Methionine Aminopeptidase Inhibitors Research Articles

Micromolar Concentrations of Methionine Aminopeptidase Inhibitor has Little Cytotoxic Effect on Cancerous and Non‐Cancerous Mammalian Cells

Tuberculosis (TB) is a disease of the lung caused by the bacterium, Mycobacterium tuberculosis (Mtb), and is the second leading cause of mortality from an infectious agent after SARS‐CoV‐2. Mtb is an opportunistic pathogen that benefits from a compromised immune system and is particularly deadly in HIV patients. New therapies targeting TB are necessary due to an increase in multi‐drug resistant strains. Our collaborators at Texas Southern University identified novel bacterial methionine aminopeptidase (MtMetAP1) inhibitors. Genetic knockout studies support the lethality of the deletion of MtMetAP1 in Mtb. Further studies support the existence of an ortholog in Homo sapiens (HsMetAP1) that performs similar essential functions. Although these novel inhibitors are known to be effective against Mtb, their cytotoxicity in mammalian cells has yet to be established. We hypothesize that there will be low levels of cytotoxicity in cancerous and primary lung cell lines at varying concentrations of the MetAP novel inhibitor, UST‐001. Using flow cytometry via a MUSE® Cell Count and Viability Kit, the cytotoxicity of UST‐001 was assayed in H1299 cancer cells. The H1299 cells were exposed to concentrations of UST‐001 ranging from 0.1μM to 750μM for 48 hours at 37℃and 5% CO2. Three negative controls were included in each replicate: DMSO (carrier), pure media, and Isoniazid (INH). INH is a common clinically effective TB treatment. The viability of H1299 cells was still over 60% at the highest inhibitor concentration tested. Trypan Blue Assays were performed on the same H1299 cells at each concentration and the controls, which corroborated flow cytometry results. In establishment of a dose‐response curve, our data showed that at UST‐001 concentrations below 100 μM, induced little to no cell death and cell viability of 90% or higher. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that we will be able to establish an IC50 curve in non‐cancerous Mlg‐2908 lung fibroblast cells at higher concentrations. Current flow cytometry testing on Mlg‐2908 cells, using the same protocol as the H1299 cells, shows cell viability of 89% or higher in 0.1 μM to 10 μM UST‐001 concentrations. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that we will be able to establish an IC50 curve in Mlg‐2908 cells at higher concentrations. Further studies using a Muse Annexin V apoptosis assay will focus on the UST‐001 mechanism of cell death. Our ultimate goal is to characterize the toxicity of this drug in mammalian systems, with hopes of potentially identifying a new class of antibiotics and pharmaceutical targets.

Design, synthesis and biological evaluation of substituted 3-amino-N-(thiazol-2-yl)pyrazine-2-carboxamides as inhibitors of mycobacterial methionine aminopeptidase 1

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the number one cause of deaths due to a single infectious agent worldwide. The treatment of TB is lengthy and often complicated by the increasing drug resistance. New compounds with new mechanisms of action are therefore needed. We present the design, synthesis, and biological evaluation of pyrazine-based inhibitors of a prominent antimycobacterial drug target - mycobacterial methionine aminopeptidase 1 (MtMetAP1). The inhibitory activities of the presented compounds were evaluated against the MtMetAP1a isoform, and all derivatives were tested against a broad spectrum of myco(bacteria) and fungi. The cytotoxicity of the compounds was also investigated using Hep G2 cell lines.Overall, high inhibition of the isolated enzyme was observed for 3-substituted N-(thiazol-2-yl)pyrazine-2-carboxamides, particularly when the substituent was represented by 2-substituted benzamide. The extent of inhibition was strongly dependent on the used metal cofactor. The highest inhibition was seen in the presence of Ni2+. Several compounds also showed mediocre in vitro potency against Mtb (both Mtb H37Ra and H37Rv). Despite the structural similarities of bacterial and fungal MetAP1 to mycobacterial MtMetAP1, title compounds did not exert antibacterial nor antifungal activity. The reasons behind the higher activity of 2-substituted benzamido derivatives, as well as the correlation of enzyme inhibition with the in vitro growth inhibition activity is discussed.

