Photoinducible Oncometabolite Detection

Abstract

Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.

Keywords: bioorthogonal chemistry; cycloaddition; epigenetics; fluorescent detection; metabolism.

Figure 1.

Photoinducible detection of the oncometabolite fumarate. (a) General schematic for bioorthogonal detection of fumarate-rich metabolomes using fluorogenic dipoles. (b) Diaryl tetrazoles are photoinducible precursors that form nitrileimines which undergo fluorogenic reactions with fumarate.

Figure 2.

Fluorogenic detection of fumarate by tetrazole 1. (a) Absorbance spectra of tetrazole 1 following photolysis in the presence or absence of 10 mM fumarate. (b) Fluorescence spectra (λ_ex = 410 nm) of tetrazole 1 following photolysis in the presence or absence of 10 mM fumarate. Reaction conditions: 100 μM tetrazole 1, 10 mM fumarate, 5% DMSO in PBS (pH 7.2), hν= 302 nm, 2 min. (c) Comparison of fluorescent detection of fumarate (10 mM) by tetrazole 1 (100 μM, hν=302 nm, 2 min) and previously described hydrazonyl chloride 3 (1 (100 μM, 1 h). (d) Limit of detection of fumarate by tetrazole 1. Data is representative of 3 replicates. (e) Fluorescent detection of other metabolite dipolarophiles by tetrazole 1 (λ_ex = 410 nm, λ_em = 540 nm) Reaction conditions: 100 μM tetrazole 1, 200 μM metabolite, sodium phosphate buffer pH 7.0 (1:1), hν= 302 nm, 2 min. Statistical significance was determined by unpaired t test (n = 3, ***P < 0.001).

Figure 3.

Detection of FH activity and endogenous dipolarophiles from tumor specimens using tetrazole 1. Analysis was performed from 3 different HLRCC tumors and kidney cortex samples from same patient, and 3 separate xenografts from 3 separate mice. (a) Schematic for signal-amplified FH activity assay. (b) Detection of differences in endogenous FH activity in tumor samples by tetrazole 1. Tumor lysates were first incubated with L-malic acid (25 mM, 1 h), then treated with tetrazole 1 (150 μM), photoirradiated (302 nm, 2 min), and analyzed by fluorimetry (λ_ex = 380 nm, λ_em = 540 nm). Lower fluorescence corresponds to lower FH activity, as expected upon FH mutation. (c) Differences in endogenous dipolarophile levels in tumor samples by tetrazole 1. Endogenous metabolites extracted from tumors were treated with tetrazole 1 (250 μM), photoirradiated (302 nm, 2 min) and analyzed by fluorimetry (λ_ex = 410 nm, λ_em = 540 nm). Higher fluorescence corresponds to higher endogenous dipolarophile concentrations, as expected upon FH mutation. Statistical significance was analysed by unpaired t test (n = 3, **P < 0.01).

Figure 4.

Detection of dipolarophiles in living cells using tetrazoles. (a-t) Images from confocal microscopy experiments. HK2 cells were simultaneously treated with tetrazole 1 (100 μM, 2 h) and vehicle (DMSO, 2 h, a-e) or dimethylfumarate (DMF) (100 μM, 2 h, f-j). UOK268 (k-o) and UOK268WT (p-t) cells were treated with tetrazole 2 (100 μM, 2 h). All cells (a-t) were treated with MitoTracker Green FM (100 nM, 30 min) and DRAQ5 (200 nM, 30 min), washed, and, photoirradiated (302 nm, 2 min). Confocal images displayed are obtained for DRAQ5 (633 nm HeNe633 laser, false-colored blue, a,f,k,p), MitoTracker Green (488 nm argon laser, false-colored red, b,g,l,q), and tetrazole (820 nm two-photon laser, green, c,h,m,r). (d,i,n,s) represents overlay of confocal images and (e,j,o,t) represents differential interference contrast (DIC) microscopy images with a 50 μm scale bar. (u) Mean fluorescence intensities of tetrazole fluorescence quantified from confocal images for UOK262 (FH −/−) relative to UOK262WT (FH +/+), and UOK268 (FH −/−) relative to UOK268WT (FH +/+). Total 10 cells were analyzed per cell line. Error bars denote SEM. Statistical significance analysed by unpaired t test (n = 3, ***P < 0.001). (v) Histograms obtained via flow cytometry analysis of UOK262 (FH −/−) and UOK262WT (FH +/+) cells treated with tetrazole 2 (100 μM, 2 h; followed by washing and photoirradiation).

Scheme 1.

Synthesis of diaryl tetrazoles used in this study. (a) EtOH, reflux, 4 h, 95%. (b) Pyridine, 0 °C to rt, 24 h, 63%. (c) 2-azidoethylene diamine, HBTU, NMM, DMF, rt, 2 h, 91%. (d) CuI, DCM, reflux, 72h, 45%.