Design and synthesis of inhibitors incorporating beta -amino acids of metalloendopeptidase EC 3.4.24.15.

Endopeptidase EC 3.4.24.15 (EP 24.15) is a thermolysin-like metalloendopeptidase which is expressed widely throughout the body, with the highest concentrations in the brain, pituitary and testis. While the precise role of EP 24.15 remains unknown, it is thought to participate in the regulated metabolism of a number of specific neuropeptides. Of the limited number of inhibitors described for EP 24.15, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-amino benzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but is unstable in vivo due to its cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). The cpp-Ala-Ala N-terminal product of this cleavage is a potent inhibitor of angiotensin converting enzyme, which further limits the use of CFP in vivo. To generate specific inhibitors of EP 24.15 that are resistant to in vivo proteolysis by EP 24.11, beta-amino acids have been incorporated into the structure of CFP. We have prepared racemic mixtures of beta-amino acids containing proteogenic side chains, which are 9-fluorenylmethoxycarbonyl (Fmoc)-protected, and several analogues of CFP containing beta-amino acids have been synthesized by solid phase peptide synthesis. The results of stability and inhibitory studies of these new analogues show that the incorporation of beta-amino acids adjacent to the scissile bond can indeed stabilize the peptides against cleavage by EP 24.11 and still inhibit EP 24.15. The results obtained in these studies demonstrate the potential of these amino acids in the synthesis of peptidomimetics and in the design of new stable and specific therapeutics.     

Effect of Covalent Dimer Conjugates of Angiotensin II on Receptor Affinity and Activity in Vitro

Abstract

Angiotensin II [1-8 or 2-8] analogues and [4–8] fragments were dimerized through the amino-or carboxy-terminal groups in order to try to increase their potency as reported for other hormones. The binding affinity to the angiotensin II receptor subtypes A (A II_A) and B (A II_B) was tested and compared to the potency in rabbit aortic ring. The [2–8] dimers coupled through the N-terminus show no significant change in potency in aortic ring. The [4–8] fragments coupled through the N-terminus are inactive in the ring. They have however a significantly increased affinity for the A II^A receptor, the specific function of which has not yet been reported. When angiotensin II analogues or fragments are coupled through the C-terminus, there was a significant drop in affinity and potency, confirming the importance of the free carboxyl group in position 8 for binding and activity. It is concluded that binding to the A II_B receptor correlates well with the effectiveness in aortic ring. However, in contrast to the beneficial effect reported for a large number of other hormones, dimerization of angiotensin II or its fragments is not accompanied by an increased biological activity in aortic ring.     

Structural characterization and auxin properties of dichlorinated indole-3-acetic acids

The dichlorinated indole-3-acetic acids: 4,5-Cl2-IAA, 4,6-Cl2-IAA, 4,7-Cl2-IAA, 5,6-Cl2-IAA, 5,7-Cl2-IAA and 6,7-Cl2-IAA were synthesized and characterized by X-ray structure analysis to unambiguously identify the substances for bioassays required to establish structure activity relationships of auxins and their analogues. Straight-growth tests were performed on Avena sativa coleoptiles to correlate their auxin activity with molecular properties which could reveal information on the topology of the auxin binding site. Structure/activity correlations revealed that the 5,6-Cl2-IAA molecule, by virtue of its size and shape, fits particularly well into the active site cavity of the receptor protein. The main contact of the substrate or inhibitor in the receptor active site via the carboxylic group determines their orientation in the active site cavity. As a consequence, the 5,6-substituted sites protrude into the widest part of the active site whereas the 7-, 4-, and 5-substituted sites are oriented towards the narrowest part of the active site. These topological parameters are in agreement with the high auxin activity of 5,6-Cl2-IAA and the low activity of 4,7-Cl2-IAA.     

