key: cord-351322-mdes28jg
authors: Bauvois, Brigitte; Dauzonne, Daniel
title: Aminopeptidase‐N/CD13 (EC 3.4.11.2) inhibitors: Chemistry, biological evaluations, and therapeutic prospects
date: 2005-10-07
journal: Med Res Rev
DOI: 10.1002/med.20044
sha: 
doc_id: 351322
cord_uid: mdes28jg

Aminopeptidase N (APN)/CD13 (EC 3.4.11.2) is a transmembrane protease present in a wide variety of human tissues and cell types (endothelial, epithelial, fibroblast, leukocyte). APN/CD13 expression is dysregulated in inflammatory diseases and in cancers (solid and hematologic tumors). APN/CD13 serves as a receptor for coronaviruses. Natural and synthetic inhibitors of APN activity have been characterized. These inhibitors have revealed that APN is able to modulate bioactive peptide responses (pain management, vasopressin release) and to influence immune functions and major biological events (cell proliferation, secretion, invasion, angiogenesis). Therefore, inhibition of APN/CD13 may lead to the development of anti‐cancer and anti‐inflammatory drugs. This review provides an update on the biological and pharmacological profiles of known natural and synthetic APN inhibitors. Current status on their potential use as therapeutic agents is discussed with regard to toxicity and specificity. © 2005 Wiley Periodicals, Inc. Med Res Rev

Aminopeptidase N (EC 3.4.11.2, APN) is a metallo-dependent integral membrane protease. 1 The enzyme belongs to the M1 family of the MA clan of peptidases 2 also called gluzincins. 3 Aminopeptidase N consists of 967 amino acids with a short N-terminal cytoplasmic domain, a single transmembrane part, and a large cellular ectodomain containing the active site. 4 This enzyme was first isolated in 1963 by Pfleiderer and Celliers from pig kidney 5 and is known under several different names (alanine aminopeptidase; microsomal aminopeptidase; microsomal leucine aminopeptidase aminopeptidase M; amino oligopeptidase; GP 150). In the last few years, certain surface molecules identified as cluster differentiation (CD) antigens were found to be identical to some membrane proteins. Thus, CD13 is identical to APN. 6, 7 Soluble APN is detectable in plasma/serum and urine [8] [9] [10] [11] but the mechanism of release of membrane APN remains unknown.

Membrane-bound APN/CD13 is widely distributed outside the hematopoietic system (epithelial-, endothelial-, fibroblast-cell types) with main sources being brush border membranes of kidney proximal tubule cells and enterocytes, and in the hematopoietic compartment is not confined to a particular lineage. 1, 12, 13 APN/CD13 is predominantly expressed on stem cells and on cells of the granulocytic and monocytic lineages at distinct stages of differentiation and is therefore considered as a marker of differentiation. 14, 15 Dysregulated expression of membrane and/or soluble forms of APN/CD13 is observed in many diseases. Compiled observations indicate enhanced APN levels in tumor cells such as melanoma, 16, 17 renal, 18 pancreas, 19 colon, 20 prostate, 21 gastric, 22 and thyroid 23 cancers. Tumor-infiltrating T cells in renal and lung cancers are CD13-positive. 24,25 APN activity is elevated in plasma and effusions of cancer patients. 11 APN activity on neutrophils from patients affected by a rare adrenal gland tumor, adrenal pheochromocytoma, is significantly increased as compared with healthy controls. 26 CD13 is overexpressed in acute and chronic myeloid leukemias 1, 12, [27] [28] [29] and in anaplastic large cell lymphomas. 30, 31 Overexpression of APN/CD13 in T lymphocytes or neutrophils occurs in several inflammatory diseases (chronic pain, various forms of joint effusions, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, polymyositis/dermatomyosytis, pulmonary sarcoidosis). [32] [33] [34] [35] [36] [37] [38] [39] APN/CD13 may be therefore considered as a useful clinical marker. Whether this protease critically contributes to the pathological behavior remains however unknown. In this review, we briefly summarise knowledge on the structure and the mechanisms of cleavage of APN/CD13 to integrate current knowledge in natural and synthetic APN inhibitors. The reader is referred to excellent reviews for the characteristics of APN/CD13 and substrate specificity. 25, [40] [41] [42] [43] [44] [45] Various aspects on the roles of APN/CD13 are reviewed here in the context of the in vitro and in vivo use of certain APN inhibitors.

APN is anchored to the plasma membrane, via an uncleaved signal sequence, by the C-terminus (type II) facing extracellularly. 1 Membrane APN/CD13 is found as a dimer of two non covalently associated subunits with a relative molecular mass of 160 kDa (Fig. 1A) . 40, 41, 43 The human CD13 gene, cloned in 1989 6 and subsequently mapped to chromosome 15 q25-26, 46 possesses two promoters (Fig. 1B) . [46] [47] [48] [49] [50] [51] The cDNA sequence reveals the presence of the amino acid sequence His-Glu-Xaa-Xaa-His which is a Zn þþ binding motif found in one class of metallo-peptidases. 3 Site-directed mutagenesis indicates that extracellular cysteines in the molecule confer correct structure and consequently enzymatic activity and surface expression of APN. 52 Mutation of glutamic acid 355 in an aminopeptidase conserved region (the GAMEN motif) leads to an inactive enzyme 53 indicating that this glutamic acid belongs to the anionic binding site in APN and interacts with the N-terminal aamino group of the substrate. APN/CD13 cleaves preferentially neutral amino acids (with the exception of proline) (Fig. 1C) from the unsubstituted N-terminus of oligopeptides. 1, 12 Biologically active peptide substrates cleaved by APN/CD13 are neuropeptides (Met-and Leu-enkephalins, neurokinin A, Met-lys-bradykinin, and endorphins such as spinorphin), 41,54-59 vasoactive peptides (kallidin, somatostatin, and angiotensins) 60-67 and chemotactic peptides (monocyte chemotactic protein//MCP-1 and N-formyl methionine leucine phenylalanine/f-MLP). 40, 68 Apart from its hydrolytic ability, APN serves as a receptor for coronaviruses. [69] [70] [71] [72] In humans, the 229E corona virus uses APN to enter alveolar cells and establish an upper respiratory tract infection. 72 

Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 ) are natural peptides capable of inhibiting APN in micromolar concentrations. 73 Similarly, elevated concentrations of leucine, proline, L-alanine, L-arginine, L-glutamine, L-methionine, as well as divalent cations (Co 2þ , Zn 2þ , Mn 2þ , Ca 2þ , Ni 2þ ) inhibit APN activity. 40 (for review) Moreover, molecules with a broad spectrum of action such as KCN, NaN 3 , ammonium oxalate, N-ethyl-maleimide, and 8-hydroxyquinoline inhibit APN/CD13. 40 (for review) APN activity is also inhibited by puromycin (1), 74,75 lapstatin (2), 76 some N-phenylphthalimide derivatives such as compound 3, 77-80 several N-phenylhomophthalimide derivatives like PIQ-22 (4) 77,78 which has later been described as a rather puromycin-sensitive aminopeptidase (PSA) inhibitor by the same group, [80] [81] [82] [83] phosphinate dipeptide analogues illustrated by hPheP[CH 2 ]Tyr (5), 84 pseudoglutamyl aminophosphinic peptides such as GluC(PO 2 CH 2 )Leu-Ala (6), 85 several variously substituted 3-amino-2-oxobutyramide exemplified by compound 7, 86 a-aminoboronic derivatives such as the benzyl derivative 8, 87 or a-aminobenzaldehydes illustrated by (S) 2-amino-5methylpentanal (9) . 88 An eclectic set of compounds has been described and used for the biochemical characterization or/and inhibition of other proteases-e.g.: urokinase-type plasminogen activator, dipeptidylpeptidase IV (DPPIV/CD26), or other different aminopeptidases including human enkephalin degrading aminopeptidase (HEDA), cytosolic leucine aminopeptidase (LAPc), glutamyl aminopeptidase (APA), and arginyl aminopeptidase (AP-B). In this context, it is also worth mentioning two systematic studies devoted to hydroxylated naturally occurring flavonoids such as baicalein (10), apigenin (11) , or myricetin (12) and related compounds which, aside their activity on neutral endopeptidase (NEP/CD10) or angiotensin-converting enzyme (ACE/CD143), exhibited a significant in vitro inhibitory effect toward APN. 89, 90 Formulas, Ki, IC 50 or inhibition percentages of enzymes for compounds 1-12 are depicted in Figure 2 . Two recent publications describing either the irreversible inhibition of both APN/CD13 and DPP IV/CD26 enzymatic activities by aqueous extracts of a Cistus incanus L. 91 or ACE, NEP, and APN inhibition by extracts of Epilobium angustifolium 92 deserve also quotation.

