Key analogs of the tetrapeptide subunit of RA-VII and deoxybouvardin

The synthesis and evaluation of two key analogs 3 and 4 of the potent antitumor antibiotics deoxybouvardin (1) and RA-VII (2) which contain fundamental modifications in the tetrapeptide subunit are described. Unlike the natural products, these agents 3 and 4, which substitute (Gly)4 and (Gly)3 for the D-Ala-Ala-NMe-Tyr(OMe)-Ala tetrapeptide subunit, adopt conformations in which the central amide in the cycloisodityrosine subunit adopts its inherently preferred trans stereochemistry and both were found to be biologically inactive.     

Quantum molecular modeling of the elastinic tetrapeptide Val-Pro-Gly-Gly

The free Val-Pro-Gly-Gly tetrapeptide belonging to the Proline-rich sequences of elastin has been studied both theoretically and experimentally. The molecular modelisation was carried out using AM1 and ab initio quantum computations while the conformation in solution was ascertained by circular dichroism spectroscopy performed on the synthesized tetrapeptide. Experimental and theoretical investigations lead to the conclusion that the most probable structure is constituted by a type II beta-turn.     

Key amino acids in the gamma subunit of the gamma-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site

Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.     

Development of a sensitive and specific assay system for cholecystokinin tetrapeptide

Cholecystokinin is a gastrointestinal and neuropeptide which has been implicated in a wide range of physiological and behavioral processes. We have developed a sensitive and specific assay system to measure the various forms of cholecystokinin (CCK) in human plasma. This 3-step system involves i) extraction of CCK fragments from plasma using reverse phase chromatography; ii) separation of peptides by high performance liquid chromatography; and iii) detection and quantification of peptides with a double-antibody radioimmunoassay, using an antibody raised against cholecystokinin tetrapeptide (CCK-4) coupled to thyroglobulin and 125I Bolton-Hunter CCK-4 as tracer. The antibody detects CCK-4, sulfated CCK-8 (CCK-8S) and nonsulfated CCK-8 (CCK-8ns) with equal affinity. The lower limit of detection is 2.7 fmol, with an ED50 of 10.6 +/- 2.2 fmol. Mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was determined to be 12.9 +/- 2.1 pM CCK-4 equivalents. Concentrations of each individual peptide in plasma were determined to be 1.0 +/- 0.2 pM, 3.4 +/- 0.8 pM and 1.9 +/- 0.4 pM for CCK-4, CCK-8s and CCK-8ns respectively.     

Assessment of the psychotropic activity of analogs of cholecystokinin tetrapeptide fragment]

BACKGROUND: Both dermabrasion and high-energy pulsed carbon dioxide (CO2) laser resurfacing can improve the appearance of surgical scars. Although the results of these two procedures have been compared using historical data, a prospective evaluation has never been performed in humans. OBJECTIVE: To prospectively compare the clinical effects of dermabrasion and high-energy pulsed CO2 laser resurfacing in the revision of surgical scars. METHODS: Facial surgical scars in four patients were prospectively revised using a split scar model. One half of the scar was dermabraded and the other half was resurfaced with the high-energy pulsed CO2 laser. Comparisons of the two treatment modalities were performed through clinical assessment, photographic evaluation, and textural analysis of the scars. RESULTS: The high-energy pulsed CO2 laser-resurfaced halves of the scar were bloodless with less postoperative crusting in comparison with the dermabraded halves. Reepithelialization time and degree and duration of postoperative erythema were similar for both treatment halves. Photographic evaluation and textural analysis showed comparable improvement in the clinical appearance and surface texture of the scars with both treatment modalities. CONCLUSIONS: Both the high-energy pulsed CO2 laser and dermabrasion can achieve comparable clinical improvement in the revision of surgical scars. The high-energy pulsed CO2 laser offers the advantage of a bloodless field and a more precise method of tissue ablation. Postoperative erythema, however, is an expected finding with both treatment modalities.     

On the conformation of tiazofurin analogues

Tiazofurin, an important inhibitor of inosine 5'-monophosphate dehydrogenase, has been argued to possess a restricted glycosylic bond due to an energetically favorable intramolecular (1-4) electrostatic interaction between the partial positive sulfur and the negative oxygen of the ribose. This rigidity has been appointed as a plausible cause that leads to activity in the sulfur containing compounds as opposed to the inactive oxazofurin-like analogues (i.e. S is replaced by an oxygen) that lack this favorable interaction. We reinvestigated this notion by using computational methods to report that although the above interaction (or its lack) is likely to contribute to the low-energy conformation of these classes of molecules, the flexibility of the glycosylic bond is ultimately determined by steric interaction of the heteroatoms with the C2'-H and O4' of the ribose. Application of this theory in the design of new analogues is presented as well.     

