Methionine Aminopeptidase Type Research Articles

Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity.

Methionine aminopeptidase type 2 (METAP2) is a ubiquitous, evolutionarily conserved metalloprotease fundamental to protein biosynthesis which catalyzes removal of the N-terminal methionine residue from nascent polypeptides. METAP2 is an attractive target for cancer therapeutics based upon its over-expression in multiple human cancers, the importance of METAP2-specific substrates whose biological activity may be altered following METAP2 inhibition, and additionally, that METAP2 was identified as the target for the anti-angiogenic natural product, fumagillin. Irreversible inhibition of METAP2 using fumagillin analogues has established the anti-angiogenic and anti-tumor characteristics of these derivatives; however, their full clinical potential has not been realized due to a combination of poor drug-like properties and dose-limiting central nervous system (CNS) toxicity. This report describes the physicochemical and pharmacological characterization of SDX-7320 (evexomostat), a polymer-drug conjugate of the novel METAP2 inhibitor (METAP2i) SDX-7539. In vitro binding, enzyme, and cell-based assays demonstrated that SDX-7539 is a potent and selective METAP2 inhibitor. In utilizing a high molecular weight, water-soluble polymer to conjugate the novel fumagillol-derived, cathepsin-released, METAP2i SDX-7539, limitations observed with prior generation, small molecule fumagillol derivatives were ameliorated including reduced CNS exposure of the METAP2i, and prolonged half-life enabling convenient administration. Multiple xenograft and syngeneic cancer models were utilized to demonstrate the anti-tumor and anti-metastatic profile of SDX-7320. Unlike polymer-drug conjugates in general, reductions in small molecule-equivalent efficacious doses following polymer conjugation were observed. SDX-7320 has completed a phase I clinical safety study in patients with late-stage cancer and is currently being evaluated in multiple phase Ib/II clinical studies in patients with advanced solid tumors.

Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis.

Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not necessarily involved in metal transport across membranes. The identification of these mechanisms provides a new opportunity to improve metal-use efficiency and develop plant foodstuffs with increased concentrations of bioavailable metal nutrients. Here, we investigate the function of two distinct subfamilies of the nucleotide-dependent metallochaperones (NMCs), named ZNG1 and ZNG2, that are found in plants, using Arabidopsis thaliana as a reference organism. AtZNG1 (AT1G26520) is an ortholog of human and fungal ZNG1, and like its previously characterized eukaryotic relatives, localizes to the cytosol and physically interacts with methionine aminopeptidase type I (AtMAP1A). Analysis of AtZNG1, AtMAP1A, AtMAP2A, and AtMAP2B transgenic mutants are consistent with the role of Arabidopsis ZNG1 as a Zn transferase for AtMAP1A, as previously described in yeast and zebrafish. Structural modeling reveals a flexible cysteine-rich loop that we hypothesize enables direct transfer of Zn from AtZNG1 to AtMAP1A during GTP hydrolysis. Based on proteomics and transcriptomics, loss of this ancient and conserved mechanism has pleiotropic consequences impacting the expression of hundreds of genes, including those involved in photosynthesis and vesicle transport. Members of the plant-specific family of NMCs, ZNG2A1 (AT1G80480) and ZNG2A2 (AT1G15730), are also required during Zn deficiency, but their target protein(s) remain to be discovered. RNA-seq analyses reveal wide-ranging impacts across the cell when the genes encoding these plastid-localized NMCs are disrupted.

Abstract 1656: Evexomostat: A novel therapeutic in development for the treatment of prostate cancer