Although the borderline is not easy to position, leaving out the above-mentioned studies dealing with non-specific compounds targeting other enzymes and, incidentally, revealing an inhibitory activity on APN, we have chosen to focus the present review on the data tightly dedicated to natural and synthetic inhibitors of APN/CD13 itself.

The most widely used among the naturally occuring APN/CD13 inhibitors are microorganismproduced and have been purified from microbial culture filtrates. A large part of them are generated by bacteria belonging to the order Actinomycetales, especially of the genera Streptomyces: (13) was first isolated by R. Green and R. Bhagwan Singh from a Malayan strain of Actynomycetes. This compound was then listed as Streptomyces Cutter C/2 (N.C.I.B. 8845). 93 About 20 years later, actinonin was also obtained from another strain referenced MG848-hF6 and its inhibition against APN was found to be competitive with the substrate. 94 The structural study and the chemical synthesis of 13 and some analogues have aroused numerous works [95] [96] [97] [98] [99] [100] [101] [102] completed by a structure-activity relationship investigation dealing with anti bacterial properties observed in this actinonin series. 103 

(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valine) (14) ) and two closely related derivatives: AHPA-Val-Pro-Hyp (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-(trans-4hydroxy-L-proline) (MR387A) (15) and AHPA-Val-Pro-Pro (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-L-proline) (MR387B) (16) were obtained from the culture broth of Streptomyces neyagawaensis SL-387. [104] [105] [106] The preparation of several novel synthetic AHPA derivatives (exemplified by 17) bearing, for most of them, heterocyclic moieties and exhibiting interesting in vivo antitumor potencies (30-40% inhibitory rate on S180 sarcoma) has been recently reported. 107 

(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-L-valine-L-valine-L-aspartic acid) (18) has been reported to be a slow-binding competitive inhibitor of APN. 108 It was first isolated from the culture filtrate of Streptomyces sp. ME98-M3. 109 and its structure has been unambiguously determined. 110 Several enantioselective syntheses of this tetrapeptide have been reported, 110, 111 and some of its analogues have also been prepared in the context of a SAR study. 112 

is an inhibitor of various leucine and arginine aminopeptidases, 113 and an efficient inhibitor of LTA 4 hydrolase. [114] [115] [116] [117] However, in spite of its marked toxicity and of its relative lack of selectivity toward exopeptidases, it is one of the most used compound for its APN/CD13 inhibitory effects. 118 Bestatin has been described as a slow-binding competitive inhibitor of APN, 108 and a schematic representation of 19 within the active site of APN 53,84 is depicted in Figure 3 . Bestatin was first isolated from a culture filtrate of Streptomyces olivoreticuli (MD976-C7) 119 and its chemical structure has been subsequently ascertained. 120 Several stereoselective total syntheses of 19 have been reported, 121-130 the preparation of its stereoisomers has been performed 131 and some ubenimex derivatives or analogues such as the para-hydroxybestatin (20), 132 the 2-thiolbestatin (21), 133,134 the bestatin thioamide (22), 133, 135 or the reduced bestatin 23 136 have also been prepared.

(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-phenylalanine) (24) is a tripeptide produced by Streptomyces sp. MJ716-m3. 137 Some stereoselective syntheses of 24 have been recently reported. 125, 128, 129 

(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-L-proline) (25) is a tetrapeptide isolated from the culture of Streptomyces azureus (MH663-2F6) 138 and its structure has been unambiguously established. 139 Probestin has been described as a competitive inhibitor of APN 138 and, here also, some total syntheses have been lately described. 125, 128, 129 An overview of the formulas of compounds 14-25 reveals that, except the synthetic analogue 23 prepared in its racemic form, they all possess the absolute configuration (2S,3R) which appears crucial for activity. 136 A comparable chiral framework is also existent in the side chain of the pharmacologically important series constituted by taxoids and, in this context, it is worth pointing out that numerous and various synthetic approaches to building blocks liable to lead to enantiomerically pure (2S,3R)-3-amino-2-hydroxyalkanoic structures and/or their diastereomers have attracted considerable attention. 7. Leuhistin (2R,3S)-3-amino-2-hydroxy-2-1H-(imidazol-4-ylmethyl)-5-methylhexanoic acid (26) has been isolated in 1991 by Takeuchi and co-workers from the culture broth of a bacteria belonging to the phylum Firmicutes: Bacillus laterosporus BM156-14F1. 186, 187 This compound inhibits APN in a competitive manner with the substrate. 186 The structure of 26 and its absolute configuration have been thereafter ascertained by the same group. 188 Several naturally occuring APN inhibitors are of vegetal origin:

Benzo[c]phenantridines such as 1,2-Dimethoxy-12-methyl 1,3 dioxolo[4 0 ,5 0 :4,5]benzo[1,2-c]phenanthridin-12-ium chloride or Chelerythrine (27) and some closely related alkaloids have recently been isolated from extracts of the Papaveraceae Macleaya cordata (Wild.) R. Br. Some of these compounds showed an efficacy against APN similar to that of amastatin (18) or bestatin (19) . A weaker inhibitory effect on DPP-IV has also been reported. 189 

(E,E-1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) (28) is a yellow natural phenolic compound isolated from the rhizomes of asian perennial herbs extensively cultivated in tropical areas and belonging to the Zingiberaceae family. All these plants are of the genera Curcuma.

The most exploited representative is Curcuma longa L., whose dried rhizome is the source of the spice turmeric which is widely employed in food and has a long tradition of use in folk medicine. In addition to its irreversible APN/CD13 inhibition potencies, 190 curcumin is now considered by oncologists as a potential cancer chemopreventive agent, 191, 192 and clinical trials in this context are carried out in several laboratories. 193 Furthermore, curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes (e.g. lipooxygenase/cyclooxygenase-2, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase). 194 Curcumin hinders also the initiation of carcinogenesis by inhibiting the cytochrome P-450 enzyme activity and increasing the levels of glutathione-S-transferase. Its anti-tumor effect in the promotion and progression stages has been attributed, in part, to the arrest of cancer cells in S, G2/M cycle phase, and induction of apoptosis. 195 It has also been proposed that curcumin may suppress tumor promotion by blocking signal transduction pathways in the target cells. 196 Curcumin is a potent inhibitor of protein kinase C, EGF-receptor tyrosine kinase and I-kB kinase. In addition, curcumin inhibits the activation of NF-kB and the expression of c-jun, c-fos, c-myc. 194, 197 Last, curcumin has been proposed as a HIV-1 or HIV-2 protease inhibitor, 198 as a HIV-1 integrase inhibitor, 199 and proved to be radioprotectant. 200, 201 Several chemical synthesis of 28, involving 2,4-pentanedione and vanillin, have been reported [202] [203] [204] [205] [206] as well as the preparations of some of its analogues designed as angiogenesis inhibitors 207 through their ability to inhibit endothelial cell proliferation. 208 

