Aminopeptidase A (APA) is a highly selective peptidase, which cleaves the N-terminal Glu or Asp residues of biologically active peptides, and has therefore been proposed to be involved in angiotensin II and CCK8 metabolism. Highly potent and selective APA inhibitors are consequently required to study the physiological regulation of these two peptides. Using, as a model, Glu-thiol (4-amino-5-mercaptopentanoic acid), which was the first efficient APA inhibitor described but is however equipotent on APA (0.14 microM) and aminopeptidase N (APN) (0.12 microM), several beta-amino thiol inhibitors have been synthesized. In these molecules, the length of the side chain was varied and the carboxylate group of Glu-thiol was replaced by other negatively charged groups, such as phosphonate, sulfonate, hydroxamate, and thiol. The inhibitory potency of one of these compounds, 22h (S)-3-amino-4-mercaptobutanesulfonate, was found to be nearly 100-fold better for APA than for APN, with an affinity (0.29 microM) almost equivalent to that of Glu-thiol. Hence, this compound is the first selective APA inhibitor reported, and as such, it should be an interesting probe to explore the physiological involvement of APA in the metabolism of neuropeptides like angiotensin II and CCK8.

In order to evaluate the possible advantages of potentiating the effects of the endogenous enkephalins, to obtain analgesia without the serious drawbacks of morphine, it was essential to design systemically active compounds which inhibit the two metabolizing enzymes, aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP). A new concept combining the idea of "prodrug" and "mixed inhibitor" was therefore developed. Given the high efficiency of beta-mercaptoalkylamines as APN inhibitors and of N-(mercaptoacyl) amino acids as NEP inhibitors, compounds associating these molecules through disulfide or thioester bonds, which are known to increase lipophilicity and to favor passage across the blood-brain barrier, have been synthesized. An HPLC study indicated that the disulfide bridge was resistant to serum enzymes but was cleaved by brain membrane homogenates, suggesting that the active inhibitors were released in the central nervous system. The validity of the approach was verified by the efficient antinociceptive responses obtained in the hot plate test in mice after iv administration of disulfide-containing inhibitors (ED50s of from 4 to 26 mg/kg on the jump latency time). The analgesic potencies of the "mixed inhibitor-prodrug" RB 101 [H2NCH(CH2CH2SCH3)CH2SSCH2CH(CH2Ph)CONHCH( CH2Ph)COOCH2Ph] after iv administration were three times greater than those of a similar combined dose of its two constitutive moieties. The separation of the two diastereoisomers constituting RB 101 showed that the analgesia has a stereochemical dependence, the (S,S,S)-isomer being more active than the (S,R,S)-isomer. Furthermore, in the tail flick test in the rat, RB 101 gave 38% analgesia at a dose of 80 mg/kg. Due to its high efficiency and its longer pharmacological effect, RB 101 was selected for a complete study of its analgesic properties.

Aminopeptidase A (APA) and aminopeptidase N (APN) are two metallopeptidases which have been suggested to be involved in the enzymatic cascade of the renin-angiotensin system. APA liberates angotensin III from angiotensin II by releasing the N-terminal aspartate, and APN participates in the inactivation of angiotensin III. As the role of angiotensin III in the regulation of blood pressure in the central nervous system and at the periphery is controversial, it was of interest to develop selective and efficient inhibitors of APA. Starting from Glu-thiol(1), which was the first efficient APA inhibitor described, but however is equipotent on APA (Ki = 0.14 microM) and APN (Ki = 0.12 microM), beta-amino thiols bearing various carboxyalkyl chains have been synthesized and their inhibitory potencies measured on both purified enzymes. Compounds containing a carboxylated aromatic ring inhibited APA and APN with Ki values in the micromolar range but were slightly more active on APA. Conversely, inhibitors containing a cyclohexyl ring were more efficient on APN. Various modifications of the structure of Glu-thiol decreased inhibitory activity on both enzymes but increased the selectivity for APA, and compound 9d ((S)-4-amino-6-mercaptohexanoic acid) was 23 times more potent on APA (Ki = 2.0 microM) than on APN (Ki = 45 microM).

The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search for new analgesics devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general formula H(3)N(+)-CH(R(1))-P(O)(OH)-CH(2)-CH(R(2))-CONH-CH(R(3))-COO(-), able to act as transition-state analogues and to fit the S(1), S(1)', and S(2)' subsites of both enzymes were designed. Selection of the R(1), R(2), and R(3) residues for optimal recognition of these enzymes led to the first dual competitive inhibitors with K(i) values in the nanomolar range for neprilysin and aminopeptidase N. These compounds induce potent analgesic responses after intracerebroventricular or intravenous administrations in mice (hot plate test), and several of them were shown to be, at least, 10 times more potent than the previously described dual inhibitors.

