Acute Changes in Matrix Metalloproteinase Following Exercise in Smokers and Non-Smokers

Matrix metalloproteinases (MMPs), also known as matrixins, are involved in the degradation of extracellular matrix of the tissue. Cigarette smoking and exercise may influence these molecules in the circulation. PURPOSE: The current study investigated the responses of MMP-1, -2, and -9 before and after a single bout of exercise performed at lower- and higher-intensity in smokers and non-smokers. METHODS: Physically inactive (N=18, physical activity \u3c 2 days per week) male smokers (N=8, carbon monoxide ≥ 16 ppm, smoking history \u3e 2 years, smoking \u3e 10 cigarettes per day) and non-smokers (N=10), the ages between 20 and 30, participated in the study. Participants exercised on a treadmill for 3 miles at two different intensities in random order (lower-intensity: 55% VO2max and higher-intensity: 75% of VO2max). Overnight fasting blood samples were collected before (PRE), immediately post-exercise (IPE), and 1-hr PE to examine the responses of MMP-1, -2, and -9. All data were analyzed using an ANOVA with repeated measure. If necessary, the Sidak’s pairwise multiple comparisons and a follow-up simple effects test were used as post-hoc tests (p \u3c 0.05). RESULTS: Only smokers significantly increased MMP-1 at IPE (1.88±0.19 ng/mL) by 22.08% from PRE (1.54±0.16 ng/mL, p=0.014), which then returned to the baseline value at 1-hr PE (1.45±0.15 ng/mL, p=0.001). The significant main effect for time indicated that MMP-2 at IPE (43.71±5.65 ng/mL) was significantly higher than PRE (25.68±3.27 ng/mL, p= 0.011) and 1-hr PE (28.04±3.34 ng/mL, p=0.036). Additionally, smokers had significantly higher MMP-9 (45.77±6.48 ng/mL, p=0.037) as compared with non-smokers (31.17±3.41 ng/mL). CONCLUSION: Regardless of exercise intensity, a single bout of exercise acutely increased both MMP-1, particularly in smokers, and MMP-2. However, this exercise-induced acute elevation of MMP-1 and MMP-2 returned to baseline values at 1-hour post exercise. A significantly higher level of MMP-9 observed in smokers may indicate that habitual cigarette smokers, as compared with non-smokers, may be more susceptible to a structural damage of extracellular matrix, endothelial inflammation, and an atherosclerotic event

Relationship between Cell Adhesion Molecules and Cigarette Smoking Before and After Exercise

Cell adhesion molecules (CAMs) found on endothelial membranes are integral membrane proteins that bind with other cells and promote immune cell transmigration. The responses of CAMs to exercise have not been fully examined in cigarette smokers. PURPOSE: The current study investigated the relationship between cell adhesion molecules [soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble E-selectin (sE-selectin)] and cigarette smoking before and after a single bout of exercise at different intensities. METHODS: Physically inactive (physical activity \u3c 2 days per week) male smokers (N=8, carbon monoxide ≥ 16 ppm, smoking history \u3e 2 years, and cigarette smoking \u3e 10 cigarettes per day) and non-smokers (N=10), the ages between 20 and 30, volunteered for the study. Participants exercised on a treadmill for 3 miles at two different intensities in random order (low: 55% and high: 75% of VO2max) on a separate occasion. Overnight fasting blood samples were collected at three different time points [before (PRE), immediately post-exercise (IPE), and 1-hr PE] and analyzed for sVCAM-1, sICAM-1, and sE-selectin. Data were analyzed using a 2 (groups) X 3 (time) ANOVA with repeated measure. The Sidak’s pairwise multiple comparisons and a follow-up simple effects test as a post-hoc test were employed if necessary. A p-value was set at \u3c 0.05 for the statistical significance. RESULTS: The significant main effect for group indicated that smokers had significantly higher sICAM-1 (137.37±5.78 ng/mL, p=0.001) than non-smokers (110.45±5.17 ng/mL). Regardless of exercise intensity, sVCAM-1 at IPE (160.09±3.88 ng/mL) significantly increased by 21.5% from PRE (145.87±3.19 ng/mL, p=0.001) and returned to baseline value at 1-hr PE (145.09±4.39, p=0.006). Similarly, sICAM-1 at IPE (131.73±4.47 ng/mL) was significantly elevated by 9.7% from PRE (120.06±3.86 ng/mL, p=0.001) but returned to baseline value at 1-hr PE (119.31±4.27, p=0.001). There was no change in sE-selectin following exercise. CONCLUSION: Exercise may temporarily increase both sICAM-1 and sVCAM-1, but not sE-selectin, which then return to baseline values after 1 hour of exercise. Smokers had significantly higher sICAM-1 as compared with their counterparts, indicating that smokers are more susceptible to inflammatory or cardiovascular diseases

Omega-3 Supplementation, Menstrual Cycle, and Acute Eccentric Exercise on Plasma Lipid Profiles