(3b-hydroxylup-20(29)-en-28-oic acid) (29) is a pentacyclic compound widely present in the plant kingdom. This oxidized derivative of betulin owes its trivial name to the fact that this class of lupane type triterpenes was first isolated from Betula ssp. (birch trees). Afterwards, betulinic acid has been obtained from various other vegetal species including Ancistrocladus ssp.,Arbutus ssp., Diospyros ssp., Paeonia ssp., Picramnia ssp., Syzygium ssp., Tetracera ssp., Tryphillum spp., Zizyphus ssp. One of the main current sources of betulinic acid from natural origin is the bark of plane trees (e.g. Platanus acerifolia) by employing a patented procedure. 209 In addition to its APN inhibitory activity in a dose-dependent manner, 210 and possibly as a partial consequence of this inhibitory potency, betulinic acid has been shown to modulate the immune response, to exhibit anti-inflammatory properties and to block HIV-1 entry into cells. It has also been reported to be a selective inhibitor of DNA polymerase b and to induce apoptosis in tumor cells. The wide range of biological properties linked to betulinic acid have recently been recapitulated and analyzed in three excellent revues. [211] [212] [213] Several hemisynthesis of 28 starting from betulin via betulonic acid [214] [215] [216] [217] or from various naturally occuring betulinic acid derivatives such as glycosides, [218] [219] [220] [221] sulfates, 222 or dihydroxycinnamic esters 223 have been reported. To our knowledge, only one naturally occuring APN inhibitor originates from animal kingdom:

is a symmetrical disulfide compound bearing two hydroxyimino functional groups. This bis-bromotyrosine derivative was, almost simultaneously, first isolated in 1987 by three groups: from an unidentified marine sponge (probably of the Verongidae family) collected in Guam, 224 from a Psammaplysilla sp., 225 and from Thorectopsamma xana. 226 Its structure has been unambiguously and independently established by these different authors. Thereafter, psammalin A has also been extracted from other sponges: Psammaplysilla purpurea, 225, 227 Dysidea spp. (in this case, the authors have erroneously named «bisprasin»-the misspelled name of the psammalin A dimer-a compound which is obviously the psammalin A itself as judged by the reported formula) 228 Aplysinella rhax, [229] [230] [231] Pseudoceratina purpurea, 232 or from a two-sponge association: Poecillastra wondoensis and Jaspis wondoensis. 233, 234 A biosynthetic pathway has been proposed for the formation of 30 involving modified cysteine and bromotyrosine 227, 232 and, to our knowledge, only one laboratory preparation of psammalin A has been carried out starting from L-tyrosine through its its N,N 0 -bis-(tetrahydropyran-2-yl)oxime derivative. 235 It is also worth pointing out that a library comprising about two hundred psammalin A type derivatives has recently been prepared by Nicolaou and his co-workers by using solution phase combinatorial synthesis with the aim to evaluate their antibacterial activity. 235, 236 In addition to its very recently reported ability to inhibit APN in a non-competitive manner thus inducing a suppression of in vitro angiogenesis, 237 30 has been shown to induce a variety of biological effects: (i) a significant in vitro antibacterial activity against Staphylococcus aureus 226 and methicillin-resistant Staphylococcus aureus 235, 236, 238 which is assumed to be due to its ability to inhibit DNA gyrase, 238 (ii) a cytotoxicity against various human tumor cell lines, 229, [231] [232] [233] (iii) an increase in Ca 2þ release from the heavy fraction of skeletal muscle sarcoplasmic reticulum, 228 (iv) an inhibition of topoisomerase II, 239 Leucine aminopeptidase and farnesyl protein transferase, 229 , mycothiol-S-conjugate amidase, 240 chitinase, 231 histone deacetylase and DNA methyltransferase, 232 and DNA replication by targeting polymerase a-primase. 241 Some antifungal and insecticidal activities have been further reported. 231 Chemical structures of APN inhibitors 13-30, and enzyme inhibition values are depicted in Figure 4 .

Several synthetic small molecules belonging to various chemical families have been reported to inhibit APN activity.

a-Aminomethylketones such as (S)-3-Amino-4-methylpentan-2-one hydrochloride (valine methyl ketone hydrochloride) (31) 242 

Alkyl D-cysteinates display also efficient competitive APN inhibitions. Among the five esters tested, an optimal inhibitory activity has been observed with the n-butyl derivative (32). 244 

3-amino-2-tetralone derivatives such as the 2-amino-1,4-dihydro-2H-phenanthren-3-one hydrochloride (33) have been reported to be efficient and selective competitive inhibitors of APN. These compounds do not affect AP-A or AP-B and poorly inhibit LAPc. 245 

3-Amino-2-hydroxypropionaldehyde and 3-amino-1-hydroxypropan-2-one derivatives such as 34 and 35, respectively. These competitive inhibitors of APN are very moderately active on LAPc or APB. 246 

Flavone-8-acetic acid derivatives constitute a class of products whose the parent compound showed antiangiogenic properties. 247 In this series, products bearing a nitro group in the 2-position such as the 2 0 ,3-dinitroflavone-8 acetic acid (36) proved the most potent APN inhibitors and act by reversibly binding to the catalytic site of the enzyme. These compounds present the advantage to exhibit no toxicity towards cultured human cells, to induce no apoptosis, and to be inactive on other proteases such as MMP-9, ACE, NEP, g-glutamyl transpeptidase, cathepsin G, or DPPIV. 248 6. N-Hydroxy-2-(naphthalene-2-ylsulfanyl)Acetamide N-Hydroxy-2-(naphthalene-2-ylsulfanyl)acetamide (37) has recently been identified as a potent APN inhibitor. It acts in a dose-dependent manner and is inactive on metalloenzymes MMP-2, MMP-9, MMP-14, or A-LAP. 249 The design of synthetic APN inhibitors has often been relied to structure-activity studies based on active site models derived from structural data obtained on the zinc-dependent protease thermolysin crystallized with a variety of inhibitors. 250 Molecules capable of interacting with at least the S 1 subsite of APN and which have a strong zinc-chelating group 251,252 were designed. According to these criteria, some a-aminophosphinic acids and derivatives such as 38 or 39 253 have been prepared and proved to be very potent APN inhibitors. According to the patterns of these models, synthesis of analogs such as the iodo derivative 40 (RB 129) have next been performed to give rise to the radiolabelled ( 125 I)RB 129 254 which represents a useful probe to investigate the physiological role of APN. 13, 255, 256 In the same context, several b-aminothiols exemplified by 41 257 or 42 251 have been conceived and synthesized. The research in this field has then been extended to more elaborated series by Roques and co-workers, and novel sulfur-containing molecules capable of inhibiting APN such as 43, 44, 258 45, 46 259 or 47 253 were prepared. From these works on b-aminothiols, two products emerged: PC 18 (S)(2-amino-4-methylthiobutanethiol) (48) 253 The similarities between the active sites of APN and the membrane-bound protease neutral endopeptidase 24.11 (EC3.4.24.11, CD10, NEP) led to the idea that mixed inhibitors could be developed by selecting frameworks bearing a strong zinc-chelating group and a residue able to interact with at least one subsite (S 1 , S 1 0 , and S 2 0 ) of each peptidase. 65,251,262 -265 The first dual E24.11/APN inhibitors developed were hydroxamate-containing molecules such as Kelatorphan (50) or RB 38A (51) 262,266,267 whose several analogs have been synthesized and found to be also potent inhibitors of leukotriene A 4 hydrolase. 268 However, the important water solubility of these compounds is an impediment for crossing the blood-brain barrier and, consequently, for obtaining a good bioavailability. Another strategy, involving more lipophilic derivatives, led to the synthesis of RB 101 (N-((R,S)-2-benzyl-3((S)(2-amino-4-methylthio)butyldithio)-1-oxopropyl)-L-phenylalanine benzyl ester (52) and RB 120 (N-((S)-2-benzyl-3((S)(2-amino-4-methylthio)butyldithio)-1oxopropyl)-L-alanine benzyl ester (53), two dual inhibitors in which a disulfide bridge links the APN inhibitor PC 18 with analogs (the phenylalanine analog (ST 43) in the case of 52, or the alanine analogue in the case of 53) of the benzyl ester of Thiorphan, a specific NEP inhibitor 269 (Fig. 6) . 251, 263, 270 Such mixed inhibitors present the advantage to possess the above-mentioned disulfide bond which is relatively stable in plasma, in contrast to its rapid cleavage in brain, thus allowing the delivery of the NEP and APN inhibitors in their active form toward their respective target. 263 The development of such mixed inhibitors has constituted an important advance in the research of new antihypertensives and novel antinociceptive drugs devoid of opioid side effects. 264, 271 (for reviews) More recently, a new generation of phosphinic acid derivatives have been prepared as NEP/APN dual inhibitors, and compounds such as 54 have been successfully tested in this context. 252, 272 Chemical structures of APN inhibitors 50-54, and enzyme inhibition values are outlined in Figure 7 . 