Abstract 108: Inhibiting methionine aminopeptidase 2 prevents liver fibrosis and hepatocellular carcinoma

Abstract Background: The methionine aminopeptidase 2 (MetAP2) pathway, a key regulator of angiogenesis progress, has been implicated in common pathologic conditions including diabetes, non-alcoholic steatohepatitis (NASH) and cancer. MetAP2 inhibition suppresses cancer cell proliferation and xenograft tumor growth. In this study we investigated the hypothesis that MetAP2 inhibition can prevent liver fibrosis and hepatocellular carcinoma (HCC) induced with diethylnitrosamine (DEN) in rat models by inhibiting neo-vascularization. Methods: Male Wistar rats received weekly intraperitoneal injections of DEN at 50 mg/kg for 18 weeks. After 8 weeks, rats were randomly assigned to treatment with one of two MetAP2 inhibitors ZGN1345 or ZGN1136 versus control vehicle (n = 10 per group): 1) water (control); or 2) ZGN-1345 (3 mg/kg) by daily gavage; 3) 0.15% DMSO (control); or 4) ZGN-1136 (0.3 mg/kg) by daily subcutaneous injection. All rats were sacrificed at 19 weeks. Serum chemistries were performed to evaluate the liver function. Liver fibrosis was assessed with hydroxyproline measurement, Sirius Red and H&E staining with review by a liver pathologist. Liver tumor nodules were recorded and analyzed with IHC staining. Results: ZGN1345 treatment improved ALT and AST on DEN-induced cirrhosis in rat models. Both inhibitors improved the liver fibrosis, including lowering collagen deposition (hydroxyproline measurement and amount of collagen measured with Sirius Red staining) and improving the Ishak fibrosis score. Both inhibitors reduced the number of tumor nodules and suppressed PCNA expression in the liver. More importantly, both inhibitors suppressed neo-vascularization and angiogenesis by inhibiting VEGF expression. Conclusions: MetAP2 inhibitors ZGN1345 and ZGN1136 effectively prevent DEN-induced liver fibrosis and HCC in rat models, potentially by inhibiting neo-vascularization. These data suggest that inhibition of MetAP2 may represent a new prevention strategy for HCC. Table. Effect of ZGN1345 and ZGN1136 on rat liver fibrosis and HCC.GroupsASTALTHydroxyprolineCollagen (%)Fibrosis score rateNodule numbers (>5 mm)PCNA expressionVEGF expressionWater (Control), PO100.0 ± 35.5100.0 ± 29.5100.0 ± 39.913.1 ± 3.8F4:25.0%,F3:37.5%,F2:37.5%27.4 ± 14.2100.0± 36.4100.0 ± 44.6ZGN-1345, PO45.5 ± 11.1 ***44.7 ± 14.3 ***36.9 ± 10.5 ***4.7 ± 2.2 ***F2:11.1%,F1:77.8%,F0:11.1%5.7 ± 3.9 ***40.1 ± 13.3 ***21.9 ± 9.8 ***0.15% DMSO (Control), SC100.0 ± 28.1100.0 ± 21.1100.0 ± 63.99.2 ± 3.5F3:42.9%,F2:42.9%,F1:14.3%41.1 ± 40.2100.0 ± 22.8100.0 ± 29.4ZGN-1136, SC89.1 ± 11.3107.1 ± 37.242.9 ± 8.6 *2.9 ± 1.7 **F2:14.3%,F1:42.9%,F0:42.9%4.1 ± 2.8 *29.9 ± 9.4 ***52.3 ± 21.6 *** Citation Format: YONGTAO WANG, Mozhdeh Sojoodi, Guoliang Qiao, Zenan Lin, Stephen C. Barrett, Lawrence Zukerberg, Michael Lanuti, Motaz Qadan, Kenneth K. Tanabe. Inhibiting methionine aminopeptidase 2 prevents liver fibrosis and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 108.

