Synthesis of N-succinyl-L,L-diaminopimelic acid mimetics via selective protection.

The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient preparation of mono-N-acylated 2,6-diaminopimelic acid (DAP) derivatives bearing the different carboxyl groups or lipophilic moieties on their amino group.

Inhibitors of N(alpha)-acetyl-L-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency.

A series of N (alpha)-acyl (alkyl)- and N (alpha)-alkoxycarbonyl-derivatives of L- and D-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N (alpha)-acetyl-L-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N (alpha)-acetyl-L-ornithine to L-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC(50) values in the muM range toward ArgE, indicating that they are moderately strong inhibitors. N (alpha)-chloroacetyl-L-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC(50) value of 85 microM while N (alpha)-trifluoroacetyl-L-ornithine (1f), N (alpha)-ethoxycarbonyl-L-ornithine (2b), and N (alpha)-acetyl-D-ornithine (1a) weakly inhibited ArgE activity providing IC(50) values between 200 and 410 microM. Weak inhibitory potency toward Bacillus subtilis-168 for N (alpha)-acetyl-D-ornithine (1a) and N (alpha)-fluoro- (1f), N (alpha)-chloro- (1g), N (alpha)-dichloro- (1h), and N (alpha)-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC(50) values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.

Gene expression responses in larvae of the fleshfly Sarcophaga bullata after immune stimulation.

Insect larvae develop in decaying organic matter and their defence against various microorganisms must therefore be highly efficient. In the present study, we explored the transcriptional kinetics and induction levels of eight genes in Sarcophaga bullata larvae after infection or aseptic injury. Using real-time PCR, we studied the time-dependent immune response of larvae of the fleshfly S. bullata. We compared the mRNA levels of eight selected genes in induced and non-induced larvae. The third-instar larvae of S. bullata were induced by injecting a bacterial suspension of Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa, or by simple aseptic injury with an entomological pin. We used intact larvae as a control for basal mRNA expression. Total RNA was isolated from the whole body, fat body and haemocytes. We determined the mRNA levels of genes encoding sapecin, transferrin, prophenoloxidase 1 and 2, storage-binding protein, cathe psin L, sarcocystatin, and 26/29 kDa protease. We found that there was massive up-regulation of genes encoding the fleshfly peptide sapecin, as well as the protein transferrin. We also detected down-regulation of, or no change in, the expression of genes that encode prophenoloxidase 1 and 2, storage-binding protein, cathepsin L, sarcocystatin, and 26/29 kDa protease.

Side reactions during photochemical cleavage of an alpha-methyl-6-nitroveratryl-based photolabile linker.

The mechanisms of reactions causing irreversible inhibition of the activity of enzymes when irradiated in the presence of the recently developed alpha-methyl-6-nitroveratryl-based photolinker [Holmes CP. J. Org. Chem. 1997; 62: 2370-2380] have been investigated. Several experiments based on the interaction of the photolinker with model peptides or n-butylamine have been accomplished. A complexity of products, resulting from the side reactions competing with the 'normal' photocleavage of the linker, have been found. The amino and thiol groups of the molecules present in the solvents upon irradiation were recognized as having a major influence on the course of photolysis. Some of these side products resulting from the interaction with amines were identified and the mechanisms by which they can be generated are discussed. The mechanism of the interaction of the thiol groups present in peptides or proteins with the photolinker is unclear and it remains to be further elucidated. It was found that the undesirable effects are favored by a basic pH and are largely reduced by a slightly acidic pH, together with the presence of dithiothreitol. Significant positive effects of dithiothreitol have been observed on the rate as well as the yield of the photocleavage. These results demonstrate that the use of photolabile linkers in biological media can be accompanied by undesired effects, which can be largely reduced by choosing appropriate conditions and additives.

Phosphinic acid compounds in biochemistry, biology and medicine.

This review summarizes our knowledge of biochemical, biological and medical applications and properties of phosphinic acid compounds. Phosphinic acid compounds (phosphinates) are derivatives of phosphinic acid H2P(O)(OH). The major attention of this article is focused on applications of phosphinates of a pseudopeptide character, however interesting examples of phosphinates of a non-peptide nature are mentioned too. Phosphinic acid peptides (phosphinic pseudopeptides) are peptide isosteres where one peptide bond is substituted by the nonhydrolysable phosphinate moiety -P(O)(OH)-CH2- or -P(O)(OH)-. This substitution represents a very convenient mimic of a substrate in the transition state for at least two distinct classes of hydrolytic enzymes, Zn-metalloproteinases and aspartic acid proteinases. In this review we discuss about thirty different protein targets for which the phosphinates have found applications as modulators of their functions in vitro and/or in vivo. These proteins are mainly proteinases, however other types of proteins such as transferases, synthetases, ligases or even receptors are also discussed. Genome sequencing projects have been identifying protein sequences faster than it is possible to discover their functions. The development of combinatorial chemistry in the past few years has boosted up the interest in the use of chemistry to address biological problems. We believe that phosphinates, especially in conjunction with combinatorial chemistry approaches, can represent an extremely versatile tool in the search for proteome and its function.