Inhibitors of metalloendopeptidase EC 3.4.24.15 and EC 3.4.24.16 stabilized against proteolysis by the incorporation of beta-amino acids

The enzyme EC 3.4.24.15 (EP 24.15) is a zinc metalloendopeptidase whose precise function in vivo remains unknown but is thought to participate in the regulated metabolism of a number of specific neuropeptides. The lack of stable and selective inhibitors has hindered the determination of the exact function of EP 24.15. Of the limited number of EP 24.15 inhibitors that have been developed, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but it is unstable in vivo due to cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). This cleavage by EP 24.11 generates a potent inhibitor of angiotensin converting enzyme, thereby limiting the use of CFP for in vivo studies. To develop specific inhibitors of EP 24.15 that are resistant to in vitro and potentially in vivo proteolysis by EP 24.11, this study incorporated beta-amino acids replacing the Ala-Tyr scissile alpha-amino acids of CFP. Both C2 and C3 substituted beta-amino acids were synthesized and substituted at the EP 24.11 scissile Ala-Tyr bond. Significant EP 24.15 inhibitory activity was observed with some of the beta-amino acid containing analogues. Moreover, binding to EP 24.11 was eliminated, thus rendering all analogues containing beta-amino acids resistant to degradation by EP 24.11. Selective inhibition of either EP 24.15 or EP 24.16 was also observed with some analogues. The results demonstrated the use of beta-amino acids in the design of inhibitors of EP 24.15 and EP 24.16 with K(i)'s in the low micromolar range. At the same time, these analogues were resistant to cleavage by the related metalloendopeptidase EP 24.11, in contrast to the alpha-amino acid based parent peptide. This study has therefore clearly shown the potential of beta-amino acids in the design of stable enzyme inhibitors and their use in generating molecules with selectivity between closely related enzymes.     

Cell-free synthesis of mycolic acids in Mycobacterium aurum: radioactivity distribution in newly synthesized acids and presence of cell wall in the system

Distribution of radiolabelling in different parts of the newly synthesized mycolic acids, by a cell-free system from Mycobacterium aurum previously described, is examined, [1-14C]acetate being the precursor. By oxidation cleavage of mycolic acids and examination of the fragments, it was shown that acetate was not uniformly incorporated into the molecule: the methyl terminal part was not labelled, while the central fragments--between unsaturations or between oxygenated functions (oxo or ester) and unsaturations--presented the major part of radioactivity, suggesting the elongation of a preformed compound that the cell-free extract was unable to synthesize. Moreover, the side-chain R2-CH2-COOH was only weakly labelled compared to the central fragments. Since non-hydroxylated fatty acids were not synthesized by the system, it is suggested that de novo C18 fatty acids may be elongated with C2 units by the cell-free extract into C22 fatty derivative, only a low level of labelling being recorded (two C2 units for all the molecule). A scheme is proposed to summarize the main results. Identification of meso-DAP which is a characteristic amino-acid of the peptidoglycan in Actinomycetes and analysis of the profiles of total fatty esters, demonstrated that the cell-free extract is partly constituted by fragments of the cell wall as has already been noticed by examination of micrographs of the extract.     

The additivity principle in the reaction of angiotensin II and its analogs with antibodies and receptors of glomerular cells from the rat adrenal cortex

The binding of angiotensin II and its analogues (13) to rabbit antibodies and glomerular cell receptors from rat adrenal cortex was studied, using the radioimmunoassay method and radioreceptor analysis. Double modifications introduced into the angiotensin structure were found to increase in an additive fashion its binding to the antibodies and renal cell receptors. The relative binding activity of the analogues carrying a double modification can be assessed if the activities of the analogues with the appropriate single modifications are known. It was concluded that the testing of modifications in the peptide structure for their additivity may provide some insight into the conformational properties of peptides during their binding to the protein.     

Activity and regulation of an amidase (acylamide amidohydrolase,EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.