The effects of some of these above described inhibitors on cell behavior have been assayed in in vitro approaches. Table I provides a summary of most relevant studies in the human system.

Actinonin, bestatin, probestin, and psammaplin A (at 1-100 mM concentrations) were shown to reduce the growth of human T/B lymphocytes, dendritic and cord blood CD34 þ cells [273] [274] [275] [276] and human myeloid and lymphoid cell lines, 273, 274, [276] [277] [278] [279] [280] [281] [282] as well as the proliferation of 283, 284 and various tumor and endothelial cell lines. 237, [285] [286] [287] A question central to APN inhibition studies is how cell growth can be turned off by APN inhibitors. APN inhibitors may alter the processing of (unknown) growth factors directly involved in the regulation of growth. In addition, several studies indicate that inhibitors like actinonin and probestin may transmit intracellular-transduction signals by interfering with the MAP kinase signaling pathway. 25, 279, 288, 289 A second cell signaling pathway involving the Wnt-5a proto-oncogene appears also affected by inhibition of APN by actinonin. 290 It has to be pointed out that actinonin (at a 10 mM concentration) inhibited the growth of both CD13-positive myeloid and CD13-negative lymphoma cell lines 287 suggesting that the effects induced by actinonin are not likely to be mediated by CD13. Moreover, amastatin at a concentration which inhibits APN activity was found without any effect on the growth of human myeloid cell lines 274, 291 .

Bestatin-mediated cell growth arrest is associated with an induction of cell maturation of clonogenic GM-CFU (granulocyte-macrophage colony forming unit) cells from human immature derived-bone marrow cells. 292, 293 Similarly, treatment of human myeloid U937 and NB4 cell lines with bestatin induced phenotypic changes characteristic of macrophage (U937) or neutrophil (NB4) maturation. 280, 293, 294 

Cell growth arrest induced by APN inhibitors correlates with alternated secretion of proinflammatory and immunosuppressive cytokines involved in pathophysiological processes. Bestatin (2.9 mM) increased the levels of IL-8 secreted by endothelial cells, 295 and of IL-1 release from mouse peritoneal macrophages and IL-2 release from concanavalin-stimulated T cells. 296 Probestin induces the synthesis and release of TGF-b1. 41,297

Recent observations point to the involvement of APN in the process of apoptosis (programmed cell death). Bestatin and actinonin (starting 30 mM) induce apoptosis in a large variety of cell lines, i.e. myeloid (P39/TSU, HL-60, U937, NB4) and lymphoid (Jurkat, BJAB, NALM6, BOE) cells, and carcinoma (fibrosarcoma, cervical, and lung carcinoma). 274, 282, 287, 291, 298, 299 Betulinic acid induces apoptosis in the HT29 colon cancer cell line (26 mM) 84 and in acute leukemia cells (50 mM). 300

In a general way, cell motility (migration and invasion) may be influenced by the processing of chemokines and/or degradation of the extracellular matrix (ECM). The two small proteins with chemotactic activity, MCP-1 and f-MLP, are in vitro hydrolyzed by APN/CD13. With regard to MCP-1, there is no current data reporting the potential action of APN inhibitors on the MCP-1-mediated migration. Actinonin and amastatin were able to enhance the chemotactic response of human neutrophils toward f-MLP. 301 One explanation of the effects of actinonin or amastatin would be that both inhibitors prevent the inactivation of f-MLP by APN, to further enhance the f-MLP-mediated chemotactic response. It has however to underline that both inhibitors weakly inhibited APN enzymatic activity over the range from 10 À8 to 10 À4 M, concentrations that are effective on neutrophil migration. 301 APN inhibition by actinonin or bestatin significantly enhanced the in vitro migration of eosinophils across HUVEC monolayers. 302 Moreover, actinonin, bestatin as well as leuhistin (50-150 mM) significantly blocked the invasion of various human metastatic tumor cells into reconstituted basement membranes 303, 304 or into Matrigel. 21, [305] [306] [307] These latter data suggested that APN could be indirectly involved in type IV collagen degradation by activating type IV procollagenase/proMMP-9. 17, 303, 304 Recent studies demonstrated that soluble APN/CD13 induces in vitro chemotactic migration of T lymphocytes, and that bestatin at high concentration (580 mM) abolishes this process, suggesting that the enzymatic activity of APN was responsible for the chemotactic activity. 34,36,304,308

The demonstration of the participation of APN in angiogenesis has come from recent studies in which blocking APN activity by APN inhibitors resulted in the perturbation of ''angiogenic'' assays (Table I) .

APN/CD13 is expressed on the human umbilical vein endothelial cells (HUVECs) of angiogenic, but not normal, vasculature. 309 Bestatin, betulinic acid, amastatin, curcumin, and psammaplin A (10-250 mM) abrogate the ability of the HUVECs cultured on matrigel to organize a capillary network 20, 190, 237, [310] [311] [312] without altering their proliferation rates. 310 In contrast, one study underlines the proangiogenic effect of bestatin (8-250 mM) which instead causes matrix degradation and stimulates the invasion of microvascular endothelial cells into a fibrin matrix. 313 

In the chorioallantoic membrane (CAM) assay, the angiogenic response is determined by measuring the number of avian extraembryonic capillary vessels that grow within a matrix polymer (containing an angiogenic molecule such as fibroblast growth factor-2/FGF-2) placed on the yolk sac membrane of a 4 day embryo in culture. 314 The chick vasculature expresses a phenotype APN/CD13, and subsequent treatment with bestatin or actinonin (200 mg) inhibited FGF-2-induced angiogenesis. 309 In the mouse retinal neovascularization model, bestatin (200 mg/mouse) leads to the blockade of hypoxia-induced retinal neovascularization in mice. 309 The intraperitoneal administration of bestatin (50-100 mg/kg/day) after the orthotopic implantation of B16-BL6 melanoma cells into mice reduces the number of vessels oriented toward the established primary tumor mass on the dorsal side of mice. 311 

Compiled data documenting the involvement of APN/CD13 in pathophysiological events (cancer, inflammation, infection, pain suppression) have come from studies which blocked APN activity in rodent models (Table I) .

Studies in rats indicate that administration of bestatin leads to the inhibition of fetal growth and the induction of placental apoptosis. 315, 316 The in vivo anti-cancer activities of bestatin and betulinic acid have been reported through their capacities to inhibit the growth of syngeneic tumor (leukemia/ melanoma/ovarian/hepatoma/gastric carcinoma) cells implanted in mice 16, 213, 309, 310, [317] [318] [319] [320] [321] [322] [323] [324] [325] and rats. 319, 326, 327 Doses as low as 0.5 mg/kg for bestatin and 5 mg/kg for betulinic acid were used in these studies. Moreover, high doses (up to 500 mg/kg) did not lead to any cytotoxic effect in mice.

Bestatin, leuhistin, and betulinic acid have been investigated for anti-inflammatory properties. Betulinic acid possessed moderate ant-inflammatory abilities at relatively high concentrations (100 mg/kg/mouse, i.v.). 213 In contrast, bestatin and leuhistin inhibit acute inflammation associated the accumulation of polymorphonuclear neutrophils in a mouse model (2 mg/kg, i.v.). 57, 328 Moreover, oral administration of bestatin (5 mg/kg) in carcinoma-bearing mice induces generation of cytotoxic T cells and NK (natural killer) cells. 317 Angiotensins II and III are two peptide effectors of the brain rennin-angiotensin system that participate in the control of blood pressure, increase water consumption and vasopressin release. In hypertensive rats, infusion of amastatin (16 nmol/min i.v.) prevents degradation of angiotensins associated with blood pressure decrease. 67,329 . In the mouse brain, APN inhibition by PC18 or EC27 (10-300 mg injected intracerebroventricularly) increases the half life of angiotensin III, resulting in enhanced vasopressin release. 61, 66, 260 Several studies report that bestatin exerts anti-infectious properties by augmenting host resistance to bacterial, viral or fungal experimental infections in mice by inducing neutrophil and macrophage activation 330, 331 and enhancing antibody production. [330] [331] [332] [333] [334] [335] Finally, in the central nervous system, enkephalins which modulate responses to painful stimuli, are inactivated by APN and the membrane-bound protease neutral endopeptidase 24.11 (EC3.4.24.11, CD10). This led to the idea that inhibition of these enzymes (alone or in combination) could achieve clinically efficient analgesia. Actinonin as well as the dual inhibitors RB101 and RB120 (9 mg/kg, i.v.; 80 mg/kg, i.p.) exhibited analgesic properties against chronic pain in rats and mice. 261, 263, 267, 336, 341 