Abstract 1068: SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®)

Abstract Small molecule MetAP2 inhibitor TNP-470 previously showed clinical anti-tumor activity, however CNS side effects and poor drug-like properties limited its development. SDX-7320 is a polymer-drug conjugate of a novel MetAP2 inhibitor (SDX-7539), intended to improve biodistribution (limit CNS penetration) and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 has recently completed a phase I trial in late-stage cancer patients (NCT02743637). SDX-7320 was previously shown to inhibit the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (“metabo-oncology”). In addition SDX-7320 synergized with the PI3Kα inhibitor alpelisib (Piqray®) to block the growth of ER+/Her2- MCF-7 xenografts. Here, we show that SDX-7320 inhibits the growth of MCF-7 xenografts alone and in combination with the CDK4/6 inhibitor Palbociclib (Ibrance®). Nude mice were implanted with slow release estrogen pellets and then injected with MCF-7 cells in the fourth mammary gland. Treatment began (n=10/group) when tumors exceeded 100 mm³: SDX-7320 (sc/q4d, 8 mg/kg), palbociclib (po, qd, 20 or 40 mg/kg), SDX-7320 plus palbociclib (20 mg/kg), SDX-7320 plus palbociclib (40 mg/kg). MCF-7 tumor growth inhibition at day 31 relative to vehicle-treated mice: SDX-7320, 29% (NS); palbociclib, 24% and 54% at 20 and 40 mg/kg respectively (NS); SDX-7320 plus palbociclib (20 mg/kg), 64% (p<0.05); and SDX-7320 plus palbociclib (40 mg/kg), 82%, (p<0.01). Tumor samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors for analysis of proteins (by WES). Cell-cycle proteins (cyclin E1, E2, cdk2, cdk4) were decreased in tumor tissue from SDX-7320-treated mice relative to vehicle, and were typically lower in tumors from mice treated with SDX-7320 plus palbociclib (40 mg/kg) compared to either vehicle-treated or SDX-7320-treated mice. A key growth factor-signaling protein Akt was reduced in SDX-7320-treated groups (alone and in combination) as was estrogen receptor alpha (ERα). pAkt (S473) was lowest in SDX-7320-treated tumors and in tumors treated with SDX-7320 plus palbociclib (40 mg/kg). The autophagy marker LC3B was elevated in response to palbociclib (20 and 40 mg/kg) but in combination with SDX-7320, the increase was significantly attenuated. Intracellular pathways and proteins linked to emergence of resistance to palbociclib include cell cycle (cyclin E1), growth factor and hormone signaling (Akt, ERα) and autophagy (LC3B). The protein changes observed (representative of these pathways) suggest that combining SDX-7320 with palbociclib (Ibrance®) may extend the period of progression-free survival in patients with ER+/Her2- breast cancer relative to treatment with palbociclib (Ibrance®) alone. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Sara Little, Andrew Slee, Raphael Nir, Adam Nir, Bradley J. Carver, James Shanahan. SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1068.

Potential Cytotoxicity of Methionine Aminopeptidase Inhibitors on Mammalian Cells

