Positioning of Inhibitors in Next Generation Cancer Therapy

Sarina M Hitzerd, Sue Ellen Verbrugge, Gert Ossenkoppele, Gerrit Jansen, Godefridus J. Peters.

7 Amino Acids, accepted for publication Submitted

Departments of Medical Oncology, Hematology, Rheumatology VU University Medical Center, Amsterdam, The Netherlands

149 Abstract

Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in of bonds. operate downstream of intracellular degradation either by trimming proteasome-generated the ubiquitin-proteasome pathway and are implicated in the final step of for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function aminopeptidase (LAP), puromycin-sensitive aminopeptidase (PuSA), and subcellular location of five key aminopeptidases (aminopeptidase N (APN), A4 (LTA 4) and endoplasmic reticulum aminopeptidase 1/2 (ERAP1/2)) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. entered the clinic 35 years ago and is still used for the treatment of lung cancer. Historically, bestatin was the first prototypical aminopeptidase inhibitor that More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anticancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy growing body of data point to aminopeptidase inhibitors as attractive tools for and/or loss of efficacy due to drug resistance-related phenomena. Together, a combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients.

150 Introduction

More than fifty years ago, Rutenburg et al. and Willighagen and Planteydt cancer.1,2 Rutenburg et al. demonstrated that patients with pancreatic cancer were the first to report the potential relevance of aminopeptidase activity for and urine, whereas patients with a malignant lymphoma or leukemia had had significantly increased leucine aminopeptidase (LAP) activity in serum increased LAP activity in urine.1 aminopeptidase activity in tumor cells and stroma in sixty surgically removed Willighagen and Planteydt also observed higher human neoplasms.2 Twenty years later, Umezawa et al. discovered one of the group of gram-positive bacteria.3 first aminopeptidase inhibitors; bestatin, being produced by actinomycetes, a Aminopeptidases, a class of (zinc) metalloenzymes, catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, facilitating hydrolysis of the peptide bond.4 Binding of one or two metal ions, mostly zinc, is required for the activity of the aminopeptidases. Some of these require two metal the second metal ion can modulate the activity either positively or negatively. ions for full activity, for others only one metal ion is sufficient for , while Aminopeptidases are widely distributed throughout plants, animals, bacteria and fungi, and function in many cellular processes.5 Their function is implicated produced by the ubiquitin-proteasome pathway either for antigen presentation in the final step of intracellular protein degradation by trimming peptides or for full hydrolysis into free amino acids, which can be reutilized for renewed protein synthesis (Figure 1).6 The critical relevance of these functions for cancer progression paved the way to explore inhibitors of aminopeptidases for application as anti-cancer therapeutic drugs. The mechanistic rationale and current status of established and experimental aminopeptidase inhibitors for next generation cancer therapy is discussed hereafter.

151 1. Positioning of Aminopeptidases 1.1 Mechanism of action downstream of the ubiquitin-proteasome pathway The ubiquitin-proteasome pathway is the major proteolytic system in the cytosol of eukaryotic cells, and plays an important role in protein homeostasis, or misfolded . Intracellular ubiquitin-mediated protein degradation is degradation of specific short-lived proteins and rapid elimination of damaged minutes to several days and up to a few years. This process is tightly regulated highly selective; different proteins can have a half-life that varies from a few and has been implicated in numerous key processes such as DNA repair, cell- cycle progression, , transcriptional regulation, receptor down-regulation, and gene expression. The ubiquitin system is also essential for immune response, development, and programmed cell death.7,8 Two main steps are involved in the degradation of a protein via the ubiquitin- proteasome pathway: 1) labeling of the protein by the covalent attachment of multiple ubiquitin (Ub) molecules and 2) degradation of the labeled protein by the 26S proteasome complex. These steps require the sequential action of three short peptides, ultimately generated by the ubiquitin-proteasome processes enzymes; E1 (Ub-activation), E2 (Ub-conjugation) and E3 (Ub-ligation). The are short-lived and do not accumulate in cells, but are further degraded to free amino acids by cytosolic peptidases, such as aminopeptidases.9–15 Aminopeptidases directly degrade the smallest products (2-6 amino acids) released from the proteasome complex, whereas larger peptides (6-24 amino acids) are primarily cleaved by , such as thimet (TOP) and II (TPPII), into shorter peptides (2-6 amino acids), which subsequently can be fully hydrolyzed by aminopeptidases (Figure protein synthesis.6,16 1; Complete hydrolysis) to free amino acids being available again for new A very small fraction of the proteasome products can escape the complete hydrolysis and is utilized for major histocompatibility complex (MHC) class are transported from the cytosol into the endoplasmic reticulum (ER) by I antigen presentation (Figure 1; Antigen presentation). These peptides the transporter associated with antigen processing (TAP). Additionally, the N-terminal extension of peptides can be processed before transportation

(cytosol) or after transportation (ER) by specific aminopeptidases. This 152 Figure. 1. Protein degradation pathway; role of proteasome and aminopeptidases. Abbreviations: aa, ; TOP, thimet oligopeptidases; MHC, major histocompatibility complex. Modified from Saric et al.200455.

phenomenon is also called ‘trimming’.6,11,17 molecules bind the (trimmed) peptides (8-10 amino acids) and expose them Subsequently, the MHC class I on the cell surface for recognition by cytotoxic T lymphocytes and initiating an immune response.18 The role of aminopeptidase activity in antigen presentation has been subject of various reviews.19–25 This review will primarily focus on the role of aminopeptidases in the peptide hydrolysis to amino acids from a cancer perspective.

1.2 Function and location of different aminopeptidases and their association with different diseases

(aminopeptidase N (APN), leucine aminopeptidase (LAP), puromycin-sensitive The function and subcellular location of at least five aminopeptidases aminopeptidase (PuSA), leukotriene A4 (LTA 4) hydrolase and endoplasmic reticulum aminopeptidase 1/2 (ERAP1/2)) has been associated with the pathophysiology of different non-malignant diseases (discussed in this

153 paragraph) as well as with (different types of) cancer (discussed in paragraph 2.3). Aminopeptidases can be subdivided into three general groups, based on iminoacyl-peptide ), 2) remove dipeptides from polypeptide chains their function, those that: 1) hydrolyze the first peptide bond (aminoacyl- and (dipeptidyl-peptide hydrolases), and 3) only act on tripeptides (tripeptidyl- peptide hydrolases).26 They can also be subdivided based on structural

4 hydrolase and ERAP1/2) belong to the M1 zinc-aminopeptidases subfamily, which harbor characteristics. Four of the five aminopeptidases (APN, PuSA, LTA a consensus HEXXH(18X)E motif for zinc binding. This zinc ion binding is essential for the enzymatic activity of aminopeptidases.27 LAP is a member of the peptidase M17 family.

