Methionine Aminopeptidase-2 Research Articles

MetAP2 inhibitor treatment of high-fat and -fructose-fed dogs: impact on the response to oral glucose ingestion and a hyperinsulinemic hyperglycemic clamp.

We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.

Identification of Methionine Aminopeptidase-2 (MetAP-2) Inhibitor M8891: A Clinical Compound for the Treatment of Cancer.

The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound 1a suffered from enterohepatic circulation, preventing further development. Nevertheless, 1a served as a starting point for further optimization. Maintaining potent antiproliferation activity, while improving other compound properties, enabled the generation of an attractive array of new MetAP-2 inhibitors. The most promising derivatives were identified by a multiparameter analysis of the compound properties. Essential for the efficient selection of candidates with in vivo activity was the identification of molecules with a long residence time on the target protein, high permeability, and low efflux ratio not only in Caco-2 but also in the MDR-MDCK cell line. Orally bioavailable, potent, and reversible MetAP-2 inhibitors impede the growth of primary endothelial cells and demonstrated antitumoral activity in mouse models. This assessment led to the nomination of the clinical development compound M8891, which is currently in phase I clinical testing in oncology patients.

Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

Structural features of methionine aminopeptidase2-active core peptide essential for binding with S100A4

Methionine aminopeptidase 2 (MetAP2) is one of the effector proteins of S100A4, a metastasis-associated calcium-binding protein. This interaction is involved in angiogenesis. The region of MetAP2 that interacts with S100A4 includes amino acids 170 to 208. A peptide corresponding to this region, named as NBD, has potent anti-angiogenic activity and suppresses tumor growth in a xenograft cancer model. However, the binding mode of NBD to S100A4 was totally unknown. Here we describe our analysis of the relationship between the inhibitory activity and the structure of NBD, which adopts a characteristic helix-turn-helix structure as shown by X-ray crystallographic analysis, and peptide fragments of NBD. We conducted physicochemical analyses of the interaction between S100A4 and the peptides, including surface plasmon resonance, microscale thermophoresis, and circular dichroism, and performed docking/molecular dynamics simulations. Active peptides had stable secondary structures, whereas inactive peptides had a little secondary structure. A computational analysis of the interaction mechanism led to the design of a peptide smaller than NBD, NBD-ΔN10, that possessed inhibitory activity. Our study provides a strategy for design for a specific peptide inhibitor against S100A4 that can be applied to the discovery of inhibitors of other protein-protein interactions.

Abstract 1226: A novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor SDX-7320 inhibits the growth of EO771 mammary gland tumors and ameliorates the immunosuppressive tumor immune micro-environment (TIME)

Abstract Obese patients with certain cancers face a greater risk of dying from their cancer compared to non-obese patients. Obesity may contribute to cancer progression via multiple mechanisms: increased leptin, decreased adiponectin, increased adipose tissue estrogen, elevated insulin (secondary to peripheral insulin resistance), increased cytokines, and alteration of the TIME which suppresses host anti-tumor immune responses. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. However, clinical development in some cases (e.g. TNP-470) has been altered CNS toxicity. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone. This is intended to limit CNS penetration and reduce CNS toxicity relative to small molecules. SDX-7320 was tested ± 5-FU in a syngeneic model of post-menopausal, obesity-accelerated breast cancer. Ovariectomized C57Bl/6 mice were fed a high-fat diet (to induce obesity) or low-fat diet for 16 weeks prior to injection of EO771 tumor cells into the mammary gland. Tumors in obese mice grew significantly faster than in low fat-fed (normal) mice. SDX-7320 (8 mg/kg, SC, Q4D) significantly attenuated tumor growth in normal and obese mice relative to vehicles (-33% and -43% respectively). 5-FU (10 mg/kg, IP, 2X/week) had no effect on tumor growth in normal or obese mice, while the combination of 5-FU with SDX-7320 inhibited tumor growth in obese mice greater than SDX-7320 alone (-61% vs -43% respectively), suggestive of a synergistic interaction. In a separate study, the effect of SDX-7320 on the EO771 TIME in obese versus normal-weight mice was assessed. Tumors in obese mice grew significantly faster than in normal mice. SDX-7320 (24 mg/kg, SC, Q4D) decreased tumor growth in both normal and obese mice (-53% and -52% change in tumor volume relative to lean and obese vehicle groups, respectively). SDX-7320 also reduced the immunosuppressive state of the TIME in obese mice. Relative to tumors from untreated mice, MDSC content (flow cytometry), Arginase 1 staining (by IHC) and FoxP3 staining (by IHC) were all decreased. Analysis of cytokines showed that IL-10 was elevated in plasma of obese mice relative to normal mice, and SDX-7320 significantly reduced the plasma levels of IL-10 in obese mice. SDX-7320 exerted favorable effects on the TIME in a model of post-menopausal obesity-accelerated breast cancer suggesting it may potentiate the efficacy of immunotherapy in breast cancer. SDX-7320 is being developed to treat cancers whose growth is accelerated by obesity and metabolic dysfunction. Now nearing completion of Phase I (NCT02743637), further clinical trials with SDX-7320 in combination with other therapies are planned for 2019. Citation Format: Peter Cornelius, Benjamin Mayes, Sara Little, Andrew Slee, David Turnquist, James Shanahan, Bradley Carver. A novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor SDX-7320 inhibits the growth of EO771 mammary gland tumors and ameliorates the immunosuppressive tumor immune micro-environment (TIME) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1226.

