D-alanine In Position Research Articles

N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties.

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of β-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.

Gram-positive cell wall structure of the A3 gamma type in heliobacteria.

The amino acid composition and structure of the peptidoglycan from Heliobacillus mobilis was determined by one- and two-dimensional thin-layer chromatography of completely and partially hydrolysed cell wall preparations. The structure was found to be of the A3 gamma type, with L,L-diaminopimelate in position 3, D-alanine in position 4 and a glycine interpeptide bridge, as found in certain groups of Gram-positive bacteria including Clostridium perfringens and Nocardioides simplex. The presence of a Gram-positive type of cell wall in heliobacteria is consistent with their phylogenetic relationship to the 'low G + C' Gram-positive bacteria, as previously demonstrated by 16S rRNA sequencing.

Identification of a D-alanine-containing polypeptide precursor for the peptide opioid, dermorphin.

The naturally occurring amphibian skin peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) are highly potent and selective agonists at the mu- and the delta-opioid receptors, respectively. For peptides synthesized by animal cells, they have a rather peculiar structural feature of containing a D-amino acid residue in their sequence which imparts biological activity on them. The cloned cDNA encoding the prodermorphin precursor contains the usual alanine and methionine codons at positions where D-alanine and D-methionine are present in the mature products. In this study, dermorphin precursor was characterized in extracts from amphibian skin by antisera recognizing distinct epitopes within the predicted structure of pro-dermorphin. Proteolytic digestion of purified endogenous pro-dermorphin generated a peptide containing a D-alanine in position 2, identified as prepro-dermorphin-(80-89), i.e. Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Glu-Ala. In addition, analysis of skin extracts by enzyme immunoassays coupled to high performance liquid chromatography separations revealed the presence of, besides dermenkephalin, novel dermenkephalin-related peptides, i.e. [L-Met2]dermenkephalin, dermenkephalin-OH, and [Met(O)6]dermenkephalin. [L-Met2]dermenkephalin was present in frog skin in a concentration of about 100 times that of dermenkephalin. These observations confirm that, despite the presence of D-amino acid residues, dermorphin and dermenkephalin are genuine products of post-translational processing of a ribosomally made precursor. They suggest that D-Ala and D-Met develop from a dehydrogenation/hydrogenation stereoinversion of their corresponding L isomers incorporated into pro-dermorphin, a process that occurs with low efficiency at an early stage of biosynthesis.

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.

CDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.

We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin.

Antibodies to cyclosporine A (CsA) by a novel route and their use to monitor cyclosporine levels by radioimmunoassay (RIA)

Anti-cyclosporine antibodies were generated in rabbits, using an antigen derived from CsA. CsA was linked to carrier proteins by means of a photoactive cross-linking reagen, 4-benzoylbenzoic acid (BBa), an α,β-unsaturated ketone. In the presence of CsA, photolysis of BBa results in hydrogen abstraction and random insertion into the cyclosporine molecule, generating a population of CsA with carboxyl groups at various positions. Immunization with CsA-BBa-bovine serum albumin gave rise to high affinity antibodies to CsA, with K d = 9.8 ± 2.8 × 10 −11 M, was determined by Scatchard analysis. Specificity was determined by competition experiments in a radioimmunoassay (RIA), using a panel of six cyclosporine derivatives, substituted at different positions. The derivatives could be arranged into three groups according to their affinities. One derivative with the lowest affinity had a bulky O-t- butyl- D- serine in place of D-alanine in position 8. Serum CsA levels in 25 transplant patients were measured by RIA, and compared to levels determined with a commercially available polyclonal antibody, which is routinely used clinically. The rabbit antiserum gave values that correlated well with the results using the commercial antibody.

Cyclosporins--new analogues by precursor directed biosynthesis.

Cyclosporin A (ciclosporin), a potent and clinically important immunosuppressive drug (Sandimmun), represents the main component of a group of over 25 closely related, cyclic undecapeptides produced by the fungus Tolypocladium inflatum. By feeding experiments using DL-alpha-allylglycine as precursor, specific incorporation in position 2 was attained leading to [Allylgly2]cyclosporin A. Exogenously supplied L-beta-cyclohexylalanine results in the almost exclusive production of [MeCyclohexylala1]cyclosporin A (replacement of methylbutenyl-methylthreonine-1). D-Alanine in position 8 can be successfully substituted by D-serine. The new [D-Ser8] analogues of the cyclosporins A, C, D and G as well as [Allylgly2]cyclosporin A exhibit high immunosuppressive effects.

Potent tetrapeptide enkephalins

Small peptides with opiate-like activity have generally had structures closely resembling that of the opioid pentapeptide enkephalin: Tyr-Gly-Gly-Phe-Met-COOH. Single deletions of any one of the amino acids has been demonstrated to reduce opiate activity drastically. In this work we show that the potency losses resulting from the removal of glycine 3 can be fully attenuated by substitution of D-alanine in position two and derivatization of the acid to the amide. This tetrapeptide (Tyr-DAla-Phe-Met-NH 2) has narcotic activity similar to the parent pentapeptide in the guinea pig ileum and mouse tail-flick tests. This enhanced potency, relative to the unaltered tetrapeptide, is theorized to arise from increased resistance to enzymatic destruction. the data presented show that a five amino acid sequence is not mandatory for the expression of opiate activity in enkephalin analogs.

