Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis

Send Orders for Reprints to [email protected] Current Topics in Medicinal Chemistry, 2016, 16, 1478-1488

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Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis Tedman Ehlers2, Scott Furness3, Thomas Philip Robinson4, Haizhen A. Zhong5, David Goldsmith6, Jack Aribser7, and J. Phillip Bowen1,* 1

Center for Drug Design, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, 3001 Mercer University Drive, Atlanta, Georgia 30341, USA; 2Dassault Systèmes', BIOVIA Corp. 5005 Wateridge Vista Drive, San Diego, California 92121, USA; 3Office of New Drug Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, USA; 4Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA; 5Department of Chemistry, University of Nebraska at Omaha, 6001 Dodge Street, Omaha, Nebraska 68182, USA; 6Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA; 7Atlanta Veterans Affairs Medical Center and Department of Dermatology, Emory University School of Medicine, 5007 Woodruff Memorial Building, Emory University, Emory University, Atlanta, Georgia 30322, USA

J. Phillip Bowen

Abstract: Angiogenesis has been identified as a crucial process in the development and spread of cancers. There are many regulators of angiogenesis which are not yet fully understood. Methionine aminiopeptidase is a metalloenzyme with two structurally distinct forms in humans, Type-1 (MetAP-1) and Type-2 (MetAP-2). It has been shown that small molecule inhibitors of MetAP-2 suppress endothelial cell proliferation. The initial discovery by Donald Ingber of MetAP-2 inhibition as a potential target in angiogenesis began with a fortuitous observation similar to the discovery of penicillin activity by Sir Alexander Fleming. From a drug design perspective, MetAP-2 is an attractive target. Fumagillin and ovalicin, known natural products, bind with IC50 values in low nanomolar concentrations. Crystal structures of the bound complexes provide 3-dimensional coordinates for advanced computational studies. More recent discoveries have shown other biological activities for MetAP-2 inhibition, which has generated new interests in the design of novel inhibitors. Semisynthetic fumagillin derivatives such as AGM-1470 (TNP-470) have been shown to have better drug properties, but have not been very successful in clinical trials. The rationale and development of novel multicyclic analogs of fumagillin are reviewed.

Keywords: ADME, Angiogenesis, Angiogenic inhibitors, Anti-angiogenic compounds, Cancer, Drug design, Fumagillin, Methionine aminiopeptidase, MetAP-2, Ovalicin, Pharmacophore, Cancer, TNP-470. 1. INTRODUCTION Cancer is a complex disease. It is not one disease but exists as a diverse collection of forms. The challenges related to the treatment of cancer are numerous. Treatments range from surgery, radiation, and pharmacotherapy to some combination therapy. Neoplasms, cancerous cells, undergo rapid proliferation as a result of having lost their ability to undergo controlled division. Cancer mortality is second only to heart disease in the United States. Successful chemotherapeutic approaches historically have concentrated on cytotoxic agents that target any rapidly dividing cell [1]; however, the side effects of these therapies often leave much to be desired. In many cases the antineoplastic drugs available for internal tumors often give rise to side effects so harmful that they compromise the benefits of treatment. As a result, these side effects limit the doses that can be administered, which, in turn, restricts aggressive intervention. While strategies for *Address correspondence to this author at the Center for Drug Design, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, 3001 Mercer University Drive, Atlanta, Georgia 30341, USA; E-mail: [email protected] 1873-5294 /16 $58.00+.00

eliminating these side effects are being developed, classical chemotherapy has another limitation. Neoplasms, like bacteria which may become resistant to antibiotics, are increasingly able to survive the anticancer drugs used to treat them [2]. Certain neoplasms have been resistant from the outset, whereas others developed resistance with repeated treatments. This is an extremely serious problem, as some tumors can develop resistance to multiple drugs after only one drug has been administered to the patient. Improving existing drugs keeps us one step ahead of resistance emergence. Drug rejuvenation, however, is unfortunately threatened in the long term by the same resistance emergence factors that plagued its predecessors [3]. Alternatives to the direct attack of unstable cells are the most promising long-term solutions to the problem; they are being pursued as more information about the nature of cancer is revealed. One alternative target is the vascular system. The need to obtain a blood supply for nutrients and the removal of waste products is common to all types of cancer. In 1972, Judah Folkman published an exciting hypothesis: the control of the growth and spread of cancers might be achieved via the inhibition of angiogenesis [4]. Initially, the hypothesis was © 2016 Bentham Science Publishers

Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis

greeted with skepticism from the medical and scientific communities. This radical approach to cancer treatment required decades of study before gaining acceptance. Presently, antiangiogenic research is an active field of biomedical investigation. The first angiogenesis inhibitor, bevacizumab (Avastin®) (http://www.drugs.com/history/avastatin.html). was approved by the U.S. Food and Drug Administration in 2004 for the treatment of patients with metastic colorectal cancer Currently there are several compounds in current clinical trials that target angiogenesis. This review provides a brief overview of small molecule inhibitors that interact with MetAP-2 and produce antiangiogenic effects. The design of fumagillin analogs as antiangiogenic compounds is discussed. Other biological activities, stemming from small molecule inhibitors of MetAP-2, are reviewed.

Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 13

Table 1.

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Positive and negative regulators of angiogenesis. Angiogenesis Regulators

Positive regulation

Negative regulation

Angiogenin

Angiostatin

Angiopoietin-1

Endostatin

Epidermal growth factor Fibroblast growth factor (a & b)

Genistein

Hepatocyte growth factor

Interferon alpha

Interleukin 8 Platelet-derived EC growth factor

Metallo-proteinase inhibitors

2. ANGIOGENESIS AND CANCER Scatter factor

The vascular system, comprised of nearly 100,000 miles of capillary blood vessels, is responsible for delivering oxygen and nutrients to every cell in the body, as well as removing metabolic waste products from them. The inner lining of blood vessels is made up of endothelial cells. These cells form a barrier between tissues and the circulating blood supply. Endothelial cells are genetically stable and generally proliferate slowly; however, they do have the unique ability to increase proliferation and spur the formation of new blood capillaries from the preexisting vasculature. This is known as angiogenesis. This normal bodily process is complex, and it is under the strict regulation of numerous angiogenic promoters and inhibitors (Table 1). The initiating event is the degradation of the basement membrane structure by activated matrix metalloproteinases. A stimulus then directs the outward migration of endothelial cells, which results in rapid endothelial cell proliferation. This is followed by the formation of a lumen and then the extracellular matrix. Angiogenesis is crucial to the developing embryo, but remains relatively inactive in the adult body. In adults, tissues are fully developed, and the need for new blood vessels is limited to healing responses to injury and processes associated with the menstrual cycle [5, 6]. The pathological conversion of benign tumors to a malignant state involves passing a critical point of signal transduction. Tumors send out signals to nearby endothelial cells calling for them to exit quiescence and become angiogenic. The balance between endogenous angiogenic promoters and inhibitors is disrupted, and the angiogenic switch is said to be “turned on” at this stage. Newly recruited blood vessels feed diseased tissues with oxygen and nutrients, allowing them to thrive. Also, the new blood supply provides the tumor with a means to spread to other locations in the body (metastasize). It has been shown that tumors will remain dormant and grow only to approximately a 1-2 mm diameter if new blood supplies are not acquired [7]. The original postulation of Judah Folkman was that the inhibition of angiogenesis would limit tumor growth and the probability of metatheses [4, 8]. A drug-like compound capable of restoring the natural balance of angiogenic regulators has the potential to be a minimally toxic, non-competing therapy which could complement current cytotoxic treatments. This is the major goal of antiangiogenic therapy.

Tumor necrosis factor alpha

Thrombospondin-1

Vascular endothelial growth factor

Vasostatin

3. METHIONINE AMINOPEPTIDATE TYPE-2 The discovery of MetAP-2 inhibition as a potential target in angiogenesis began with a fortuitous observation by Donald Ingber, similar to the one made by Alexander Fleming. During routine culture of endothelial cells, Ingber noted that a culture was contaminated with a fungus, and, like penicillin, there was a zone of clearing around the fungal contamination. Upon examination it was noted that endothelial cell rounding had occurred. Closer to the fungus, rounding became more pronounced, and endothelial cells eventually were detached from the cell surface. Resisting the usual tendency to discard a fungally contaminated sample, the fungus was cultured and found to be Aspergillus fumigatus; the putative active metabolite was the natural product fumagillin (Fig. 1, 1) [9, 10]. Fumagillin was originally identified by Eble and Hanson in 1951, and was found to be a powerful antibiotic [11]. The sesquiterpene-like structure, easily attained from fumagillol, consists of a functionalized cis-diol, two epoxides, and a total of six contiguous stereocenters. There was little interest in this compound until the discovery that fumagillin, as described above, and related compounds turned out to be highly potent inhibitors of angiogenesis [12]. Fumagillin caused the characteristic endothelial rounding observed in fungally contaminated endothelial cells, but it was toxic in the murine dorsal air sac model of angiogenesis, resulting in significant weight loss. Consequently, an extensive library of semisynthetic derivatives was prepared, and a candidate compound, AGM-1470 or TNP-470 (Fig. 1, 2) was selected for further evaluation. AGM-1470 was found to be a potent inhibitor of tumor growth in vivo, demonstrating reduction in Lewis lung carcinoma and B16 melanoma with acceptable toxicities [9]. Further studies confirmed antiangiogenic effects of AGM-1470 on other neoplastic disorders and inflammatory conditions such as murine models of arthritis, i.e. collagen induced arthritis. The efficacy of AGM-1470 was demonstrated over the next few years in several hundred papers, including studies on carcinomas, sarcomas, and he-

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matopoietic tumors. Metabolic studies demonstrated hepatic conversion of AGM-1470 to six compounds, followed by epoxide hydrolase opening of the epoxide ring.