Abstract Background: Prostate cancer (PCa) is the second-most diagnosed malignancy among men and is a leading cause of cancer death worldwide (WHO, 2020). Elevated expression of the enzyme methionine aminopeptidase type 2 (MetAP2) in prostate cancer was recently shown to be associated with higher grade tumors and with worse clinical outcomes (Xie, 2021). Evexomostat/SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor attached to a polymer backbone via a cleavable linker. This design alters biodistribution (limits CNS penetration) and improves pharmacokinetics relative to small molecule fumagillin-derived MetAP2 inhibitors. Evexomostat completed a phase I safety study in late-stage cancer patients and is in two phase 2 breast cancer studies (NCT05455619, NCT05570253). Methods: To evaluate the efficacy of evexomostat in a model of prostate cancer, NSG mice were injected subcutaneously with 2×106 LNCaP cells. When tumors reached ≈200 mm3, mice were randomized into 4 groups. A cohort of intact mice (Group 1; n=4/group) received treatment with SDX-7320 (12 mg/kg, s.c. Q4D) or vehicle (5% mannitol/water). Group 2 were castrated and randomized to SDX-7320 (6 mg/kg, 12 mg/kg) or Vehicle (5% mannitol) upon recovery (n=4/group). Group 3 (n=13/group) and Group 4 (n=14/group) were castrated and randomized to Vehicle or SDX-7320 at recurrence of tumor growth with Group 4 additionally receiving enzalutamide (10 mg/kg, p.o., QD). Tumor growth and body weight were assessed twice/week. Upon necropsy, tumors were dissected, weighed and snap frozen for RNAseq analysis or formalin fixed along with major organs for histological processing (H&E staining, and tumor CD31, CD34 IHC analysis). Results: Evexomostat elicited a significant reduction in tumor growth in the absence of significant changes in body weight. Survival in evexomostat-treated mice was doubled relative to vehicle-treated mice (12.5 vs 23.5 days). In castrated mice (Group 2), evexomostat (6 and 12 mg/kg) attenuated tumor growth with no change in body weight, and in CRPC mice (Group 3), evexomostat also significantly inhibited tumor growth. Treatment of CRPC mice (Group 4) with enzalutamide alone elicited a mixed response, with 6/14 mice showing sustained response to enzalutamide (responders). At endpoint, tumors from enzalutamide non-responders averaged 0.54g (± 0.09g) and enzalutamide responders 0.27g (± 0.07g), p&lt0.05, while tumors from SDX-7320 plus enzalutamide-treated mice weighed 0.19g (± 0.07g), p&lt0.005 compared to enzalutamide non-responders. Conclusions: MetAP2 inhibition has potential as a new approach for the treatment of prostate cancer. The clinical-stage MetAP2 inhibitor evexomostat/SDX-7320 significantly inhibited the growth of LNCaP tumors in intact, castrated and CRPC mice. Clinical evaluation of evexomostat in castration-sensitive as well as castration-resistant prostate cancer patients is warranted. Citation Format: Peter Cornelius, Benjamin Mayes, Bradley J. Carver, James Shanahan, Jennifer Gunter, Colleen Nelson. Evexomostat: A novel therapeutic in development for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1656.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Potential inhibitors of methionine aminopeptidase type II identified via structure-based pharmacophore modeling.

Methionine aminopeptidase (MetAP2) is a metal-containing enzyme that removes initiator methionine from the N-terminus of a newly synthesized protein. Inhibition of the enzyme is crucial in diminishing cancer growth and metastasis. Fumagillin-a natural irreversible inhibitor of MetAP2-and its derivatives are used as potent MetAP2 inhibitors. However, because of their adverse effects, none of them has progressed to clinical studies. In search for potential reversible inhibitors, we built structure-based pharmacophore models using the crystal structure of MetAP2 complexed with fumagillin (PDB ID: 1BOA). The pharmacophore models were validated using Gunner-Henry scoring method. The best pharmacophore consisting of 1 H-bond donor, 1 H-bond acceptor, and 3 hydrophobic features was used to conduct pharmacophore-based virtual screening of ZINC15 database against MetAP2. The top 10 compounds with pharmacophore fit values > 3.00 were selected for further analysis. These compounds were subjected to absorption, distribution, metabolism, elimination, and toxicity (ADMET) prediction and found to have druglike properties. Furthermore, molecular docking calculations was performed on these hits using AutoDock4 to predict their binding mode and binding energy. Three diverse compounds: ZINC000014903160, ZINC000040174591, and ZINC000409110720 with respective binding energy/docking scores of - 9.22, - 9.21, and -817kcal/mol, were submitted to 100ns (MD) simulations using Nanoscale MD (NAMD) software. The compounds showed stable binding mode over time. Therefore, they may serve as a scaffold for further computational and experimental optimization toward the design of more potent and safer MetAP2 inhibitors.