In first clinical trials, bestatin (30 mg/daily) has been used to treat patients with acute and chronic myeloid leukemias (AML, CML) and lymphomas. [342] [343] [344] [345] [346] Therapeutic efficacy was demonstrated by a prolongation of survival in patients with AML 345, 346 and lymphomas, 342, 343, 347 and in promoting graft versus leukemia effects in patients following allogeneic bone marrow transplant. 348 In a phase Ib trial, activation of blood monocytes and increase in the CD4/CD8 lymphocyte ratio were observed in Hodgkin's and non-Hodgkin's lymphoma patients treated orally with high doses of bestatin (90-180 mg/daily/60 days) following autologous bone marrow transplantation. 330, 334, 349 In phase III trials in resected stage I squamous cell lung carcinoma, survival was statistically better for patients who were treated with bestatin (30 mg/daily/2 years) as a post-operative adjuvant therapy than those who received a placebo. 350, 351 

APN/CD13, is useful in defining clinical subgroups of patients with various malignancies or inflammatory diseases. The use of natural and synthetic APN inhibitors has revealed that APN/CD13 participates to the control of major biological processes such as proliferation, secretion and apoptosis. Dysregulation of APN/CD13 in tumors is often linked to tumor invasion and angiogenesis. Studies on non-hematopoietic cells suggest that APN/CD13 may influence cell migration and invasion. APN/ CD13 inhibitors have been shown to alter angiogenesis in in vitro and in vivo assays. Documented evidence underlines both the antiangiogenic and proangiogenic effects of bestatin. 309, 310, 313 Figure 8 summarizes our current understanding of the involvement of APN inhibitors in the modulation of these events. The detailed molecular mechanisms underlying these effects are however yet unclear.

Importantly, the requirement for APN in these processes has been mostly confirmed with studies in which APN/CD13 expression was blocked by neutralizing CD13 antibodies 20, 285, 303, 309, 310 or antisense CD13 oligonucleotides, 20, 41, 352 or enhanced by the use of CD13 transfectants. 17 It has however to be pointed out that most of APN inhibitors lack tight specificity by inhibiting other membrane-bound metalloproteases or secreted matrix metalloproteinases (MMPs) ( Table I) . For example, bestatin interacts with leucyl-aminopeptidase (EC3.4.11.1, oxytocinase, Leu-AP), aminopeptidase B (EC 3.4.11.6, AP-B) and aminopeptidase W (EC 3.4.11.16, AP-W) 136,353-357 thus suggesting that some of the observed chemotherapeutic actions of bestatin may be due to inhibition of other cell surface peptidases. Actinonin was recently shown to interact with human peptide deformylase, 358,359 meprin a (EC 3.4.24.18, endopeptidase 24.18), 360 and MMP-2. 361 Amastatin and probestin in the low micromolar range (1.5-20 mM) inhibit aminopeptidase A (EC 3.4.11.2, AP-A) and AP-W. 109, 355, 362 Leuhistin inhibits AP-A and AP-B to the same degree than APN. 186 Curcumin and betulinic acid block MMP-9 expression and collagenase activity through inhibition of NF-kB activation. [363] [364] [365] [366] [367] In addition, the use of available APN inhibitors in some experimental situations has revealed complex effects on cell behavior. As mentioned in paragraph 4.A, CD13-positive and CD13negative cell lines are equally sensitive to the growth-inhibitory effect of actinonin (50-260 mM) 287 thus emphasizing that actinonin may induce unspecific cytotoxic side-effects. Moreover, betulinic acid inhibits tube formation of bovine aortic endothelial cells at a concentration which had no effect on the cell viability and in vivo APN activity of endothelial cells, thus indicating an APN-independent mode of action of betulinic acid. 312 Together, these observations emphasize the need for more specific and targeted APN inhibitors to (re)evaluate the actions of APN/CD13 in pathophysiological processes. Future consideration has to be given to the obtention of the three-dimensional structure of APN determined by NMR spectroscopy to help APN inhibitor design strategy. Further in vitro and in vivo studies with promising non cytototoxic APN inhibitors (such as psammaplin A, phosphonic derivatives, flavone-8-acetic acid derivatives) are also required before clinically prescribing an APN inhibitor as an anti-cancer or anti-inflammatory agent. 

Ectopeptidases in pathophysiology

MEROPS: The peptidase database

Families of zinc metalloproteases

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

Isolation of an aminopeptidase from kidney particles

Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N

Hematopoietic differentiation antigens that are membrane-associated enzymes: Cutting is the key!

Characterization of particulate and soluble variants of the brush-border enzymes alanine aminopeptidase, alkaline phosphatase and gamma-glutamyltransferase in human urine

CD13 (GP150; aminopeptidase-N): Predominant functional activity in blood is localized to plasma and is not cell-surface associated

High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients

Soluble aminopeptidase N/CD13 in malignant and nonmalignant effusions and intratumoral fluid

Transmembrane proteases as disease markers and targets for therapy

Ontogenic and adult whole body distribution of aminopeptidase N in rat investigated by in vitro autoradiography

The significance of aminopeptidases and haematopoietic cell differentiation

Induction of CD13 expression on fresh myeloid leukaemia: Correlation of CD13 expression with aminopeptidase-N activity

Biochemical and functional characterization of aminopeptidase N expressed by human melanoma cells

Human melanoma invasion and metastasis enhancement by high expression of aminopeptidase N/CD13

Urinary excretion of glycine.prolile dipeptidile aminopeptidase, N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase, and low molecular protein in patients with renal cell carcinoma

Clinical significance of aminopeptidase N/CD13 expression in human pancreatic carcinoma

Aminopeptidase N is involved in cell motility and angiogenesis: Its clinical significance in human colon cancer

Inhibition of aminopeptidase N (AP-N) and urokinase-type plasminogen activator (uPA) by zinc suppresses the invasion activity in human urological cancer cells

Determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-N that is distinct from the enzymatic site

Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV

Further characterization of aminopeptidase-N as a receptor for coronaviruses

Human aminopeptidase N is a receptor for human coronavirus 229E

Substance P and bradykinin are natural inhibitors of CD13/ aminopeptidase N

Alanyl aminopeptidase from human seminal plasma: Purification, characterization, and immunohistochemical localization in the male genital tract

Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: Purification and characterization

Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases

Novel potent nonpeptide aminopeptidase N inhibitors with a cyclic imide skeleton

Novel small molecule nonpeptide aminopeptidase n inhibitors with a cyclic imide skeleton

Nonpeptide small-molecular inhibitors of dipeptidyl peptidase IV: N-phenylphthalimide analogs

Preparation of novel specific aminopeptidase inhibitors with a cyclic imide skeleton

Potent homophthalimide-type inhibitors of B16F10/L5 mouse melanoma cell invasion

Specific inhibitor of puromycin-sensitive aminopeptidase with a homophthalimide skeleton: Identification of the target molecule and a structure-activity relationship study

Specific nonpeptide inhibitors of puromycin-sensitive aminopeptidase with a 2,4(1H,3H)-quinazolinedione skeleton

The most potent organophosphorus inhibitors of leucine aminopeptidase. Structure-based design, chemistry, and activity

Potent and selective inhibition of zinc aminopeptidase A (EC 3.4.11.7, APA) by glutamyl aminophosphinic peptides: Importance of glutamyl aminophosphinic residue in the P1 position

alpha-Keto amide inhibitors of aminopeptidases

alpha-Aminoboronic acid derivatives: Effective inhibitors of aminopeptidases

alpha-aminoaldehydes: Transition state analogue inhibitors of leucine aminopeptidase