MetAP2 inhibition increases energy expenditure through direct action on brown adipocytes

Inhibitors of methionine aminopeptidase 2 (MetAP2) have been shown to reduce body weight in obese mice and humans. The target tissue and cellular mechanism of MetAP2 inhibitors, however, have not been extensively examined. Using compounds with diverse chemical scaffolds, we showed that MetAP2 inhibition decreases body weight and fat mass and increases lean mass in the obese mice but not in the lean mice. Obesity is associated with catecholamine resistance and blunted β-adrenergic receptor signaling activities, which could dampen lipolysis and energy expenditure resulting in weight gain. In the current study, we examined effect of MetAP2 inhibition on brown adipose tissue and brown adipocytes. Norepinephrine increases energy expenditure in brown adipose tissue by providing fatty acid substrate through lipolysis and by increasing expression of uncoupled protein-1 (UCP1). Metabolomic analysis shows that in response to MetAP2 inhibitor treatment, fatty acid metabolites in brown adipose tissue increase transiently and subsequently decrease to basal or below basal levels, suggesting an effect on fatty acid metabolism in this tissue. Treatment of brown adipocytes with MetAP2 inhibitors enhances norepinephrine-induced lipolysis and energy expenditure, and prolongs the activity of norepinephrine to increase ucp1 gene expression and energy expenditure in norepinephrine-desensitized brown adipocytes. In summary, we showed that the anti-obesity activity of MetAP2 inhibitors can be mediated, at least in part, through direct action on brown adipocytes by enhancing β-adrenergic-signaling-stimulated activities.

149-LB: The MetAP2 Inhibitor, ZGN-1061, Improves Insulin Sensitivity and Beta-Cell Function in Patients with Type 2 Diabetes

ZGN-1061 is a second-generation methionine aminopeptidase 2 (MetAP2) inhibitor in development for treatment of T2D. Full results of a Phase 2 clinical trial of ZGN-1061 vs. placebo (PBO) administered SC for 12 weeks on change from baseline in A1C in subjects with T2D are reported elsewhere. Baseline (ITT population N=175) A1C 8.6±1.1%, BMI 37±7 kg/m2, T2D duration 8±6 y. ZGN-1061 was generally well tolerated; 93% completed the trial. LS mean±SE A1C change was 0.2±0.1% for PBO vs. ±0.4±0.1% and -0.9±0.2% for 0.9 and 1.8 mg ZGN-1061 (both p<0.001 vs. PBO). PBO weight change was -0.6±0.3 kg vs. -1.4±0.3 kg (p=ns) and ±2.8±0.5 kg (p<0.001) for 0.9 and 1.8 mg ZGN-1061. This analysis included a subset of subjects who participated in a mixed-meal tolerance test (MMTT) at baseline and week 12. ZGN-1061 produced dose-dependent improvements in fasting and postprandial glucose (p<0.05 vs. PBO). Minimal model analysis showed improvements in insulin sensitivity (SI) and disposition index (DI_OB; a measure of β-cell compensation) from baseline to week 12 with ZGN-1061 (Table). In an exploratory combined analysis of 0.9+1.8 mg ZGN-1061, SI and DI_OB increased (p=0.02 and p=0.07). SI and DI_OB were unchanged with PBO. In patients with T2D, improved glycemic control with ZGN-1061 may be driven by changes in insulin sensitivity and β-cell function. These results support future assessments using other definitive methods. Disclosure R. Fabunmi: None. R.N. Bergman: Consultant; Self; Zafgen, Inc. F. Piccinini: None. T. Kim: Employee; Spouse/Partner; Celgene Corporation. Employee; Self; Zafgen, Inc. D. Zhuang: Employee; Self; Zafgen, Inc. K. Taylor: Employee; Self; Zafgen, Inc. D.D. Kim: Employee; Self; Zafgen, Inc. Funding Zafgen, Inc.