1.2.1 Aminopeptidase N (APN)

28 Upon Aminopeptidase N (APN, also known as CD13) has been referred to as a ligand binding, APN can operate as an , a receptor and/or signaling ‘moonlighting ectoenzyme’, because it can fulfill a multitude of functions. molecule. Each of these three functions is associated with their own mechanism

Subsequently, each of these three mechanisms elicited different biological of action; peptide cleavage, endocytosis and signal transduction, respectively. from hydrolysis of proteins and polypeptides, in particular those involved in effects. Generally, APN plays a role in the final digestion of peptides generated the metabolism of various regulatory peptides that impact the function of small intestinal and tubular epithelial cells, macrophages, granulocytes and synaptic 28,30 Moreover, APN can cleave antigenic peptides 20 membranes from the CNS. prior to binding to MHC class II molecules of antigen presenting cells. 1.2.2 Leucine aminopeptidase (LAP) Unlike APN, leucine aminopeptidase (LAP, also known as cytosol aminopeptidase) is less well characterized. It catalyzes the removal of unsubstituted N-terminal amino acids from various peptides and is presumably involved in the processing and regular turnover of intracellular proteins.30 Subsequently, it processes 31 LAP is antigenic peptides for presentation by the MHC class I molecules. been implicated in a small variety of pathophysiological states, including HIV located in the cytoplasm, but data on tissue specificity are scarce. LAP has

154 infection, systemic lupus erythematosus and malaria.32–34

1.2.3 Puromycin-sensitive aminopeptidase (PuSA) several peptides and is involved in proteolytic events essential for cell growth Puromycin-sensitive aminopeptidase (PuSA) has broad substrate specificity for and viability.35 As for APN, PuSA is thought to act as a regulator of neuropeptide activity36 and plays an important role in the antigen-processing pathway for 25,37,38 PuSA is also able to digest polyglutamine (polyQ) peptides found in many cellular proteins.39 MHC class I molecules. PuSA is localized in both the cytoplasm and cellular membranes, and was found in liver, epithelium of renal tubules, epithelium of small and large intestine, gastric epithelial cells, and alveoli of the lung.40 PuSA was found to be involved in the degradation of tau, which does it patients.41,42 more efficiently in normal brain compared to brain from Alzheimer disease

1.2.4 Leukotriene A4 (LTA4) hydrolase

Leukotriene A4 (LTA 4) hydrolase is involved in the removal of a single N-terminal amino acid residue and exhibits a variety of important biological functions, including the processing of cell surface antigens and involvement in tumor angiogenesis. LTA4 hydrolysis catalyzes the formation of the chemotaxin

Leukotriene B4 (LTB4 stimulates adhesion of circulating neutrophils to vascular endothelium and ), a key lipid mediator of the innate immune response; it 43–45 LTA hydrolase is compartmentalized in lipid-rich organelles (lipid droplets) directs4 their migration to sites of inflammation. residing in the cytoplasm and is expressed in monocytes, lymphocytes, 46–48 LTB neutrophils,4 reticulocytes, platelets and fibroblasts. due to these biological activities of LTB , LTA hydrolase is involved in a variety plays an important role in a variety4 of allergic4 and inflammatory reactions, chronic obstructive pulmonary disease and asthma.47–51 of acute and chronic inflammatory diseases, e.g., nephritis, arthritis, dermatitis,

1.2.5 Endoplasmic reticulum aminopeptidase 1/2 (ERAP1/2) also known as puromycin-insensitive leucine aminopeptidase (PILSAP) and The main function of endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1;

155 ERAP2) involves trimming of HLA class I-binding precursors in the endoplasmic monomer, but mostly function as heterodimers allowing them to combine reticulum (ER) for MHC class I antigen presentation. ERAP1 and 2 can act as their restricted specificities to remove complex N-terminal extensions. In substrates between nine and sixteen amino acid residues long and thereby addition, ERAP1 has a unique substrate preference; it strongly prefers peptide covers the formation of about one third of peptide–MHC class I complexes. substrates. Both enzymes are also thought to play a role in the inactivation of ERAP2 presents distinct specificity for the N-terminal residue of the peptide peptide hormones.52–57 ERAP1 is also found to be involved in blood pressure regulation by inactivation of angiotensin II.58 ERAP1 and 2 are localized on the ER membrane and are ubiquitously expressed, mostly in spleen and leukocytes.52

1.3 Relation between aminopeptidases and cancer Aminopeptidases are essential for physiologically important processes such as protein maturation, degradation of peptides, and cell cycle control. For cancer cells, the supply of cellular free amino acids, regulated by aminopeptidases, is of utmost importance for their survival and proliferation. Importantly, many amino acids has a greater impact on cancer cells than normal cells.59 Moore tumor cells are dependent on specific amino acids and depletion of these et al. indicated that myeloid leukemia and multiple myeloma cells were highly dependent on the unfolded protein response in which aminopeptidases play an important role.60 resulted in marked inhibition of myeloma cell growth and survival, and thus of Consistently, this study showed that aminopeptidase inhibition potential therapeutic interest. Martinez et al. documented both up- and down regulation of selective aminopeptidase activities in breast cancer tissue. These alterations were dependent on local hormonal status, indicating that tumor microenvironment plays a role in regulating aminopeptidase expression.61 expression and tissue distribution of endoplasmic reticulum aminopeptidase Cifaldi et al. discussed the outcome of six different studies that assessed the 1 and 2 (ERAP1 and ERAP2) in tumor cells of lymphoid and non-lymphoid origin compared to their normal counterparts.62 In one study including eleven different tumor cell lines (melanomas, leukemia-lymphomas and carcinomas of breast, colon, lung, chorion, skin, prostate, cervix, kidney and bladder),

156 ERAP1 and 2 were expressed at highly variable levels. In a second study, the expression of ERAP1 and ERAP2 was either lost, acquired or retained in 150 surgically removed neoplastic lesions as compared to their normal histotype counterparts. Down-regulation of ERAP1 and/or ERAP2 expression was mainly found in ovarian, breast and lung carcinomas, whereas an up-regulation of these enzymes was observed in colon and thyroid carcinomas. A third study reported heterogeneous expression of ERAP1 and ERAP2, ranging from high to very low levels, in 28 melanoma cell lines as compared to primary melanocytes. Three other studies demonstrated ERAP1 expression in 64% of endometrial carcinomas, in which ERAP1 may function to suppress angiogenesis and endothelial cell migration. Both ERAP1 and 2 are present in leukemia, lymphoma, carcinoma, and melanoma cell lines.63–66 Mostly ERAP1 is involved in a few types of cancer, such as endometrial carcinoma and cervical carcinoma.66,67 Most studies on aminopeptidase activity in correlation with cancer are focused on aminopeptidase N (APN), a zinc-dependent membrane-bound ectopeptidase that degrades preferentially proteins and peptides with a N-terminal neutral amino acid. Tokuhara et al. investigated the clinical significance of show a relationship between APN expression and poor prognosis of patients with aminopeptidase N in non−small cell lung cancer (NSCLC) and were the first to 68 Van Hensbergen et al. found elevated soluble APN activity in plasma and effusions of cancer patients, which was strongly correlated with tumor load.69 NSCLC. APN also appeared to be involved in cell motility of thyroid carcinoma cells,70 of which undifferentiated anaplastic thyroid carcinomas had a higher APN expression than differentiated thyroid carcinomas. Increased APN expression neck squamous cell carcinoma,71 acute myeloid leukemia, prostate cancer and was associated in the pathophysiology of additional types of cancer; head and colon cancer.72–75 Beyond this, APN was also found to be selectively expressed in the vasculature of tissues that undergo angiogenesis, e.g. in malignant gliomas and lymph node metastases from multiple tumor types, but not in blood vessels of normal tissues.76 In line with these observations, APN serves as a receptor vasculature.77 for a specific motif (NGR), which is expressed on endothelial cells of angiogenic was related to a more malignant phenotype. Collectively, it can be concluded that an increased APN expression Other than for APN and ERAP, cancer-related involvement of additional