Inhibition of zinc metallopeptidases by flavonoids and related phenolic compounds: Structure-activity relationships

Inhibition of metallopeptidases by flavonoids and related compounds

Inhibition of human leukocyte function, alanyl aminopeptidase (APN, CD13) and dipeptidylpeptidase IV (DP IV, CD26) enzymatic activities by aqueous extracts of Cistus incanus L. ssp. incanus

Compounds from Epilobium angustifolium inhibit the specific metallopeptidases ACE, NEP, and APN

Actinonin: An antibiotic substance produced by an actinomycete

Production of actinonin, an inhibitor of aminopeptidase M, by actinomycetes

Studies concerning the antibiotic actinonin. Part I. The constitution of actinonin. A natural hydroxamic acid with antibiotic activity

Studies concerning the antibiotic actinonin. Part II. Total synthesis of actinonin and some structural analogues by the isomaleimide method

Studies concerning the antibiotic actinonin. Part III. Synthesis of structural analogues of actinonin by the anhydride-imide method

Studies concerning the antibiotic actinonin. Part IV. Synthesis of structural analogues of actinonin by the mixed anhydride method

Studies concerning the antibiotic actinonin. Part V. Synthesis of structural analogues of actinonin by the anhydride-ester method

Studies concerning the antibiotic actinonin. Part VI. Synthesis of structural analogues of actinonin by dicyclohexylcarbodiimide coupling reactions

Studies concerning the antibiotic actinonin. Part VII. Mass spectra of actinonin and related compounds

Asymmetric synthesis of (À)-actinonin and (À)-epi-actinonin

Studies concerning the antibiotic actinonin. Part VIII. Structure-activity relationships in the actinonin series

Bestatin analogue from Streptomyces neyagawaensis SL-387

MR-387A and B, new aminopeptidase N inhibitors, produced by Streptomyces neyagawaensis SL-387

Biosynthesis of peptide inhibitor MR-387 by Streptomyces neyagawaensis

synthesis and anti-cancer activity of AHPA derivatives

Inhibition of aminopeptidases by amastatin and bestatin derivatives

Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes

Structure and chemical synthesis of amastatin

Synthesis of (2S, 3R)-3-amino-2-hydroxy-5-methylhexanoic acid derivatives. Application to the synthesis of amastatin, an inhibitor of aminopeptidase

Synthesis and structure-activity relationships of amastatin analogues, inhibitors of aminopeptidase A

Leucine aminopeptidase: Bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis

Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes

Bestatin inhibits covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase

Effects of metalloproteinase inhibitors on leukotriene A4 hydrolase in human airway epithelial cells

Leukotriene A4 hydrolase: A critical role of glutamic acid-296 for the binding of bestatin

Bestatin as an experimental tool in mammals

Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes

The structure of bestatin

The chemical synthesis of bestatin

A stereocontrolled synthesis of (À)-bestatin from an acyclic allylamine by iodocyclocarbamation

Synthesis of [beta]-amino-[alplha]-hydroxy acids via aldol condensation of a chiral glycolate enolate. Synthesis of (À)-bestatin

A stereospecific synthesis of (À)L-Bestatin from -malic acid

Synthesis of the peptidic [alpha]-hydroxy amides phebestin, probestin, and bestatin from [alpha]-keto amide precursors

Acylnitrene route to vicinal amino alcohols. Application to the synthesis of (À)-bestatin and analogues

A new one-pot method for the synthesis of [alpha]-siloxyamides from aldehydes or ketones and its application to the synthesis of (À)-bestatin

New stereoselective synthesis of the peptidic aminopeptidase inhibitors bestatin, phebestin and probestin

Application of acyl cyanophosphorane methodology to the synthesis of protease inhibitors: Poststatin, eurystatin, phebestin, probestin, and bestatin

Chirospecific synthesis of the(2S,3R)-and(2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acids from sugar: Application to (À)-bestatin

Synthesis and structure-activity relationships of bestatin analogues, inhibitors of aminopeptidase B

Synthesis of p-hydroxyubenimex

Synthesis of sulfur-containing analogues of bestatin. Inhibition of aminopeptidases by alpha-thiolbestatin analogues

Design of novel inhibitors of aminopeptidases. Synthesis of peptide-derived diamino thiols and sulfur replacement analogues of bestatin

Development of selective tight-binding inhibitors of leukotriene A4 hydrolase

Inhibition of arginine aminopeptidase by bestatin and arphamenine analogues

Phebestin, a new inhibitor of aminopeptidase N, produced by Streptomyces sp. MJ716-m3

Probestin, a new inhibitor of aminopeptidase M, produced by Streptomyces azureus MH663-2F6. I. Taxonomy, production, isolation, physico-chemical properties, and biological activities

Probestin, a new inhibitor of aminopeptidase M, produced by Streptomyces azureus MH663-2F6. II. Structure determination of probestin

X-ray structure determination of (2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoic acid, a new amino acid component of bestatin

Regio-and stereo-specific synthesis of threo-3-amino-2-hydroxy-acids, novelamino-acids contained in aminopeptidase inhibitors of microbial origin

Diastereo-and enantioselective synthesis of 1,2-amino alcohols from glycol aldehyde hydrazones; asymmetric synthesis of (R,R)-statin

Versatile synthetic routes to threo-[beta]-amino hydroxy carboxylic acids, statine and its analogues

Synthesis of taxol and taxotere side chains by 2-(trimethylsilyl)thiazole based homologation of L-phenylglycine

A catalytic asymmetric synthesis of cyclohexylnorstatine

Catalytic asymmetric aminohydroxylation (AA) of olefins

N-halocarbamate salts lead to more efficient catalytic assymetric aminohydroxylation

The enantioselective synthesis of [beta]-aminoacids, their [alpha]-hydroxy derivatives, and the N-terminal components of bestatin and microginin

An enantioselective, stereodivergent approach to anti-and syn-[alpha]-hydroxy-[beta]-amino acids from anti-3-amino-1,2-diols. Synthesis of the ready for coupling taxotere(R) side chain

Stereoselective synthesis of (À)-N-Boc-statine and (À)-N-Boc-Norstatine

Metal halide-mediated opening of three membered rings: Enantioselective synthesis of (2S,3R)-3-amino-2-hydroxydecanoic acid and (3R)-3-aminodecanoic acid

Enantiospecific and diastereoselective synthesis of syn-[beta]-amino-[alpha]-hydroxy acids

Practical synthesis of (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, a key component of HIV protease inhibitors

A convenient preparation of (2SR, 3S)-3amino-2-hydroxy-4-phenylbutanoic acid; an important peptide bond isostere

A novel synthesis of allophenylnorstatine from (R)-aspartic acid

Asymmetric synthesis of 3-amino-2-hydroxy-4-phenylbutanoate

Diastereoselective synthesis of N-protected [beta]-amino-[alpha]-hydroxyacids (norstatines) from urethane N-carboxyanhydrides (UNCAs)

Process for producing erythro-3-amino-2-hydroxybutyric acid derivatives

The S-thioester enolate/imine condensation: A shortcut to [beta]-lactams

New synthetic technology for efficient construction of [alpha]-hydroxy-[beta]-amino amides via the Passerini reaction

Analysis of amide bond formation with an [alpha]-hydroxy-[beta]-amino acid derivative, 3-amino-2-hydroxy-4-phenylbutanoic acid, as an acyl component: Byproduction of homobislactone

A short and enantioselective synthesis of N-terminal components of bestatin, amastatin, and microginin

Stereoselective synthesis of [alpha]-hydroxy-[beta]-amino acids: The chiral pool approach

Leuhistin, a new inhibitor of aminopeptidase M, produced by Bacillus laterosporus BMI156-14F1. I. Taxonomy, production, isolation, physico-chemical properties, and biological activities

Biosynthetic study of leuhistin, a new inhibitor of aminopeptidase M

Leuhistin, a new inhibitor of aminopeptidase M, produced by Bacillus laterosporus BMI156-14F1. II. Structure determination of leuhistin

Quaternary benzo[c]phenanthridine alkaloids as inhibitors of aminopeptidase N and dipeptidyl peptidase IV