21-OR: Final Results of a Phase 2, Randomized, Double-Blind, Placebo-Controlled Clinical Trial of the MetAP2 Inhibitor ZGN-1061 in Patients with Type 2 Diabetes

ZGN-1061 is a second-generation methionine aminopeptidase 2 (MetAP2) inhibitor in development for treatment of T2D. This clinical trial evaluated ZGN-1061 (0.05, 0.3, 0.9, or 1.8 mg) vs. placebo (PBO) administered SC every 3 days for 12 weeks on change from baseline in A1C in individuals with T2D at multiple sites in Australia/New Zealand. Stable noninsulin diabetes therapy was permitted. Baseline (ITT population N=175): 52% female, 77% white, (mean±SD) age 54±8 y, A1C 8.6±1.1%, BMI 37±7 kg/m2, T2D duration 8±6 y. Interim analysis results (0.05, 0.3, 0.9 mg ZGN-1061) were presented previously. Here, we report additional results with 0.9 and 1.8 mg ZGN-1061. The LS mean±SE A1C change for PBO was 0.2±0.1% (N=42) vs. -0.4±0.1% (N=44) and -0.9±0.2% (N=23) for 0.9 and 1.8 mg ZGN-1061, respectively. The LS mean±SE difference vs. PBO was -0.6±0.2% and 1.1±0.2% for 0.9 mg (p=0.0003) and 1.8 mg (p<0.0001). The LS mean±SE weight change for PBO was -0.6±0.3 kg vs. -1.4±0.3 kg (p=ns) and -2.8±0.5 kg (p<0.001) for 0.9 and 1.8 mg ZGN-1061. Dose-dependent fasting and postprandial glucose reductions were observed for ZGN-1061 (p<0.05 vs. PBO); β-cell function and insulin sensitivity were improved with 1.8 mg ZGN-1061 (p<0.05 vs. PBO). ZGN-1061 also improved triglycerides, hsCRP, adiponectin, and leptin (p<0.05 vs. PBO). Across all ZGN-1061 treatment groups, adverse events (AEs) were primarily mild/moderate without any notable trends. The completion rate was 93% (163/175). Three subjects in ZGN-1061 groups withdrew due to AEs (sensory disturbance, 0.05 mg; upper abdominal pain, 0.9 mg; injection site urticaria, 1.8 mg). There were no reports of venous thrombosis or changes in thrombosis markers indicative of a thrombosis event. ZGN-1061 (1.8 mg) demonstrated statistically significant and clinically meaningful efficacy; the largest reductions in A1C and weight were observed at 12 weeks with no evidence of a waning effect and no notable safety signals. Disclosure T. Kim: Employee; Spouse/Partner; Celgene Corporation. Employee; Self; Zafgen, Inc. D. Zhuang: Employee; Self; Zafgen, Inc. T.L. Haugen: Employee; Self; Zafgen, Inc. D.D. Kim: Employee; Self; Zafgen, Inc. K. Taylor: Employee; Self; Zafgen, Inc.

Synthesis, molecular modeling, in vivo study, and anticancer activity of 1,2,4-triazole containing hydrazide-hydrazones derived from (S)-naproxen.

