Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry.

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4 ± 11.5 and 10.1 ± 4.0 nmol/mg protein, respectively.

Synthesis and Pharmacological Characterization of a Difluorinated Analogue of Reduced Haloperidol as a Sigma-1 Receptor Ligand.

Reduced haloperidol (1) was previously reported to act as a potent sigma-1 receptor (S1R) ligand with substantially lower affinity to the dopamine D2 receptor (D2R) compared to haloperidol. It was also found to facilitate brain-derived neurotrophic factor (BDNF) secretion from astrocytic glial cell lines in a sigma-1 receptor (S1R)-dependent manner. Although an increase in BDNF secretion may have beneficial effects in some neurological conditions, the therapeutic utility of reduced haloperidol (1) is limited because it can be oxidized back to haloperidol in the body, a potent dopamine D2 receptor antagonist associated with well-documented adverse effects. A difluorinated analogue of reduced haloperidol, (±)-4-(4-chlorophenyl)-1-(3,3-difluoro-4-(4-fluorophenyl)-4-hydroxybutyl)piperidin-4-ol (2), was synthesized in an attempt to minimize the oxidation. Compound (±)-2 was found to exhibit high affinity to S1R and facilitate BDNF release from mouse brain astrocytes. It was also confirmed that compound 2 cannot be oxidized back to the corresponding haloperidol analogue in liver microsomes. Furthermore, compound 2 was distributed to the brain following intraperitoneal administration in mice and reversed the learning deficits in active avoidance tasks. These findings suggest that compound 2 could serve as a promising S1R ligand with therapeutic potential for the treatment of cognitive impairments.

A chemoselective, one-pot transformation of aldehydes to nitriles.

This paper describes a procedure for direct conversion of aldehydes to nitriles using O-(diphenylphosphinyl)hydroxylamine (DPPH). Aldehydes are smoothly transformed to their corresponding nitriles by heating with DPPH in toluene. The reaction can be accomplished in the presence of alcohol, ketone, ester, or amine functionality.

Clinical development of metabolic inhibitors for oncology.

Metabolic inhibitors have been used in oncology for decades, dating back to antimetabolites developed in the 1940s. In the past 25 years, there has been increased recognition of metabolic derangements in tumor cells leading to a resurgence of interest in targeting metabolism. More recently there has been recognition that drugs targeting tumor metabolism also affect the often acidic, hypoxic, immunosuppressive tumor microenvironment (TME) and non-tumor cell populations within it, including immune cells. Here we review small-molecule metabolic inhibitors currently in clinical development for oncology applications. For each agent, we evaluate the preclinical studies demonstrating antitumor and TME effects and review ongoing clinical trials. The goal of this Review is to provide an overview of the landscape of metabolic inhibitors in clinical development for oncology.

Lipid Peroxidation Produces a Diverse Mixture of Saturated and Unsaturated Aldehydes in Exhaled Breath That Can Serve as Biomarkers of Lung Cancer-A Review.

The peroxidation of unsaturated fatty acids is a widely recognized metabolic process that creates a complex mixture of volatile organic compounds including aldehydes. Elevated levels of reactive oxygen species in cancer cells promote random lipid peroxidation, which leads to a variety of aldehydes. In the case of lung cancer, many of these volatile aldehydes are exhaled and are of interest as potential markers of the disease. Relevant studies reporting aldehydes in the exhaled breath of lung cancer patients were collected for this review by searching the PubMed and SciFindern databases until 25 May 2022. Information on breath test results, including the biomarker collection, preconcentration, and quantification methods, was extracted and tabulated. Overall, 44 studies were included spanning a period of 34 years. The data show that, as a class, aldehydes are significantly elevated in the breath of lung cancer patients at all stages of the disease relative to healthy control subjects. The type of aldehyde detected and/or deemed to be a biomarker is highly dependent on the method of exhaled breath sampling and analysis. Unsaturated aldehydes, detected primarily when derivatized during preconcentration, are underrepresented as biomarkers given that they are also likely products of lipid peroxidation. Pentanal, hexanal, and heptanal were the most reported aldehydes in studies of exhaled breath from lung cancer patients.

D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II.

Glutamate carboxypeptidase-II (GCPII) is a zinc-dependent metalloenzyme implicated in numerous neurological disorders. The pharmacophoric requirements of active-site GCPII inhibitors makes them highly charged, manifesting poor pharmacokinetic (PK) properties. Herein, we describe the discovery and characterization of catechol-based inhibitors including L-DOPA, D-DOPA, and caffeic acid, with sub-micromolar potencies. Of these, D-DOPA emerged as the most promising compound, with good metabolic stability, and excellent PK properties. Orally administered D-DOPA yielded high plasma exposures (AUCplasma = 72.7 nmol·h/mL) and an absolute oral bioavailability of 47.7%. Unfortunately, D-DOPA brain exposures were low with AUCbrain = 2.42 nmol/g and AUCbrain/plasma ratio of 0.03. Given reports of isomeric inversion of D-DOPA to L-DOPA via D-amino acid oxidase (DAAO), we evaluated D-DOPA PK in combination with the DAAO inhibitor sodium benzoate and observed a >200% enhancement in both plasma and brain exposures (AUCplasma = 185 nmol·h/mL; AUCbrain = 5.48 nmol·h/g). Further, we demonstrated GCPII target engagement; orally administered D-DOPA with or without sodium benzoate caused significant inhibition of GCPII activity. Lastly, mode of inhibition studies revealed D-DOPA to be a noncompetitive, allosteric inhibitor of GCPII. To our knowledge, this is the first report of D-DOPA as a distinct scaffold for GCPII inhibition, laying the groundwork for future optimization to obtain clinically viable candidates.

Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement.

BACKGROUND: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. OBJECTIVE: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. METHODS: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. RESULTS: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. CONCLUSION: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.