatic/aromatic amino acids (such as Gly, β-Ala, Val, Lys, Arg, Phe, Tyr, DOPA, etc.) and dipeptides (such as GlyGly, β-AlaHis) led to the identification of three derivatives that possess tumor growth inhibitory properties against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, and breast cancer cell lines in vitro. The new derivatives were prepared by reaction of 4-toluenesulfonyl isocyanate with (protected) amines, amino acids or dipeptides. The mechanism of antitumor action with these new derivatives is not known at the moment but it may imply uncoupling of mitochondria, as for the structurally related diarylsulfonylurea sulofenur, an investigational anticancer agent.     

Dipeptides as inhibitors of the gelation of sickle hemoglobin

To examine in detail a class of peptides that inhibit the polymerization of deoxyhemoglobin S, we assayed the L-amino acids and 22 dipeptides for their effect on deoxyhemoglobin S solubility. Of the amino acids, the aromatics (phenylalanine, tyrosine, and tryptophan) significantly increased deoxyhemoglobin S solubility, as did high concentration of arginine. Combinations of the hydrophobic (specifically the aromatic) amino acids with a hydrophilic amino acid, such as arginine or lysine, resulted in dipeptides which were much more soluble than the hydrophobic or aromatic amino acid alone, and also inhibited polymerization. Furthermore, samples of deoxyhemoglobin S at 26 to 27 g/dl containing some of these dipeptides such as Arg-Trp, Arg-Phe, and Lys-Trp in excess of 50 to 100 mM did not polymerize, indicating a 1.4- to 1.6-fold increase in deoxyhemoglobin S solubility. The enhancement of polymerization, i.e., decrease in deoxyhemoglobin S solubility, observed by the addition of aspartic acid, glycine, or lysine was observed or was reduced in the dipeptides containing these hydrophilic amino acids combined with hydrophobic amino acids (valine, leucine, isoleucine, or the aromatic amino acids). The effects of these dipeptides on deoxyhemoglobin S solubility were mostly linear with concentration. However, the changes in deoxyhemoglobin S solubility by addition of a dipeptide was not simply the sum of the effects observed with the individual amino acids as exemplified by the differential effect of reversing the dipeptide sequence (e.g., Arg-Phe and Phe-Arg, or Arg-Tyr and Tyr-Arg). These data provide further evidence as to the stereospecific nature of this class of noncovalent inhibitors of deoxyhemoglobin S polymerization.     

Viral NS3 helicase activity is inhibited by peptides reproducing the Arg-rich conserved motif of the enzyme (motif VI)

The NTPase/helicase of Flaviviridae viruses is one of the essential components of their replication complex. The enzyme is defined by the presence of seven highly conserved amino acid motifs. Random screening of numerous hepatitis C virus (HCV) derived peptides, revealed a basic amino acid stretch corresponding to motif VI of the HCV NTPase/helicase (amino acids 1487-1500 of the HCV polyprotein). This peptide inhibited the unwinding activity of the enzyme with an IC(50)=0.2 microM. Peptides corresponding to motif VI of HCV, West Nile virus (WNV) and Japanese encephalitis virus (JEV) were synthesized and tested as inhibitors of NTPase and unwinding reactions mediated by the viral enzymes. Peptides distinguished in regard to their length and structure. Between the peptides tested HCV(1487-1500) reproducing the sequence of motif VI was the most potent inhibitor of helicase activities of investigated enzymes. Other respective peptides were rather modest inhibitors. The examined peptides inhibited the Flaviviridae helicases in the following order of potency: HCV(1487-1500)>WNV(1959-1572)>JEV(1962-1975). Interestingly, the susceptibility of the helicase activity to the inhibition by the peptides was similar and in the row: HCV>WNV>JEV. The inhibition results from binding and blockade of the active site of the enzyme lyes beyond the NTP-binding and hydrolyzing site. The kinetic analyses indicated that the binding of the peptides do not interfere with the NTPase activity of the enzymes. The peptide may serve as effective and selective tool to reduce the virus propagation.     