Synthesis and opioid activities of stereoisomers and other D-amino acid analogs of methionine-enkephalin

A series of D-amino acid-substituted analogs of the opiate peptide, methionine 5-enkephalin, were synthesized by solid-phase methods and tested for their abilities to inhibit electrically-evoked contractions of mouse vasa deferentia and to compete with tritiated enkephalin for opiate receptors on particulate fractions isolated from homogenates of rat brain. [D-Ala 2]-enkephalin and [D-Ala 2]-enkephalin amide were found to be the most potent peptides in both assay systems, being about 1000% active in the vas deferens bioassay and 120% and 150% active, respectively, in the stereospecific binding test relative to methionine 5-enkephalin itself. In comparison, [D-Met 5]-, [D-Tyr 1]-, [D-Leu 2]-, [D-Phe 2]-, [D-Ala 3]-, and [D-Phe 4]-enkephalin had not more than 10% activity. The stabilization of the β-bend conformation of methionine 5-enkephalin by the substitution of D-alanine in position 2 of the peptide chain may contribute to the high activities of the [D-Ala 2]-analogs.

Anti-Luteinizing Hormone (LH)-Releasing Activity of Several Analogues of LH-Releasing Hormone

The anti-LH-releasing activity of several analogues of LH-RH was tested by 2-hour infusion in ovariectomized, estrogen-progesterone-pretreated rats and in immature male rats. [Leu3]LH-RH, [Leu3, desGly10]-LH-RH ethylamide, [desHis2, Leu3,desGly10]-LH-RH ethylamide, [Gly2, Leu3,desGly10]-LH-RH ethylamide, [Leu1, desGly10]-LH-RH ethylamides, [desHis2,D-Ala6,desGly10]-LH-RH ethylamide, [desHis2,Leu3,D-Ala6, desGly10]-LH-RH ethylamide, and [D-pGlu1,desGly10]-LH-RH ethylamide, and and [D-pGlu1,desHis2,desGly10]-LH-RH ethylamide showed some anti-LH-releasing activity, but never completely inhibited the increase in serum LH in response to LH-RH. No significant differences were found among the analogues tested and [desHis2,desGly10]-LH-RH ethylamide, and inhibitory potency was not improved with those peptides containing D-alanine in position 6 of the chain. None of the analogues tested, including [desHis2,desGly10]-LH-RH ethylamide, was able to block the LH-RH-induced FSH release in these systems.

Polyfluoroalkylamine derivatives of luteinizing hormone-releasing hormone.

A series of C-terminally fluorinated analogs of luteinizing hormone-releasing hormone (LH-RH) was synthesized by a combination of solid-phase and classical techniques and found to have interesting properties when assayed for LH- and FSH-releasing activities over a prolonged period of time in immature male rats. One of the peptides, desGly-10-LH-RH-2,2,2-trifluoroethylamide, was found to be about nine times more effective than LH-RH in releasing LH, or approximately twice as active as the corresponding alkylamide peptide, desGly-10-LH-RH-ethylamide. However, desGly-10-LH-RH-2,2,3,3,3-pentafluoropropylamide was only slightly more active than LH-RH and considerably less active than the corresponding propylamide analog. A family of peptides was also prepared containing D-alanine in position six of the chain in conjunction with the C-terminal modifications. D-Ala-6,desGly-10-LH-RH-2,2,2-trifluoroethylamide, surprisingly, gave patterns of gonadotropin release which were only as intense and virtually identical with those obtained with D-Ala-6,desGly-10-LH-RH-ethylamide which in turn releases about 20 times more LH than a similar dose of LH-RH. D-Ala-6,desGly-10-LH-RH-propylamide also gave almost identical patterns of gonadotropin release. D-Ala-6,desGly-10,LH-RH-2,2,3,3,3-pentafluoropropylamide was considerably less potent, being only five times more effective than LH-RH.

On the Specificity of Phospho-N-acetylmuramyl-pentapeptide Translocase: THE PEPTIDE SUBUNIT OF URIDINE DIPHOSPHATE-N-ACETYLMURAMYL-PENTAPEPTIDE

Abstract Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-γdGlu-Lys-dAla-dAla:undecaprenylphosphate phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. In addition, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurNAc-pentapeptide. A specificity profile towards the peptide subunit of UDP-MurNAc-pentapeptide has been established for the exchange and transfer reactions catalyzed by the translocase from Staphylococcus aureus Copenhagen. A good correlation between Vmax/Km in the transfer reaction and Rmax/Km in the exchange reaction was observed. On the basis of glycine substitution, the enzyme has a high specificity for l-alanine in position 1 (R1) and d-alanine in position 4 (R4) and a low specificity for d-alanine in position 5 (R5). For example, when compared with UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla, glycine replacement in either R1 or R4 results in a 9- and 15-fold decrease in Vmax/Km, respectively. In contrast, glycine substitution in R5 results in only a 1.5-fold decrease in Vmax/Km. These results are further supported by comparing UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla, -Ala-dGlu-Lys-dAla, and -Ala-dGlu-Lys. For UDP-MurNAc-tetrapeptide a 4-fold decrease in Vmax/Km was observed whereas a 77-fold decrease was found for the -tripeptide. Replacement of both R1 and R4 by glycine reduces Rmax/Km 135-fold. The translocase has a low specificity for the diamino acid in R3. Substitution of lysine with meso-diaminopimelic acid results in a 1.5-fold decrease in Rmax/Km and with ornithine in no change. The established specificity profile of the translocase is consistent with the in vivo effects of elevated concentrations of glycine on the growth of S. aureus Copenhagen (Hammes, W., Schleifer, K.H., and Kandler, O. (1973) J. Bacteriol., 116, 1029–1053). Thus, it is suggested that the translocase has a key role in selecting analogs of UDP-MurNAc-Ala-γdGlu-Lys-dAla-dAla for peptidoglycan synthesis.