Tuberculosis (TB) is a disease of the lung caused by the bacterium, Mycobacterium tuberculosis (Mtb), and is a leading cause of mortality from infectious disease. Mtb is an opportunistic pathogen that benefits from a compromised immune system and can lead to a statistically increased risk of HIV/TB co-infection, which leads to a significant increase in the mortality rate of HIV patients. Treatment has become difficult due to an increase in multi-drug resistant strains. Our collaborators at Texas Southern University have identified novel bacterial methionine aminopeptidase (MtMetAP1) inhibitors. Genetic knockout studies support the lethality of the deletion of MtMetAP1 in Mtb. Further studies support the existence of an ortholog of MtMetAP1 in Homo sapiens (HsMetAP1). Humans have several paralogs of MetAP, which perform similar essential functions to MetAP1. Although these novel inhibitors are known to be effective against Mtb bacteria, their cytotoxicity in mammalian cells has yet to be established. We hypothesize that there will be low cytotoxicity in cancerous and primary lung cell lines at varying concentrations of the MetAP novel inhibitor, UST-001. Using flow cytometry via a MUSE Cell Count and Viability Kit, the cytotoxicity of UST-001 was tested in H1299 human lung carcinoma cells. The H1299 cells were exposed for 48 hours to concentrations of UST-001 ranging from 1μM to 75μM. Three controls were included in each replicate: DMSO (carrier), pure media, and Isoniazid (INH), which is a common clinically effective TB treatment. When compared to the controls, even at the highest inhibitor concentration, 75 µM, the viability of H1299 cells was over 60%. Trypan Blue Assays were performed on the same H1299 cells for each concentration and controls which corroborated flow results. In establishment of a dose-response curve, our data showed that at UST-001 concentrations below 10 μM, there was little to no cell death and a greater than 90% viability. To see a significant decrease in percent viability, albeit to only 60%, the concentrations had to be increased to 75μM. We are currently establishing the cytotoxicity of UST-001 in healthy non-cancerous cells, with a second cell line, Mlg-2908, a non-cancerous mouse lung fibroblast line. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that 1) we will be able to establish anIC50 and 2) it will be closer to 10 μM in non-cancerous cells. Further studies using a Muse Annexin V apoptosis assay will focus on the UST-001 mechanism of cell death. Our ultimate goal is to characterize the toxicity of this drug in mammalian systems leading to its potential of being a new class of antibiotics and ultimately its promise of yet a new drug target.

MetAP2 inhibition modifies hemoglobin S to delay polymerization and improves blood flow in sickle cell disease

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and βS-globin. Kinetic studies with MetAP2 show that βS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood–derived erythroid progenitor 2 cells shows more extensive modification of α-globin than β-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on βS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor–treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2–affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

The methionine aminopeptidase 2 inhibitor, TNP-470, enhances the antidiabetic properties of sitagliptin in mice by upregulating xenin

The therapeutic mechanism of action of methionine aminopeptidase 2 (MetAP2) inhibitors for obesity-diabetes has not yet been fully defined. Xenin, a K-cell derived peptide hormone, possesses an N-terminal Met amino acid residue. Thus, elevated xenin levels could represent a potential pharmacological mechanism of MetAP2 inhibitors, since long-acting xenin analogues have been shown to improve obesity-diabetes. The present study has assessed the ability of the MetAP2 inhibitor, TNP-470, to augment the antidiabetic utility of the incretin-enhancer drug, sitagliptin, in high fat fed (HFF) mice. TNP-470 (1 mg/kg) and sitagliptin (25 mg/kg) were administered once-daily alone, or in combination, to diabetic HFF mice (n = 10) for 18 days. Individual therapy with TNP-470 or sitagliptin resulted in numerous metabolic benefits including reduced blood glucose, increased circulating and pancreatic insulin and improved glucose tolerance, insulin sensitivity, pyruvate tolerance and overall pancreatic islet architecture. Further assessment of metabolic rate revealed that all treatments reduced respiratory exchange ratio and increased locomotor activity. All sitagliptin treated mice also exhibited increased energy expenditure. In addition, treatment with TNP-470 alone, or in combination with sitagliptin, reduced food intake and body weight, as well as elevating plasma and intestinal xenin. Importantly, combined sitagliptin and TNP-470 therapy was associated with further significant benefits beyond that observed by either treatment alone. This included more rapid restoration of normoglycaemia, superior glucose tolerance, increased circulating GIP concentrations and an enhanced pancreatic beta:alpha cell ratio. In conclusion, these data demonstrate that TNP-470 increases plasma and intestinal xenin levels, and augments the antidiabetic advantages of sitagliptin.