Peptide substrate specificity of the α-amidating enzyme isolated from rat medullary thyroid CA-77 cells

The kinetic parameters were obtained for enzymatic α-amidation of peptides of the form N-dansyl-(Gly)₄-X-Gly-OH, in which the amino acid at position × was substituted with each of the 20 natural amino acids. The enzyme used in these studies was a highly enriched preparation of α-amidating enzyme secreted by a clonal (CA-77) cell line which actively expresses mature α-amidated peptides. A 130-fold and 11-fold variation respectively in apparent K_m and V_max values was observed. The effect of the amino acid side chain at position × in stabilization of the enzyme-substrate complex decreased through the series ×= planar aromatic or sulfur containing > neutral aliphatic > polar and basic > cyclic aliphatic or acidic.     

Opiate aromatic pharmacophore structure-activity relationships in CTAP analogues determined by topographical bias, two-dimensional NMR, and biological activity assays

Topographically constrained analogues of the highly mu-opioid-receptor-selective antagonist CTAP (H-D-Phe-c[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH(2), 1) were prepared by solid-phase peptide synthesis. Replacement of the D-Phe residue with conformationally biased beta-methyl derivatives of phenylalanine or tryptophan (2R,3R; 2R,3S; 2S,3R; 2S,3S) yielded peptides that displayed widely varying types of biological activities. In an effort to correlate the observed biological activities of these analogues with their structures, two-dimensional (1)H NMR and molecular modeling was performed. Unlike the parent (1), which is essentially a pure mu antagonist with weak delta agonist activities in the MVD bioassay, the diastereomeric beta-MePhe(1)-containing peptides exhibited simultaneous delta agonism and mu antagonism by the (2R,3R)-containing isomer 2; mu antagonism by the (2R,3S)-containing isomer 3; weak mu agonism by the (2S,3R)-containing isomer 4; and delta agonism by the (2S,3S)-containing isomer 5. Incorporation of beta-MeTrp isomers into position 1 led to peptides that were mu antagonists (2R,3R), 8; (2R,3S), 9, or essentially inactive (<10%) in the MVD and GPI assays (2S,3R), 10; (2S,3S), 11. Interestingly, in vivo antinociceptive activity was predicted by neither MVD nor GPI bioactivity. When D-Trp was incorporated in position 1, the result (7) is a partial, yet relatively potent mu agonist which also displayed weak delta agonist activity. Molecular modeling based on 2D NMR revealed that low energy conformers of peptides with similar biological activities had similar aromatic pharmacophore orientations and interaromatic distances. Peptides that exhibit mu antagonism have interaromatic distances of 7.0-7.9 A and have their amino terminal aromatic moiety pointing in a direction opposite to the direction that the amino terminus points. Peptides with delta opioid activity displayed an interaromatic distance of <7 A and had their amino terminal aromatic moiety pointing in the same direction as the amino terminus.     

Potent V2/V1a vasopressin antagonists with C-terminal ethylenediamine-linked retro-amino acids.

We report the solid-phase synthesis and antagonistic potencies of 25 analogues (1-25) of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-ethyl-D-tyrosine,4-valine]arginine-vasopressin (d(CH2)5D-Tyr(Et)2-VAVP) (A) and of the related Ile4 (D) and [D-Phe2,Ile4] (E) analogues, potent antagonists of the antidiuretic (V2-receptor) and of the vasopressor (V1a-receptor) responses to arginine-vasopressin (AVP). Six of these peptides (1, 13, 17, 19, 21, and 23) have the Pro-Arg-Gly-NH2 tripeptide side chain fully or partially replaced or extended by ethylenediamine (Eda). The remaining 19 peptides have L- or D-amino acids retrolinked to these six C-terminal Eda peptides. Peptides 1, 13, 17, and 19 all have the ring structure of (A). Their side-chain structures are as follows: 1, Eda; 13, Pro-Eda; 17, Pro-Arg-Eda; 19, Arg-Gly-Eda. Peptide 21 is the Pro-Arg-Eda analogue of D; peptide 23 is the Pro-Arg-Gly-Eda analogue of E. Peptide 2 is the retro-Arg analogue of 1. Its side-chain structure is Eda<--Arg. Peptides 3-6 are analogues of 2 which have the D-Tyr-(Et)2 residue replaced by L-Tyr(Et)2 (3), D-Phe2 (4), D-Ile2 (5), or D-Leu2 (6), respectively. Peptides 7-12 are analogues of 2 which have the C-terminal retro-Arg replaced in retrofashion by D-Arg (7), Gly (8), Orn (9), D-Orn (10), D-Lys (11), or Arg-Arg (12). Peptides 14-16 have D-Orn (14), D-Lys (15), and D-Arg (16) retrosubstituted to peptide 13. Peptides 18, 20, and 22 are the retro-Arg-substituted analogues of 17, 19, and 21, respectively. Peptides 24 and 25 have Val and D-Val in retrolinkage with 23, respectively. All 25 peptides were examined for agonistic and antagonistic potencies in AVP V2/V1a assays. With the exception of peptides 5 and 6, all exhibit potent anti-V1a antagonism, with anti-V1a pA2 values in the range 7.64-8.33.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential inhibition of HIV-1 and SIV envelope-mediated cell fusion by C34 peptides derived from the C-terminal heptad repeat of gp41 from diverse strains of HIV-1, HIV-2, and SIV