Zero-length crosslinking of the beta subunit of phosphorylase kinase to the N-terminal half of its regulatory alpha subunit

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer containing four copies each of four distinct subunits: alpha, beta, gamma, and delta. By intramolecular zero-length crosslinking with transglutaminase, we have previously demonstrated that the regulatory alpha and beta subunits abut one another in the holoenzyme [Nadeau, O. W., and Carlson, G. M. (1994) J. Biol. Chem. 269, 29670-29676]. Selective partial proteolysis of the 138 kDa alpha subunit in holoenzyme that had been crosslinked by transglutaminase has revealed a high molecular weight conjugate corresponding to full-length beta subunit crosslinked to a 60 kDa N-terminal fragment of alpha (determined by SDS-PAGE, Western blotting and N-terminal sequencing). This conjugate was also observed when the enzyme was first activated by partial proteolysis of alpha and then crosslinked by transglutaminase. Both forms of the kinase, generated by either sequential crosslinking and proteolysis or the reverse, coeluted with non-crosslinked hexadecameric control enzyme in size exclusion chromatography, indicating that the crosslinking was intramolecular, i.e., within hexadecamers. This is the first demonstration of any intersubunit interaction involving the N-terminal domain of the alpha subunit and the first region of any subunit shown to interact with the beta subunit. The results are consistent with the predicted path of the polypeptide backbone of the alpha subunits within the holoenzyme and with the proposed location of the beta subunits.     

Structure-activity relationship of lepidimoide and its analogues

The structure-activity relationship of lepidimoide and its analogues was investigated by means of the Amaranthus caudatus L. hypocotyl elongation test. In addition, the activities of alpha-D-galacturonic acid and L-(+)-rhamnose, which are component sugars of lepidimoide, were also studied. The carboxylic acid free type of lepidimoide showed growth-promoting activity as high as the original lepidimoide (sodium type). The acetylated compound showed considerably higher activity than lepidimoide, whereas the methylated lepidimoide did not show any activity. The hydroxylated lepidimoide without a double bond in the C-4,5 position showed lower activity. The sugar alcohol type of lepidimoide [2-O-(alpha-D-glucopyranosyl)-L-rhamnose] showed the highest activity in all the compounds studied. alpha-D-Galacturonic acid, L-(+)-rhamnose and their mixtures, which are component sugars of lepidimoide, exhibited only slight or no activity, respectively. D-Glucose and the mixture of D-glucose and L-(+)-rhamnose were also slightly active or inactive. These data suggest that the active sites in the chemical structure of the lepidimoide are the uronic acid derivative bearing an alpha,beta-unsaturated carboxylate bonded to rhamnose via an alpha-glucoside linkage and a double bond in the C-4,5 position in the uronic acid.     

Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase

In the investigation of the sequences of myosin phosphatase target subunit 1 (MYPT1) involved in binding the substrate and catalytic subunit of protein phosphatase type 1 (PP1c), fragments of MYPT1 were prepared and characterized. The shortest fragment capable of full activation of PP1c contained the sequence of residues 1-295. Within this fragment, the N-terminal sequence of residues 1-38 is involved in activation of PP1c (kcat) and the ankyrin repeats (residues 39-295) were involved in substrate binding (Km). The ankyrin repeats alone (residues 39-295) and the C-terminal fragment of residues 667-1004 did not activate PP1c. Using gel filtration, an interaction with PP1c was detected for the sequences of residues 1-295, 17-295, and 1-170. Affinity columns were prepared with various fragments to assess binding of PP1c. Binding to the column with residues 1-295 was strongest, followed by the binding to the column with residues 1-170. A weak interaction was observed with the column with residues 1-38. The column with residues 1-295 was used to isolate PP1c from gizzard. The purified PP1c was activated by MYPT1 and fragments to a greater extent than previous preparations. These results suggest that the N-terminal sequence (residues 1-38) and the ankyrin repeats are involved in binding PP1c. The C-terminal ankyrin repeats appear to be dominant, but there is an interaction of PP1c with the N-terminal ankyrin repeats. The N-terminal peptide has two apparent functions, the binding of PP1c via the consensus binding sequence and activation of PP1c by the sequence of residues 1-16.     

Identification of a potent analogue of Nazumamide A through iteration of combinatorial tetrapeptide libraries

Five sets of N-acylated tetrapeptide libraries and sublibraries related to Nazumamide A have been prepared using 25 natural and unnatural amino acids. They were evaluated in antithrombin assay, in order to quantify inhibition at each step of the tetrapeptide sublibrary iteration. The studies led to the identification of 2,5-dihydroxybenzoyl-lysyl-isoleucyl-phenylalanyl-arginine as a novel inhibitor of thrombin and was found to be at least 25 times more potent than the natural tetrapeptide 2,5-dihydroxybenzoyl-arginyl-prolyl-isoleucyl-alpha-aminobutyric acid (NAZA).     