Ehlers et al.

have been undertaken by several research groups. Our work is highlighted. His 231

O O

Co 481 OH

O O

O

O

O

O

fumagillin (1)

O O

O O O

O

O

O

O

O N H

Cl O OH O

O

O TNP-470 (2)

O

ovalicin (3)

Fig. (1). Structures of fumagillin, AGM-1470, and ovalicin. These small molecules form covalently-bound inhibitors of MetAP-2.

Subsequent research revealed that fumagillin formed a covalent adduct with the cellular enzyme methionine aminopeptidase (MetAP-2), which is highly conserved between human and Saccharomyces cerevisiae. This was accomplished using a biotinylated analog, where the biotin was placed in a noncritical region of fumagillin. MetAP-2 was covalently labeled by the biotinylated analog, while the related analog MetAP-1 was not affected [13]. Independently, a second group discovered that fumagillin and the closely related compound ovalicin (Fig. 1, 3) also bound MetAP-2 through the conserved spiro epoxide moiety [14]. MetAP-2 cleaves N-terminal methionines from multiple cellular targets, and the possible specificity of MetAP-2 blockade may result from a downstream effect on the src family kinases. This requires removal of the N-terminal methionine prior to myristoylation, which is required ultimately for plasma membrane localization of src family kinases. Of interest, a myristoyltransferase inhibitor, tris DBA palladium, was shown by one of us to be a potent inhibitor of tumor growth and angiogenesis in vivo [15]. In 1997, the provocative finding that female reproduction was also angiogenesis dependent made using AGM-1470 the primary angiogenesis inhibitor [16]. The molecular mechanism of action of these compounds has been studied extensively. Unambiguous crystallographic studies by Clardy demonstrated that a nucleophilic attack on the spiro-epoxide by a histidine of the methionine aminopeptidase type-2 (MetAP-2) enzyme had formed a covalent bond between MetAP-2 and Fumagillin [13, 17]. (Figs. 2 and 3). The semi-synthetic derivative AGM-1470 has been shown to be a more potent inhibitor than fumagillin [9] and does not induce drug resistance [18]; thus, confirming the potential to develop a more effective drug that inhibits MetAP-2. Synthetic work within our lab has resulted in novel multicyclic analogs of fumagillin [19]. With the early development of novel analogs, it is important to verify the feasibility of molecular binding and to provide a rational basis for subsequent modification to produce more effective enzyme inhibitors. With the crystal structure of the bound complex in hand, the rational design of fumagillin analogs

Fig. (2). Proposed nucelophilic attack by the histidine 232 residue of MetAP-2 on the spiro epoxide of fumagillin, which yields a covalently bound adduct.

4. STRUCTURAL FEATURES AMINOPEPTIDASE TYPE-2

OF

METHIONE

Methionine aminopeptidase (MetAP) is the enzyme which removes the N-terminal methionine residue from newly synthesized proteins. The removal of this translation initiator is often crucial to the stability and function of the protein. There are two forms of methionine aminopeptidase (Type-1 and Type-2). The crystal structures of both forms have been solved and deposited in the protein data bank, which is currently housed by the Research Collaboratory for Structural Bioinformatics (RCSB) [20]. The published crystal structures of MetAP-2 show an active site pocket, which is located at the center of a large β-sheet with two small βsheets capping the top and bottom (Fig. 3). The active site contains two metal cofactors reported as being Co2+. Crystal structures show that besides Co2+[21, 22], other metal ions are also found in the MetAP-2 active sites, including Zn2+ [23], Mn2+ [24, 25], and Fe3+ [26]. It should be pointed out that the presence of Fe3+ has been observed in Encephalitozoon cuniculi MetAP-2, not in human MetAP-2 [26]. D’souza and Holz suggested that Fe2+ may be involved in E. Coli MetAP [27]. A study by D’souza and Holz showed that Co2+, Fe2+, Mn2+, and Zn2+ provided detectable levels of enzymatic activity in anaerobic enzymatic assays and that excess metal ions were found to be inhibitory. Presently, Fe2+ and Fe3+ have not been observed in human MetAP-2 crystal structures. Quantum mechanics studies at the DFT/B3LYP level of theory demonstrated that the zinc di-cation model yielded the lowest activation energy for the hydrolysis reaction where the nucleophilic addition of the hydroxide on the substrate carbon has occurred [28]. The energy barriers of Co2+, Fe2+, and Mn2+ appeared to be much higher. This suggests that MetAP-2 may be classified as a di-zinc enzyme [28]. The fact that most observed MetAP-2 crystal structures contain either Co2+ or Mn2+ may be attributed to the crystallization conditions that were adopted. In the presence of excess Co2+, a third Co2+ could occupy the active site region and become inhibitory [29]. Therefore, it is essential not to use an excess amount of ions during the biological testing and/or crystallization.