157 aminopeptidases is less well documented. Notwithstanding this fact, leucine aminopeptidase (LAP) activity appeared to play a role in prostate carcinoma and head and neck cancer, PuSA activity in clear cell renal cancer and prostate adenocarcinoma, and LTA4 hydrolase in lung cancer, esophageal adenocarcinoma, colon cancer and pancreatic cancer.78–85 In conclusion, there is a growing body of evidence that pinpoint aminopeptidase activity, especially APN and ERAP1/2, to a great variety of cancer types and their progressive and proliferative state.

2. Mechanism of action of aminopeptidase inhibitors discovered by Umezawa et al., was originally designed as an immune- The first clinically approved aminopeptidase inhibitor bestatin (), modulating agent.3 Follow up research demonstrated that bestatin also harbored antiproliferative effects and displayed activity as an anti-cancer drug, corroborating the relevance of aminopeptidases in cancer tissue.1,2,86,87 As a mechanism of action, Taylor revealed that bestatin was tightly bound to the aminopeptidases LAP and APN.4 Each subunit of LAP was capable of bestatin exert an inhibitory effect. Additionally, Botbol and Scornik noted that bestatin binding, but the binding of one bestatin molecule was already sufficient to induced the accumulation of di- and tripeptide intermediates, again indicating aminopeptidase inhibition as a mechanism of action.88 Almost two decades later, Krige et al. provided evidence that for the aminopeptidase inhibitor prodrug of intracellular amino acids, which suppressed cell growth.89 tosedostat (CHR2797), its main mechanism of action was to provoke a depletion earlier studies, also tosedostat exposure introduced intracellular accumulation Consistent with of small peptides. Intracellular amino acid depletion triggers the so-called amino acid deprivation response (AADR), which is involved in transcriptional and post-transcriptional regulatory mechanisms, such as upregulation of amino acid synthetic genes, amino acid transporters, and tRNA synthetases (Figure 2). Lastly, Krige et al observed that aminopeptidase inhibition reduced the phosphorylation of mammalian target of rapamycin (mTOR) substrates to suppress rates of protein synthesis.89 mTOR is a protein kinase known as the master regulator of protein synthesis, cell growth and proliferation.90 The mTOR protein exists of two distinct multi-protein complexes; mTOR complex

158 Figure. 2. Mechanism of action of aminopeptidase inhibitors bestatin and tosedostat. Aminopeptidase inhibitors elicits two main effects: 1) amino acid deprivation response (AADR) and 2) inhibition of mTOR. Modified from: Löwenberg et al. 2010.96

synthesis by phosphorylating the eukaryotic initiation factor 4E-binding protein 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 stimulates protein 1 (4E-BP1) and the p70 ribosomal 6S kinase 1 (S6K1). Phosphorylation of 4E- BP1 prevents its binding to eukaryotic initiation factor 4E (eIF4E), enabling eIF4E to promote cap-dependent translation. The induction of S6K1 activity by and elongation, and the translation of ribosomal proteins through regulation of mTORC1 leads to an increase in mRNA biogenesis, cap-dependent translation the activity of many proteins. Amino acids can strongly regulate mTORC1 activity, largely unresolved. Recent evidence suggested that the amino acid leucine is but the mechanism by which intracellular amino acids signal to mTORC1 is still glutamine-dependent fashion.91 Next, the Rag proteins, a family of four related essential for mTORC1 activation, levels of which rely on transport into cells in a

92 In contrast small GTPases, also interact with mTORC1 in an amino acid-sensitive manner and are also necessary for the activation of the mTORC1 pathway.

159 to mTORC1, mTORC2 activity is less directly affected by amino acid depletion, though through facilitating phosphorylation of Akt, it can promote mTORC1 survival, metabolism, proliferation and cytoskeleton organization.91,93,94 activity as a compensatory mechanism. As such, mTORC2 plays key roles in cell

AADR and reduction of mTOR activity, which ultimately results in the inhibition Collectively, aminopeptidase inhibitors elicit their effect mainly by induction of of cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription.90,95,96 This unique mechanism of action merits further clinical exploitation and implementation in current cancer chemotherapy.

3. Aminopeptidase inhibitors in cancer therapy

Drug Administration (FDA) have approved any aminopeptidase inhibitor Currently, neither the European Medicines Agency (EMA) nor the Food and in an anticancer treatment setting. However, some clinical trials are ongoing or completed, being most advanced for bestatin (Ubenimex) and tosedostat is a hydrophobic aminopeptidase inhibitor prodrug that is rapidly taken up (CHR2797). Whereas bestatin is a direct aminopeptidase inhibitor, tosedostat by cells and then intracellular activated by de-esterification into a hydrophilic pharmacologically active acid product (CHR79888). This hydrophilic aminopeptidases, with preference for LTA hydrolase, APN and LAP.89 Below, metabolite is efficiently retained in cells to exert4 an inhibitory effect to multiple different aminopeptidases will be discussed in the context of clinical cancer therapy and the development of next generation experimental aminopeptidase inhibitors.

3.1 Aminopeptidase inhibitors tested in the clinic Bestatin was used in Japan as an immunomodulator and antitumor drug (lung cancer and acute myeloid leukemia), under the trademark Ubenimex (Nippon 86 Its broader clinical development proceeds at a low scale. The outcome of recent clinical studies in solid tumors and leukemia with single Kayaku Co, Tokyo). agent bestatin and tosedostat are shown in Table 1.

