The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.

Tumor angiogenesis is a cancer hallmark, and its therapeutic inhibition has provided meaningful, albeit limited, clinical benefit. While anti-angiogenesis inhibitors deprive the tumor of oxygen and essential nutrients, cancer cells activate metabolic adaptations to diminish therapeutic response. Despite these adaptations, angiogenesis inhibition incurs extensive metabolic stress, prompting us to consider such metabolic stress as an induced vulnerability to therapies targeting cancer metabolism. Metabolomic profiling of angiogenesis-inhibited intracranial xenografts showed universal decrease in tricarboxylic acid cycle intermediates, corroborating a state of anaplerotic nutrient deficit or stress. Accordingly, we show strong synergy between angiogenesis inhibitors (Avastin, Tivozanib) and inhibitors of glycolysis or oxidative phosphorylation through exacerbation of anaplerotic nutrient stress in intracranial orthotopic xenografted gliomas. Our findings were recapitulated in GBM xenografts that do not have genetically predisposed metabolic vulnerabilities at baseline. Thus, our findings cement the central importance of the tricarboxylic acid cycle as the nexus of metabolic vulnerabilities and suggest clinical path hypothesis combining angiogenesis inhibitors with pharmacological cancer interventions targeting tumor metabolism for GBM tumors.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against T. brucei ENO (TbENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC50 value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC50 value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of −6.8 and −7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of −4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against T. brucei, with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC50 values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against T. brucei and that further development of prodrug HEX analogs is warranted.

Purpose Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[F-18]fluoroglutamine ([F-18]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [F-18]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results In UMRC3 cells, [F-18]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60-90% in the presence of excess glutamine in the media and by 20-50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [F-18]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [F-18]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [F-18]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [F-18]FGln retention did not change significantly upon treatment with CB-839. Conclusions We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [F-18]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [F-18]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

Purpose Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2 S ,4 R )-4-[ 18 F]fluoroglutamine ([ 18 F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [ 18 F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results In UMRC3 cells, [ 18 F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60–90% in the presence of excess glutamine in the media and by 20–50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [ 18 F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [ 18 F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [ 18 F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [ 18 F]FGln retention did not change significantly upon treatment with CB-839. Conclusions We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [ 18 F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [ 18 F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

The phosphonate group is a key pharmacophore in many anti-viral, anti-microbial, and anti-neoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods is challenging and requires derivatization with hazardous reagents. Given the emerging importance of phosphonate-containing drugs, developing a practical, accessible, and safe method for their quantitation in pharmacokinetics (PK) studies is desirable. NMR-based methods are often employed in drug discovery but are seldom used for compound quantitation in PK studies. Here, we show that proton-phosphorous ( 1 H- 31 P) heteronuclear single quantum correlation (HSQC) NMR allows for quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissue at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in <1 hr), the plasma half-life of HEX was more than 1hr in rats and nonhuman primates. This slower clearance rate affords a significantly higher exposure of HEX in rat models compared to mouse models while maintaining a favorable safety profile. Similar results were observed for the phosphonate-containing antibiotic, fosfomycin. Our study demonstrates the applicability of the 1 H- 31 P HSQC method to quantify phosphonate-containing drugs in complex biological samples and illustrates an important limitation of mice as preclinical model species for phosphonate-containing drugs.

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B-two essential components of the autophagosome maturation machinery-with mid-nanomolar affinities and disrupted protein-protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

Hyperpolarized magnetic resonance spectroscopy enables quantitative, non-radioactive, real-time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single- and double-13 C labels and hyperpolarized by dynamic nuclear polarization with 4.7 % and 3 % polarization, respectively. The longitudinal relaxation constants (T1 ) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by 13 C-NMR spectroscopy. Hyperpolarized, double-labeled aspirin was well tolerated in mice and could be observed by both 13 C-MR imaging and 13 C-NMR spectroscopy in vivo.

he mechanistic details of the aldol addition of N-amino cyclic carbamate (ACC) hydrazones is provided herein from both an experimental and computational perspective. When the transformation is carried out at room temperature the anti-aldol product is formed exclusively. Under these conditions the anti- and syn-aldolate intermediates are in equilibrium and the transformation is under thermodynamic control. The anti-aldolate that leads to the anti-aldol product was calculated to be 3.7 kcal mol(-1) lower in energy at room temperature than that leading to the syn-aldol product, which sufficiently accounts for the exclusive formation of the anti-aldol product. When the reaction is conducted at -78 degrees C it is under kinetic control and favors formation of the syn-aldol addition product. In this case, it was found that a solvent separated aza-enolate anion and aldehyde form a sigma-intermediate in which the lithium cation is coordinated to the aldehyde. The sigma-intermediate collapses with a very small activation barrier to form the beta-alkoxy hydrazone intermediate. The chiral nonracemic lithium aza-enolate discriminates between the two diastereotopic faces of the pro-chiral aldehyde, and there is no rapid direct pathway that interconverts the two diastereomeric intermediates. Consequently, the reaction does not follow the Curtin-Hammett principle and the stereochemical outcome at low temperature instead depends on the relative energies of the two sigma-intermediates.

The α-alkylation of ketones is a fundamental synthetic transformation. The development of asymmetric variants of this reaction is important given that numerous natural products, drugs, and related compounds exist as α-functionalized ketones or derivatives thereof. We previously reported our preliminary studies on the development of a new enantioselective ketone α-alkylation procedure using N-amino cyclic carbamate (ACC) auxiliaries. In comparison to other auxiliary-based methods, ACC alkylation offers a number of advantages and is both highly enantioselective and high yielding. Herein, we provide a full account of our studies on the enantioselective ACC ketone α-alkylation method.

A chiral auxiliary-based diastereoselective transformation that entirely avoids the stereochemically mismatched pairing, providing equally high levels of asymmetric induction in the formation of each diastereomer is described. In particular, we show that chiral nonracemic methyl ketones undergo α,α-bisalkylation using phenylalanine-derived N-amino cyclic carbamate (ACC) auxiliaries with essentially perfect diastereoselectivity, as well as excellent yield and regioselectivity. Significantly, with the use of a single enantiomer of the auxiliary, either diastereomeric product can be synthesized with an equally high level of asymmetric induction.

The asymmetric alpha-allylation of 3-pentanone using several different N-amino cyclic carbamate (ACC) auxiliaries is described. The level of asymmetric induction was found to range from er = 93:7 to er = 99:1. The factors that lead to compromised selectivity for the various auxiliaries have been determined. Significantly, we have established that using the easily accessible and inexpensive valine-derived ACC auxiliary, it is possible to obtain synthetically useful levels of asymmetric induction (er = 96:4). (C) 2016 Published by Elsevier Ltd.

Chiral nonracemic N-amino cyclic carbamates (ACCs) are important auxiliaries for certain asymmetric transformations. In the past they have been synthesized from oxazolidinones using methods that require the preparation and use of non-atom economical aminating agents that can be difficult to prepare, and often strong bases, In what follows we describe a mild and operationally simple method for the direct N-amination of oxazolidinones that use NH2Cl derived from commercial bleach. (C) 2016 Elsevier Ltd. All rights reserved.