A new series of 1,2,4-triazole containing hydrazide-hydrazones derived from (S)-naproxen ( 7a-m) was synthesized in this study. The structures of these compounds were characterized by spectral (Fourier-transform infrared spectroscopy, 1 H-nuclear magnetic resonance (NMR), 13 C-NMR, and high-resolution electron ionization mass spectrometry) methods. Furthermore, molecular modeling of these compounds was studied on human methionine aminopeptidase-2. All synthesized compounds were screened for anticancer activity against three prostate cancer cell lines (PC3, DU-145, and LNCaP) using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium colorimetric method. Compound 7a showed the best activity against the PC3, DU-145 and LNCaP cancer cell lines with IC50 values of 26.0, 34.5, and 48.8 μM, respectively. Compounds 7b, 7k, and 7m showed anticancer activity against cancer cell lines PC3 and DU-145 with IC50 values of 43.0, 36.5, 29.3 μM and 49.8, 49.1, 31.6 μM, respectively. Compounds 7f and 7g showed anticancer activity against PC3 cells with IC50 values of 43.4 and 34.5 μM, respectively. To assess the biodistribution in mice of IRDye800, dye-labeled compound 7a or 100 μM of free dye was injected intravenously into the mice's tail. In vivo images were taken with in vivo imaging system spectrum device at 60, 120, 180, 240, 300, and 360 min after injection. At the end of 360 min, ex vivo studies were carried out to determine in which organs the dye was accumulated in the urogenital system. Ex vivo studies showed that the accumulation of compound 7a in the prostate is greater than that of the free dye, and it is concluded that compound 7a may be promising for the treatment of prostate cancer.

Discovery and Structure-Based Optimization of Next-Generation Reversible Methionine Aminopeptidase-2 (MetAP-2) Inhibitors.

Co- and post-translational processing are crucial maturation steps to generate functional proteins. MetAP-2 plays an important role in this process, and inhibition of its proteolytic activity has been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitors of MetAP-2 may be promising options for the treatment of cancer. This work describes the discovery and structure-based hit optimization of a novel MetAP-2 inhibitory scaffold. Of critical importance, a cyclic tartronic diamide coordinates the MetAP-2 metal ion in the active site while additional side chains of the molecule were designed to occupy the lipophilic methionine side chain recognition pocket as well as the shallow cavity at the opening of the active site. The racemic screening hit from HTS campaign 11a was discovered with an enzymatic IC50 of 150 nM. The resynthesized eutomer confirmed this activity and inhibited HUVEC proliferation with an IC50 of 1.9 μM. Its structural analysis revealed a sophisticated interaction pattern of polar and lipophilic contacts that were used to improve cellular potency to an IC50 of 15 nM. In parallel, the molecular properties were optimized on plasma exposure and antitumor efficacy which led to the identification of advanced lead 21.

Blocking of methionine aminopeptidase-2 by TNP-470 induces apoptosis and increases chemosensitivity of cholangiocarcinoma.

Resistance of cancer cells to chemotherapeutic drugs is a major pitfall of the failure of chemotherapy treatment for cholangiocarcinoma (CCA). A new therapeutic strategy that can improve treatment efficacy is mandatory for CCA patients. Our previous findings demonstrated the overexpression of methionine aminopeptidase-2 (MetAP2) in CCA patients. In addition, supplementation of TNP-470, a MetAP2 inhibitor, significantly inhibited the growth and metastatic activities of CCA cell lines. However, the molecular mechanism of antitumor activity of TNP-470 and the synergistic antitumor activity of TNP-470 combined with chemotherapeutic drugs are still unknown. The aim of this study is to evaluate the molecular mechanism of anticancer activity and the potential use of TNP-470 as a chemosensitizing agent in CCA cell lines. Cell cycle and apoptosis of CCA cell lines were evaluated using flow cytometry with propidium iodide staining. Expression of apoptosis regulatory proteins was measured by Western blotting. The chemosensitizing effect of TNP-470 was determined using combination index. TNP-470 inhibited the growth of CCA cells via induction of apoptosis through activation of the p38-phosphorylation and up- and down-regulation of Bax and Bcl-xL, respectively. Furthermore, TNP-470 significantly enhanced the antitumor activity of 5-fluorouracil, cisplatin, doxorubicin, and gemcitabine. The present results show that TNP-470 could be a potential therapeutic or adjuvant agent for CCA.

Molecular characterization of Nosema bombycis methionine aminopeptidase 2 (MetAP2) gene and evaluation of anti-microsporidian activity of Fumagilin-B in silkworm Bombyx mori.