The spectrum of inhibition of human (HIV) and simian (SIV) immunodeficiency virus envelope (Env)-mediated cell fusion by C34, a 34 residue peptide corresponding to the C-heptad repeat of gp41 (residues 628-661 of HIV-1 Env), has been examined using a panel of five envelope glycoproteins, three from HIV-1 (LAV, SF162 and 89.6) and two from SIV (mac239 and mac316), and six C34 peptides derived from three strains of HIV-1 (LAV, N CM, and O CM), two strains of HIV-2 (EHO and ALI), and one strain of SIV (African Green Monkey, AGM). A quantitative vaccinia-based reporter gene cell fusion assay was employed. The inhibition data from the panel of 30 C34/envelope glycoprotein combinations, which can be fit to a simple activity relationship with IC(50) values spanning a range of over 4 orders of magnitude from 4 nM to 70 microM, permits one to rationalize both the potency and broadness of the inhibitory properties of the C34 peptides in terms of computed interaction free energies between the C34 peptides and the N-helical trimeric coiled-coil of gp41 and the helical propensities of the free C34 peptides. Of particular interest is the finding that the C34 peptide derived from the EHO strain of HIV-2 is a broad spectrum, highly potent inhibitor of Env-mediated cell fusion with IC(50) values spanning a very narrow range from only 4 to 25 nM over the entire panel of HIV-1 and SIV envelope glycoproteins tested. This result suggests that C34 from HIV-2 EHO may present a potentially useful therapeutic agent against diverse and/or resistant strains of HIV-1.     

In vivo immunopharmacological properties of tuftsin (Thr-Lys-Pro-Arg) and some analogues

Tuftsin (Thr-Lys-Pro-Arg) is part of the Fc fragment of a leukophilic IgG and is a stimulator of the phagocytic activity of macrophages and polymorphonuclear cells (PMN) when cleaved from its carrier molecule. Tuftsin was shown to stimulate in vitro all PMN and macrophage functions examined through binding to specific cell surface receptors. In the present work, we provide further evidence that synthetic tuftsin administered to mice may act as an immunomodulator and that its effects on immune functions may result from a primary action on macrophages. After i.v. injection at a dosage of 25 micrograms/mouse, tuftsin stimulated effector (phagocytosis) and regulatory (IL1 production) functions of macrophages and potentiated DTH reaction. Lymphocyte functions (proliferative response to mitogens, T cell-mediated cytotoxicity, IL2 and gamma IFN production) were depressed at times at which macrophage activities were maximally enhanced, suggesting that negative regulatory functions of these latter cells were also stimulated. Tuftsin analogues were synthetized representing substitution or derivatization of the threonyl residue. The relative potencies of these analogues in augmenting phagocytosis-induced chemiluminescence of macrophages were tuftsin greater than or equal to (Gly1)-tuftsin greater than for-tuftsin greater than (for-Met1)-tuftsin greater than (Met1)-tuftsin. Concerning potentiation of DTH reaction the order was (Gly1)-tuftsin greater than or equal to (for-Met1)tuftsin greater than tuftsin greater than (Met1)-tuftsin greater than for-tuftsin. In contrast to tuftsin, none of the analogues induced depression of spleen cell reactivity to mitogens. In addition, (for Met1)-tuftsin administration resulted in an increased production of IL2 and IFN by ConA-stimulated spleen cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification.

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.     