Abstract CT153: SDX-7320 elicits improvements in tumor-related and metabolic biomarkers: Results of a phase 1 dose-escalation study in patients with advanced refractory or late-stage solid tumors

Abstract Background and Rationale: Metabo-oncology is the study of specific tumor types whose growth and/or metastasis is accelerated by metabolic dysfunction, often associated with hyper-adiposity. In this setting the metabolic hormones leptin, adiponectin and insulin are potential mediators. SDX-7320 is a novel methionine aminopeptidase type 2 inhibitor (MetAP2) being developed for use in combination with other anti-cancer agents to treat the metabo-oncology patient population. Study Design: This first-in-human study used a 3+3 design with dose expansion at the RP2D. Measurements included standard assessments of safety, tolerability, as well as inhibition of MetAP2 in whole blood, plasma levels of protein biomarkers and drug exposure. Tumor response was measured using RECIST v.1.1. Results: 32 patients with an average of 5.3 prior treatment regimens were dosed. Sub-cutaneous dosing of SDX-7320 began at 1.7 mg/m2 once per week (Q7D, 28 days/cycle) and was escalated to 49 mg/m2. Repeated Q7D administration at 49 mg/m2 resulted in dose-limiting (G3-G4) thrombocytopenia. Expansion at 49 mg/m2 confirmed thrombocytopenia as the dose-limiting toxicity (DLT), reversible upon cessation of dosing. Dose-escalation was re-initiated at 36 mg/m2 every two weeks (Q14D). Repeated dosing at 65 mg/m2 (Q14D) resulted in G3 reversible thrombocytopenia. Expansion at 49 mg/m2 (Q14D) confirmed that this was the maximum tolerated dose (MTD) on a Q14D schedule. No deaths or Grade 5 TEAEs attributable to SDX-7320 were reported. Six patients had a combined total of 9 Grade 3/4 TEAEs considered by the Investigator to be at least possibly SDX-7320-related: thrombocytopenia (4 patients) and single cases of anemia, anorexia, fatigue, QTc prolonged, and vasculitis. Fourteen patients (44% had stable disease &gt2 cycles and three patients had stable disease for ≥6 cycles. Inhibition of MetAP2 in whole blood was 100% at all doses evaluated while the time to reach 100% inhibition was inversely related to dose. When stratified for elevated baseline levels, the following biomarkers declined after initiating SDX-7320 (average of each patient's maximum % change relative to baseline +/- SD): bFGF (-53 +/- 76%), VEGF-C (-35 +/- 39%), and insulin (-55 +/- 30%). Without stratification, leptin levels declined (-51 +/-29%) and levels of adiponectin increased after SDX-7320 (+74 +/- 63% relative to baseline). Conclusions and Next Steps : SDX-7320 was well-tolerated with prolonged stable disease in an open-label, phase I first-in-human study. Improvements were observed in both tumor-related and metabolic biomarkers. Additional studies of SDX-7320 (in combination with other anti-cancer agents) in patients with solid tumors sensitive to metabolic hormones are scheduled. SDX-7320 is the first novel agent in clinical development to treat the metabo-oncology patient population. Citation Format: Monica M. Mita, Johanna Bendell, Alain C. Mita, Michael Gordon, Jasgit Sachdev, Bradley J. Carver, James Shanahan, Benjamin Mayes, Kris Awerkamp, David Browning, Neal Salomon, Kimberly Sullivan, Alfred Anderson-Villaluz, Joe Johnson, John S. Petersen, David J. Turnquist, Peter Cornelius. SDX-7320 elicits improvements in tumor-related and metabolic biomarkers: Results of a phase 1 dose-escalation study in patients with advanced refractory or late-stage solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT153.

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications.

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.