3.2 New compounds in development or via prodrugs, which are enzymatically metabolized into a pharmacologically There are two main approaches for aminopeptidase targeting; by direct inhibition

160 Table 1. Overview clinical studies with aminopeptidase inhibitors bestatin and tosedostat.

Phase n Age Design/ Schedule Activity Ref. (years) Cohort I 40 Advanced solid 24-80 10 mg (oral) tose- Most commonly observed toxici- 111 (f.i.m.) tumor dostat daily for 7, ties: fatigue, diarrhea, peripheral 14, 21 or 28 days edema, nausea, dizziness, and (increased duration constipation. 1 patient had study). 10-320 mg partial response (renal cell carci- (oral) tosedostat noma) and 4 patients had stable daily for 28 days disease (>6 months). Acceptable (dose escalation safe dose is 240 mg/day. study) I 16 Elderly and/ 45-84 60-180 mg (oral) Most commonly reported severe 96 or relapsing/ tosedostat daily adverse event was reduction in refract-tory for 84 days (MTD the platelet count (56%). 130 mg patients with determined during tosedostat is well tolerated. AML or MDS or MM first 28 days) II 41 Elderly and/ 34-82 130 mg (oral) Objective response rate of 27% 96 or relapsing/ tosedostat daily for (age > 60 years) and 79% had refract-tory 84 days relapsed/refractory AML. The patients with median duration of responses AML or MDS was 95 days (range 28-478 or MM antileukemic activity. days). Tosedostat has significant III 400 Resected stage 41-76 30 mg (oral) 5-year survival rate was 81.0% 105 (P.R.) I squamous- bestatin or placebo of bestatin group and 74.2% of cell carcinoma daily for 2 years placebo group. 5-year disease (adjuvant therapy) free survival rate was 71.6% of bestatin group and 62.0% of placebo group. Postoperative ad-

improvement juvant setting yields a significant Abbreviations; f.i.m.: first-in-man, P.R.: prospective randomized, AML: acute myeloid leukemia, MDS: high-risk myelodysplastic syndrome, MM: multiple myeloma, MTD: maximum tolerated dose. active acid products 77. In order to improve on selectivity, pharmacokinetics/ dynamics, most rationally designed novel aminopeptidase inhibitors build on bestatin as prototypical compound. Remarkably, most of newly generated compounds came out as inhibitors of APN rather than of other aminopeptidases.

A selection of recently identified experimental aminopeptidase inhibitors; their chemical structure and activity profile is listed in Table 2.

161 TABLE 2. New compounds in development with aminopeptidase inhibitor function.

Aminopepti- Structure Drug ap- Structure Activity (Ref) dase Inhibi- proach based on tor Tosedostat Prodrug Batimastat Anti-proliferative effects against a (CHR-2797) (BB-94) range of tumor cell lines in vitro and in vivo. Selectivity for transformed over non-transformed cells, anti-prolifera- tive effects are much more potent than bestatin.(89) LYP Direct Bestatin Greater inhibitory effect of LYP com- inhibitor pared to bestatin at same concentra- tions in different human ovarian carcinoma cell lines and xenograft mouse models. (104) LYP3 Direct Bestatin LYP3 better water-soluble and much inhibitor higher inhibitory effect of APN com- pared to bestatin in multiple cell lines. (107) 3-amino- Direct Produced by Greater activity than bestatin in an 2-hydroxy- inhibitor the strepto- APN inhibition assay. Not yet been 4-phenylbu- myces strain implicated in cancer therapy.(110) tanoylvalyli- so-leucine HCCB10043 Series of Direct D24 Three compounds 4m, 4t, and 4cc, synthetic inhibitor exhibited most potent APN inhibitory compounds targeting anti-metastasis and anti-angiogenesis APN effectsactivities in vitroand displayed and in vivo, significant compared to bestatin.(112) Series of Direct Synthetic Most of amino acid ureido derivatives amino acid inhibitor compounds exhibited good inhibition against ureido de- designed by APN, several better than bestatin. rivatives (112) Most active compound, 12j, exhibited

cancer cell invasion.(113) significant inhibitory effect of human Novel lead Direct Virtual 24 molecules were selected for structures inhibitor screening of as potent commercial exhibited highest inhibitory effect and APN inhibi- database con- goodenzyme anti-proliferative inhibition assay. activity Compound against 2 tors taining about broad spectrum of human cancer cell 160,000 lines. It induced cell cycle arrest at G1 molecules phase and eventual apoptosis.(103) CIP-13F Direct X-ray struc- - inhibitor ture of APN/ cant delay of Lewis lung carcinoma CIP-13F treatment resulted in a signifi structure pulmonary metastasis and induction bestatinCD13 and (LLC) growth, decrease in number of- creases in microvessel density (MVD) andof LLC angiogenic apoptosis. factors(109). CIP-13F caused de

162 3.3 Aminopeptidase inhibitory profiles of classical and novel experimental aminopeptidase inhibitors Following medicinal chemistry, preclinical evaluation of classical and novel experimental aminopeptidase inhibitors includes assessment of their inhibitory and rodent sources. Table 3 provides an overview of inhibitory potency of potency against one or multiple crude/purified aminopeptidases from human bestatin, tosedostat and selected novel aminopeptidase inhibitors against APN

(most commonly tested), LAP, PuSA, LTA4

-hydrolase and ERAP1. With bestatin against APN, LAP, LTA hydrolase and PuSA, most novel inhibitors displayed APN and tosedostat/CHR798884 as a reference, displaying potent inhibitory effects inhibitory capacity, with the 4cc compound being more potent that bestatin and tosedostat. Overall, natural inhibitors showed lower toxicity, broad spectrum 28 It activity and poor tissue specificity as compared to synthetic inhibitors. remains a challenge to design inhibitors that could selectively target specific diseases (e.g. LTA hydrolase). aminopeptidases,4 which can be implicated in cancer or chronic inflammatory

4. Resistance modalities for aminopeptidase inhibitors Prolonged drug administration often comes along with the onset of acquired drug resistance. Also for aminopeptidase inhibitors, therapy resistance may occur as observed in a phase I/II clinical studies with tosedostat.96 However, the molecular basis for resistance remains elusive. Some mechanisms that resistance. may confer resistance are briefly discussed below just as options to overcome 4.1 Possible mechanisms of resistance One general mechanism of drug resistance relates to cellular extrusion of 97. In fact Grujic et al.,98 showed that inhibition of multidrug resistance-associated protein (MRP) and drugs, mediated by ATP-dependent drug efflux pumps P-glycoprotein (P-gp) enhanced the activity of both bestatin and actinonin,

Additionally, activation of mTOR by free amino acids can induce resistance as suggesting that these compounds may be substrates for these efflux pumps. part of overcoming the amino acid deprivation response.91 These amino acids may be delivered through upregulated expression of amino acid transporters.