Structure-based optimisation of orally active & reversible MetAP-2 inhibitors maintaining a tight ‘molecular budget’

Structure-based led optimisation of orally active reversible Methionine Aminopeptidase-2 (MetAP-2) inhibitors utilising a ‘molecular budget’ medicinal chemistry strategy is described. The key physicochemical parameters of target molecules (cLogP, molecular size and H-bond donor count) were monitored through straightforward and intuitive use of atom count and distribution. The balance between structure-based design and an awareness of the physicochemical properties of the compounds synthesised enabled the rapid identification of a potent molecule with good oral pharmacokinetic (PK) characteristics by making fewer, higher quality compounds. The resulting candidate quality molecule was validated in a mechanistic cellular assay and a rodent secondary immunisation model.

The Research of New Inhibitors of Bacterial Methionine Aminopeptidase by Structure Based Virtual Screening Approach of ZINC DATABASE and In Vitro Validation.

The great emergence of multi-resistant bacterial strains and the low renewal of antibiotics molecules are leading human and veterinary medicine to certain therapeutic impasses. Therefore, there is an urgent need to find new therapeutic alternatives including new molecules in the current treatments of infectious diseases. Methionine aminopeptidase (MetAP) is a promising target for developing new antibiotics because it is essential for bacterial survival. To screen for potential MetAP inhibitors by in silico virtual screening of the ZINC database and evaluate the best potential lead molecules by in vitro studies. We have considered 200,000 compounds from the ZINC database for virtual screening with FlexX software to identify potential inhibitors against bacterial MetAP. Nine chemical compounds of the top hits predicted were purchased and evaluated in vitro. The antimicrobial activity of each inhibitor of MetAP was tested by the disc-diffusion assay against one Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli & Pseudomonas aeruginosa) bacteria. Among the studied compounds, compounds ZINC04785369 and ZINC03307916 showed promising antibacterial activity. To further characterize their efficacy, the minimum inhibitory concentration was determined for each compound by the microdilution method which showed significant results. These results suggest compounds ZINC04785369 and ZINC03307916 as promising molecules for developing MetAP inhibitors. Furthermore, they could therefore serve as lead molecules for further chemical modifications to obtain clinically useful antibacterial agents.

Methionine Aminopeptidase Inhibitors in Mammalian Cells

Mycobacterium tuberculosis (Mtb) is the leading cause of mortality from an infectious disease, worldwide. Mtb benefits from an HIV‐weakened immune system, and leads to a statistically increased risk of HIV/TB co‐infection. The rise of multi‐drug resistant Mtb strains makes treatment difficult, thus a novel drug may be required to eliminate Mtb infection. Our collaborators at Texas Southern University have identified novel bacterial methionine aminopeptidase (MtMetAP1) inhibitors. Genetic knockout studies support the lethality of the deletion of MtMetAP1 in Mtb. Further studies support the existence of an ortholog of MtMetAP1 in Homo sapiens (HsMetAP1). In addition, humans have several paralogs of MetAP, which perform similar essential functions to MetAP1. Although these novel inhibitors are effective against Mtb, the cytotoxicity in mammalian cells has not been established. To address this, we tested a MtMetAP1 inhibitor (UST‐001) on H1299 human lung carcinoma cells and Mlg‐2908 mouse lung fibroblast cells. Using flow cytometry and a WST‐1 assay to assess cell viability, we determined the LC50 to be 1.652μM in the H1299 lung carcinoma cell line. The consistent non‐significance of our data supports our hypothesis regarding the non‐fatal nature of this drug in a human cell line, as there were no significant differences between controls and experimental conditions. Initial results from WST‐1 assays with Mlg‐2908 cells indicate that the LC50 for Mlg‐2908 is between 5 and 10μM, higher than that of H1299. Further studies using flow cytometry analysis are required to solidify this comparison, while another apoptotic assay may also be beneficial to determine an exact LC50 and ultimately the efficacy of this drug.Support or Funding InformationU.S. DOE, Hispanic‐Serving Institutions, STEM Articulation Grant P031C110128, the Cullen‐Smith Foundation, and the UST Committee for Student Research for their financial support