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5. CURRENT STATUS OF DEVELOPMENT OF METAP-2 INHIBITORS Other than the covalently-bound fumagillin, AGM-1470, and ovalicin (Fig 1, 1, 2, and 3), as well as several noncovalently-bound MetAP-2 inhibitors have been developed. Having the His231 residue in MetAP-2 is essential for ligand binding since it forms a covalent bond with fumagillin and its analogs. To exploit molecular interactions with this important residue, researchers have synthesized a wide variety of carboxylic acid-based compounds that presumably are able to form a hydrogen bond with the imidazole ring of His231 and generrate electrostatic interactions with Co2+ in the active site.

The sequence identity in MetAP between the catalytic domain of Type-1 and that same domain of Type-2 human MetAP is low (27%); however, the active sites (the catalytic domains) of these two proteins have a high degree of similarity [21]. This allows all MetAPs to have the ability to carry out the same function – to cleave the NH2-terminal methionine from proteins. Nevertheless, the insertion of residues 381 to 444 distinguishes the MetAP-2 family of proteins from those of MetAP-1. The tyrosine 444 (Tyr444) residue is conserved in all MetAP-2 proteins but is absent from MetAP-1. Tyr444, Ile338, His339, and Phe219 provide important hydrophobic contacts with the epoxide-bearing ring system in fumagillin and its analogs. Many MetAP-2 Xray structures show that Tyr444 provides hydrophobic interactions with ligands. In addition, His231 in MetAP-2 forms a covalent bond with fumagillin, ovalicin, and AGM-1470. The corresponding residue in MetAP-1 is His212, which is conserved in the MetAP-1 family. The His212 residue in MetAP-1, however, has moved 1.1 Å from the active site, which makes the binding of fumagillin to MetAP-1 impossible because binding to the same position as that in MetAP-2 would induce several steric clashes [21]. The NH2-terminal residues appear to be quite different among these two families of human proteins: in MetAP-1, the N-terminal residues preceding the catalytic domain are zinc finger residues and serve as a link between the zinc finger domain and the catalytic domain; in MetAP-2, the N-terminal contains acidic and basic residues. The size of the binding pocket in MetAP1 is smaller than that of MetAP-2 [29]. All these observations explain why fumagillin, ovalicin and AGM-1470 only bind to MetAP-2 but not MetAP-1. This difference provides a basis for selective inhibitor design targeting MetAP-2.

It was reported that A41 (Fig. 4, 4) inhibited MetAP-2 with an IC50 of 1.4 μM. Replacement of the phenyl ring with a tetrahydro naphthalic acid yielded A75 (Fig. 4, 5) with an IC50 of 0.010 μM, an increase of more than 100-fold in potency [24, 32]. In addition to the carboxylic acid derivatives, other MetAP-2 binding compounds include triazoles and hydrozines. The X-ray structure of the thiopheno-anilinotriazole (Fig. 4, 6) MetAP-2 complex shows that the two continuous triazole nitrogens (N1 and N2 of the triazole ring) provide coordination to two Co2+ ions. The third nitrogen of the triazole provides H-bond interactions with His231. Moreover, the aniline and thiophene rings form hydrophobic interactions with Try444 and His231, respectively. The binding of triazole 6 was shown to be iondependent. In the presence of Co2+, the IC50 of 6 with MetAP-2 is 2 nM; in the presence of Mn2+, the IC50 was drastically changed to 1,500 nM [22]. In addition to MetAP2, glycosidase is another target for angiogenesis inhibition. Inhibitors of α-glucosidases, e.g. N-methyl-1deoxynojirimycin (DNJ) (Fig. 4, 7) affect the biosynthesis of glycans on endothelial cell surfaces, subsequently inhibiting angiogenesis. The hybridization of 1-deoxynojirimycin, 7, and aryl-1,2,3-triazoles yielded a hybrid compound (Fig. 4, 8). Compound 8 was an angiogenesis inhibitor more potent than parent compounds: DNJ alone or the 1,2,3-triazole alone. The extent of angiogenesis inhibition was measured by the ability to inhibit the proliferation of bovine aortic endothelial cells (BAEC). Compound 8 inhibited αglucosidases and BAEC growth with an IC50 of 1.15 μM and 105 μM, respectively [33]. It remains to be seen whether compound 8 is able to inhibit MetAP-2. A screening of a library of approved drugs and phase 2 clinical inhibitors in human umbilical vein endothelial cells (HUVEC) identified the antifungal drug itraconazole (Fig. 4, 9) as an angiogenesis inhibitor. Itraconazole has two triazole rings in both terminals of the molecule. The IC50 of itraconazole with the HUVEC cell proliferation, a measurement of angiogenesis inhibition, was 0.16 μM [34].

