Design of novel and potent cPLA2α inhibitors containing an α-methyl-2-ketothiazole as a metabolically stable serine trap

We report the design of novel, potent cPLA₂α inhibitors that possess an α-methyl-2-ketothiazole that acts as a serine-reactive moiety. We describe the optimization of the series for potency and metabolic stability towards ketone reduction. This was achieved by attenuating the reactivity of the ketone using a combination of electronic and steric effects.     

Engineering novel cell surface receptors for virus-mediated gene transfer.

The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.     

New norbornyl derivatives as woody fragrant materials

The optimization of a Diels-Alder reaction to prepare a novel ketone bearing a 2,3-dimethylnorbornyl group is presented together with the structure elucidation of the isomers. Employing this new ketone as starting material, derivatives with new woody odor notes as well as attempts to obtain ambery-musky odorants are reported.     

Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body β-hydroxybutyrate (β-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM β-HB significantly induced transforming growth factor-β1 expression, with a marked increase in collagen I expression. β-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.     

Carbonylbisphosphonate and (diazomethylene)bisphosphonate analogues of AZT 5(')-diphosphate

A novel nucleotide analogue is described, in which the alpha,beta-phosphoric anhydride oxygen of a nucleoside 5(')-diphosphate is replaced by a carbonyl group: the carbonylbisphosphonate analogue 5 of 2('),3(')-dideoxy-3(')-azidothymidine 5(')-diphosphate (AZT 5(')-diphosphate). 5 was synthesized from tetramethyl (diazomethylene)bisphosphonate 1 via the trimethyl ester 4 of the corresponding AZT 5(')-(diazomethylene)bisphosphonate 6, which is also a new type of nucleotide analogue. The ultimate product 5 was isolated by reverse-phase HPLC, and characterized by 31P, 13C, and 1H NMR; and by high-resolution mass spectrometry. The ketone group of 5 is a visible chromophore (yellow) and reversibly forms a colorless hydrate. The ketone hydrate 'pK' is about 4.2 when excess of magnesium ion is present. The potential of such analogues as novel inhibitors of enzymes mediating nucleotide-dependent biochemical processes is discussed.     

Synthesis and Bioactivity of Novel Acetylenic Compounds

The synthesis of novel acetylenic ketone compounds and anti-inflammatory and antimicrobial activities are herein described.     

Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant omega-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, omega-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using omega-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.     

Ketone body utilization drives tumor growth and metastasis

We have previously proposed that catabolic fibroblasts generate mitochondrial fuels (such as ketone bodies) to promote the anabolic growth of human cancer cells and their metastasic dissemination. We have termed this new paradigm “two-compartment tumor metabolism.” Here, we further tested this hypothesis by using a genetic approach. For this purpose, we generated hTERT-immortalized fibroblasts overexpressing the rate-limiting enzymes that promote ketone body production, namely BDH1 and HMGCS2. Similarly, we generated MDA-MB-231 human breast cancer cells overexpressing the key enzyme(s) that allow ketone body re-utilization, OXCT1/2 and ACAT1/2. Interestingly, our results directly show that ketogenic fibroblasts are catabolic and undergo autophagy, with a loss of caveolin-1 (Cav-1) protein expression. Moreover, ketogenic fibroblasts increase the mitochondrial mass and growth of adjacent breast cancer cells. However, most importantly, ketogenic fibroblasts also effectively promote tumor growth, without a significant increase in tumor angiogenesis. Finally, MDA-MB-231 cells overexpressing the enzyme(s) required for ketone re-utilization show dramatic increases in tumor growth and metastatic capacity. Our data provide the necessary genetic evidence that ketone body production and re-utilization drive tumor progression and metastasis. As such, ketone inhibitors should be designed as novel therapeutics to effectively treat advanced cancer patients, with tumor recurrence and metastatic disease. In summary, ketone bodies behave as onco-metabolites, and we directly show that the enzymes HMGCS2, ACAT1/2 and OXCT1/2 are bona fide metabolic oncogenes.     

Identification of the new UV filter compound cysteine-L-3-hydroxykynurenine O-beta-d-glucoside in human lenses

UV filters protect the human lens and retina from UV light-induced damage. Here, we report the identification of a new UV filter, cysteine-l-3-hydroxykynurenine O-beta-d-glucoside, which is present in older normal human lenses. Its structure was confirmed by independent synthesis. It is likely this novel UV filter is formed in the lens by nucleophilic attack of cysteine on the unsaturated ketone derived from deamination of 3-hydroxykynurenine O-beta-d-glucoside. Quantitation studies revealed considerable variation in normal lens levels that may be traced to the marked instability of the cysteine adduct. The novel UV filter was not detected in advanced nuclear cataract lenses.     