Nosema bombycis is a spore-forming parasite causing microsporidiosis in silkworm Bombyx mori. Methionine aminopeptidase 2 (MetAP2), an essential gene of N. bombycis, is a target for the anti-microsporidian drug Fumagillin, an antibiotic derived from Aspergillus fumigatus. In this study, a 1077bp full-length cDNA of the MetAP2 gene of N. bombycis was cloned and characterized. Furthermore, the expression study of the MetAP2 gene revealed a ubiquitous expression during all the developmental stages of the silkworm B. mori. The phylogenetic analysis of the MetAP2 gene of N. bombycis revealed the MetAP2 gene sequences to be highly conserved in nature. The present study also includes the validation of the anti-microsporidian drug Fumagillin against the MetAP2 gene of N. bombycis. The findings revealed that Fumagilin-B could also suppress the N. bombycis multiplication in the silkworm B. mori, thereby proving the therapeutic role of Fumagillin against microsporidian infection. This is the first-ever report regarding the characterization of the MetAP2 gene in the Indian isolate of N. bombycis and also towards the usage of Fumagillin in the control of microsporidiosis in B. mori.

Efficacy and safety of methionine aminopeptidase 2 inhibition in type 2 diabetes: a randomised, placebo-controlled clinical trial.

This multicentre randomised double-blind placebo-controlled clinical trial assessed the efficacy and safety of a methionine aminopeptidase 2 (MetAP2) inhibitor, beloranib, in individuals with obesity (BMI ≥30kg/m2) and type 2 diabetes (HbA1c 53-97mmol/mol [7-11%] and fasting glucose <15.6mmol/l). Participants were randomised (via a centralised interactive web response system) to placebo, 1.2 or 1.8mg beloranib s.c. twice weekly for 26weeks. Participants, investigators and the sponsor were blinded to group assignment. The primary endpoint was the change in weight from baseline to week 26. The trial was terminated early when beloranib development was stopped because of an imbalance of venous thromboembolism events in beloranib-treated individuals vs placebo that became evident during late-stage development of the drug. In total, 153 participants were randomised, 51 to placebo, 52 to 1.2mg beloranib and 50 to 1.8mg beloranib. In participants who completed week 26, the least squares mean ± SE weight change (baseline 111kg) was -3.1 ± 1.2% with placebo (n = 22) vs -13.5 ± 1.1% and -12.7 ± 1.3% with 1.2 and 1.8mg beloranib, respectively (n = 25; n = 19; p < 0.0001). The change in HbA1c (baseline 67mmol/mol [8.3%]) was -6.6 ± 2.2mmol/mol (-0.6 ± 0.2%) with placebo vs -21.9 ± 2.2mmol/mol (-2.0 ± 0.2%) or -21.9 ± 3.3mmol/mol (-2.0 ± 0.3%) with 1.2 or 1.8mg beloranib (p < 0.0001), respectively. The most common beloranib adverse events were sleep related. One beloranib-treated participant experienced a non-fatal pulmonary embolism. MetAP2 inhibitors represent a novel mechanism for producing meaningful weight loss and improvement in HbA1c. ClinicalTrials.gov NCT02324491 FUNDING: The study was funded by Zafgen, Inc.

Results of an Interim Analysis of a Phase 2, Randomized, Double-Blind, Placebo-Controlled Clinical Trial of ZGN-1061 in Patients with Obesity and Type 2 Diabetes

ZGN-1061 (1061) is a methionine aminopeptidase 2 (MetAP2) inhibitor being developed to improve glycemic control in type 2 diabetes. This clinical trial investigated the effect of 12 weeks of subcutaneous (SC) 1061 (0.05, 0.3, 0.9 mg or placebo) administered every 3 days on A1C, safety, and tolerability. Stable noninsulin diabetes therapy was permitted. The study included 129 patients; an interim analysis was conducted on a subset of patients at week 8. Baseline characteristics of the interim ITT population (N=57): 54% male, 77% white, (mean±SD) age 54±8 years, A1C 8.7±1.0%, BMI 37.2±6.4 kg/m2. At week 8, the LS mean±SE change in A1C for placebo was 0.2±0.1% (N=13) vs. -0.4±0.2% with 0.9 mg 1061 (N=13, LS mean±SE difference -0.6±0.2%, p&amp;lt;0.02). In patients with week 12 measurements, the change in A1C was 0.4±0.2 (N=12) vs. -0.5±0.2 (N=8, difference -0.9±0.3, p&amp;lt;0.01). There were trends for weight loss and improved fasting plasma glucose with 0.9 mg 1061 vs. placebo as well as improvements in biomarkers, including hsCRP (week 8 -2.1±2.2 mg/L vs. 1.0±2.1, ns), adiponectin (1.0±0.3 ug/mL vs. -0.1±0.3, p&amp;lt;0.05), leptin (-6.9±1.9 ng/mL vs. 2.1±1.7, p&amp;lt;0.05), FGF-21 (0.026±0.014 ng/mL vs. 0.000±0.013; ns), and postprandial glucose AUC (p&amp;lt;0.01). Improvements in efficacy measures with the lower doses of 1061 were variable. There were no serious or severe adverse events (AEs) and no subjects withdrew due to an AE. The most common AE (1061&amp;gt;placebo) was upper respiratory tract infection. The 1061 pharmacokinetic profile indicated that exposure was within target levels for efficacy and safety and there were no changes in thrombosis markers (e.g., D-dimer). In this interim analysis, 1061 produced improvements in glycemic control and metabolic biomarkers consistent with MetAP2 inhibition. 1061 was also well tolerated with no safety signals in all doses tested. Week 12 results from the full analysis dataset will be presented. Disclosure T. Kim: Employee; Self; Zafgen. D. Zhuang: Employee; Self; Zafgen. T.E. Hughes: Board Member; Self; Zafgen. Employee; Self; Zafgen. Stock/Shareholder; Self; Zafgen. D.D. Kim: Employee; Self; Zafgen. K. Taylor: Employee; Self; Zafgen.