Alteration in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV in quinolone-resistant clinical isolates of Staphylococcus epidermidis

We examined 22 clinical isolates of Staphylococcus epidermidis to analyze the association of alterations in GyrA and ParC with fluoroquinolone resistance. The simultaneous presence of GyrA and ParC alterations was associated with a high level of fluoroquinolone resistance in the clinical isolates of S. epidermidis.     

Structure-activity study of the nociceptin(1-13)-NH2 N-terminal tetrapeptide and discovery of a nociceptin receptor antagonist

In the present study, the minimal fragment sequence required to fully activate the nociceptin (NC) receptor, namely NC(1-13)-NH2, was used as template for the design of a series of new compounds. Changes were made in the N-terminal tetrapeptide Phe-Gly-Gly-Phe, which has been shown to be essential for receptor occupation and activation. The new compounds were tested for their ability to inhibit the electrically evoked contraction of the mouse vas deferens, a pharmacological preparation sensitive to NC. Results obtained indicate that (a) the replacement of Gly2 or Gly3 with an aromatic residue (Phe) of L or D chirality eliminates the ability of the peptide to occupy the NC receptor; (b) the distance between Phe1 and Phe4 of NC appears to be critical, since any alteration of it leads to a marked decrease or a total elimination of biological activity; and (c) the insertion of a pseudopeptide bond between Phe1 and Gly2 maintains affinity but eliminates the ability of the peptide to activate the NC receptor and leads to antagonism. The peptide [Phe1psi(CH2-NH)Gly2]-NC(1-13)-NH2 acts as a selective NC receptor antagonist and is inactive on opioid receptors. The results summarized in this paper confirm and extend our previous findings by showing that the structural requirements for NC binding to its receptor are clearly different from those of opioids; in addition, this structure-activity study has led to the identification of the first NC receptor selective antagonist.     

Immunocytochemical demonstration of peptidergic cells in the pond snail Lymnaea stagnalis with an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide

With an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide immunoreactive perikarya and nerve fibers were identified in the central and peripheral nervous system of the pond snail Lymnaea stagnalis. Their localization is described. The same antiserum yielded reactive product in particular cells of the epithelium of the alimentary tract. The use of two different fixatives, glutaraldehyde, and a mixture of glutaraldehyde, picric acid, and acetic acid (GPA) showed that certain nerve cells can be identified only in material fixed with either the one or the other of these two fixatives, a result which indicates that in Lymnaea more than one FMRF-amide-like substances may occur. "Positive" axon endings were found in the periphery of various nerves, i.e., in places where neurohormones are released into the blood. Other fibers were found to end, probably synaptically, on other neurons, on epithelial cells in the stomach, and between muscle cells in various parts of the body, e.g., in the heart. In these cases the FMRF-amide-like substance may function as a neurotransmitter or a neuromodulator.     

The molecular specificity of action of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in the control of hematopoietic stem cell proliferation

Nutritional effects have traditionally focused on outcomes, such as nitrogen balance, wound healing, or muscle strength. Little emphasis has been placed on how biochemical or physical improvements translate into functional changes as perceived by the patient. Because glutamine (GLN)-supplemented nutrition promotes protein synthesis and improves nitrogen balance, we assessed the mood of individuals participating in a randomized controlled blinded trial receiving GLN solutions. Patients (n = 23) undergoing marrow transplantation were randomized by the research pharmacist to receive either standard total parenteral nutrition (TPN) (control) or GLN-containing TPN (40 g of glutamine total). The solutions were isocaloric and isonitrogenous and were administered until the patient was eating 50% of estimated requirements. Before TPN and on admission to the hospital, the patient completed the Profile of Mood States questionnaire, a standardized test quantifying the degree of tension, depression, anger, vigor, fatigue, and confusion. The patient completed the questionnaire again at the end of TPN near discharge. The tests were scored and the change from baseline for each mood for both groups of patients was calculated at the completion of TPN. The scores for vigor in the control group (delta scores) decreased over the course of hospitalization as would be expected with a serious illness. The group receiving glutamine TPN, however, essentially showed little change in vigor from baseline and the delta score was significantly different from the control group (delta vigor score -0.85 +/- 2.1 in the glutamine group vs. -5.90 +/- 1.7 in the control group; p = .07).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis

The gyrA and parC genes of 31 clinical isolates of Enterococcus faecalis, including fluoroquinolone-resistant isolates, were partially sequenced and analyzed for target alterations. Topoisomerase IV may be a primary target in E. faecalis, but high-level fluoroquinolone resistance was associated with simultaneous alterations in both GyrA and ParC.