Abstract 4919: Preclinical activity of SDX-7320 in mouse models of obesity and obesity-driven cancer

Abstract Cancer patients who are obese face a greater risk of dying from cancer compared to nonobese patients (Calle, 2003). Obesity is believed to contribute to metastasis and progression of cancer via multiple mechanisms: increased secretion of the adipose tissue hormone leptin, decreased secretion of adiponectin, increased production of estrogen in adipose tissue, and elevated insulin (secondary to peripheral insulin resistance) as well as the local effects of inflammatory cytokines (Gucalp, 2016). Small-molecule inhibitors of methionine aminopeptidase type 2 (MetAP2) have previously demonstrated clinical activity in oncology (Kudelka, 1998) as well as obesity/type 2 diabetes (Hughes, 2013; Kim, 2015). However, development of some small-molecule MetAP2 inhibitors has been hampered by CNS side effects (Bhargava, 1999). SDX-7320 is a copolymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone, intended to limit CNS penetration and therefore reduce CNS toxicity. In vitro MetAP2 binding assays showed that SDX-7320 was unable to bind to MetAP2 while SDX-7539 was a potent binder (apparent IC50 = 0.13 nM), indicating that cleavage of SDX-7320 is required for biologic activity. SDX-7539 inhibited proliferation of human umbilical vein endothelial cells (HUVECs) with an apparent IC50 = 0.2 nM. The preclinical efficacy of SDX-7320 was evaluated in mouse models of obesity/insulin resistance and also in syngeneic tumor models coupled with high fat diet-induced obesity (DIO)/metabolic dysfunction. Obesity and insulin resistance were induced in C57Bl/6 mice by feeding them a high-fat diet (HFD) for at least 12 weeks. SDX-7320 was dosed subcutaneously every 4 days (for 28 days) into DIO mice or control mice fed a low-fat diet. SDX-7320 caused significant reduction in body weight and fat mass in obese mice while also reversing insulin resistance relative to controls. In addition, SDX-7320 caused greater reduction in body weight in obese mice relative to lean mice. Circulating levels of leptin and insulin were reduced while levels of adiponectin were increased in response to SDX-7320. In mouse models of obesity-accelerated tumor growth, SDX-7320 was more efficacious in obese relative to lean mice harboring subcutaneous B16F10 melanoma or EO771 mammary gland tumors. These effects occurred without any signs of neurotoxicity. SDX-7320, currently in phase I (solid tumors; all-comers; NCT02743637), is being developed for the treatment of cancers whose progression is accelerated in the setting of obesity and host metabolic dysfunction, termed “metabo-oncology.” Citation Format: Peter Cornelius, John S. Petersen, Benjamin Mayes, David Turnquist, Kimberly Sullivan, Alfred Anderson-Villaluz, Robert Lutz, Sara Little, Andrew Slee, Bradley J. Carver, James Shanahan. Preclinical activity of SDX-7320 in mouse models of obesity and obesity-driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4919.

Neutral metalloaminopeptidases APN and MetAP2 as newly discovered anticancer molecular targets of actinomycin D and its simple analogs

The potent transcription inhibitor Actinomycin D is used with several cancers. Here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type 2. These metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. We show that the peptide portions of Actinomycin D and Actinomycin X2 are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. Two structurally less complex Actinomycin D analogs containing the phenoxazone chromophores, Questiomycin A and Actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (Ki in the micromolar range). The mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. This knowledge gives new perspectives to Actinomycin D's action on tumors and suggests new avenues and molecules for medical applications.

Evaluation of Fumagilin-B® and other potential alternative chemotherapies against Nosema ceranae-infected honeybees (Apis mellifera) in cage trial assays

Fumagilin-B® is the only currently registered chemical treatment available to combat nosema disease in apiculture. Fumagillol, the basic hydrolysis product of fumagillin, two semisynthetic fumagillin analogues, and four in-house purely synthetic compounds which were designed to mimic the mode of action of fumagillin against the methionine aminopeptidase type 2 (MetAP-2) enzyme, was observed to exhibit statistically significant biological activity against Nosema ceranae-infected caged bees. None of these compounds were, however, as effective as Fumagilin-B®. The commercially available thymol and enilconazole also exhibited activity against N. ceranae, with thymol being the most promising chemical treatment other than Fumagilin-B®. High cumulative bee mortality was associated with the therapeutic dosage of Fumagilin-B® during our study, suggesting the need for continued investigation.