Though the strong correlation between tight binding of MetAP-2 and increased antiangiogenic activity exists [30], the exact mechanism of endothelial cell growth inhibition remains unclear [31]. It is with the hope that devising a more tightly binding structure containing a multicyclic core will yield a compound with potent antiangiogenic activity while avoiding the known toxicity problems that were encountered with some of the originally discovered inhibitors (e.g., fumagillin and AGM-1470).

X-ray structures of MetAP-2 revealed that the active site was large enough to accommodate small molecules much larger than methionine and allow for flexible molecules with extended aliphatic chains. Molecules with larger side chains showed greater selectivity toward MetAP-2 over MetAP-1. This agrees with the observation that the MetAP-1 active site pocket is smaller. A series of acylhydroxyamides and acylhydrazines were synthesized and reported as inhibitors of MetAP-2 [35]. A-357300 (Fig. 4, 10) is an acylhydrazine

Fig. (3). Ribbon structure of MetAP-2 showing bound fumagillin and His231


Ehlers et al.

derivative that inhibited MetAP-2 and the cell proliferation of HMVEC at 0.12 and 0.10 μM, respectively [36]. This suggested that compound A-357300 is both a MetAP-2 inhibitor and antiangiogenic. The crystal structure of MetAP2/A-357300 shows that in spite of the flexibility in the hydrazine side chain, this ligand is still able to bind well with the active site, interacting with His231, His331, and Tyr444. Moreover, the protonated amino group forms electrostatic interactions (new interactions for this class of compounds with MetAP-2) with Asp251 and Asp262 of MetAP-2 (PDB id: 1R58) [35].

AGM-1470 exhibited potent activity against chloroquine resistant strains W2 and C2B of P. falciparum, the most lethal and common source of malaria infection. Compound 13 (Fig. 5) is a fumagillin analog from P. falciparum cell growth with an IC50 of 26.3 nM. Compound 13-treated mice infected with malaria P. berghei survived beyond 12 days post infection, whereas control mice died after seven days of infection [39]. These results show significant in vivo efficacy in the treatment of 13 against malaria. A structural analogue of compound 13 was discovered by modification of AGM1470, yielding PPI-2458 (Fig. 5, 14). PPI-2458 inhibited endothelial cell proliferation in HUVECs and BAECs with IC50 values of 0.095 and 0.046 nM, respectively. It also inhibited 97.2% of Met-AP2 activity when added to MetAP-2 with 200 nM concentration for 8 hours (i.e. only 2.8% of MetAP-2 remained active after 8 hours of treatment). Docking studies of compound 14 and fumagillin showed that the core structures of both compounds overlaid very well with each other, occupying the identical binding pocket. The long tail of fumagillin extended beyond the carbamate group of compound 14 [40]. Notably, compound 14 showed efficacy in animal models inhibiting cell proliferation in nonHodgkin’s lymphoma [40], melanoma [41], and in rheumatoid arthritis [42-44].

In addition to carboxylic acids, triazoles, hydrazines and hydroxyamides, sulfonic acid NC-2213 (Fig. 4, 11), a molecule structurally divergent from fumagillin but similar to curcumin, was found to be a potent MetAP-2 inhibitor. NC2213 inhibited human colon cancer cell lines (HCCL) in a dose-dependent manner with an IC50 of 1.2 μM. At the concentration of 1.0 μM NC-2213 was able to block the expression of MetAP-2 [37]. A study of the MetAP inhibition by a sulfonamide derivative (Fig. 4, 12) in the presence of different ions (Mn2+, Co2+, Fe2+, Ca2+ and Mg2+) revealed that 12 inhibited E. Coli MetAP the most when Co2+ ions were present, followed by Mn2+, Fe2+, Mg2+, and Ca2+ [38].