163 Table 3.

IC50 (nmol/L) values of aminopeptidase inhibitors involved in cancerAminopeptidase therapy forStructure five differentAPN targetsLAP PuSa LTA4 ERAP1 Ref. Inhibitor hydro- lase Bestatin 300 4 350 200 >5,000 89

Tosedostat 220 100 150 >10,000 >5,000 89 (CHR-2797)

CHR-79888 190 30 850 8 >5,000 89

Actinonin 160-5000 860-1190 116, 98

Amastatin 980 102, 108

Amino acid 1,100 113 ureido derivate (12j) AG-205/ 3,700 103 36450018

PAQ-22 >100,000 3800 106

PAQ-22/36c >100,000 500 106

LYP3 7,200 107

4cc 50 112

99 showed that L-amino acid transporter (LAT1) was upregulated in human ovarian cancer cells and that the inhibition of LAT1 Consistently, Fan et al. like tosedostat, it may be anticipated that down-regulation of carboxylesterases, sensitized cells for bestatin. Conceivably, for aminopeptidase inhibitor prodrugs

164 could be a contributing factor in loss of activity of tosedostat.89 These implicated in the conversion of tosedostat to the active metabolite CHR79888, systems and in a clinical setting. mechanisms warrant further exploration and confirmation in preclinal model 4.2 Combination therapy to bypass resistance Personalized medicine has received considerable attention in current cancer chemotherapeutic approaches. Aminopeptidase inhibitors can either be used in combination with other drugs to enhance their own activity and reduce toxicity, or could constitute synergistic interactions with other chemotherapeutic or therapies. Table 4 depicts an overview of completed and ongoing clinical studies of combination therapies with aminopeptidase inhibitors. As an example,99 increased bestatin activity in human ovarian cancer cells, which may thus be showed that the combination of bestatin and a LAT1 inhibitor significantly considered as an improved treatment option for ovarian cancer patients.100 demonstrated that inhibition of APN by Ubenimex enhanced radiosensitivity of cervical cancer cells in vitro as well as in vivo (mouse models). In the clinical setting tosedostat is combined with standard chemotherapy regimens in order to determine whether this would improve their efficacy. Moreover, inhibition rationale for these combinations. E.g. for the synthesis of RNA and DNA of aminopeptidases leading to amino acid deprivation has a clear scientific precursors amino acids are essential, so that combination with antimetabolites, 101 Moreover, inhibition of either aminopeptidases or the proteasome will prevent degradation of such as with cytarabine in AML is likely to be beneficial. enzymes involved in DNA repair, apoptosis and signaling. Therefore interaction with drugs such as anthracyclines, also used in the treatment of AML are likely to be synergistic. All these options need further investigations. Together, the unique mechanism of action of aminopeptidase inhibitors has attractive options to further explore synergistic drug/therapeutic combinations.

Concluding remarks

A growing body of data has underscored the critical role of aminopeptidases in various types of cancer tissues. One role involves protein/peptidase degradation

165 TABLE 4. aminopeptidase inhibitors Clinical studies of approved cancer therapies combined with Phase n Cohort Age Design/ Schedule Activity(Ref.) (years) P.R. 242 AML & 65-80 Ubenimex group complete daily (combined with showed marginal remission several200 mg/m2 other (iv) cancer BHAC therapeutics) with or improvement of OS without 30 mg ubeni- (115)benefit in RFS and no mex daily Ib 23 Advanced/ >18 135–175mg m2 (iv) refractory paclitaxel once every tolerated. 3 patients solid tumors 3 weeks for 6 cycles hadCombination partial response, is well 12 combined with 90–240 patients stable disease mg tosedostat daily (>3 months) (114) II 60 AML or MDS >18 - Ongoing abine daily (5 days) combinedCytarabine with or decit tose- dostat daily (21 days) (NCT01567059) I/II 96 AML or MDS >18 - Ongoing dine daily (5 days) com- binedCytarabine with tosedostator 5-azaciti daily (21 days) (NCT01636609) II 253 AML >18 45 mg/m2 (iv) Ongoing (Hovon103)

AML or RAEB >66 daunorubicin daily 3 days) with 200 mg/ m2 (iv) cytarabine daily (7 days) and assigned dose of tosedostat (oral) daily (21 days) Abbreviations; P.R.: prospective randomized, AML: acute myeloid leukemia, iv: intravenous, BHAC: behenoyl cytarabine, RFS: relapse-free survival, OS: overall survival, MDS: high-risk myelodysplas- tic syndrome, RAEB: refractory anemia with excess blasts. to free amino acid which is required for renewed protein biosynthesis, while the

Although aminopeptidases represent attractive candidates for therapeutic other role involves trimming of antigenic peptides for MHC class I presentation. intervention, development of aminopeptidase inhibitors is in a relatively early stage, compared to the development of inhibitors of the proteasome, which functions upstream of aminopeptidases in protein degradation. This may be related to the broad spectrum of functions regulated by individual aminopeptidases preventing a specific inhibition. Moreover aminopeptidase 166 inhibition may affect various physiological processes (e.g. cell adhesion, enzymatic regulation of peptides, differentiation, proliferation, chemotaxis, antigen presentation, cholesterol metabolism, phagocytosis and angiogenesis). However, in a cancer therapeutic setting, two of the most advanced studied aminopeptidase inhibitors (i.e. bestatin and tosedostat) were generally well tolerated with most of the patients only experiencing mild adverse events (grade 1-2). Another unresolved issue relates to the fact that aminopeptidases have a broad and overlapping substrate specificity, and therefore inhibition design selective inhibitors for individual aminopeptidases and explore may not always be specific. It is a challenge for medicinal chemists to rationally or even non-malignant diseases (e.g. HIV, malaria, Alzheimer disease, chronic whether this could elicit differential effects against specific types of cancer, inflammatory diseases). Expanded knowledge of the mechanism of action and aminopeptidase inhibitors in future cancer chemotherapy. Lessons learned putative resistance modalities may also help to define optimal application of from (pre)clinical investigations with tosedostat highlighted the impact of the amino acid depletion and inability to deal with the associated amino acid deprivation response as a critical factor in suppressing cancer cell growth.89 Hence, promoting compensatory effects for amino acid depletion could dictate their unique mechanism of action, it is anticipated that the most successful the efficacy of aminopeptidase inhibitors as stand-alone drugs. However, given application will adhere to combinations with other chemotherapeutic drugs. As such, aminopeptidases and their inhibitors hold promise for future rationally designed chemotherapeutic applications.

167 References 1

Rutenburg A, Goldbarg J, Pineda E. Leucine aminopeptidase activity; observations in patients 3 Umezawawith cancer H, of Aoyagi the pancreas T, Suda H.and Bestatin, other diseases. an inhibitor N Engl of aminopeptidaseJ Med 1958;259:469–72. B, produced by actino- 2 Willighagen R, Planteydt H. Aminopeptidase activity in cancer cells. Nature 1959;183:263–4.

mycetes. J Antibiot (Tokyo) 1976;XXIX:97–9. 4 Taylor A. Aminopeptidases: structure and function. FASEB J 1993;32:290–8. 5 Lowther WT, Matthews BW. Metalloaminopeptidases: common functional themes in disparate structural surroundings. Chem Rev 2002;102:4581–608. 6 32.Saric T, Graef CI, Goldberg AL. Pathway for degradation of peptides generated by proteasomes: a key role for thimet and other metallopeptidases. J Biol Chem 2004;279:46723–

87 GoldbergHershko A, AL. Ciechanover Protein degradation A. The ubiquitin and protection system for against protein misfolded degradation. or damaged Annu Rev proteins. Biochem 1992;61:761–807.