Inhibition and reversal of endotoxin-, aggregated IgG- and paf-induced hypotension in the rat by SRI 63-072, a paf receptor antagonist

Platelet activating factor (paf) given intravenously produces systemic hypotension in the rat. Similar effects can be induced using endotoxin or heat-aggregated IgG challenges, which are thought to involve endogenous paf release. Extending this concept, we have examined the ability of the paf antagonist SRI 63-072 to inhibit or reverse systemic hypotension induced with paf, heat-aggregated IgG or endotoxin 0111-B4 in rats. At 100 ng kg-1 paf, there occurred a 38.6 +/- 5.1% decrease in carotid mean arterial pressure (MAP) followed by a 3.2 +/- 0.7 min recovery period (RP) to return to normal pressure values. The ED50 of SRI 63-072 was 0.16 mg kg-1 i.v. (MAP) and 0.25 mg kg-1 (RP) when given 1-5 min before the paf challenge. Endotoxin (15 mg kg-1 i.v.) produced a hypotensive response (54 +/- 8% decrease in MAP) and a corresponding 80% decrease in mesenteric artery blood flow. When given 2-8 min after endotoxin, 1.0 mg kg-1 i.v. SRI 63-072 totally restored blood pressure and artery blood flow. SRI 63-072 similarly reversed heat-aggregated IgG (10 mg kg-1) induced reduction of MAP, with an ED50 of 0.05 mg kg-1 i.v. The observations that SRI 63-072 can inhibit or reverse systemic vascular effects produced from paf and other provocators of endogenous paf release strongly implicates paf as a common final mediator of hypotension and shock. As SRI 63-072 is a competitive receptor antagonist, the hypotensive effects of these provocators appear to be mediated by vascular receptors for paf.     

Modulation of the effector functions of cytolytic T-lymphocytes with synthetic peptide inhibitors of protein kinases.

The hypothesis was tested that it is possible to influence cellular responses of intact cells using synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases. Using cytotoxic T-cells (CTL), we demonstrate here that some basic amino acid-containing synthetic peptide substrates of protein kinases [e.g., of cGMP-dependent protein kinase (peptide PKG-S), synthetic peptide inhibitor of cGMP-dependent protein kinase (peptide PKG-I), and peptide corresponding to the tyrosine phosphorylation site in pp60src (peptide RR-src)] were strongly inhibitory in T-cell receptor (TCR) and T-cell growth factor, interleukin 2 (IL-2)-triggered proliferation of CTL. These peptides also inhibited other cellular responses of CTL. Peptides which contain basic amino acids, but do not have substrate specificity determinants for protein kinase, were not inhibitory. The inhibition with peptides is not due to their toxicity, since no cell death was observed by the trypan blue exclusion test and by lactate dehydrogenase release. Use of the granule exocytosis assay provided opportunities to clarify the mechanism of the peptide action. Tested peptides inhibited not only cell-surface ligand-induced CTL activation, but also affected cell-surface receptor-independent CTL activation (granule exocytosis and gamma-interferon secretion) induced by the synergistic action of the protein kinase C activator (PMA) and ionophore A23187. It was found that minor changes in amino acid composition or amino acid position in the synthetic peptides dramatically change their ability to affect lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prodrugs as drug delivery systems. XXX. 4-Imidazolidinones as potential bioreversible derivatives for the α-aminoamide moiety in peptides

The hydrolysis kinetics of five 4-imidazolidinones derived from acetone and the dipeptides Ala-Gly, Ala-Ala, Phe-Leu, Leu-Gly and Asp-Phe methyl ester were studied to assess their suitability as prodrug forms for the α-aminoamtde moiety occurring in peptides. The imidazolidinyl peptides were found to undergo a complete hydrolysis in the pH range 1–10 at 37°C and most of them showed a sigmoidal pH-rale profile with maximum rates at pH > 4. The stability of the derivatives varied widely, the following half-lives being obtained at pH 7.40 and 37°C: 0.9 h (Asp-Phe methyl ester), 3.4 h (Phe-Leu), 24.6 h (Ala-Ala). 410 h (Ala-Gly) and 530 h (Leu-Gly). The major structural factor influencing the stability appeared to be the stcric properties within the C-terminal amino acid residue. The 4-imidazolidinones are much weaker bases (pK_a about 3.1) than the parent dipeptides and, as determined by partition experiments in octanol-aqueous buffer systems with the Phe-Leu derivative, they are more Hpophilic than the parent compounds. It is suggested that 4-imidazolidinone formation in principle may Become a useful approach to bioreversible dehvatization of dipeptides or other peptides containing an α aminoamide function with the aim of solving delivery problems fur peptide drugs.     

Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS‐TTEGDA support for the investigation of HCV infection in human blood

Abstract: Four peptides were designed and synthesized on a highly solvating copolymer of tetraethyleneglycol diacrylate cross‐linked polystyrene (PS‐TTEGDA) support with very high purity and yield. The polymer was synthesized in various cross‐linking densities (1, 2, 3, 4, 5 and 10%) using radical aqueous suspension polymerization. Four per cent PS‐TTEGDA resin showed rigidity and mechanical characteristics comparable with those of divinylbenzene cross‐linked polystyrene (PS‐DVB) support. Swelling and solvation characteristics of PS‐TTEGDA were much higher than PS‐DVB support in all solvents used in solid‐phase peptide synthesis. Forty‐eight hour treatment of the support with neat trifluoroacetic acid did not show any change in its infrared spectra. PS‐TTEGDA could be functionalized with chloromethyl, aminomethyl and hydroxymethyl functional groups under various controlled conditions. Synthetic utility of the support was demonstrated by the synthesis of four peptides selected from the envelope and nonstructural protein region of the prototype hepatitis C virus (HCV). These peptides were later used successfully to develop a peptide‐based immunoassay (PBEIA) for the detection of HCV immunity. Peptides designed from the NS1 and NS4 protein regions were found to be very promising for the development of a new diagnostic kit to detect HCV infection in human blood. Peptide purity was tested by RP‐FPLC and the peptide identity was confirmed by amino acid analysis.     

N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands

Previous structure-activity studies on nociceptin/orphanin FQ (N/OFQ) identified [Phe(1)Psi(CH(2)NH)Gly(2)]N/OFQ(1-13)-NH(2) and [Nphe(1)]N/OFQ(1-13)-NH(2) as a N/OFQ peptide receptor (NOP) partial agonist and pure antagonist, respectively. The addition of fluorine to the Phe(4) or the insertion of a further pair of basic amino acids Arg(14)-Lys(15) generate potent agonists. On the basis of these findings, we combined in the N/OFQ-NH(2) template the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) that increase the agonist potency with those conferring partial agonist (Phe(1)Psi(CH(2)NH)Gly(2)) or pure antagonist (Nphe(1)) properties. Twelve peptides were synthesized and pharmacologically evaluated in Chinese hamster ovary cells expressing the human recombinant NOP and in electrically stimulated mouse vas deferens and guinea pig ileum assays. All peptides behaved as NOP ligands; the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) increased ligand affinity/potency. Peptides with the normal Phe(1)-Gly(2) peptide bond behaved as full agonists, and those with the Phe(1)Psi(CH(2)NH)Gly(2) modification behaved as partial agonists, while those with the Nphe(1) modification behaved as partial agonists or pure antagonists depending on the presence or absence of the (pF)Phe(4) modification, respectively. The full agonist [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), the partial agonist [Phe(1)Psi(CH(2)NH)Gly(2),(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), and the pure antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) represent the most potent peptide ligands for NOP.     

Transdermal Delivery of Bioactive Peptides: The Effect of n-Decylmethyl Sulfoxide,pH, and Inhibitors on Enkephalin Metabolism and Transport