The methionine aminopeptidase 2 inhibitor ZGN-1061 improves glucose control and weight in overweight and obese individuals with type 2 diabetes: A randomized, placebo-controlled trial.

The methionine aminopeptidase 2 (MetAP2) inhibitor ZGN-1061 lowered weight and improved glucose in preclinical studies. We sought to determine its efficacy and safety by performing a multicentre, phase 2, randomized controlled trial involving overweight and obese adults with type 2 diabetes and HbA1c between 7% and 11% inclusive. Participants were randomized to receive subcutaneous treatment with placebo or 0.05, 0.3, 0.9 or 1.8 mg ZGN-1061 every third day for 12 weeks. The primary outcome was change in HbA1c at week 12. Relative to placebo, the 0.9 and 1.8 mg doses induced clinically meaningful reductions in HbA1c of 0.6% (95% CI 0.2% to 0.9%; P = 0.0006) and 1.0% (95% CI 0.6% to 1.4%; P < 0.0001), respectively. The 1.8 mg dose also induced weight loss of 2.2% (95% CI 1.1% to 3.3%; P = 0.0002). The incidence of adverse events was balanced across the treatment groups. We conclude that MetAP2 inhibition with ZGN-1061 for 12 weeks improved glucose control and aided weight loss in overweight and obese people with type 2 diabetes. However, given safety issues, Zafgen has discontinued MetAP2 inhibitor development.

Identification of potential inhibitors of human methionine aminopeptidase (type II) for cancer therapy: Structure-based virtual screening, ADMET prediction and molecular dynamics studies

Methionine Aminopeptidases MetAPs are divalent-cofactor dependent enzymes that are responsible for the cleavage of the initiator Methionine from the nascent polypeptides. MetAPs are classified into two isoforms: namely, MetAP1 and MetAP2. Several studies have revealed that MetAP2 is upregulated in various cancers, and its inhibition has shown to suppress abnormal or excessive blood vessel formation and tumor growth in model organisms. Clinical studies show that the natural product fumagillin, and its analogs are potential inhibitors of MetAP2. However, due to their poor pharmacokinetic properties and neurotoxicities in clinical studies, their further developments have received a great setback. Here, we apply structure-based virtual screening and molecular dynamics methods to identify a new class of potential inhibitors for MetAP2. We screened Otava’s Chemical Library, which consists of about 3 200 000 tangible-chemical compounds, and meticulously selected the top 10 of these compounds based on their inhibitory potentials against MetAP2. The top hit compounds subjected to ADMET predictor using 3 independent ADMET prediction programs, were found to be drug-like. To examine the stability of ligand binding mode, and efficacy, the unbound form of MetAP2, its complexes with fumagillin, spiroepoxytriazole, and the best promising compounds compound-3369841 and compound-3368818 were submitted to 100 ns molecular dynamics simulation. Like fumagillin, spiroepoxytriazole, and both compound-3369841 and compound-3368818 showed stable binding mode over time during the simulations. Taken together, these uninherited-fumagillin compounds may serve as new class of inhibitors or provide scaffolds for further optimization towards the design of more potent MetAP2 inhibitors -development of such inhibitors would be essential strategy against various cancer types.