RXP 407, a phosphinic peptide, is a potent inhibitor of angiotensin I converting enzyme able to differentiate between its two active sites.

The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-Lysyl-Proline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L)Phepsi(PO2-CH2)(L)Ala-Ala-NH2, called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (Ki = 12 nM), RXP 407, which is metabolically stable in vivo, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.

Theory of the correlation between capillary and free-flow zone electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative processes. Application to analysis and preparation of fragments of insulin.

A basic theoretical description of the correlation between capillary zone electrophoresis (CZE) and free-flow zone electrophoresis (FFZE) is presented. The theory of the correlation between CZE and FFZE results from the fact that both methods are based on the same separation principle, zone electrophoresis, and both are performed in the carrierless separation medium with the same composition of the background electrolyte. The equations describing the movement of the charged and noncharged particles in the d.c. electric field applied in the capillary and in the flow-through electrophoretic chamber are presented and used for the quantitative description of the correlation between CZE and FFZE. Based on the theory of the correlation between CZE and FFZE a procedure has been developed for conversion of analytical, microscale CZE separations into continuous preparative separation processes realized by FFZE. Practical application of the developed procedure is demonstrated by CZE analysis and FFZE preparation of an octapeptide fragment of human insulin.

Contribution of endopeptidase 3.4.24.15 to central neurotensin inactivation.

The tridecapeptide, neurotensin elicits naloxone-insensitive analgesia after its intracebroventricular administration in mice. We used this central pharmacological effect to assess the putative contribution of the endopeptidase 3.4.24.15 to central inactivation of the peptide. By means of combinatorial chemistry, we previously designed the first potent endopeptidase 3.4.24.15 inhibitor. This agent, Z-(L,D)Phe psi(PO2CH2)(L,D)Ala-Lys-Met (phosphodiepryl 21), is shown here to behave as a fully specific endopeptidase 3.4.24.15 inhibitor, as demonstrated by the absence of effect on a series of other exo- and endopeptidases belonging to various classes of proteolytic activities present in murine brain membranes. Furthermore, central administration of phosphodiepryl 21 drastically prolongs the forepaw licking latency of mice tested on the hot plate and injected with sub-maximally active doses of neurotensin. Altogether, our results demonstrated that, in addition to endopeptidase 3.4.24.16, endopeptidase 3.4.24.15 likely contributes to the physiological termination of the neurotensinergic message in murine brain.

Effect of a novel selective and potent phosphinic peptide inhibitor of endopeptidase 3.4.24.16 on neurotensin-induced analgesia and neuronal inactivation.

1. We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. 2. The most selective inhibitor, Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. 3. The effect of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. 4. Pro-Phe-psi(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. 5. Intracerebroventricular administration of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. 6. Altogether, our study has established Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin.

Examination of the role of endopeptidase 3.4.24.15 in A beta secretion by human transfected cells.

1. We have taken advantage of our recent development of highly potent and specific phosphinic inhibitors of endopeptidase 3.4.24.15 to examine the putative contribution of the enzyme in the secretion of A beta by HK293 transfected cells overexpressing the wild type and the Swedish (Sw) double mutated form of beta APP751. 2. First, we showed that HK293 cells contain a peptidase activity, the inhibition profile of which fully matches that of purified endopeptidase 3.4.24.15. Second, we established that the treatment of HK293 cells with specific phosphinic inhibitors leads to about 80% inhibition of intracellular endopeptidase 3.4.24.15 activity, indicating that these inhibitors penetrate the cells. 3. Metabolic labelling of wild type and Sw beta APP751-expressing cells, followed by immunoprecipitation of A beta-containing peptides, revealed the secretion of A beta and the intracellular formation of an A beta-containing 12 kDa product. 4. A beta secretion by Sw beta APP751 transfected cells was drastically enhanced when compared to cells expressing wild type beta APP751. This production was not affected by endopeptidase 3.4.24.15 inhibitors in either cell type. This correlates well with the observation that endopeptidase 3.4.24.15 does not cleave recombinant baculoviral Sw beta APP751, in vitro. 5. Our previous data indicated that endopeptidase 3.4.24.15 activity was reduced in the parietal cortex of Alzheimer's disease affected brains and that the enzyme probably participated, in this brain area, to the catabolism of somatostatin 1-14. However, the present work indicates that endopeptidase 3.4.24.15 does not seem to behave as a beta-secretase in HK293 transfected cells. Therefore, it is suggested that endopeptidase 3.4.24.15 could participate in the symptomatology, but probably not in the aetiology of Alzheimer's disease.