10 HershkoNature 2003;426:895–9. A. The ubiquitin system for protein degradation and some of its roles in the control of 9 Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem 1998;67:425–79.

the cell division cycle. Cell Death Differ 2005;12:1191–7. 1211 KisselevGlickman A, MH, Akopian Ciechanover T, Goldberg A. The A. ubiquitin-proteasomeRange of sizes of peptide proteolytic products pathway: generated destruction during degra for- the sake of construction. Physiol Rev 2002;82:373–428.

dation of different proteins by archaeal proteasomes. J Biol Chem 1998;273:1982–9. 1413 EmmerichKisselev A, NP,Akopian Nussbaum T, Woo AK, K, Stevanovicet al. The sizes S, et of al. peptides The human generated 26 S and from 20 Sprotein proteasomes by mammalian gener- ate26 andoverlapping 20 S proteasomes. but different J Biol sets Chem of peptide 1999;274:3363–71. fragments from a model protein substrate. J Biol

Chem 2000;275:21140–8. 1615 BotbolLecker V,SH, Scornik Goldberg OA. AL, Degradation Mitch WE. of Protein abnormal degradation proteins byin intactthe ubiquitin-proteasome mouse reticulocytes: pathway accumu - in normal and disease states. J Am Soc Nephrol 2006;17:1807–19. 17 Groettrup M, Soza A, Kuckelkorn U, et al. Peptide antigen production by the proteasome: com- lation of intermediates in the presence of bestatin. Proc Natl Acad Sci U S A 1979;76:710–3.

plexity provides efficiency. Immunol Today 1996;17:429–35. 18 Rock K, Goldberg A. Degradation of cell proteins and the generation of MHC class I-presented mediatedpeptides. Annutrimming Rev Immunolof major histocompatibility 1999;17:739–79. complex class II-bound peptides. J Exp Med 19 Larsen SL, Pedersen LO, Buus S, et al. T cell responses affected by aminopeptidase N (CD13)-

1996;184:183–9. 20 Dong X, An B, Salvucci Kierstead L, et al. Modification of the amino terminus of a class II epitope 21 Reitsconfers E, Griekspoorresistance to A, degradation Neijssen J, et by al. CD13 Peptide on dendriticdiffusion, cells protection, and enhances and degradation presentation in nu to- T cells. J Immunol 2000;164:129–35.

22 Yewdellclear and J, cytoplasmicPrinciotta M. compartments Proteasomes Get before By withantigen Lots presentation of Help from by Their MHC Friends. class I. Immunity 2003;18:97–108. 23 Rock K, York I, Goldberg A. Post-proteasomal antigen processing for major histocompatibility 2004;:362–3.

complex class I presentation. Nat Immunol 2004;5:670–7.

168 24 Hattori A, Tsujimoto M. Processing of Antigenic Peptides by Aminopeptidases. Biol Pharm Bull

- 2004;27:777–80. 25 Kim E, Kwak H, Ahn K. Cytosolic aminopeptidases influence MHC class I-mediated antigen presen tation in an allele-dependent manner. J Immunol 2009;183:7379–87. 26 Sanderink GJ, Artur Y, Siest G. Human aminopeptidases: a review of the literature. J Clin Chem Clin Biochem 1988;26:795–807. 27 Sato Y. Role of aminopeptidase in angiogenesis. Biol Pharm Bull 2004;27:772–6. 28 Mina-Osorio P. The moonlighting enzyme CD13: old and new functions to target. Trends Mol Med - 2008;14:361–71. 3029 MatsushimaSantos AN, Langner M, Takahashi J, Herrmann T, Ichinose M, et M, al. et Aminopeptidase al. Structural and N/CD13 immunological is directly evidence linked to fort signal he identrans- tityduction of prolyl pathways aminopeptidase in monocytes. with Cell leucyl Immunol aminopeptidase. 2000;201:22–32. Biochem Biophys Res cummunications

31 Beninga J, Rock KL, Goldberg A. Interferon-gamma can stimulate post-proteasomal trimming 1991;178:1459–64.

of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J Biol Chem 1998;273:18734–42. 3332 InokumaPulido-Cejudo S, Setoguchi G, Conway K, Ohta B, Proulx T, et al. P, Serumet al. Bestatin-mediated leucine aminopeptidase inhibition as an of activityleucine aminopeptidaseindicator in sys- may hinder HIV infection. Antivir Res 1997;36:167–77.

temic lupus erythematosus: a study of 46 consecutive cases. Rheumatol (Oxford) 1999;38:705–8. 34 Lee JY, Song SM, Seok JW, et al. M17 leucine aminopeptidase of the human malaria parasite - Plasmodium vivax. Biochem Parasitol 2010;170:45–8. 35 Constam DB, Tobler AR, Rensing-Ehl A, et al. Puromycin-sensitive aminopeptidase. Sequence anal- ysis, expression, and functional characterization. J Biol Chem 1995;270:26931–9. 36 Tobler AR, Constam DB, Schmitt-Gräff A, et al. Cloning of the human puromycin-sensitive amino- peptidase and evidence for expression in neurons. J Neurochem 1997;68:889–97. 37 Towne C, York I, Neijssen J. Puromycin-sensitive aminopeptidase limits MHC class I presenta tion in dendritic cells but does not affect CD8 T cell responses during viral infections. J Immunol 2008;180:1704–12. 3938 BhutaniStoltze L, N, Schirle Venkatraman M, Schwarz P, Goldberg G, et al. Twoa L. Puromycin-sensitive new in the aminopeptidase MHC class I processing is the major pathway. pepti - daseNat Immunol. responsible Nat for Immunol digesting 2000;1:413–8. polyglutamine sequences released by proteasomes during protein

40 Yamamoto Y, Li YH, Ushiyama I, et al. Puromycin-sensitive alanyl aminopeptidase from human liver degradation. EMBO J 2007;26:1385–96. 41 Sengupta S, Horowitz PM, Karsten SL, et al. Degradation of tau protein by puromycin-sensitive cytosol: purification and characterization. Forensic Sci Int 2000;113:143–6. - aminopeptidase in vitro. 2006;45:15111–9. 4342 TholanderHui K-S. Brain-specific F, Muroya A, aminopeptidase: Roques B-P, et al. from Structure-based dissection to protector of the against active neurodegenerasite chemistry of tion. Neurochem Res 2007;32:2062–71.

44 Fitzpatrickleukotriene FA, A4 Lepleyhydrolase: R, Orning implications L, et al. forSuicide M1 aminopeptidases inactivation of leukotriene and inhibitor A4 hydrolase/aminodesign. Chem Biol- 2008;15:920–9. 45 Radmark O, Shimizus T, Jornvall H, et al. Leukotriene A4 hydrolase in human leukocytes. peptidase. Ann N Y Acad Sci 1994;744:31–8. - Purification and properties. J Biol Chem 1984;259:12339–45. 4746 HaeggströmBozza PT, Magalhães JZ, Nordlund KG, Weller P, Thunnissen PF. Leukocyte MMGM. lipid Functional bodies - propertiesBiogenesis andand molecularfunctions inarchitecture inflamma tion. Biochim Biophys Acta 2009;1791:540–51.