The Brevibacterium R 312 strain has an amidase with a wide substrate spectrum previously named acetamidase. The study of its activity showed that this enzyme was able to hydrolyze a large number of amides into their corresponding organic acids. The affinity of this enzyme for the substrates varied according to the length of the carbon chain and the spatial crowding of the molecule. The comparison of the specific rates of hydrolysis showed that propionamide was the amide substrate most quickly hydrolyzed.We confirmed the inducible feature of this enzyme and noted that only acetamide and N-methylacetamide were inducers of this enzyme among the compounds tested. Thioacetamide and N-methylpropionamide, both as amide analogues, were shown to inhibit the biosynthesis of acetamidase. Similarly, the organic acids, products of the hydrolysis reaction, showed a strong repression action on the biosynthesis of the enzyme.     

Anticonvulsant activity of cyclopentano amino acids

The hypothesis that certain amino acid analogues possessing a five-membered ring structure or amino acid analogues that can be viewed as fragments derived from such a ring would have anticonvulsant activity was proposed and tested. The compounds 1-aminocyclopentane carboxylic acid, 1-amino-3-methylcyclopentane carboxylic acid, 3-aminotetrahydrothiophene carboxylic acid, and -aminoisobutyric acid were found to protect rats against seizures in the maximal electroshock test but offered no protection against metrazol-(pentylenetetrazol) induced seizures in mice. The structural feature of this class of anticonvulsants that allows for hydrophobic interactions at the receptor site is considered to be a major physical factor necessary in promoting the activity of this class of anticonvulsants.     

Solid phase isotope exchange with spillover hydrogen in amino acids, peptides, and proteins

We summarize here information on the theoretical and experimental study of high-temperature (150-200 degrees C) solid phase catalytic isotope exchange (HTSPCIE) carried out with amino acids, peptides, and proteins under the action of spillover hydrogen. Main specific features of the HTSPCIE reaction, its mechanism, and its use for studying spatial interactions in polypeptides are discussed. A virtually complete absence of racemization makes this reaction a valuable preparative method. The main regularities of the HTSPCIE reaction with the participation of spillover tritium have been revealed in the case of peptides and proteins, and the dependence of reactivity of peptide fragments on the spatial organization of their molecules has been studied. An important peculiarity of this reaction is that HTSPCIE proceeds at 150-200 degrees C with a high degree of chirality retention in amino acids and peptides. This is provided by its reaction mechanism, which consists in a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond between the carbon and the incoming and outgoing hydrogen atoms.     

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.     

Isolation and structural and functional characterization of two stable peptic fragments of human alpha-fetoprotein.

Short-time limited peptic hydrolysis of ligand-free human alpha-fetoprotein (AFP) gave two main fragments with molecular masses of 38 and 32 kDa, which had been produced by splitting of the molecule at the position Leu(312)-Asn(313). A more prolonged proteolysis led to the further degradation of these fragments and appearance of highly proteolytically resistant 23-kDa (P23) and 26-kDa (P26) fragments, corresponding to N- and C-terminal parts of the AFP molecule, respectively. Comparative study of intact free of ligands AFP and isolated stable P23 and P26 fragments by circular dichroism, differential scanning calorimetry, and immunoprecipitation techniques demonstrated that these fragments conserved native secondary, tertiary; and antigenic structure, characteristic of the intact molecule. It was concluded that, free of ligands, the AFP molecule could be considered as a three-domain molecule, in which two compact rigid domains (N-terminal domain I and C-terminal domain III) are connected by relatively labile domain II. The structure of domain II could be approximated by a "molten globule" state, characterized by the absence of rigid tertiary structure but having a pronounced secondary structure. Tumor-suppressive activity via induction of apoptosis was recently shown for AFP [Dudich, E. I., et al. (1998) Tumor Biol. 19, 261-272]. We studied here the ability of isolated proteolytic AFP fragments to induce apoptosis in the AFP-sensitive Raji cell line, to determine possible localization of the active site responsible for apoptosis signaling. Unlike intact AFP, neither isolated fragments nor their equimolar mixture was able to induce apoptosis in a human lymphoma Raji cell line. However, it was demonstrated that both fragments P23 or P26 and their equimolar mixture P23 + P26 operated synergistically with intact AFP in suppression of Raji cell proliferation. These data suggested that two structurally determined requirements are necessary for AFP-mediated triggering of apoptosis: (i) dimerization of AFP to form the heterodimeric complex of C- and N-terminal domains and (ii) participation of the central part of AFP molecule (domain II).     