ZGN-1061 Improves Metabolic Parameters and Hepatic Pathology in an Obese Mouse Model of Diet-Induced and Biopsy-Confirmed Nonalcoholic Steatohepatitis

ZGN-1061 (1061) is a methionine aminopeptidase 2 (MetAP2) inhibitor being developed to improve glycemic control in type 2 diabetes. In diet-induced obese mice, 1061 reduces fat mass and improves glycemic control, lipid metabolism, and other metabolic parameters. This study investigated 8 weeks of treatment with 1061 (0.3 mg/kg, SC, N=11) or vehicle (N=10) on metabolic parameters, hepatic pathology, and nonalcoholic fatty liver disease (NAFLD) activity score (NAS; composite measure of steatosis, inflammation, and ballooning degeneration; range 0-8) in male mice with diet-induced and biopsy-confirmed nonalcoholic steatohepatitis (DIO-NASH). At week 8, there was a vehicle corrected weight loss of 13.1% with 1061 (p&amp;lt;0.001). 1061-treated mice had reduced liver weight (mean±SE: 2.6±0.4 g) vs. vehicle (3.8±0.8 g, p&amp;lt;0.001) and 21% reduction of liver triglyceride (p&amp;lt;0.05). There was no change in food intake. NAS was unchanged in vehicle-treated mice (BL 6.2±0.1, Wk8 6.4±0.2, ns). 1061-treated mice had reduced NAS (BL 6.2±0.2, Wk8 5.0±0.3, p=0.002). Two of the NAS component measures improved: steatosis in vehicle-treated mice was unchanged (BL 3.0±0.0, Wk8 3.0±0.0, ns), whereas ZGN-1061 reduced steatosis (BL 2.9±0.1, Wk8 2.5±0.2, p=0.02), and hepatocellular ballooning was unaffected in vehicle-treated mice (BL 0.7±0.2, Wk8 0.6±0.2, ns), whereas 1061 significantly reduced ballooning severity (BL 0.6 ± 0.2, Wk8 0.0 ± 0.0, p=0.006). There was no treatment effect on fibrosis stage or liver collagen 1A. However, liver galectin-3 was reduced with 1061 vs. vehicle. 1061 produced a reduction in terminal plasma ALT and AST vs. vehicle. In DIO-NASH mice, 1061 markedly reduced body weight in conjunction with liver weight and triglyceride content. Importantly, 1061 improved liver function, steatohepatitis and NAS composite score. These findings introduce 1061 as a promising therapy for obesity-related NASH. Disclosure B.F. Burkey: Employee; Self; Zafgen. M. Illemann: None. S. Veidal: None. M. Feigh: None. J. Vath: Employee; Self; Zafgen. Stock/Shareholder; Self; Zafgen. T.E. Hughes: Board Member; Self; Zafgen. Employee; Self; Zafgen. Stock/Shareholder; Self; Zafgen.