Development of the first potent and selective inhibitor of the zinc endopeptidase neurolysin using a systematic approach based on combinatorial chemistry of phosphinic peptides.

A new systematic approach, based on combinatorial chemistry of phosphinic peptides, is proposed for rapid development of highly potent and selective inhibitors of zinc metalloproteases. This strategy first evaluates the effects on the inhibitory potency and selectivity of the following parameters: 1) size of the phosphinic peptides, 2) position of the phosphinic bond in the sequence, and 3) the state (free or blocked) of the peptide extremities. After this selection step, the influence of the inhibitor sequence is analyzed in order to determine the identity of the residues that optimized both the potency and the selectivity. We demonstrate the efficiency of this novel approach in rapid identification of the first potent inhibitor of the mammalian zinc endopeptidase neurolysin(24-16), able to discriminate between this enzyme and the related zinc endopeptidase thimet oligopeptidase(24-15). The most potent and selective inhibitor developed in this study, Pro-LPhePsi(PO2CH2)Gly-Pro, displays a Ki value of 4 nM for 24-16 and is 2000 times less potent on 24-15. The specific recognition of such a free phosphinic tetrapeptide by 24-16, as well as the unique specificity of the 24-16 S2 and S2' subsites for proline, unveiled by this study, are discussed in terms of their possible significance for the function of this enzyme and its related zinc endopeptidase activities.

Development of highly potent and selective phosphinic peptide inhibitors of zinc endopeptidase 24-15 using combinatorial chemistry.

Several hundred phosphinic peptides having the general formula Z-(L,D)Phe psi (PO2CH2)(L,D)Xaa'-Yaa'-Zaa', where Xaa' = Gly or Ala and Yaa' and Zaa' represent 20 different amino acids, have been synthesized by the combinatorial chemistry approach. Peptide mixtures or individual peptides were evaluated for their ability to inhibit the rat brain zinc endopeptidases 24-15 and 24-16. Numerous phosphinic peptides of this series act as potent (Ki in the nanomolar range) mixed inhibitors of these two peptidases. However, our systematic and comparative strategy led us to delineate the residues located in P2' and P3' positions of the inhibitors that are preferred by these two peptidases. Thus, endopeptidase 24-15 exhibits a marked preference for inhibitors containing a basic residue (Arg or Lys) in the P2' position, while 24-16 prefers a proline in this position. The P3' position has less influence on the inhibitory potency and selectivity, both peptidases preferring a hydrophobic residue at this position. On the basis of these observations, we have prepared highly potent and selective inhibitors of endopeptidase 24-15. The Z-(L,D)Phe psi-(PO2CH2)(L,D)Ala-Arg-Met compound (mixture of the four diastereoisomers) displays a Ki value of 70 pM for endopeptidase 24-15. The most selective inhibitor of endopeptidase 24-15 in this series, Z-(L,D)Phe psi (PO2-CH2)(L,D)Ala-Arg-Phe, exhibits a Ki value of 0.160 nM and is more than 3 orders of magnitude less potent toward endopeptidase 24-16 (Ki = 530 nM). Furthermore, at 1 microM this selective inhibitor is unable to affect the activity of several other zinc peptidases, namely endopeptidase 24-11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and B. Therefore, Z-(L,D)Phe psi (PO2CH2)(L,D)Ala-Arg-Phe can be considered as the most potent and specific inhibitor of endopeptidase 24-15 developed to date. This new inhibitor should be useful in assessing the contribution of this proteolytic activity in the physiological inactivation of neuropeptides known to be hydrolyzed, at least in vitro, by endopeptidase 24-15. Our study also demonstrates that the combinatorial chemistry approach leading to the development of phosphinic peptide libraries is a powerful strategy for discovering highly potent and selective inhibitors of zinc metalloproteases and should find a broader application in studies of this important class of enzymes.

Semisynthetic insulin analogues modified in positions B24, B25 and B29.

New semisynthetic analogues of human insulin, modified in the C-terminal region of the B-chain, were prepared to refine our understanding of the importance of particular amino acid residues in the expression of hormone biological properties. The following insulin analogues were synthesized by trypsin-catalyzed peptide-bond formation between the C-terminal arginineB22 of des-octapeptide(B23-B30)-insulin and synthetic octapeptides with the epsilon-amino group of lysineB29 protected by a phenylacetyl group: [L-Lys(Pac)B29]insulin, [D-PheB24,B25,L-Lys(Pac)B29]insulin and [D-Phe(p-Et)B24, L-Lys(Pac)B29]insulin. Enzymatic deprotection using immobilized penicillin amidohydrolase yielded: human insulin, [D-PheB24,B25]insulin and [DPhe(p-Et)B24]-insulin. Biological in vitro potencies (specific binding to cultured human lymphocytes IM-9 and lipogenic potency in isolated rat adipocytes) of the semisynthetic analogues were estimated, ranging from 0.2 to 100% relative to porcine insulin.