Irreversible inhibition of CD13/aminopeptidase N by the antiangiogenic agent curcumin

Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances

Role of chemopreventive agents in cancer therapy

Anticancer potential of curcumin: Preclinical and clinical studies

Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: Down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation

Chemotherapeutic potential of curcumin for colorectal cancer

Cell signaling pathways altered by natural chemopreventive agents

The molecular mechanisms for the antitumorigenic effect of curcumin

Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes

Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection

Effect of curcumin on radiation-impaired healing of excisional wounds in mice

Role of curcumin, a naturally occurring phenolic compound of turmeric in accelerating the repair of excision wound, in mice whole-body exposed to various doses of gammaradiation

Synthesis of curcumin and related compounds

Synthesis of naturally occuring curcuminoids and related compounds

Simplified condition for synthesis of curcumin I and other curcuminoids

Curcumin analogs with altered potencies against HIV-1 integrase as probes for biochemical mechanisms of drug action

Electron ionization mass spectrometry of curcumin analogues: An olefin metathesis reaction in the fragmentation of radical cations

Design, synthesis, and biological evaluation of angiogenesis inhibitors: Aromatic enone and dienone analogues of curcumin

Hydrazinocurcumin, a novel synthetic curcumin derivative, is a potent inhibitor of endothelial cell proliferation

Method of producing betulinic acid

Betulinic acid inhibits aminopeptidase N activity

A review of natural and modified betulinic, ursolic and echinocystic acid derivatives as potential antitumor and anti-HIV agents

Betulinic acid: A promising anticancer candidate

Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection

A concise semi-synthetic approach to betulinic acid from betulin

The synthesis of betulinic acid from betulin and its solubilization with liposomes

Synthesis of betulinic acid derivatives with activity against human melanoma

Lupane triterpenes and derivatives with antiviral activity

Pavophylline, a new saponin from the stem of Pavonia zeylanica

(29)-en-28-oic acid from the stem of Dillenia pentagyna

A saponin from Asparagus gonocladus

A betulinic acid glycoside from Schefflera venulosa

Two new triterpenoid sulfates from the leaves of Schefflera octophylla

Studies on anti-inflammatory agents. V. A new anti-inflammatory constituent of Pyracantha crenulata roem

Brominated tyrosine metabolites from an unidentified sponge

Phenolic constituents of Psammaplysilla

Two bromotyrosine-cysteine derived metabolites from a sponge

Novel marine sponge derived amino acids 13. Additional psammaplin derivatives from Psammaplysilla purpurea

Bisprasin, a novel Ca(2þ) releaser with caffeine-like properties from a marine sponge, Dysidea spp., acts on Ca(2þ)-induced Ca(2þ) release channels of skeletal muscle sarcoplasmic reticulum

New bromotyrosine metabolites from the sponge Aplysinella rhax

Isolation of psammaplin A 11¢-sulfate and bisaprasin 11¢-sulfate from the marine sponge Aplysinella rhax

Psammaplin A, a chitinase inhibitor isolated from the Fijian marine sponge Aplysinella rhax

Psammaplins from the sponge Pseudoceratina purpurea: Inhibition of both histone deacetylase and DNA methyltransferase

Cytotoxic compounds from a two-sponge association

New bromotyrosine derivatives from an association of two sponges, Jaspis wondoensis and Poecillastra wondoensis

Combinatorial synthesis through disulfide exchange: Discovery of potent psammaplin A type antibacterial agents active against methicillinresistant Staphylococcus aureus (MRSA)

Optimization and mechanistic studies of psammaplin A type antibacterial agents active against methicillin-resistant Staphylococcus aureus (MRSA)

Psammaplin A, a marine natural product, inhibits aminopeptidase N and suppresses angiogenesis in vitro

Psammaplin A, a natural bromotyrosine derivative from a sponge, possesses the antibacterial activity against methicillin-resistant Staphylococcus aureus and the DNA gyrase-inhibitory activity

Psammaplin A, a natural phenolic compound, has inhibitory effect on human topoisomerase II and is cytotoxic to cancer cells

Bromotyrosine-derived natural and synthetic products as inhibitors of mycothiol-S-conjugate amidase

Cytotoxicity of psammaplin A from a two-sponge association may correlate with the inhibition of DNA replication

[alpha]-aminoketone-Ein beitrag zur synthese optish aktiver derivate von aminosäuren und peptiden

Inhibition of aminopeptidase M by alkyl D-cysteinates

3-Amino-2-tetralone derivatives: Novel potent and selective inhibitors of aminopeptidase-M (EC 3.4.11.2)

3-Amino-2-hydroxy-propionaldehyde and 3-amino-1-hydroxypropan-2-one derivatives: New classes of aminopeptidase inhibitors

Effect of flavone acetic acid on endothelial cell proliferation: Evidence for antiangiogenic properties

Synthesis and biological evaluation of novel flavone-8-acetic acid derivatives as reversible inhibitors of aminopeptidase N/CD13

N-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide, a novel hydroxamic acid-based inhibitor of aminopeptidase N and its anti-angiogenic activity

The binding of L-valyl-L-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis

Potent and systemically active aminopeptidase N inhibitors designed from active-site investigation

Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors: Synthesis, biological properties, and antinociceptive activities

Design of the first highly potent and selective aminopeptidase N (EC 3.4.11.2) inhibitor

Synthesis of 2(S)-benzyl-3-[hydroxy(1¢(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ 125 I]-iodotyrosine: A radiolabelled inhibitor of aminopeptidase N

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography

First discrete autoradiographic distribution of aminopeptidase N in various structures of rat brain and spinal cord using the selective iodinated inhibitor [ 125 I]RB 129

Potent inhibition of cerebral aminopeptidases by carbaphethiol, a parenterally active compound

Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors

Differential inhibition of aminopeptidase A and aminopeptidase N by new beta-amino thiols

PC18, a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III

Aminopeptidase A, which generates one of the main effector peptides of the brain rennin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure

Analgesic effects of kelatorphan, a new highly potent inhibitor of multiple enkephalin degrading enzymes

Mixed inhibitorprodrug as a new approach toward systemically active inhibitors of enkephalin-degrading enzymes

Dual inhibitors of enkephalin-degrading enzymes (neutral endopeptidase 24.11 and aminopeptidase N) as potential new medications in the management of pain and opioid addiction

Inhibitors of neprilysin: Design, pharmacological and clinical applications

Analgesic responses elicited by endogenous enkephalins (protected by mixed peptidase inhibitors) in a variety of morphine-sensitive noxious tests

Inhibition of the enkephalin-metabolizing enzymes by the first systemically active mixed inhibitor prodrug RB 101 induces potent analgesic responses in mice and rats

Kelatorphan and related analogs: Potent and selective inhibitors of leukotriene A4 hydrolase

Molecular pharmacology of endothelin converting enzymes

Zinc metallopeptidases: Active site structure and design of selective and mixed inhibitors: new approaches in the search for analgesics and anti-hypertensives

Peptidomimetics as receptors agonists or peptidase inhibitors: A structural approach in the field of enkephalins, ANP, and CCK

Aminophosphinic inhibitors as transition state analogues of enkephalin-degrading enzymes: A class of central analgesics

Inhibitory effect of bestatin on the growth of human leukemic cells

Induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines

CD13/N-aminopeptidase is involved in the development of dendritic cells and macrophages from cord blood CD34(þ) cells

Inhibitory effect of bestatin on the growth of human lymphocytes

Bestatin, an inhibitor of aminopeptidase B, suppresses the proliferation and differentiation of human B-cells in vitro

Inhibition of alanylaminopeptidase suppresses the activation-dependent induction of glycogen synthase kinase-3beta (GSK-3beta) in human T cells

Inhibition of alanyl aminopeptidase induces MAP-kinase p42/ERK2 in the human T cell line KARPAS-299

Effect of ubenimex on the proliferation and differentiation of U937 human histiocytic lymphoma cells

Cell cycle retardation in monocytoid cells induced by aminopeptidase N (CD13)

Aminopeptidase inhibitors inhibit proliferation and induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta pathways

Expression of CD13/aminopeptidase N and CD10/neutral endopeptidase on cultured human keratinocytes

Identification of extra-and intracellular alanyl aminopeptidases as new targets to modulate keratinocyte growth and differentiation

Expression of aminopeptidase N on human choriocarcinoma cells and cell growth suppression by the inhibition of aminopeptidase N activity

Growth inhibitory effect of bestatin on choriocarcinoma cell lines in vitro

Antitumor activity of actinonin in vitro and in vivo

Aminopeptidase N/CD13 is directly linked to signal transduction pathways in monocytes

Aminopeptidase N-mediated signal transduction and inhibition of proliferation of human myeloid cells

Modulation of WNT-5A expression by actinonin: Linkage of APN to the WNT-pathway?