Chemical shift assignments of zinc finger domain of methionine aminopeptidase 1 (MetAP1) from Homo sapiens.

Methionine aminopeptidase Type I (MetAP1) cleaves the initiator methionine from about 70% of all newly synthesized proteins in almost every living cell. Human MetAP1 is a two domain protein with a zinc finger on the N-terminus and a catalytic domain on the C-terminus. Here, we report the chemical shift assignments of the amino terminal zinc binding domain (ZBD) (1-83 residues) of the human MetAP1 derived by using advanced NMR spectroscopic methods. We were able to assign the chemical shifts of ZBD of MetAP1 nearly complete, which reveal two helical fragments involving residues P44-L49 (α1) and Q59-K82 (α2). The protein structure unfolds upon complex formation with the addition of 2M excess EDTA, indicated by the appearance of amide resonances in the random coil chemical shift region of (15)NHSQC spectrum.

Synthesis and antitumor activity of 1,3,4-oxadiazole possessing 1,4-benzodioxan moiety as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,3,4-oxadiazole derivatives containing 1,4-benzodioxan moiety (7a–7q) have been designed, synthesized and evaluated for their antitumor activity. Most of the synthesized compounds were proved to have potent antitumor activity and low toxicity. Among them, compound 7a showed the most potent biological activity against Human Umbilical Vein Endothelial cells, which was comparable to the positive control. The results of apoptosis and flow cytometry (FCM) demonstrated that compound 7a induce cell apoptosis by the inhibition of MetAP2 pathway. Molecular docking was performed to position compound 7a into MetAP2 binding site in order to explore the potential target.

Research of New Molecules Able to Starve the Tumors by Molecular Docking\'s Method

In recent years, a promising new approach uses the anti-angiogenesis properties of certain molecules to try to block cancer by depriving tumors of nutrients and oxygen they need to grow. Endothelial cells, specialized in the development of new blood vessels, are the target of most anti-angiogenic strategies, Or methionine aminopeptidase (MetAp) type 2 is a member of a family of proteins that regulates the growth of these endothelial cells. The molecular docking program, GOLD, was developed to assist in the development of molecules with therapeutic activity. It has been used to study the inhibition of 1QZY, human methionine aminopeptidase type 2, by bengamide derivatives, with the aim of discovering new anti-angiogenic drugs. The evaluation of the affinity of these molecules brought to light those presenting the best inhibitive effect. It is about the compound 16, the value of the score of which is 135.35.

Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan fragment as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,2,4-triazole derivatives containing 1,4-benzodioxan ( 5a– 5q) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential MetAP2 inhibitors. All the synthesized compounds were first reported. Among the compounds, compound 5k showed the most potent biological activity against HEPG2 cancer cell line (IC 50 = 0.81 μM for HEPG2 and IC 50 = 0.93 μM for MetAP2), which was comparable to the positive control. Docking simulation by positioning compound 5k into the MetAP2 structure active site was performed to explore the possible binding model. The results of apoptosis and Western-blot assay demonstrated that compound 5k possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 5k with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell.

Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity

BackgroundMethionine aminopeptidase (MetAP) is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins.ResultsWe cloned genes encoding two such metalloproteases (MtMetAP1a and MtMetAP1c) present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, MtMetAP1c had significantly high enzyme turnover rate as opposed to MtMetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C) of MtMetAP1a compared to that of the MtMetAP1c. Modelling of MtMetAP1a based on MtMetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of MtMetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of MtMetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of MtMetAP1c.ConclusionOur findings unequivocally emphasized that N-terminal extension of MtMetAP1c contributes towards the functionality of the enzyme presumably by regulating active site residues through "action-at-a-distance" mechanism and we for the first time are reporting this unique function of the enzyme.

