Angelw: chemical reaction based sensing method for detection of cyclooxygenase-2 in living cells

Cyclooxygenase (Cox, EC 1.14.99.1) is a key rate limiting enzyme for the synthesis of prostaglandins from arachidonic acid (AA) There are two main isoenzymes (COX-1 and COX-2) in human Cox: COX-1 is constitutively expressed in most of the lower cell types, and COX-2 can produce prostaglandins rapidly according to the composition or induction of tissue types Prostaglandins are a kind of unsaturated fatty acids with physiological activities, which are widely distributed in various tissues and body fluids of the body They affect the occurrence of physiological processes through the regulation of lipids, such as the protection of gastric epithelial cells, hemostasis and sodium metabolism In addition, COX-2 overexpression is closely related to inflammation, neurodegenerative diseases and cancer However, the current detection methods for COX-2 in biological systems are very limited Recently, Professor Jefferson Chan of the Department of chemistry at the University of Illinois Urbana Champaign designed and synthesized a fluorescent probe, Cox fluor, based on the activity-based sensing (ABS) It successfully applied to the detection of COX-2 in living cells by making use of the slight differences in the size and dynamics of Cox active sites ABS mainly refers to the use of dynamic molecular reactivity to achieve high selective detection of analyte in complex environment, with high specificity, accurate signal output and high tolerance to complex environment Relevant achievements were published on angelw Chem Int ed (DOI: 10.1002 / anie 201914845) under the title of "an activity-based sensing approach for the detection of cyclooxygenase-2in live cells" Cox fluor is composed of AA and 3,7-dihydroxybenzoxazine via cleavable amide bond The authors speculate that the lipid tail acts as a COX-2 substrate (scheme 1a) in a similar way to AA After binding to the Cox active site, the probe generates the Cox fluor pgg2 intermediate through the dioxygen and cyclization reactions, then transfers to the peroxidase active site and is oxidized by compound I or II, producing unstable Trihalogenated radicals and the disproportionation and amide hydrolysis reactions, thereby releasing the fluorescent products and pgg2 or PGH2 (reduced state, scheme 1b) (source: angelw Chem Int ed.) Cox fluor was synthesized by zinc mediated reduction of azurol, and then acetylated with acetic anhydride to obtain intermediate 1 in 63% yield Then, 1 was coupled with arachidonyl chloride under the action of potassium iodide to obtain compound 2 Finally, 2 was deacetylated in the presence of sodium methothane (in situ formation), and Cox fluor was obtained in 68% yield The total yield of the four steps was 21% (scheme 2) (source: angelw Chem Int ed.) first, the authors evaluated Cox fluor's response to recombinant human COX-2 Under the action of COX-2, the cleavage of Cox fluoror resulted in the formation of tetrazoline (573 nm, Φ f = 0.55) and pgg2, and the fluorescence enhancement was about 41 times (Figure 1a) In order to confirm that this result is caused by enzyme catalytic oxidation rather than peroxidase activity, we synthesized the control compound Ctrl Cox fluor with saturated lipid tail (arachidic acid) instead of AA lipid Fortunately, under the same conditions, there is no reaction (Figure 1b) for Ctrl coxflur, which indicates that the fluorescence enhancement of coxflur is the result of enzyme catalysis In addition, the K cat of Cox fluor oxidized to trihalolin was 19 min-1, and the kcat was 22 μ m (Figure 1c) Later, the authors found that indomethacin and celecoxib can inhibit the fluorescence response of COX-2 (Figure 1D), which makes it easier to identify or evaluate COX-2 inhibitors In order to evaluate whether COX-2 has the effect of Miss target activation, a group of enzymes with related activities (lipoxygenase, peroxidase, catalase, etc.) were selected for testing, which can cleave amide bond (esterase) or metabolize AA analog (cytochrome P450s) It is gratifying that even in the presence of 10 fold enzyme, Cox fluoror still shows good selectivity (Figure 1E) Similarly, in the presence of reactive oxygen species, reactive nitrogen or other cell oxidants / reducers, Cox fluoror did not show fluorescence enhancement (Figure 1F) (source: angelw Chem Int ed.) in order to further study the mechanism of COX-2 activating Cox fluor, molecular docking and molecular dynamics (MD) calculations were used The author stops Cox fluor at each angle along the simulated orbit to explore the most likely combination The results showed that, compared with COX-1, the cyclooxygenase active site of COX-2 had a good binding with Cox fluor (Figure 2a-c), except for the difference in quantity and region Through structural comparison, the author also found that Cox fluor can bind in an appropriate way at the active site of cyclooxygenase and generate oxidation reaction (Figure 2D) (source: angel Chem Int ed.) next, the author used Cox fluor to detect and image the activity of endogenous COX-2 in rat macrophages of raw 264.7 We used lipopolysaccharide (LPS) to induce the production of inflammatory model, so as to detect the expression of COX-2 and prostaglandins According to the results of confocal photos and fluorescence semi quantitative analysis, the fluorescence intensity of the cells stimulated by LPS was about 1.6 times of that of the control group (Figure 3) (source: angel Chem Int ed.) finally, the authors focused on whether COX-2 activity can be regulated above protein level It is speculated that oxygen can combine with heme coenzyme to affect the activity of COX-2 Therefore, the fluorescence of raw 264.7 macrophages was detected by flow cytometry under the condition of normal oxygen (~ 21%) and hypoxia (< 0.1%) Interestingly, under the condition of hypoxia, COX-2 showed higher activity (Figure 4a-c) In addition, the fluorescence intensity of cells treated with indomethacin decreased by 12% year on year, indicating that the increase of fluorescence was Cox-2-dependent (Figure 4D) In order to evaluate the dose dependence of oxygen, the author repeated the experiment in 1% and 0.1% oxygen atmosphere, and observed that the decrease of oxygen dependence of COX-2 activity (Figure 4d-e) was not related to the protein expression level (Figure 4F) This result fully shows that the local cellular microenvironment plays a key role in the regulation of COX-2 activity (source: angelw Chem Int ed.) in a word, the author synthesized the first isotype selective probe Cox fluor for detecting COX-2 activity by using ABS method, which proved that COX-2 activity can be regulated by oxygen partial pressure in living cells, providing important help for studying and evaluating the role of COX-2 in living cells.