Augmentation of death ligand-induced apoptosis by aminopeptidase inhibitors in human solid tumor cell lines

Enhancing effect of ubenimex (bestatin) on proliferation and differentiation of hematopoietic progenitor cells, and the suppressive effect on proliferation of leukemic cell lines via peptidase regulation

Modulation of bone marrow cell functions in vitro by bestatin (ubenimex)

Enhancement of sensitivity by bestatin of acute promyelocytic leukemia NB4 cells to all-trans retinoic acid

Leukemic cell-surface CD13/aminopeptidase N and resistance to apoptosis mediated by endothelial cells

Enhancement of interleukin 1 and interleukin 2 releases by ubenimex

Synergistic action of DPIVand APN in the regulation of T cell function

Aminopeptidase inhibitor Bestatin induces HL-60 cell apoptosis through activating caspase 3

Induction of apoptosis by ubenimex (Bestatin) in human non-small-cell lung cancer cell lines

Betulinic acid-induced apoptosis in leukemia cells

Effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils

Differential regulation of aminopeptidase N (CD13) by transendothelial migration and cytokines on human eosinophils

Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin)

Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion

Inhibition of tumor cell invasion and matrix degradation by aminopeptidase inhibitors

Possible contribution of aminopeptidase N (APN/CD13) to invasive potential enhanced by interleukin-6 and soluble interleukin-6 receptor in human osteosarcoma cell lines

Role of CD13/ aminopeptidase N in rat lymphocytic alveolitis caused by thoracic irradiation

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis

CD13/APN is activated by angiogenic signals and is essential for capillary tube formation

Anti-tumor angiogenesis effect of aminopeptidase inhibitor bestatin against B16-BL6 melanoma cells orthotopically implanted into syngeneic mice

Betulinic acid inhibits growth factorinduced in vitro angiogenesis via the modulation of mitochondrial function in endothelial cells

Aminopeptidase inhibitor bestatin stimulates microvascular endothelial cell invasion in a fibrin matrix

The chick embryo chorioallantoic membrane as a model for in vivo research on anti-angiogenesis

Bestatin results in pathophysiological changes similar to preeclampsia in rats via induction of placental apoptosis

Effects of bestatin on intrauterine growth of rat fetuses

Antitumor cells found in tumor-bearing mice given ubenimex

Effect of bestatin on syngeneic tumors in mice

Antitumor effect of bestatin combined with bleomycin against hepatoma AH 66 subcutaneously transplanted in rats

Preclinical approaches to the development of effective immunotherapeutic protocols for the treatment of metastasis

Inhibition of lymph node metastasis of P388 leukemia by bestatin in mice

Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis

Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells

Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis

Inhibitory effect of rikkunshi-to, a traditional Chinese herbal prescription, on tumor promotion in two-stage carcinogenesis in mouse skin

Enhancement of antitumor effect of cytotoxic agents by bestatin

The effect of ubenimex on N-methyl-N¢-nitro-N-nitrosoguanidine-induced stomach tumor in rats

Spinorphin as an endogenous inhibitor of enkephalin-degrading enzymes: Roles in pain and inflammation

Use of aminopeptidase M as a hypotensive agent in spontaneously hypertensive rats

Studies on the mechanisms of action of the immunomodulator bestatin in various screening test systems

Enhancement by ubenimex (bestatin) of host resistance to Candida albicans infection

Enhancement of antibody formation against herpes simplex virus in mice by the T-cell mitogen bestatin

Stimulation of cell-mediated immunity by bestatin correlates with reduction of bacterial persistence in experimental chronic Salmonella typhimurium infection

Study of the prophylactic effect of ubenimex on experimental pyelonephritis induced by Pseudomonas in neutropenic mice

The mode of immunopotentiating action of bestatin: enhanced resistance to Listeria monocytogenes infection

Analgesic effect of actinonin, a new potent inhibitor of multiple enkephalin degrading enzymes

Repeated systemic administration of the mixed inhibitor of enkephalin-degrading enzymes, RB101, does not induce either antinociceptive tolerance or cross-tolerance with morphine

Pain-suppressive effects on various nociceptive stimuli (thermal, chemical, electrical and inflammatory) of the first orally active enkephalin-metabolizing enzyme inhibitor RB 120

Long lasting antinociceptive properties of enkephalin degrading enzyme (NEP and APN) inhibitor prodrugs

Depressant effect on a C-fibre reflex in the rat, of RB101, a dual inhibitor of enkephalin-degrading enzymes

Facilitation of enkephalins catabolism inhibitor-induced antinociception by drugs classically used in pain management

Immunotherapy with bestatin for acute nonlymphocytic leukemia in adults

Results of follow-up studies on prognosis after immunotherapy with bestatin in acute nonlymphocytic leukemia

Monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation

The effect of bestatin on patients with acute and chronic leukemia and malignant lymphoma

Bestatin treatment of myelodysplastic syndromes and chronic myelogenous leukemia

Immunopotentiation with Ubenimex for prevention of leukemia relapse after allogeneic BMT. The Study Group of Ubenimex for BMT

Partial review of immunotherapeutic pharmacology in stem cell transplantation

Randomized double-blind placebo-controlled trial of bestatin in patients with resected stage I squamous-cell lung carcinoma

Bestatin in resected lung cancer. A randomized clinical trial

Antisense-mediated inhibition of aminopeptidase N (CD13) markedly decreases growth rates of hematopoietic tumour cells

Inhibition of aminopeptidase B and leucine aminopeptidase by bestatin and its stereoisomer

Action of ubenimex on aminopeptidase activities in spleen cells and peritoneal macrophages from mice

Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme

Enhancement of delayed-type hypersensitivity by bestatin, an inhibitor of aminopeptidase B and leucine aminopeptidase

The slow, tight binding of bestatin and amastatin to aminopeptidases

A new human peptide deformylase inhibitable by actinonin

Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics

Human meprin alpha and beta homo-oligomers: Cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors

Endostatin binds to the catalytic domain of matrix metalloproteinase-2

Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin

Curcumin exhibits antimetastatic properties by modulating integrin receptors, collagenase activity, and expression of Nm23 and E-cadherin

Curcumin (diferuloylmethane) down-regulates cigarette smoke-induced NF-kappaB activation through inhibition of IkappaBalpha kinase in human lung epithelial cells: Correlation with suppression of COX-2, MMP-9, and cyclin D1

Betulinic acid suppresses carcinogen-induced NF-kappa B activation through inhibition of I kappa B alpha kinase and p65 phosphorylation: Abrogation of cyclooxygenase-2 and matrix metalloprotease-9

Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-kappaB signaling

Curcuminoids inhibit the angiogenic response stimulated by fibroblast growth factor-2, including expression of matrix metalloproteinase gelatinase B

Characterization of aminopeptidase N from the brush border membrane of the larvae midgut of silkworm, Bombyx mori as a zinc enzyme

Metabolism of aspartame by human and pig intestinal microvillar peptidases

Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor

The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N

The aminopeptidase activity in the human T-cell lymphoma line (Jurkat) is not at the cell surface and is not aminopeptidase N (CD-13)

Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M

Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney

Enzymic and molecular properties of aminopeptidase W

Insights into peptide and protein function: A convergent approach

After a post-doctoral stay with Professor A. G. M. Barrett at the Imperial college in London, he joined the CNRS (Centre National de la Recherche Scientifique) at Institut Curie (Paris), being appointed «Attaché de Recherche», then «Directeur de Recherche

Directeur de Recherche'' in 1993. She is appointed to her current position in the INSERM laboratory of Prof