Novel aminopeptidase N inhibitors derived from 1,3,4-thiadiazole scaffold

The aminopeptidase N (APN/CD13), overexpressed in tumor cells, plays a critical role in angiogenesis. In this study, we report the synthesis and in vitro enzyme inhibition assay of 1,3,4-thiadiazole scaffold compounds. These new compounds have potent inhibitory activities toward APN with IC(50) values in the micromol rang.     

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

In the present study, we characterized in monocytes the rise in [Ca(2+)](i) evoked by monoclonal antibodies (mAbs) to aminopeptidase N (APN)/CD13, showing a two-phase calcium increase with a small-belled [Ca(2+)](i) rise due to the release of calcium from intracellular stores and a more sustained plateau due to the influx of calcium from the extracellular environment. Tyrosine kinase inhibitors were able to inhibit the rise in [Ca(2+)](i) induced by ligation APN/CD13, as were inhibitors of the phosphatidylinositol 3-kinase. For the first time we can show that mAbs to APN/CD13 provoke phosphorylation of the mitogen-activated protein kinases ERK1/2, JNK, and p38. Furthermore, we show that mRNA of the chemotactic cytokine IL-8 is upregulated under the influence of APN/CD13 ligation. Although the in vivo ligand as well as possible cooperating membrane molecules remains to be identified, our results suggest that the membrane ectoenzyme APN/CD13 is a novel signal transduction molecule in monocytes.     

Amino-benzosuberone: a novel warhead for selective inhibition of human aminopeptidase-N/CD13

This paper describes the design and synthesis of compounds belonging to a novel class of highly selective mammalian CD13 inhibitors. Racemic homologues of 3-amino-2-tetralone 1 were synthesised and evaluated for their ability to selectively inhibit the membrane-bound, zinc-dependent aminopeptidase-N/CD13 (EC 3.4.11.2). Some of these novel non-peptidic compounds are potent, competitive inhibitors of the mammalian enzyme, with K(i) values in the low micromolar range in spite of their minimal size (MW <200 Da). Moreover, they show an interesting selectivity profile against representative members of the aminopeptidase family, that is leucine aminopeptidase (EC 3.4.11.1), Aeromonas proteolytica aminopeptidase (EC 3.4.11.10) and the aminopeptidase activity of leukotriene A4 hydrolase (EC 3.3.2.6). The amino-benzosuberone derivative 4 is the most promising compound in terms of potency, stability and selectivity. A hypothetical binding mode of 4 to the catalytic zinc and several conserved active site residues is proposed, based on the observed structure-activity relationships, structural insights from aminopeptidase-N homologues of known three-dimensional structure.     

Novel β-dicarbonyl derivatives as inhibitors of aminopeptidase N (APN)

Most zinc metalloproteases are over-expressed in tumor cells and play a critical role in the genesis, development, and metastasis of tumors. Novel zinc binding groups (ZBGs) represent a novel strategy to obtain optimal potency and selectivity for zinc metalloproteases inhibitors. Here we described the design, synthesis, and biological studies of novel β-dicarbonyl derivatives as aminopeptidase N (APN/CD13) inhibitors. The results demonstrated that some compounds exhibited moderate to good inhibitory activities against APN with compound 5c being the most potent, suggesting that 5c could serve as new lead for the future APN inhibitor development. The results further confirm our design rationale of β-dicarbonyl moiety as a new ZBG, which may provide a new direction for the design and discovery of zinc metalloproteases inhibitors as new anti-tumor agents.     

Novel leucine ureido derivatives as inhibitors of aminopeptidase N (APN)

Aminopeptidase N (APN/CD13) over expressed on tumor cells, plays a critical role in tumor invasion, metastasis, and tumor angiogenesis. Here we described the design, synthesis and preliminary activity studies of novel leucine ureido derivatives as aminopeptidase N (APN/CD13) inhibitors. The results showed that compound 8c had the most potent inhibitory activity against APN with the IC₅₀ value to 0.06 ± 0.041 μM, which could be used for further anticancer agent research.     

Design, synthesis and biological evaluation of novel L-lysine ureido derivatives as aminopeptidase N inhibitors

As the exopeptidase over-expressed in the cell surface of endothelial cells, aminopeptidase N (APN/CD13) is an essential target for tumor angiogenesis and metastasis. Based on the previous work of L-lysine amide derivatives in our laboratory, we designed and synthesized two series of L-lysine ureido derivatives as APN inhibitors. Within these compounds, one compound, 5d (IC?? = 4.51 μM), showed similar inhibitory effect compared with Bestatin (IC?? = 5.87 μM).     

A novel aminopeptidase N inhibitor developed by virtual screening approach

A virtual screening was performed to discover novel lead structures as potent aminopeptidase N(APN) inhibitors. A commercial database containing about 1,60,000 molecules in SPECS was filtered by rule of five, zinc binding groups, pharmacophore models and binding pattern analysis. At last, 24 molecules were selected for enzyme inhibition assay and compound 2 exhibited the inhibition constant (K(i)) of 2.79±0.32μM against APN compared with Bestatin (K(i)= 3.37±0.24μM). Our results indicated that compound 2 exhibited good antiproliferative activities against a broad spectrum of human cancer cell lines, and induced cell cycle arrest at G1 phase and eventual apoptosis. Moreover, compound 2 can inhibit the invasion of MDA-MB-231 cells. In summary, our results suggest that compound 2, a potent APN inhibitor, is worthy of further development.     