A fumagillin analog, ZGN-433 (beloranib) (Fig. 5, 15) was found to be an effective MetAP-2 inhibitor for antidiabetes. ZGN-440 was evaluated for safety, tolerability, and efficacy in obese female volunteers in Phase I clinical trials (ClinicalTrials.gov Identifier: NCT01372761), and beloranib has moved into Phase III clinical trials (ClinicalTrials.gov Identifier: NCT02179151) [45]. Another compound, RK-805

6. OTHER APPLIATIONS OF METAP-2 INHIBITORS MetAP-2 inhibitors have antiangiogenic properties and therefore are able to inhibit cell growth in various tumor cells. The promise of MetAP-2 inhibition for cancer treatment remains unfulfilled, but other therapeutic areas have developed in recent years. For example, fumagillin and

Tyr444

O

Co2+

O

H N

OH

OH Tyr444

His231

H N SO2

H N SO2

Co2+ His231

A41 (4)

A75 (5)

OH

HO

S

S

Co2+

His331

O

H N

O

N

5

N N

OH

N

N

OH

N N 2 1

Triazole (6)

OH N

H N

N

N

O

N

OH HO

1-deoxynojirimycin (7)

O

OH

hybrid (8)

Itraconazole (9)

N N

N

O

Cl

O His231

His331

HN NH

Cl

O

O

Cl S HO

Mn2+

NH3+

Tyr444

N

NO2

Asp251 Asp262

A-357300 (10)

Fig. (4). Structures of competitive inhibitors of MetAP-2 are shown.

O NC 2213 (11)

S

NO2 SO3H

N HN

O S O

sulfonamide (12)



O

O O O O

O

N H

O

H N O O

O

O

(13)

N H

NH2 O

PPI-2458 (14) O

O O

O

R O OCH3

O O

1483

O

Beloranib(ZGN-433, 15)

N

O

RK-805 (R=-H, 16) ovalicin (R=-OH)

Fig. (5). Structures of Fumagillin analogues.

(Fig. 5, 16), is an angiogenesis inhibitor isolated from the fungus Neosartorya sp. RK-805 is a structural analog of ovalicin, and it inhibited the growth of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. The IC50 values RK-805, ovalicin, and AGM-1470 against the cell growth of HUVECs were 0.03, 0.02, and 0.06 ng/ml, respectively [46].

be used to locate abnormal peptide linkages, or other unlikely bond angles. Fig. 6 displays the Ramachandran plot for MetAP-2 (1bn5) as presented by Discovery Studio.

7. MULTICYCLIC FUMAGILLIN DERIVATIVES

It should be noted that in the structural preparation, the crystallographic waters were removed. Disorder within the crystal structure required four residues to be repaired. The side-chains of Lys110, Asp114, Asn126, and Glu136 were inserted and placed in the lowest energy rotamer conformation. Partial charges and parameters were loaded with the CHARMm force field [48], and the metal cofactor charges were fixed at 2+ [49, 50]. The protein ionization and pKa of the individual residue were set taking the local environment

In general, the first step in any computational study when macromolecular data is available requires that calculations be carried out on the enzyme and enzyme-substrate complex. For the work described herein, the Discovery Studio software suite of programs was used [47]. Crystal structures (1bn5, 1boa) were retrieved from the RCSB protein data bank and analyzed. Tools such as a Ramachandran plot can

Fig. (6). Ramachandran plot for MetAP-2 (1bn5)

It was illustrative to examine the active site of the substrate-MetAP-2 complexes. The residues around the bound conformations of either fumagillin, 1, or ovalicin, 3, (Fig. 1) are displayed and labeled in Fig. 7.


into account [51]. Full energy minimizations were carried out, and conformational stability was seen between bound and unbound structures. An overlay of resulting backbone structures is shown in Fig. 8.

Ehlers et al.

spiro epoxide was a critical pharmacophoric group, and this was later unambiguously confirmed experimentally by Clardy’s X-ray work [21]. It should be noted that in an effort to explore other potential groups (e.g., exo methylene and ketone) that were electrophilic or potential functional groups that might bind to the metal in the active site of MetAP-2, the derivatives 18-20, was undertaken. Each of these structures has a ketal functional group and methyl at the ring junction. O

O R

R O

O

O

O

R O

(17)

O

O

R O

(18)

O

O

O

(19)

O

O

(20)

O

R=

Fig. (9). Ketal Tricyclic fumagillin derivatives with epoxide side chain. Fig. (7). Residues surrounding bound fumagillin.