Ketone body utilization drives tumor growth and metastasis

We have previously proposed that catabolic fibroblasts generate mitochondrial fuels (such as ketone bodies) to promote the anabolic growth of human cancer cells and their metastasic dissemination. We have termed this new paradigm “two-compartment tumor metabolism.” Here, we further tested this hypothesis by using a genetic approach. For this purpose, we generated hTERT-immortalized fibroblasts overexpressing the rate-limiting enzymes that promote ketone body production, namely BDH1 and HMGCS2. Similarly, we generated MDA-MB-231 human breast cancer cells overexpressing the key enzyme(s) that allow ketone body re-utilization, OXCT1/2 and ACAT1/2. Interestingly, our results directly show that ketogenic fibroblasts are catabolic and undergo autophagy, with a loss of caveolin-1 (Cav-1) protein expression. Moreover, ketogenic fibroblasts increase the mitochondrial mass and growth of adjacent breast cancer cells. However, most importantly, ketogenic fibroblasts also effectively promote tumor growth, without a significant increase in tumor angiogenesis. Finally, MDA-MB-231 cells overexpressing the enzyme(s) required for ketone re-utilization show dramatic increases in tumor growth and metastatic capacity. Our data provide the necessary genetic evidence that ketone body production and re-utilization drive tumor progression and metastasis. As such, ketone inhibitors should be designed as novel therapeutics to effectively treat advanced cancer patients, with tumor recurrence and metastatic disease. In summary, ketone bodies behave as onco-metabolites, and we directly show that the enzymes HMGCS2, ACAT1/2 and OXCT1/2 are bona fide metabolic oncogenes.     

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body

Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2–1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control. Arch. Insect Biochem. Physiol. 39:9–17, 1998. © 1998 Wiley-Liss, Inc.     

Enzymatic reduction of shogaol: a novel biotransformation pathway for the alpha,beta-unsaturated ketone system.

A novel reductive metabolism of 1-(4-hydroxy-3-methoxyphenyl)-deca-4-ene-3-one (shogaol), a pungent principle of ginger, was investigated in rat liver in vitro. Ethyl acetate-extractable metabolites of shogaol formed by incubation of this alpha,beta-unsaturated ketone with rat liver cytosolic fraction fortified with NADPH or NADPH-generating system were isolated, and two major metabolites were identified as 1-(4-hydroxy-3-methoxyphenyl)-decan-3-one (paradol) and 1-(4-hydroxy-3-methoxy)-decan-3-ol (reduced paradol). 1-(4-hydroxy-3-methoxyphenyl)-deca-1-ene-3-one (dehydroparadol), a non-pungent analog of shogaol, formed the same metabolites as did shogaol under similar incubation conditions. Paradol appears to be an intermediate in the reductive metabolism of the alpha,beta-unsaturated ketone moiety of shogaol to the corresponding saturated alcohol.     

Acetoacetyl-CoA synthetase gene is abundant in rat adipose, and related with fatty acid synthesis in mature adipocytes

Acetoacetyl-CoA synthetase (AACS, acetoacetate-CoA ligase, EC 6.2.1.16) is a novel cytosolic ketone body (acetoacetate)-specific ligase, the physiological role of which remains to be elucidated. We examined the expression profiles of AACS mRNA in adult rat tissues, finding that it was particularly abundant in male subcutaneous white adipose tissue after weaning. In white adipose tissue, AACS mRNA was preferentially detected in mature adipocytes but not in preadipocytes. The AACS mRNA expression in primary preadipocytes increased during the adipocyte differentiation. These expression profiles were similar to that of acetyl-CoA carboxylase-1, but not like to that of 3-hydroxy-3-methylglutaryl-CoA reductase. These results suggest that AACS in adipose tissue plays an important role in utilizing ketone body for the fatty acid-synthesis during adipose tissue development.     

Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors

We describe novel peptide-based caspase inhibitors. Potent and comparatively selective compounds containing a dipeptide scaffold and a substituted oxymethyl ketone as a warhead were developed. The newly synthesized compounds were tested for inhibition in in vitro enzymatic assays of caspases-1, -3, -6, -8, and -9. The benzyloxycarbonyl-phenylglycyl-aspartyl benzoyloxymethyl ketone (Z-Phg-Asp-CH2OCO-Ph, coded as HU44) was the most potent inhibitor of caspase-1 and caspase-3. Of several analogs of HU44 that were made, the beta-Asp methyl ester (2) is an effective inhibitor against caspase-3 and caspase-8, and less effective against caspase-1. These compounds did not inhibit caspase-6 and caspase-9 significantly.     

Phosphorylation and dephosphorylation of the Ca2+ pump of human red cells in the presence of monovalent cations

A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.     

Candidate sex pheromones of the New World screwworm Cochliomyia hominivorax

Five novel homologous acetate derivatives of long-chain secondary alcohols and a related ketone were tested for their efficacy as contact mating stimulants for Cochliomyia hominivorax Coquerel (Diptera: Calliphoridae). Full copulatory behaviour at a high percentage was found in tests with racemic 6-acetoxy-19-methylnonacosane at 2.5-20 microg using fertile males from three strains. Males of two strains responded nearly as well to 7-acetoxy-15-methylnonacosane, but an older strain first colonized in 1992 did not respond to this compound. Few or no copulatory responses were obtained to the other secondary alcohol acetates and a related ketone. These two acetate derivatives are the first sex pheromones identified in a calliphorid fly. The threshold of response was also tested, but could not be pinpointed.     

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with “stemness,” more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This “two-compartment” metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert “low-risk” breast cancer patients to “high-risk” status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCT4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.     

Fused bicycles as arylketone bioisosteres leading to potent, orally active thiadiazole H3 antagonists

A structure-activity relationship study was undertaken to address the lack of oral exposure of the H3 antagonist 1, which incorporated an arylketone. Among a number of sub-series, the 4H-pyrido[1,2-a]pyrimidin-4-one analog 21 showed an improved PK profile in rat and mouse and was active in an obesity model. The pyrimidin-4-one proved to be a novel and useful ketone bioisostere.