Design, synthesis and biological evaluation of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as aminopeptidase N/CD13 inhibitors

A series of novel 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were designed, synthesized and assayed for their activities against aminopeptidase N (APN/CD13) and MMP-2. The results showed that most compounds exhibited higher inhibitory activities against APN than that of MMP-2. Within this series, compound 12h (IC(50)=6.28 ± 0.11 μM) showed similar inhibitory activities compared with Bestatin (IC(50)=5.55 ± 0.01 μM), and it could be used as novel lead compound for the future APN inhibitors development as anticancer agents.     

Design and synthesis of novel chloramphenicol amine derivatives as potent aminopeptidase N (APN/CD13) inhibitors

Herein we report a series of novel chloramphenicol amine derivatives as aminopeptidase N (APN)/CD13 inhibitors. All compounds were synthesized starting from commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol. The preliminary biological screening showed that some compounds exhibited potent inhibitory activity against APN. It should be noted that one compound, 13b (IC₅₀ = 7.1 μM), possess similar APN inhibitory activity compared with Bestatin (IC₅₀ = 3.0 μM).     

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC?? of 12.2 μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC??) of 7.3 μM), but also had a potent antiproliferative activity against human cancer cell lines cells.     

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

A series of phosphinic compounds mimicking the transition state of substrates hydrolysed by aminopeptidase N (EC 3.4.11.2) were synthesized. These new compounds have potent inhibitory activities with Ki values in the nanomolar range. These derivatives behave as the most potent APN inhibitors designed to date.     

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication.     

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC₅₀ of 12.2?μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC₅₀) of 7.3?μM), but also had a potent antiproliferative activity against human cancer cell lines cells.     

Novel aminopeptidase N inhibitors derived from antineoplaston AS2–5 (Part I)

Overexpression of zinc-dependent metalloproteinase, aminopeptidase N (APN/CD13), is considered to be involved in the process of tumor invasion and metastasis. Herein we describe the synthesis and in vitro enzymatic inhibition assay of antineoplaston AS2–5 scaffold peptidomimetic compounds. The results demonstrated that most of these l-iso-glutamine derivatives displayed selective inhibitory activity against APN as compared with MMP-2, with IC₅₀ values in the micromole range. The structure–activity relationships were also briefly discussed.     

Design, synthesis and biological evaluation of novel amino acid ureido derivatives as aminopeptidase N/CD13 inhibitors

A series of amino acid ureido derivatives as aminopeptidase N (APN/CD13) inhibitors were synthesized and evaluated for their APN inhibitory activities and anti-cancer effects. The results showed that most of these amino acid ureido derivatives exhibited good inhibition against APN, several of which were better than Bestatin. The most active compound 12j (IC(50) = 1.1 μM, compared with Bestatin IC(50) = 8.1 μM) not only possessed much better APN inhibitory activity and anti-proliferation effect on cancer cells, but also exhibited significant block effect of human cancer cell invasion compared with the positive control, Bestatin. These amino acid ureido derivatives could be possibly developed as new APN inhibitors for cancer chemotherapy in the future.     

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

In the course of our screening, N-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide (1), which contains a metal-chelating hydroxamate group, has been identified as a potent inhibitor of aminopeptidase N (APN, EC 3.4.11.2). Compound 1 potently inhibited APN activity with a K(i) value of 3.5 microM. It also inhibited the basic fibroblast growth-factor-induced invasion of bovine aortic endothelial cells at low micromolar concentrations.     

Enkephalin-degrading enzyme inhibitors. Crucial role of the C-terminal residue on the inhibitory potencies of retro-hydroxamate dipeptides

The retro-inversion of the amide bond in kelatorphan and analogs, the first series of complete inhibitors of enkephalin metabolism, led to compounds highly efficient only against the neutral endopeptidase 24-11 (NEP). In order to increase the recognition of the aminopeptidase N (APN) and dipeptidylaminopeptidase (DAP), without loss of affinity for NEP, the malonyl group of these retro-inhibitors was replaced by diversely substituted succinyl moieties. All the molecules synthesized are highly efficient NEP inhibitors with Ki's in the 0.2-1 nM range, indicating that NEP possesses a relatively large and not very selective S'2 subsite. In contrast, inhibition of DAP activity is crucially dependent on the size and the position of the substituent in the succinyl moiety. Inhibitory potencies in the nanomolar range are obtained with compounds containing a benzyl group in the alpha-position related to the retro amide bond. Finally, a relatively modest inhibition of APN was observed with Ki's in the 0.5-1 microM range for compounds with benzyl or cyclohexyl group in P'2 position. However, these data demonstrate that efficient and complete inhibition of enkephalin degradation can be obtained with hydroxamate dipeptides containing a retro amide bond. The analgesic potency of the most active inhibitors was measured using the hot plate test in mice. Significant antinociceptive responses were obtained but these effects were rather weaker than those expected from the in vitro inhibitory potencies of these compounds on the three enkephalin-degrading enzymes.     

Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a.

Rapid increases in the membrane expression of C3 receptors on granulocytes and monocytes in response to the anaphylatoxin C5a have previously been described. In this study we demonstrate increases in the membrane expression of neutral endopeptidase (NEP, CD10, CALLA), aminopeptidase N (APN, CD13), tyrosine phosphatase (CD45/CD45Ro) and the Fc R Fc gamma-RIII (CD16) on granulocytes within minutes of treatment with human C5a. Monocytes responded to C5a with increases in CD13 and CD45/CD45Ro. These membrane modulations could be prevented by preincubating the C5a preparations with anti-C5a mAb C17/5 but not by pretreating the cells with cycloheximide. Increases of CD10, CD13, and CD11b but not CD11a (LFA-1) were also observed in leukocytes from patients undergoing hemodialysis with cuprophan membranes. The increase of CD16 on granulocytes was dependent on the presence of plasma during in vitro activation with C5a indicating that plasma contains inhibitors which prevent the previously described loss of Fc gamma-RIII upon stimulation of the cells.