Characterization of a highly specific NQO1-activated near-infrared fluorescent probe and its application for in vivo tumor imaging

Abstract

The Near-infrared Fluorescence (NIRF) molecular imaging of cancer is known to be superior in sensitivity, deeper penetration, and low phototoxicity compared to other imaging modalities. In view of an increased need for efficient and targeted imaging agents, we synthesized a NAD(P)H quinone oxidoreductase 1 (NQO1)-activatable NIR fluorescent probe (NIR-ASM) by conjugating dicyanoisophorone (ASM) fluorophore with the NQO1 substrate quinone propionic acid (QPA). The probe remained non-fluorescent until activation by NQO1, whose expression is largely limited to malignant tissues. With a large Stokes shift (186 nm) and a prominent near-infrared emission (646 nm) in response to NQO1, NIR-ASM was capable of monitoring NQO1 activity in vitro and in vivo with high specificity and selectivity. We successfully employed the NIR-ASM to differentiate cancer cells from normal cells based on NQO1 activity using fluorescence microscopy and flow cytometry. Chemical and genetic approaches involving the use of ES936, a specific inhibitor of NQO1 and siRNA and gene transfection procedures unambiguously demonstrated NQO1 to be the sole target activating the NIR-ASM in cell cultures. NIR-ASM was successfully used to detect and image the endogenous NQO1 in three live tumor-bearing mouse models (A549 lung cancer, Lewis lung carcinoma, and MDMAMB 231 xenografts) with a high signal-to-low noise ratiometric NIR fluorescence response. When the NQO1-proficient A549 tumors and NQO1-deficient MDA-MB-231 tumors were developed in the same animal, only the A549 malignancies activated the NIR-ASM probe with a strong signal. Because of its high sensitivity, rapid activation, tumor selectivity, and nontoxic properties, the NIR-ASM appears to be a promising agent with clinical applications.

Small-molecule fluorescence-based probes for interrogating major organ diseases

“…39). 57 Reaction with NQO1 in the presence of NADH, results in a significant increase in the fluorescence intensity of NIR-ASM in PBS buffer. The proposed mode of action involves cleavage of the amide bond, which triggers the spontaneous release of strong fluorescent dicyanoisophorone (ASM).…”

Nutritional Stress in Head and Neck Cancer Originating Cell Lines: The Sensitivity of the NRF2-NQO1 Axis

“…These cells significantly differ with respect to basal NQO1 activity (FaDu > Detroit 562 > Cal 27) [36]. IMR-90 fibroblasts, which are considered as NQO1 non-expressing cell lines [37], were used as representative of an untransformed cell line.…”

Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response

“…We also detected an increase in the ratio between oxidized versus reduced GSH (GSSH/GSH) in lysates of cKO BMDMs ( Figure 5—figure supplement 3B–D ). Additionally, abundance of NQO1 was significantly higher in cKO BMDMs as determined by a specific NQO1-activated fluorescent probe ( Figure 5—figure supplement 3E and F ; Punganuru et al, 2019 ). Altogether, these findings indicate that cKO BMDMs are basally exposed to higher oxidative stress, and suggest that upregulation of redox homeostasis factors is required to maintain their viability ( Tal et al, 2009 ).…”

“…The NQO1 activity probe was generated in-house as described in Punganuru et al, 2019 . The endogenous NQO1 activity of WT and cKO BMDMs was determined by flow cytometry after exposure to the probe.…”