Structure-activity relationship of human calcitonin-gene-related peptide.

The calcitonin-calcitonin-gene-related peptide (CGRP) gene complex encodes a small family of peptides: calcitonin, CGRP and katacalcin. Calcitonin is a circulating hormone that prevents skeletal breakdown by inhibiting the resorption of bone by osteoclasts. CGRP, a potent vasodilator, is involved in normal regulation of blood flow. The calcitonins structurally resemble the CGRP peptides, and both are known to cross-react at each others' receptors. The present study was undertaken to examine the structural prerequisites for biological activity of the intact CGRP molecule. We therefore prepared eight chymotryptic and tryptic fragments of CGRP and synthesized its acetylated and S-carboxyamidomethylcysteinyl analogues. The analogues were purified by h.p.l.c. and their structures were confirmed by fast-atom bombardment mass spectrometry. We have examined the effects of structurally modified analogues and fragments of human CGRP in a calcitonin-receptor-mediated assay, the osteoclast bone resorption assay, and in one or two CGRP-receptor-mediated assays, the rabbit skin blood flow assay and the oedema formation assay. The results showed that (1) in the osteoclast bone resorption assay, both CGRP peptides, alpha and beta, were equipotent, and were both at least 1000-fold were both approx. 1000-fold more potent than salmon calcitonin; human calcitonin had no effect; (3) the bis- and N-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxyamidomethylcysteinyl-human CGRP was without activity; and (4) all tryptic and chymotryptic fragments of CGRP were without biological activity, with the exception of hCGRP-(Ala1-Lys35): this fragment had much reduced activity compared with the intact peptide in inhibiting osteoclastic bone resorption and increasing blood flow in the rabbit skin. The results suggest that: (1) calcitonin and CGRP act at distinct receptors to mediate different physiological effects; (2) minor amino acid substitutions, as between the alpha and beta forms of CGRP (these two forms have 94% structural similarity) do not result in differences in biological activity; (3) the intact peptide is required for full biological activity of the CGRP molecule, and even the loss of two amino acids at the C-terminus of the molecule results in a marked decrease in activity; (4) the disulphide bridge appears to play an important role in the interaction of the intact CGRP molecule with its receptor; and (5) the C-terminal region is probably necessary for the peptide to assume the right conformation in the interaction with the receptor.     

Structural features of linear and cyclic analogs of angiotensin, affecting secretion of histamine from rat mast cells

Linear and cyclic analogues of angiotensin were studied to clarify the structural properties of peptides possessing a histamine-releasing action. It was shown that an increase in the angiotensin basicity or its cyclization leads to the appearance of the histamine-releasing activity which is not characteristic of the natural hormone. This increase in the basicity of the angiotensin cyclic analogs results in highly active compounds with the EC50 exceeding by 2 to 3 orders of magnitude that of polymyxin B or substance 48/80. The data obtained confirm the hypothesis postulating a high degree of amphiphilicity for histamine-releasing peptides. As a result of cyclization of angiotensin analogues, a block of positively charged amino acids with an oppositely located hydrophobic region is formed. This finding can be of importance for the effective interaction of peptides with cellular structures as well as for the stimulation of secretory processes.     

Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.

The origin-specific DNA-binding domain of simian virus 40 large T antigen was analyzed, and its C-terminal boundary was found to be at or before amino acid 259. This does not include the zinc finger structural motif located at amino acids 302 to 320 (J. M. Berg, Science 232:485-486, 1986). Interestingly, N-terminal fragments of 266 and 272 amino acids and larger displayed dramatically reduced origin-binding activity. In addition, the specific DNA-binding properties of truncated proteins purified from both bacterial and mammalian sources were compared. Truncated T antigens from mammalian cells bound specific DNA fragments more efficiently than did their bacterial counterparts. These results implicate posttranslational modification with a role in regulating the DNA-binding activity of large T antigen.     

