Illuminating the Disease Landscape: Human Oxidoreductase-Targeted Imaging and Quantification Agents for Precision...

Electron-specific reduction reactions afforded by quinone oxidoreductases have long been recognized as essential for safeguarding healthy mammalian cells against oxidative stress induced by environmental insults. The presence of reactive oxygen species (ROS) initiates the upregulation of intracellular redox enzyme activities used to battle the effects of ROS in healthy cells. At the same time, diseased human cells hijack the pathways of certain oxidoreductases, such as the two-electron-specific human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1), and use the overexpressed enzymes as standalone weapons or in concert with other proteins as an arsenal to advance disease progression and defeat therapies. Fortunately, in numerous cancers, inherent overexpression of hNQO1 activity is successfully targeted to trigger quinone-bearing, pro-therapeutic agents whose hNQO1 reduction yields cytotoxic agents only within hNQO1 activity-overexpressing cells. Furthermore, the development of diverse new approaches for controlling and leveraging expression of intracellular hNQO1 activity for disease treatment is being propelled by the growing evidence about the significant role of hNQO1 in cardiovascular diseases, central nervous system disorders, and autoimmune conditions. While oxidoreductases like hNQO1 are pivotal in many diseases and their development, there is a limited toolset for nonintrusive, selective, and low-limit-of-detection determination and quantification of enzyme activity in human tissue or mammalian models of disease. Our research team used tailored design criteria and computational routes to develop fluorescence-based molecular probes for highly selective, rapid measurement and visualization of hNQO1 activity through the latter's catalyzed production of reporters that are spectrally distinct from their parent probes. In this Account, we report on low- and tunable-brightness probes designed to be straightforwardly made and offer rapid intracellular accessibility to and fast activation by target enzymes with high selectivity to generate high-brightness reporters having large Stokes shifts. The probe/reporter pairs exhibit high photophysical, chemical, and environmental stabilities, low cellular toxicities, and emission energies that enable mucosal/epithelial and subsurface tissue imaging. These off-on and on-on probe-reporter pairs afford determination of intracellular enzyme presence and activity in simple cell cultures, complex tumor mimics, and an animal model of human cancer. These approaches offer unparalleled image contrast (target-to-background ratio, TBR), dynamic range, and limit of detection. The pivotal results presented in this Account stem from the focused development of fluorogenic molecular probes designed to assess hNQO1 activity across diverse cellular structures. The generalizable strategies for probe development described herein have broad implications for chemical biology and diagnostic/theranostic imaging with significant translational potential spanning various diseases.