In the treatment of cardiovascular diseases, it could be of therapeutic interest to associate the hypotensive effects resulting from the inhibition of angiotensin II formation, ensured by endothelial angiotensin-converting enzyme (ACE), with the diuretic and natriuretic responses due to the protection of the endogenous atrial natriuretic peptide (ANP) from inactivation by epithelial neutral endopeptidase (NEP). However, an investigation of this hypothesis requires an orally active compound able to jointly inhibit ACE and NEP. Dual inhibitors have therefore been designed by a rational approach, based on the characteristics of the active sites of both enzymes, which belong to the same family of zinc metallopeptidases, and on the structures of their most potent and selective inhibitors. As both NEP and ACE contain a large S'1-S'2 domain able to accommodate aromatic residues, the cyclic ACE inhibitor 3-(mercaptomethyl)-3,4,5,6-tetrahydro-2-oxo-1H-1-benzazocine-1-ace tic acid was selected as a template. Various aliphatic constraints were introduced on the benzyl moiety of the potent NEP inhibitor N-[2-(mercaptomethyl)-3-phenylpropanoyl]-L-tyrosine (IC50 NEP = 2 nM, IC50 ACE = 25 nM) to improve the fit between the computed most stable conformers of these molecules and the ACE template. New dual inhibitors, of general formula, N-[2(R,S)-(mercaptomethyl)-3(R,S)-phenylbutanoyl]-L-amino acid with IC50 values in the nanomolar range for both enzymes were generated by this approach. The separation of the four stereoisomers using chiral amines and the stereoselective synthesis of the 2-(mercaptomethyl)-3-phenylbutanoyl moiety showed that inhibitors with the 2S,3R configuration are the most potent on both NEP and ACE. The "in vivo" potency of various prodrugs of these inhibitors to inhibit ACE activity in lung and NEP activity in kidney was measured after oral administration in mice. From this pharmacokinetical study the most potent dual inhibitor RB 105 (N-[(2S,3R)-2-(mercaptomethyl)-3-phenylbutanoyl-L-alanine (compound 44c) (KI NEP 1.7 nM, KI ACE 4.5 nM) and its most efficient in vivo prodrug mixanpril, [N-[(2S,3R)-2-[(benzoylthio)methyl]-3-phenylbutanoyl]-L-alan ine (compound 18) (ED50 NEP approximately 1 mg/kg, ED50 ACE approximately 7 mg/kg) were selected. Competition experiments with a tritiated inhibitor of ACE or NEP bound to mouse lung and kidney membranes respectively showed that mixanpril has a long duration of action (> 8 h). As expected, after i.v. administration in the spontaneously hypertensive rat (SHR), RB 105 decreased blood pressure and increased diuresis and natriuresis.(ABSTRACT TRUNCATED AT 400 WORDS)

Derivatives of amino acids bearing various zinc-coordinating moieties (SH, COOH, CONHOH, and PO3H2) were synthesized and tested for their ability to inhibit aminopeptidase N (APN). Among them, beta-amino thiols were found to be the most efficient with IC50's in the 11-50 nM range. These results suggest that the S1 subsite of APN is a deep but not very large hydrophobic pocket, optimally fitting side chains of moderate bulk endowed with some degree of freedom. The iv administration of the inhibitors, alone, did not induce antinociceptive responses on the hot plate test in mice. However, in presence of 10 mg/kg acetorphan, a prodrug of the neutral endopeptidase inhibitor thiorphan, these compounds gave a large increase in the jump latency time with ED50's of 2 and 2.4 mg/kg for the disulfides of methioninethiol H2NCH(CH2CH2SCH3)CH2S]2 and S-oxomethioninethiol [H2NCH(CH2CH2S(O)CH3)CH2S]2, respectively. These results show that the disulfide forms of beta-amino thiols are efficient prodrugs of aminopeptidase N inhibitors capable of crossing the blood-brain barrier.

Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/ Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/ Phe-rich domain forms an amphipathic ␣-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the ␣-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A؉L32A؉L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection. IMPORTANCEAgnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic ␣-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the ␣-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection.

Angiogenesis is the development of a novel vascular network from a pre-existing structure. Blocking angiogenesis is an attractive strategy to inhibit tumor growth and metastasis formation. Based on structural and mutagenesis data, we have developed novel cyclic peptides that mimic, simultaneously, two regions of the VEGF crucial for the interaction with the VEGF receptors. The peptides, displaying the best affinity for VEGF receptor 1 on a competition assay, inhibited endothelial cell transduction pathway, migration, and capillary-like tubes formation. The specificity of these peptides for VEGF receptors was demonstrated by microscopy using a fluorescent peptide derivative. The resolution of the structure of some cyclic peptides by NMR and molecular modeling has allowed the identification of various factors accounting for their inhibitory activity. Taken together, these results validate the selection of these two regions as targets to develop molecules able to disturb the development of cancer and angiogenesis-associated diseases.