Omega-3 fatty acids supplementation and the menstrual cycle may independently influence plasma lipids and lipoproteins following acute exercise. PURPOSE: The current study investigated the effects of dietary fish oil supplement and acute eccentric exercise on plasma lipids and lipoproteins during two different phases of the menstrual cycle [mid-follicular (MF) vs. mid-luteal phase (ML)]. METHODS: As a double-blind, placebo-controlled design, 22 healthy young females (age= 20.86±1.39 years) volunteered. Participants were randomly assigned to either the fish oil (N=11) or the placebo (N=11) group. The fish oil group took a total of 6 capsules of fish oil per day (6g total; each capsule with 2.4 g of eicosapentaenoic acid and 1.8 g of docosahexaenoic acid), while the placebo group took 6 capsules of safflower oil/day (6g total; 2.8 g of lauric acid, 1.1g of myristic acid, 0.4 g of caprylic acid, 0.4 g of oleic acid, and 0.09 mg of linoleic acid) for 3 weeks. Participants in each group performed an acute eccentric single-leg exercise protocol during the MF and ML phases, which consisted of 10 sets of 10 repetitions with a 3-min resting between sets at an isokinetic speed of 30⁰/sec. The leg exercised for the MF phase was randomly selected and the opposing leg exercised during the ML phase. Plasma samples were collected at pre-, 6-hours post-exercise (6-hr PE), and 24-hours post-exercise (24-hr PE)] to analyze total cholesterol (TC), high-density lipoprotein (HDL-C), lipoprotein (a), very low-density lipoprotein (VLDL-C), and low-density lipoprotein (LDL-C). Data were analyzed using a multivariate analysis of variance (p \u3c 0.05). If a significant interaction was found, a Tukey’s post-hoc test was performed. RESULTS: Plasma lipids and lipoproteins were not different between the fish oil and placebo groups or before and after the acute eccentric leg exercise. However, HDL-C was significantly higher (p = 0.041) during the ML (61.66 ± 2.44 mg/dL) phase than that of the MF (54.53 ± 2.44 mg/dL) phase. CONCLUSION: Although it didn’t reach a statistical significance, the overall lipid and lipoprotein profile tended to improve following a relatively short-term fish oil supplement in healthy, young women. Acute eccentric exercise may not negatively affect plasma lipids and lipoproteins. Elevated HDL-C during the mid-luteal phase may possibly be associated with increased estradiol levels. It is recommended for future studies that an extended period of fish oil supplement using different dosages and exercise regimen be implemented to examine a long-term benefit of fish oil supplement in a variety of sample groups

Racemases and epimerases operating through a 1,1-proton transfer mechanism:Reactivity, mechanism and inhibition

Racemases and epimerases catalyse changes in the stereochemical configurations of chiral centres and are of interest as model enzymes and as biotechnological tools. They also occupy pivotal positions within metabolic pathways and, hence, many of them are important drug targets. This review summarises the catalytic mechanisms of PLP-dependent, enolase family and cofactor-independent racemases and epimerases operating by a deprotonation/reprotonation (1,1-proton transfer) mechanism and methods for measuring their catalytic activity. Strategies for inhibiting these enzymes are reviewed, as are specific examples of inhibitors. Rational design of inhibitors based on substrates has been extensively explored but there is considerable scope for development of transition-state mimics and covalent inhibitors and for the identification of inhibitors by high-throughput, fragment and virtual screening approaches. The increasing availability of enzyme structures obtained using X-ray crystallography will facilitate development of inhibitors by rational design and fragment screening, whilst protein models will facilitate development of transition-state mimics

Identification of novel small-molecule inhibitors of α-methylacyl-CoA racemase (AMACR; P504S) and structure-activity relationships

α-Methylacyl-CoA racemase (AMACR; P504S; EC 5.1.99.4) catalyses epimerization of 2-methylacyl-CoAs and is important for the degradation of branched-chain fatty acids and the pharmacological activation of ibuprofen and related drugs. It is also a novel drug target for prostate and other cancers. However, development of AMACR as a drug target has been hampered by the difficulties in assaying enzyme activity. Consequently, reported inhibitors have been rationally designed acyl-CoA esters, which are delivered as their carboxylate prodrugs. The novel colorimetric assay for AMACR based on the elimination of 2,4-dinitrophenolate was developed for high-throughput screening and 20,387 ‘drug-like compounds’ were screened, with a throughput of 768 compounds assayed per day. Pyrazoloquinolines and pyrazolopyrimidines were identified as novel scaffolds and investigated as AMACR inhibitors. The most potent inhibitors have IC50 values of ~2 µM. The pyrazoloquinoline inhibitor 10a displayed uncompetitive inhibition, whilst 10j displayed mixed competitive inhibition. The pyrazolopyrimidine inhibitor 11k displayed uncompetitive inhibition. This is the first report of the identification of specific drug-like small-molecule AMACR inhibitors by high-throughput screening. Pyrazoloquinolines and pyrazolopyrimidines may also be useful as inhibitors of other CoA-utilizing enzymes