Abstract P3-11-13: Synergistic inhibition of MCF-7 mammary gland tumor growth with Piqray® (alpelisib) plus SDX-7320, a novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor

Abstract Mutations in the PI3K pathway have been observed in about 40% of ER+/Her2- breast cancer patients. Amplification as well as mutation of the catalytic p110α subunit of PI3K results in activation of this pathway within tumors, conferring a growth advantage due to increased transmission of intracellular growth signals. Recently a selective inhibitor of p110α, Piqray®(alpelisib) has been approved by the FDA (in combination with the estrogen receptor degrader/antagonist Faslodex/fulvestrant) for the treatment of ER+/Her2- breast cancers harboring mutation(s) in the p110α subunit of PI3K. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. 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 alter biodistribution (limit CNS penetration) and improve pharmacokinetics relative to small molecule, fumagillin-derived MetAP2 inhibitors. SDX-7320 has completed a phase I trial in late-stage cancer patients (NCT02743637). The objective of this study was to evaluate the anti-tumor efficacy of Piqray®(alpelisib) in combination with SDX-7320 in a model of PI3K-mutated, ER+ breast cancer (i.e., MCF-7). Female nude mice had estrogen pellets surgically implanted and after two weeks of recovery, MCF-7 cells were injected into the fourth mammary gland. When tumors became palpable (i.e., &amp;gt; 50 mm3) treatment with SDX-7320 (dosed subcutaneously Q4D at 8 or 16 mg/kg) and/or Piqray®(alpelisib) (dosed PO, QD at 25 or 45 mg/kg) commenced. Combinations included SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 25 mg/kg as well as SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 45 mg/kg. Endpoints included tumor volume, body weight, terminal plasma glucose, insulin, leptin and adiponectin. Analysis of tumor tissue for molecular endpoints of PI3K pathway activation were also investigated (e.g., S6, Akt, 4E-BP1). Anti-tumor efficacy was assessed by calculating the %change in tumor volume from baseline for each individual animal, then comparing the average change for each group at the end of the study (day 64). One-way ANOVA with multiple comparisons was conducted to determine significance of differences in final tumor volume on day 64 relative to vehicle. Treatment with SDX-7320 at 8 or 16 mg/kg inhibited MCF-7 tumor growth -19% and -17% relative to vehicle at day 64 (NS), respectively. Piqray®(alpelisib) inhibited tumor growth in a dose-dependent manner (-30% (NS) and -82% (p&amp;lt;0.05) for 25 and 45 mg/kg, respectively). The combination of SDX-7320 at 8 mg/kg with Piqray®(alpelisib) at 25 mg/kg resulted in synergistic efficacy, with -105% inhibition of tumor growth relative to vehicle at day 64 (p&amp;lt;0.01).The combination of SDX-7320 at 8 mg/kg with Piqray®(alpelisib) at 45 mg/kg resulted in an additive effect on tumor growth relative to vehicle (-100%; p&amp;lt;0.01). These results show that SDX-7320 (8 mg/kg) is synergistic with Piqray®(alpelisib; 25 mg/kg) in control of orthotopic MCF-7 mammary gland tumors. A clinical trial combining SDX-7320 with Piqray®(alpelisib) plus fulvestrant is planned to start in Q1/2020 and will assess the ability of SDX-7320 to enhance the efficacy of Piqray®(alpelisib) in treating PI3K-mutated, ER+/Her2- breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Sara Little, Andrew Slee, Adam Nir, Raphael Nir, Bradley Carver, James Shanahan. Synergistic inhibition of MCF-7 mammary gland tumor growth with Piqray® (alpelisib) plus SDX-7320, a novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-13.

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.

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&amp;lt;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&amp;lt;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&amp;lt;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&amp;lt;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&amp;lt;0.001) for 0.9 and 1.8 mg ZGN-1061. Dose-dependent fasting and postprandial glucose reductions were observed for ZGN-1061 (p&amp;lt;0.05 vs. PBO); β-cell function and insulin sensitivity were improved with 1.8 mg ZGN-1061 (p&amp;lt;0.05 vs. PBO). ZGN-1061 also improved triglycerides, hsCRP, adiponectin, and leptin (p&amp;lt;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.