Angiotensin II binding to mammalian brain membranes.

High affinity binding sites for angiotensin II in bovine and rat brain membranes have been identified and characterized using monoiodinated Ile5-angiotensin II of high specific radioactivity. Degradation of labeled and unlabeled peptide by washed brain particulate fractions was prevented by adding glucagon to the final incubation medium and including a proteolytic enzyme inhibitor (phenylmethylsulfonyl fluoride) in preincubation and incubation procedures. 125I-Angiotensin II binding can be studied using either centrifugation or filtration techniques to separate tissue-bound radioactivity. 125I-Angiotensin II binding to calf brain membranes is saturable and reversible, with a dissociation binding constant of 0.2 nM at 37 degrees. A similar binding constant is found in rat brain membranes. Analogues and fragments of angiotensin II compete for these brain binding sites with potencies which correlate with both their in vivo potencies and their binding inhibition protencies at adrenal cortex angiotensin II receptors. Angiotensin I is 1 to 2 orders of magnitude weaker than angiotensin II; the 3-8 hexapeptide and 4-8 pentapeptide are much weaker still. (desAsp1) angiotensin II (angiotensin III) is slightly more potent than angiotensin II, as are several antagonists of angiotensin II with aliphatic amino acids substituted at position 8. In calf brain 125I-angiotensin II binding is restricted almost exclusively to the cerebellum (cortex and deep nuclei). In rat brain, angiotensin II binding is highest in the thalamus-hypothalamus, midbrain, and brainstem, areas which are believed to be involved in mediating angiotensin II-induced central effects. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in rat and bovine brain and suggest a physiological role for angiotensin peptides in the central nervous system.     

Novel inhibitors and substrates of bilirubin: UDP-glucuronosyltransferase. Arylalkylcarboxylic acids

The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP-glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7-triphenylheptanoic acid was the most powerful inhibitor of bilirubin:UDP-glucuronosyltransferase with a lower effect on 1-naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin:UDP-glucuronosyltransferases with Kiapp values of 12.0 microM and 1.6 microM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin:UDP-glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group. These inhibitors were also tested as possible substrates of UDP-glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP-glucuronosyltransferases. However, 7,7,7-triphenylheptanoic acid was actively glucuronidated by purified bilirubin:UDP-glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin:UDP-glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin:UDP-glucuronosyltransferase. The results suggest that 7,7,7-triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.     

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid-induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BK(Ca) channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid-induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BK(Ca) channel activity is not due to Ca(2+)-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid-induced increase in BK(Ca) channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid-induced increases in BK(Ca) channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BK(Ca) channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BK(Ca) channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BK(Ca) channel activity may explain their relaxing effect on smooth muscle.     

The actions of low-molecular fragments of substrate and their analogs on the activity of isoenzymes of phospholipase C from Clostridium perfringens]

The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl. perfringens was studied by gel-diffusion in agarose-lecithin gels. It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme. The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups. However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2.     

Interaction of aminoacyl-tRNA-synthetases with amino acids

The review deals with interactions of the key enzymes of the protein biosynthesis-aminoacyl-tRNA synthetases (EC 6.1.1.) with amino acids and their analogues, considering the contribution of different groups in the process of specific complex formation and catalysis. The important role of alpha-amino group of amino acid in the enzyme recognition has been revealed. Modification of the carboxylic group does not change significantly the analogues complex formation with aminoacyl-tRNA synthetases. However this group is essential for amino acid rearrangement in the specific complex with the enzyme. The structural organization of the enzyme binding sites specific for amino acids and the enzyme interaction with the analogues of aminoacyladenylates are discussed.