Novel 2-arylthiopropanoyl-CoA inhibitors of α-methylacyl-CoA racemase 1A (AMACR; P504S) as potential anti-prostate cancer agents

α-Methylacyl-CoA racemase (AMACR; P504S) catalyses an essential step in the degradation of branched-chain fatty acids and the activation of ibuprofen and related drugs. AMACR has gained much attention as a drug target and biomarker, since it is found at elevated levels in prostate cancer and several other cancers. Herein, we report the synthesis of 2-(phenylthio)propanoyl-CoA derivatives which provided potent AMACR inhibitory activity (IC50 = 22–100 nM), as measured by the AMACR colorimetric activity assay. Inhibitor potency positively correlates with calculated logP, although 2-(3-benzyloxyphenylthio)propanoyl-CoA and 2-(4-(2-methylpropoxy)phenylthio)propanoyl-CoA were more potent than predicted by this parameter. Subsequently, carboxylic acid precursors were evaluated against androgen-dependent LnCaP prostate cancer cells and androgen-independent Du145 and PC3 prostate cancer cells using the MTS assay. All tested precursor acids showed inhibitory activity against LnCaP, Du145 and PC3 cells at 500 µM, but lacked activity at 100 µM. This is the first extensive structure-activity relationship study on the influence of side-chain interactions on the potency of novel rationally designed AMACR inhibitors

A Convenient Colorimetric Assay for α-Methylacyl-CoA Racemase (AMACR; P504S) and Testing Of Inhibitors

α-Methylacyl-CoA racemase (AMACR; P504S) catalyzes a key step in the degradation of branched-chain fatty acids and is important for the pharmacological activation of Ibuprofen and related drugs. Both the concentration and activity of AMACR are increased in prostate and other cancer cells. Diminution of AMACR levels using siRNA reduces proliferation of cancer cells. Therapeutic development of AMACR inhibitors has been hampered by the lack of a convenient assay. A novel substrate, 3-(2,4-dinitrophenoxy)-2-methylpropanoyl-CoA, was designed and synthesized. Incubation with active AMACR results in the elimination of the strongly yellow 2,4-dinitrophenoxide. A series of acyl-CoA substrates, previously reported AMACR inhibitors and non-specific protein modifying agents were tested as inhibitors using this assay. The fully-developed assay was used to determine IC50 values and other inhibitor characteristics. Medium-length straight-chain acyl-CoA esters (C6 – C10) were modest competitive inhibitors of AMACR, with IC50 values of 3 - 16 µM. R- and S-2-methyldecanoyl-CoA were ~ 3-fold more potent inhibitors than decanoyl-CoA. Ibuprofenoyl-CoA and similar compounds were reversible inhibitors with an IC50 value of ~0.5 µM. N-dodecyl-N-methylcarbamoyl-CoA (IC50 = 400 pM) was the most potent reversible inhibitor. Ebselen Oxide was a non-specific inactivator of AMACR, whilst Rose Bengal degraded the enzyme in a time and light-dependent manner. This assay will facilitate exploration of the biology of AMACR and the development of inhibitors as anti-cancer agents.This work was funded by Prostate Cancer UK (S10-03 and PG14-009), a University of Bath Overseas University Research Studentship, and a Biochemical Society Summer Vacation Studentship Award.<br/

<em>N</em>³-Alkylation during formation of quinazolin-4-ones from condensation of anthranilamides and orthoamides

Dimethylformamide dimethylacetal (DMFDMA) is widely used as a source of electrophilic one-carbon units at the formate oxidation level; however, electrophilic methylation with this reagent is previously unreported. Reaction of anthranilamide with DMFDMA at 150 °C for short periods gives mainly quinazolin-4-one. However, prolonged reaction with dimethylformamide di(primary-alkyl)acetals leads to subsequent alkylation at N3. 3-Substituted anthranilamides give 8-substituted 3-alkylquinazolin-4-ones. Condensation of anthranilamides with dimethylacetamide dimethylacetal provides 2,3-dimethylquinazolin-4-ones. In these reactions, the source of the N3-alkyl group is the O-alkyl group of the orthoamides. By contrast, reaction with the more sterically crowded dimethylformamide di(isopropyl)acetal diverts the alkylation to the oxygen, giving 4-isopropoxyquinazolines, along with N3-methylquinazolin-4-ones where the methyl is derived from N-Me of the orthoamides. Reaction of anthranilamide with the highly sterically demanding dimethylformamide di(t-butyl)acetal gives largely quinazolin-4-one, whereas dimethylformamide di(neopentyl)acetal forms a mixture of quinazolin-4-one and N3-methylquinazolin-4-one. The observations are rationalised in terms of formation of intermediate cationic electrophiles (alkoxymethylidene-N,N-dimethylammonium) by thermal elimination of the corresponding alkoxide from the orthoamides. These are the first observations of orthoamides as direct alkylating agents