ZGN-1061, a Novel MetAP2 Inhibitor, and Liraglutide Combine to Improve Glycemic Control and Reduce Body Weight in a Rat Model of Diet-Induced Obesity

ZGN-1061 (1061) is a methionine aminopeptidase 2 (MetAP2) inhibitor being developed to improve glycemic control in T2D. This study investigated 5 weeks (weeks) of once-daily SC treatment with a submaximal 0.3 mg/kg dose of 1061, a maximal 0.4 mg/kg dose of liraglutide (lira), 1061+lira, or vehicle (N=10/group) on body weight (BW), food intake/preference, and glycemic control in the Gubra diet-induced obese (DIO) rat model (ad libitum fed chow and highly palatable high-fat, high-sugar [HFHS] diet). Fasting (4 hour) blood glucose (FBG) was collected weekly and a semifasted (16 hour access to 50% of daily energy requirement) OGTT was performed at week 4. At week 5 (day 33), BW changed by +1.7% with vehicle vs. -6.0% with 1061, -9.1% with lira, and -16.9% with 1061+lira (all p&amp;lt;0.001). BW loss occurred earlier with lira (day 1) than 1061 (day 5). Starting at day 6, intake of the HFHS diet was modestly reduced (15-30%) with 1061 vs. vehicle. In contrast, lira and 1061+lira induced rapid and sustained 40-50% and 50-80% reductions in intake of the HFHS diet, respectively, over the first 8-9 days. Cumulative (day 0-35) HFHS diet intake was reduced vs. vehicle by 1061 (13.4%, p=0.07), lira (17.8%, p&amp;lt;0.01), and 1061+lira (38.2%, p&amp;lt;0.001). Cumulative chow intake was increased by 1061 and 1061+lira but not lira alone. There was a modest reduction in FBG (mean week 1-5) with 1061 vs. vehicle (p=0.08), a significant reduction with lira, and 1061+lira produced the largest reduction (both p&amp;lt;0.001) that was significantly lower than either agent alone (both p&amp;lt;0.01). In the OGTT, 1061 reduced the glucose AUC vs. vehicle by 37% (p=0.07), lira by 60% (p&amp;lt;0.001), and 1061+lira by 80% (p&amp;lt;0.001), achieving an AUC that was no different from lean rats. In this DIO rat model, 1061 and lira had complementary effects on reducing BW and normalizing glycemic control. Combination treatment with 1061 and liraglutide may yield greater weight loss and glycemic control than either agent alone in patients with T2D. Disclosure B.F. Burkey: Employee; Self; Zafgen. P.J. Pedersen: None. T.X. Pedersen: Employee; Self; Gubra. M. Feigh: None. J. Vath: Employee; Self; Zafgen. Stock/Shareholder; Self; Zafgen. T.E. Hughes: Board Member; Self; Zafgen. Employee; Self; Zafgen. Stock/Shareholder; Self; Zafgen.

Structural and functional highlights of methionine aminopeptidase 2 from Leishmania donovani

Methionine aminopeptidase 2 (MAP2) is a principal regulator of apoptosis for Leishmania donovani and a potential candidate for the design and synthesis of novel antileishmanials. The LdMAP2 gene was cloned in pET28a(+)-SUMO vector, expressed in E. coli and then purified by chromatographic methods. It was found to be a monomer and required divalent metal ion for its activity against synthetic substrates with Co(II), Mg(II), Mn(II) and Ni(II) being the major activators. Moreover, Ca(II) showed the tightest binding with Km value of 124.7 ± 9.2 μM, while Co(II) proved most efficient for catalysis with kcat value of 128.1 ± 4 min−1. The naturally occurring aminopeptidase B inhibitor bestatin was found to be a potent inhibitor of LdMAP2 with a Ki value of 0.86 μM. Further, structural studies with circular dichroism (CD) showed an increase in the α-helical and β-sheet contents and a decrease in random coils in LdMAP2 upon interactions with both bestatin and fluorogenic substrates. Finally, structural studies pointed out key differences in the structure of LdMAP2 and HsMAP2 and their interactions with inhibitor bestatin, Ala-AMC, Leu-AMC and Met-AMC. The structural differences of two orthologs and different binding modes with bestatin can be crucial for the development of novel and specific inhibitor against leishmaniasis.

In silico identification of microRNAs predicted to regulate N-myristoyltransferase and Methionine Aminopeptidase 2 functions in cancer and infectious diseases.