Fumagillin Reduces Adipose Tissue Formation in Murine Models of Nutritionally Induced Obesity

The effect of fumagillin (a methionine aminopeptidase-type 2 (Met-AP2) inhibitor, with antiangiogenic properties) was investigated in murine models of diet-induced obesity. Eleven-week-old male C57Bl/6 mice (group 1) were given fumagillin by oral gavage at a dose of 1 mg/kg/day during 4 weeks while fed a high-fat diet (HFD) (20.1 kJ/g), and control mice (group 2) received solvent and were pair-fed. At the end of the experiment, body weights in group 1 were significantly lower as compared to group 2 (P < 0.0005). The subcutaneous (SC) and gonadal (GON) fat mass was also significantly lower in group 1 (P < 0.005 and P < 0.05, respectively). Adipocytes were smaller in adipose tissues of mice in group 1, associated with higher adipocyte density. Blood vessel density normalized to adipocyte density was lower in group 1 adipose tissues. However, in mice with established obesity monitored to maintain the same body weight and fat mass as controls, short-term fumagillin administration was also associated with adipocyte hypotrophy (P = 0.01) without affecting blood vessel size or density. Thus, treatment with fumagillin impaired diet-induced obesity in mice, associated with adipocyte hypotrophy but without marked effect on adipose tissue angiogenesis.

Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470

Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity invitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

An inhibitor of methionine aminopeptidase type-2, PPI-2458, ameliorates the pathophysiological disease processes of rheumatoid arthritis

To elucidate the role of methionine aminopeptidase type-2 (MetAP-2) in the clinical pathology of rheumatoid arthritis, arthritis was induced in rats by administration of peptidoglycan-polysaccharide (PG-PS). The inhibitor of MetAP-2, PPI-2458, was administered orally at 5 mg/kg every other day during 3 distinct phases of the disease. In vitro studies were performed to clarify in vivo findings. Ankle swelling was completely alleviated by MetAP-2 inhibition. Inhibition of MetAP-2 in blood and tissues correlated with protection against PG-PS-induced arthritis. Histopathology of the tarsal joints improved following PPI-2458 administration, including a significant improvement of bone structure. In in vitro studies, osteoclast formation and activity were inhibited by PPI-2458, a mechanism not previously attributed to MetAP-2 inhibition. The important role that MetAP-2 has in the pathophysiological disease processes of PG-PS arthritis provides a strong rationale for evaluating PPI-2458 as a disease modifying antirheumatic treatment for rheumatoid arthritis.

Suppression of inflammation and structural damage in experimental arthritis through molecular targeted therapy with PPI‐2458

To determine the disease-modifying activity and mechanism of action of the orally available methionine aminopeptidase type 2 inhibitor, [(1R)-1-carbamoyl-2-methyl-propyl]-carbamic acid-(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro [2.5] oct-6-yl ester (PPI-2458), in a rat model of peptidoglycan-polysaccharide (PG-PS)-induced arthritis. Arthritis was induced in rats by administration of PG-PS, causing tarsal joint swelling and histopathologic changes characteristic of rheumatoid arthritis (RA). PPI-2458, a potent irreversible methionine aminopeptidase type 2 inhibitor, was administered orally every other day at 1, 5, or 10 mg/kg. In an in vitro osteoclastogenesis model, PPI-2458 potently inhibited osteoclast differentiation and bone resorption. In the rat PG-PS arthritis model, PPI-2458 afforded significant protection against established disease after therapeutic dosing. This in vivo activity of PPI-2458 was linked to the inhibition of methionine aminopeptidase type 2. Histopathologic assessment of affected joints showed improvement in processes of inflammation, bone resorption, and cartilage erosion, associated with significant improvement in all clinical indices. The protective effects of PPI-2458 against bone destruction in vivo, including the structural preservation of affected hind joints, correlated with improvements in bone histomorphometric markers, as determined by microfocal computed tomography and a significant decrease in systemic C-telopeptide of type I collagen, suggesting decreased osteoclast activity in vivo. Moreover, PPI-2458 prevented cartilage erosion as shown by a significant decrease in systemic cartilage oligomeric matrix protein. The findings of this study suggest that PPI-2458 exerts disease-modifying activity in experimental arthritis through its direct inhibition of several pathophysiologic processes of this disease. These results provide a rationale for assessing the potential of PPI-2458 as a novel RA therapy.

Development of Sulfonamide Compounds as Potent Methionine Aminopeptidase Type II Inhibitors with Antiproliferative Properties.

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