The compounds in Fig. 9 were reasonably challenging in terms of synthesis, so it was decided to focus on simpler derivatives without the epoxide side chain (Fig. 10, 21-24). It was recognized that the omission of the epoxide side chain would result in fewer interactions with the active site residues. Nevertheless, if the spiro epoxide or some electrophilic equivalent was present then binding to MetAP-2 and angiogenesis inhibition might be achieved. The major objective of this study was to discover lead compounds that would be antiangiogenic. O

O

O

O (21)

O

O

O

O

O

(22)

O

O

O

O

(23)

O

O

(24)

Fig. (10). Ketal Tricyclic fumagillin derivatives without the epoxide side chain.

Fig. (8). Backbone and active site consistency between bound and unbound structures.

The molecular design work described below was begun prior to the availability of crystallographic data. Efforts were undertaken to prepare and design tricyclic ring structures that resembled fumagillin, Fig. 9 [19]. The approach was justified by examination of the structural data of the MetAP-2/ fumagillin complex once available. The original idea was to explore the available space in the active site with a decalin ring system compared to the cyclohexane ring system present in fumagillin. The important pharmacophoric groups that needed to be preserved in our ring systems were the spiro epoxide, methoxy group, and epoxide side chain. Four potential derivatives (Fig. 9, 17-20) were considered. Prior to the availability of structural data, we hypothesized that the

A retrosynthetic analysis, Fig. 11, shows that the spiro epoxide ketal 21 can be prepared in two steps from ketone ketal 23, via hydrogenation and epoxide formation. In turn, ketal 23 is derived from the unsaturated 1,4-dione, 25 with methanol and a mild acid. Conditions had to be carefully controlled to avoid a beta elimination. O

O

O

O (21)

O

O

O

O

O

(23)

O

O

OH

(25)

Fig. (11). A retrosynthetic analysis of the preparation of the spiro epoxide decalin ketal 21 from unsaturated 1,4-dione 26.

Compound 25 is formally the adduct of a Diels Alder reaction between unsaturated 1,4-dione 26 and diene 27 (Fig. 12), with subsequent epimerization of the ring junction to the more stable trans isomer.

Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis O

O +

O

O

OH

(25)

O

OH

O (26)

(27)

Fig. (12). A retrosynthetic analysis of the preparation of the 26 via a Diels Alder reaction with subsequent epimerization of the ring junction to the trans isomer.

Molecular modeling studies justify the rationale behind the tricyclic core of compounds 21-24. There is a substantial pocket in the active site in which the additional ring of the decalin core can be accommodated without steric interference from the residues of MetAP-2. Presumably this steric bulk will allow for more finely tuned Van der Wäals interactions of newly designed analogs. Fig. 13 shows the overlap of fumagillin and the spiro decalin ketal 21, while Fig. 14 shows the putative binding that would occur between 21 and MetAP-2 as determined from the molecular modeling work.


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trials on AGM-1470 have been carried out in patients with locally advanced, non-metastatic adenocarcinoma of pancreas and in AIDS-associated Kaposi’s sarcoma [52]. AGM1470 entered clinical trials in 1998, with an initial study in AIDS related Kaposi’s sarcoma. As a weekly infusion, AGM-1470 was found to be well tolerated, with 18% of patients obtaining a partial response, with the average duration of the response at 11 weeks. The phase I study of AGM1470 in AIDS-associated Kaposi’s sarcoma showed that adverse events such as neutropenia, hemorrhage, and urticaria were moderate and that the median time to partial response was weeks. It was recommended that further evaluation is needed for this drug in patients with AIDS-KS as a single agent or as a combination therapy with other drugs. It is noteworthy, however, that the elimination half-life value of AGM-1470 was very short, ranging from 0.01 to 0.61 hours [52].

Fig. (14). The in silico bound conformation of the spiro decalin ketal 21 in the active site of MetAP-2.

Fig. (13). The additional ring of the decalin derivative is accommodated by the active site of MetAP-2

An endothelial cell proliferation assay carried out in the Arbiser laboratory showed promising inhibition by approximately 50% for 21 and 22 [19]. Interestingly the ketals with the spiro epoxide and exo methylene groups, both electrophilic, showed the best activity. Future studies on the experimental binding with MetAP-2 will prove interesting. The initial phase of design, synthesis, and biological testing has demonstrated in principle that the cyclohexane core of fumagillin and similar derivatives may be expanded to decalin cores. 8. CLINICAL TRIALS It is somewhat surprising to learn that no clinical studies have been reported for fumagillin and ovalicin given the long history of discovery of these two compounds. Clinical