48 Haeggströmof leukotriene JZ. A4 Leukotriene hydrolase, A4a pivotal hydrolase/aminopeptidase, catalyst of chemotactic the leukotriene gatekeeper formation. of chemotactic Sci World leukot J - 2002;2:1734–49. 169 riene B4 biosynthesis. J Biol Chem 2004;279:50639–42. 49 Holloway JW, Barton SJ, Holgate ST, et al. The role of LTA4H and ALOX5AP polymorphism in asthma and allergy susceptibility. Allergy 2008;63:1046–53. 50 Oliveira E De, Wang K, Kong H. Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema. Bioorg Med Chem Lett - 2011;21:6746–50. 51 Thunnissen M, Nordlund P, Haeggström JZ. Crystal structure of human leukotriene A 4 hydrolase , a bifunc- tional enzyme in inflammation. Nat Struct Biol 2001;8:131–5. 52 Saveanu L, Carroll O, Lindo V, et al. Concerted peptide trimming by human ERAP1 and ERAP2 aminopepti dase complexes in the endoplasmic reticulum. Nat Immunol 2005;6:689–97. 5453 BirtleyNguyen JR, TT, Saridakis Chang S-C, E, StratikosEvnouchidou E, et I, al. et The al. Structural crystal structure basis for of antigenic human endoplasmic peptide precursor reticulum processing aminopepti by - the endoplasmic reticulum aminopeptidase ERAP1. Nat Struct Mol Biol 2011;18:604–13. - dase 2 reveals the atomic basis for distinct roles in antigen processing. Biochemistry 2012;51:286–95. 55 Saric T, Chang S-C, Hattori A, et al. An IFN-gamma-induced aminopeptidase in the ER, ERAP1, trims precur- sors to MHC class I-presented peptides. Nat Immunol 2002;3:1169–76. 56 Serwold T, Gonzalez F, Kim J, et al. ERAAP customizes peptides for MHC class I molecules in the endoplas mic reticulum. Nature 2002;419:480–3. 5857 HallbergYork IA, Chang L, Michaëlsson SC, Saric T,K. et Adipocyte-derived al. The ER aminopeptidase leucine aminopeptidase ERAP1 enhances genotype or limits and antigen response presentation to antihyper by - trimming epitopes to 8-9 residues. Nat Immunol 2002;3:1177–84. 59 Scott L, Lamb J, Smith S, et al. Single amino acid () deprivation: rapid and selective death of cul- tensive therapy. BMC Cardiovasc Disord 2003;6:7–12. 60 Moore HE, Davenport EL, Smith EM, et al. Aminopeptidase inhibition as a targeted treatment strategy in tured transformed and malignant cells. Br J Cancer 2000;83:800–10.

myeloma. Mol Cancer Ther 2009;8:762–70. 61 Martínez JM, Prieto I, Ramírez MJ, et al. Aminopeptidase activities in breast cancer tissue. Clin Chem 1999;45:1797–802. 6362 FruciCifaldi D, L, Giacomini Romania P, LorenziNicotra S,MR, et etal. al. Role Altered of endoplasmic expression reticulum of endoplasmic aminopeptidases reticulum aminopeptidases in health and disease: from infection to cancer. Int J Mol Sci 2012;13:8338–52. 64 Fruci D, Ferracuti S. Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy ERAP1 and ERAP2 in transformed non-lymphoid human tissues. J Cell Physiol 2008;216:742–9.

donors and Leukemia/Lymphoma, carcinoma, and melanoma cell lines. J Immunol 2006;176:4869–79. 65 Kamphausen E, Kellert C, Abbas T, et al. Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma. Cancer Immunol Immunother 2010;59:1273–84. 66 Mehta AM, Jordanova ES, Corver WE, et al. Single nucleotide polymorphisms in antigen processing machinery component ERAP1 significantly associate with clinical outcome in cervical carcinoma. Genes Chromosom Cancer 2009;48:410–8. 67 Watanabe Y, Shibata K, Kikkawa F. Adipocyte-Derived Leucine Aminopeptidase Suppresses Angiogenesis- in Human Endometrial Carcinoma via Renin-Angiotensin System. Clin cancer Res 2003;9:6497–503. 68 Tokuhara T, Hattori N, Ishida H, et al. Clinical significance of aminopeptidase N in non-small cell lung can cer. Clin Cancer Res 2006;12:3971–8. 69 Hensbergen Y Van, Broxterman H. Soluble Aminopeptidase N / CD13 in Malignant and Nonmalignant Effusions and Intratumoral Fluid. Clin cancer Res 2002;8:3747–54. 70 Kehlen A, Lendeckel U, Dralle H. Biological Significance of Aminopeptidase N / CD13 in Thyroid Carcinomas. Cancer Res 2003;63:8500–6. 71 Pérez I, Varona A, Blanco L, et al. Increased APN/CD13 and acid aminopeptidase activities in head and neck squamous cell carcinoma. Head Neck 2009;10:1335–40. 72 Lee P, Lin C, Liu C. Acute Leukemia With Myeloid, B-, and Natural Killer Cell Differentiation. Arch Pathol Lab Med 2003;127:93–5. 7473 IshiiPiedfer K, UsuiM, Dauzonne S, Sugimura D, Tang Y, et R,al. etAminopeptidase al. Aminopeptidase-N/CD13 N regulated by is zinc a potential in human proapoptotic prostate participates target in human in myeloid tumor cells. FASEB J 2011;25:2831–42.

tumor cell invasion. Int J Cancer 2001;92:49–54. 170 75 Hashida H, Takabayashi A, Kanai M, et al. Aminopeptidase N is involved in cell motility and angio-

76 Pasqualini R, Koivunen E, Kain R. Aminopeptidase N Is a Receptor for Tumor-homing Peptides and genesis: its clinical significance in human colon cancer. Gastroenterology 2002;122:376–86. - a Target for Inhibiting Angiogenesis. Cancer Res 2000;60:722–7. 7877 RackleyWickström RR, M, Yang Larsson B, Pretlow R, Nygren TG, et P, al. et Differencesal. Aminopeptidase in the leucine N (CD13) aminopeptidase as a target for activity cancer in chemo extracts therapy. Cancer Sci 2011;102:501–8. 79 Garg LN, Yadav SP, Lal H. Serum leucine aminopeptidase in head and neck cancer. J Laryngol Otol from human prostatic carcinoma and benign prostatic hyperplasia. Cancer 1991;68:587–93. 80 Varona A, Blanco L, López JI, et al. Altered levels of acid, basic, and neutral peptidase activity and 1994;108:660–2. F788. expression in human clear cell renal cell carcinoma. Am J Physiol Renal Physiol 2007;292:F780–

8281 AbeLee S.M, Cobalt Matsuki Chloride-Induced H, Domae M, et Downregulational. Lung cancer cell of Puromycin-Sensitivelines inhibit leukotriene Aminopeptidase B4 production by Suppresses the Migration and Invasion of PC-3 Cells. J Microbiol Biotechnol 2009;19:530–6.