We investigated the effects of the nonionic surfactant, n-decylmethyl sulfoxide (NDMS), pH, and inhibitors on the metabolism and the permeation of amino acids, dipeptides, and the pentapeptide enkephalin, through hairless mouse skin. An HPLC gradient method was developed to identify the possible peptide and amino acid metabolites of leucine-enkephalin. NDMS increased the permeability of all amino acids and peptides tested. At neural pH, the enzyme activity within the skin was such that no flux of leucine-enkephalin (YGGFL) was observed and the donor cell concentration of YGGFL decreased rapidly. The major cleavage occurred at the Tyr-Gly bond. At pH 5.0 the metabolic activity was reduced significantly and a substantial flux of YGGFL was observed. Enzymatically stable YGGFL analogues, Tyr-D-Ala-Gly-Phe-Leu (YDAGFL) and its amide, exhibited significant fluxes even at neutral pH in the presence of NDMS, but with substantial metabolism. YDAGFL amide was more stable to metabolism than YDAGFL. The rates of metabolism of the peptides in the skin homogenates were in the order: FL.>>YGGFL > GFL > GGFL >> YG, YGG >> YDAGFL amide. In the skin homogenates puromycin and amastatin showed the highest inhibitory effects, while FL and GFL were only slightly active. However, in the skin diffusion experiments, FL allowed the highest amount of intact parent compound to permeate, making it the most potent inhibitor. These results show that the complex proteolytic enzyme activities occurring during skin permeation are different from those in skin homogenates and that a combination of enhancer, pH adjustment, and inhibitors can increase the transdermal delivery of peptides.     

Human PEPT1 pharmacophore distinguishes between dipeptide transport and binding

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.     

Sequence-based design and discovery of peptide inhibitors of HIV-1 integrase: insight into the binding mode of the enzyme

Integration of viral DNA into the host chromosome is an essential step in the HIV life cycle. This process is mediated by integrase (IN), a 32 kDa viral enzyme that has no mammalian counterpart, rendering it an attractive target for antiviral drug design. Herein, we present a novel approach toward elucidating "hot spots" of protein-protein or protein-nucleic acid interactions of IN through the design of peptides that encompass conserved amino acids and residues known to be important for enzymatic activity. We designed small peptides (7-17 residues) containing at least one amino acid residue that is important for IN catalytic activities (3'-processing and strand transfer) or viral replication. All these peptides were synthesized on solid phase by fluorenylmethoxycarbonyl (Fmoc) chemistry and evaluated for their inhibition of IN catalytic activities. Such specific sites of interest (i.e., protein-DNA or protein-drug interactions) could potentially be used as drug targets. This novel "sequence walk" strategy across the entire 288 residues of IN has allowed the identification of two peptides NL-6 and NL-9 with 50% inhibitory concentration (IC50) values of 2.7 and 56 microM for strand transfer activity, respectively. Amino acid substitution analysis on these peptides revealed essential residues for activity, and the rational truncation of NL-6 produced a novel hexapeptide (peptide NL6-5) with inhibitory potency equal to that of the parent dodecapeptide (peptide NL-6). More significantly, the retroinverso analogue of NL-6 (peptide RDNL-6) in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted displayed improved inhibitory potency against 3'-processing of HIV-1 IN by 6-fold relative to the parent NL-6, serving as a metabolically stable derivative for further in vitro and in vivo analyses.     

Comparison of opioid properties between D-Arg-containing dipeptides and tetrapeptides.

Since the D-Arg-containing dipeptides, H-Tyr-D-Arg-OMe (TDA) and H-Tyr(Et)-D-Arg-OMe, and D-Arg2-substituted dermorphin N-terminal tetrapeptide analogues, H-Tyr-D-Arg-Phe-Gly-OEt (TDAPG) and H-Tyr(Et)-D-Arg-Phe-Gly-OEt gave different pharmacological responses in vivo, opioid interaction and structure-activity relationships have been investigated in vitro. In the isolated guinea-pig ileum assay, the tetrapeptides were potently inhibitory, their activity markedly exceeding that of the dipeptides. In particular, the first tetrapeptide had twice the activity of morphine, while the potency of the dipeptides was less than one twentieth that of morphine. Also in the opioid receptor binding assay, tetrapeptides had a higher affinity than the dipeptides. IC50 values of tetrapeptides were 8.46 and 23.7 nM, respectively, which were lower than that of morphine. Ethylation of the Tyr residue of TDA much increased the opioid activity whereas that of TDAPG greatly decreased it. All peptides used were extremely stable to aminopeptidase-M and carboxypeptidase-Y and had an inhibitory effect on enkephalin (EK)-degrading enzymes. From these results, it appears that the effects of the tetrapeptides are due mainly to specific interaction with opioid receptors, whereas the dipeptides do not act specifically on the opioid receptors, but are involved in non-opioid mechanisms. The resistance to enzymes and inhibitory effect of the peptides used on the EK-degrading enzymes may also account for their potent and long-lasting opioid-like activities.