Protein myristoylation is a key protein modification carried out by N-Myristoyltransferase (NMT) after Methionine aminopeptidase 2 (MetAP2) removes methionine from the amino-terminus of the target protein. Protein myristoylation by NMT augments several signaling pathways involved in a myriad of cellular processes, including developmental pathways and pathways that when dysregulated lead to cancer or immune dysfunction. The emerging evidence pointing to NMT-mediated myristoylation as a major cellular regulator underscores the importance of understanding the framework of this type of signaling event. Various studies have investigated the role that myristoylation plays in signaling dysfunction by examining differential gene or protein expression between normal and diseased states, such as cancers or following HIV-1 infection, however no study exists that addresses the role of microRNAs (miRNAs) in the regulation of myristoylation. By performing a large scale bioinformatics and functional analysis of the miRNAs that target key genes involved in myristoylation (NMT1, NMT2, MetAP2), we have narrowed down a list of promising candidates for further analysis. Our condensed panel of miRNAs identifies 35 miRNAs linked to cancer, 21 miRNAs linked to developmental and immune signaling pathways, and 14 miRNAs linked to infectious disease (primarily HIV). The miRNAs panel that was analyzed revealed several NMT-targeting mRNAs (messenger RNA) that are implicated in diseases associated with NMT signaling alteration, providing a link between the realms of miRNA and myristoylation signaling. These findings verify miRNA as an additional facet of myristoylation signaling that must be considered to gain a full perspective. This study provides the groundwork for future studies concerning NMT-transcript-binding miRNAs, and will potentially lead to the development of new diagnostic/prognostic biomarkers and therapeutic targets for several important diseases.

Physiological mechanisms of sustained fumagillin-induced weight loss.

Current obesity interventions suffer from lack of durable effects and undesirable complications. Fumagillin, an inhibitor of methionine aminopeptidase-2, causes weight loss by reducing food intake, but with effects on weight that are superior to pair-feeding. Here, we show that feeding of rats on a high-fat diet supplemented with fumagillin (HF/FG) suppresses the aggressive feeding observed in pair-fed controls (HF/PF) and alters expression of circadian genes relative to the HF/PF group. Multiple indices of reduced energy expenditure are observed in HF/FG but not HF/PF rats. HF/FG rats also exhibit changes in gut hormones linked to food intake, increased energy harvest by gut microbiota, and caloric spilling in the urine. Studies in gnotobiotic mice reveal that effects of fumagillin on energy expenditure but not feeding behavior may be mediated by the gut microbiota. In sum, fumagillin engages weight loss-inducing behavioral and physiologic circuits distinct from those activated by simple caloric restriction.

Preclinical Efficacy and Safety of the Novel Antidiabetic, Antiobesity MetAP2 Inhibitor ZGN-1061.

Methionine aminopeptidase 2 (MetAP2) inhibition is a promising approach to treating diabetes, obesity, and associated metabolic disorders. Beloranib, a MetAP2 inhibitor previously investigated for treatment of Prader-Willi syndrome, was associated with venous thrombotic adverse events likely resulting from drug effects on vascular endothelial cells (ECs). Here, we report the pharmacological characterization of ZGN-1061, a novel MetAP2 inhibitor being investigated for treatment of diabetes and obesity. Four weeks of subcutaneous administration of ZGN-1061 to diet-induced obese (DIO) insulin-resistant mice produced a 25% reduction in body weight, primarily due to reduced fat mass, that was comparable to beloranib. ZGN-1061 also produced improvements in metabolic parameters, including plasma glucose and insulin, and, in HepG2 cells, initiated gene changes similar to beloranib that support observed in vivo pharmacodynamics. In vitro studies in ECs demonstrated that ZGN-1061 effects on EC proliferation and coagulation proteins were greatly attenuated, or absent, relative to beloranib, due to lower intracellular drug concentrations, shorter half-life of inhibitor-bound MetAP2 complex, and reduced cellular enzyme inhibition. In dogs, ZGN-1061 was more rapidly absorbed and cleared, with a shorter half-life than beloranib. Unlike beloranib, ZGN-1061 did not increase coagulation markers in dogs, and ZGN-1061 had a greatly improved safety profile in rats relative to beloranib. In conclusion, ZGN-1061 and beloranib demonstrated similar efficacy in a mouse model of obesity, while ZGN-1061 had a markedly improved safety profile in multiple in vitro and in vivo models. The lower duration of exposure characteristic of ZGN-1061 is expected to provide a meaningfully enhanced clinical safety profile.