human polymorphonuclear leukocytes at the level of phospholipase A2. Am J Respir Cell Mol Biol- 1996;15:565–73. 83 Chen X, Li N, Wang S, et al. Leukotriene A4 hydrolase in rat and human esophageal adenocarcino mas and inhibitory effects of bestatin. J Natl Cancer Inst 2003;95:1053–61. 84 Jeong C-H, Bode AM, Pugliese A, et al. [6]-Gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase. Cancer Res 2009;69:5584–91. 85 Oi N, Jeong C-H, Nadas J, et al. Resveratrol, a red wine polyphenol, suppresses pancreatic cancer by 85.inhibiting leukotriene A₄hydrolase. Cancer Res 2010;70:9755–64. 86 Scornik OA, Botbol V. Bestatin as an experimental tool in . Curr Drug Metab 2001;2:67– induce apoptosis of K562 and STI571-resistant K562 cell lines through the MAPK and GSK-3beta 87 Sawafuji K, Miyakawa Y, Weisberg E, et al. Aminopeptidase inhibitors inhibit proliferation and 88 Botbol V, Scornik O. Measurement of instant rates of protein degradation in the livers of intact mice pathways. Leuk Lymphoma 2003;44:1987–96.

by the accumulation of bestatin-induced peptides. J Biol Chem 1991;266:2151–7. 89 Krige D, Needham LA, Bawden LJ, et al. CHR-2797 : An antiproliferative aminopeptidase inhibitor that leads to amino acid deprivation in human leukemic cells. Cancer Res 2008;68:6669–79. 9290 SancakLaplante Y, M,Peterson Sabatini TR, DM. Shaul mTOR YD, signaling et al. The in Rag growth GTPases control bind and raptor disease. and mediate Cell 2012;149:274–93. amino acid signal- 91 Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci 2009;122:3589–94.

ing to mTORC1. Science 2008;320:1496–501. 93 Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004;18:1926–45. 94 Goberdhan D. Intracellular amino acid sensing and mTORC1-regulated growth: New ways to block 96 Löwenbergan old target? B, CurrMorgan Opin G, InvestigOssenkoppele drugs 2010;11:1360–7.G, et al. Phase I/II clinical study of Tosedostat, an inhibitor 95 Guertin D a, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell 2007;12:9–22.

97 Gottesmanof aminopeptidases, MM, Fojo inT, patientsBates SE. with Multidrug acute myeloidresistance leukemia in cancer: and role myelodysplasia. of ATP-dependent J Clin transportOncol - 2010;28:4333–8.

ers. Nat Rev Cancer 2002;2:48–58. 9998 FanGrujić X, RossM, Renko DD, Arakawa M. Aminopeptidase H, et al. Impact inhibitors of system bestatin L amino and acidactinonin transporter inhibit 1 cell (LAT1) proliferation on prolifer of - ationmyeloma of human cells predominantly ovarian cancer by cells: intracellular a possible interactions. target for combination Cancer Lett therapy 2002;182:113–9. with anti-prolifera-

tive aminopeptidase inhibitors. Biochem Pharmacol 2010;80:811–8.

171 101100 PetersTsukamoto GJ, Jansen H, Shibata G. Antimetabolites. K, Kajiyama H, In:et al.Oxford Aminopeptidase Textbook of NOncology (APN)/CD13 2nd edition. inhibitor, 2001. Ubenimex, Oxford. enhances 102 Aoyagiradiation T, Tobesensitivity H, Kojima in human F, et al. cervical , cancer. an BMCinhibitor Cancer of aminopeptidase 2008;8:74. A, produced by actinomycetes. J

103 Feng J, Jin K, Zhu H, et al. A novel aminopeptidase N inhibitor developed by virtual screening approach. Antibiot (Tokyo) 1978;31:636–8.

Bioorg Med Chem Lett 2012;22:5863–9. 104 Gao JJ, Gao ZH, Zhao CR, et al. LYP, a novel bestatin derivative, inhibits cell growth and suppresses 105 IchinoseAPN/CD13 Y, Genkaactivity K, in Koike human T, et ovarian al. Randomized carcinoma double-blind cells more potently placebo-controlled than bestatin. trial Invest of bestatin New Drugs in patients 2011;29:574–82. - with resected stage I squamous-cell lung carcinoma. J Natl Cancer Inst 2003;95:605–10. 106 Kakuta H, Tanatani A, Nagasawa K, et al. Specific nonpeptide inhibitors of puromycin-sensitive aminopepti dase with a 2,4(1H,3H)-quinazolinedione skeleton. Chem Pharm Bull (Tokyo) 2003;51:1273–82. 107 Luan Y, Ma C, Sui Z, et al. LYP3, a new bestatin derivative for aminopeptidase N inhibition. Med Chem 2011;7:32–6. 108 Menrad A, Speicher D, Wacker J, et al. Biochemical and Functional Characterization of Aminopeptidase N Expressed by Human Melanoma Cells Biochemical and Functional Characterization Human Melanoma Cells1 N Expressed by. Cancer Res 1993;53:1450–5. 109 Pei KL, Yuan Y, Qin SH, et al. CIP-13F, a novel aminopeptidase N (APN/CD13) inhibitor, inhibits Lewis lung carcinoma growth and metastasis in mice. Cancer Chemother Pharmacol 2012;69:1029–38. 110 Rao M, Li Q, Feng L, et al. A new aminopeptidase inhibitor from Streptomyces strain HCCB10043 found by UPLC-MS. Anal Bioanal Chem 2011;401:699–706. 111 Reid AHM, Protheroe A, Attard G, et al. A first-in-man phase i and pharmacokinetic study on CHR-2797 (Tosedostat), an inhibitor of M1 aminopeptidases, in patients with advanced solid tumors. Clin Cancer Res 2009;15:4978–85. 113112 Su L, JiaCao Y, J, Zhang Jia Y, et L, al. et Developmental. Design, synthesis of Synthetic and biological Aminopeptidase evaluation N/CD13 of novel Inhibitors amino acid to Overcome ureido derivatives Cancer Metastasis and Angiogenesis. ACS Med Chem Lett 2012;3:959–64.

as aminopeptidase N/CD13 inhibitors. Bioorg Med Chem 2012;20:3807–15. 114 Van Herpen CML, Eskens F a LM, de Jonge M, et al. A Phase Ib dose-escalation study to evaluate safety and tolerability of the addition of the aminopeptidase inhibitor tosedostat (CHR-2797) to paclitaxel in patients individualizedwith advanced inductionsolid tumours. therapy Br Jand Cancer use of2010;103:1362–8. ubenimex during and after consolidation therapy for elderly 115 Wakita A, Ohtake S, Takada S, et al. Randomized comparison of fixed-schedule versus response-oriented

patients with acute myeloid leukemia: the JALSG GML200 Study. Int J Hematol 2012;96:84–93. 116 Xu Y, Lai LT, Gabrilove JL, et al. Antitumor activity of actinonin in vitro and in vivo. Clin Cancer Res 1998;4:171–6.

172