A second study was carried out in patients with advanced cervical cancer, in which a complete response was observed in one patient, and stable disease was observed in three additional patients [53]. Neurotoxicity was the dose limiting toxicity, and a regimen of 60 mg/m2 every Monday/ Wednesday/Friday was proposed. Neurotoxicity, which was neurocognitive (anxiety, agitation), was reversible upon discontinuation of therapy. AGM-1470 was conjugated into a polymeric delivery system (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, Gly-Phe-Leu-Gly linker and TNP-470). This conjugate is believed to accumulate selectively in tumor vessels because of the enhanced permeability and retention (EPR) effect due to vascular permeability [54]. A multi-institutional phase II study of AGM-1470 in patients with metastatic renal carcinoma revealed that AGM1470 has manageable toxicities but does not lead to any significant objective responses [55]. A phase I study of AGM1470 in patients with metastatic and androgen-independent prostate cancer showed that the dose-limiting toxic events


included characteristic neuropsychiatric symptom complex (anesthesia, gait disturbance, and agitation), and unfortunately no definite antitumor activity of AGM-1470 was observed [56]. The safety and tolerance of PPI-2458, 14, in patients with non-Hodgkin’s lymphoma and solid tumors was examined in the phase I clinical trials between years 2004 and 2007 [44]. The synthesis of compound ZGN-433 (beloranib), 15, also called CKD-732, discussed above, and its structural analogues was reported by Lee et al. [57]. The antiproliferation activity of 15 on calf pulmonary artery endothelial (CPAE) cells and HUVEC cells in vitro were evaluated. The data were very impressive. The IC50 values of 14 against CPAE and HUVEC cells were 0.65 nM and 0.0067 nM, respectively [57]. CKD-732 at the dosage of 50 mg/kg caused a decrease of body temperature but was recovered in 6 hours. No effect on general behavior, spontaneous locomotor activity, motor coordination, analgesia, and convulsion were observed [58]. This suggested that CKD-732 showed better safety than other fumagillin derivatives, including AGM1470. The preclinical studies of A-357300 showed a wide therapeutic index. No evidence of toxicity was observed, and the progression-free survival was impressive (observed in five out of eight mice) [59]. Other clinical trials include advanced prostate cancer, where transient decreases were observed in prostate specific antigen (PSA). A combination clinical trial of AGM-1470, paclitaxel and carboplatin in which four patients (24%) had a partial response, and eight (47%) had stable disease [60, 61]. According to Pubmed, no further clinical studies of AGM1470 have been carried out. CONCLUSIONS Our studies indicated that it is very likely to develop MetAP-2 selective inhibitors by utilizing the active site differences between MetAP-2 and MetAP-1 subfamilies. Considering the important role of ions in the active site, it would be wise to include Zn2+ (based on QM-calculation) or Co2+ (based on experimental data) in future structure-based drug design of MetAP-2 inhibitors. Future molecular design could focus on the development of a new generation of irreversible AGM-1470 based ligands or competitive, reversible compounds which could feature negatively charged carboxylic acid, sulfonic acid, or neutral hydroxyamide, hydrazine, or curcumin derivatives. Several trends are visible with the biological studies that plague modern drug development. First, a seven year gap existed between the initial discovery of AGM-1470 and its first clinical trial. In the meantime, hundreds of published works demonstrated in vivo efficacy of AGM-1470, so the relative contribution of these papers in advancing to a proof of principle is small and inefficient. Second, despite some initial promising clinical studies, AGM-1470 has not elicited much enthusiasm, even in the presence of a novel and potentially more effective form of delivery, namely HPMA copolymers conjugated with AGM-1470. Most of the interest in MetAP-2 inhibition now is in the treatment of obesity (Beloranib, Zafgen Pharmaceuticals), and future trials will be necessary to assess its efficacy as an angiogenesis inhibitor for obesity. Perhaps success in obesity will lead to a re-

Ehlers et al.

evaluation of AGM-1470 as a cancer agent, since both obesity and cancer are angiogenesis dependent, but, as of now, the promise of MetAP-2 inhibition remains unfulfilled. Nevertheless, new research in this area guided by computerassisted drug design continues to develop more efficacious MetAP-2 inhibitors with better ADME (absorption, distribution, metabolism, and excretion) properties. LIST OF ABBREVIATIONS BAEC

=

Bovine aortic endothelial cells

CPAE

=

Calf pulmonary artery endothelial

DFT

=

Density Functional Theory

EPR

=

Enhanced permeability and retention

HPMA

=

(N-(2-hydroxypropyl)methacrylamide

HUVEC

=

Human umbilical vein endothelial cells

MetAP-2

=

Methionine aminopeptidase Type 2

PSA

=

Prostate Specific Angiogen

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2]

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Received: July 07, 2015

Revised: August 24, 2015

Accepted: August 25, 2015

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