Responsive NIR fluorescent probes targeting PKM2 with enzyme-activating properties for precision cancer diagnosis

Pyruvate kinase M2 (PKM2) serves as a crucial metabolic biomarker for tumor progression; however, distinguishing PKM2 from its highly homologous isoform PKM1 and dynamically monitoring its oligomeric state in living systems remain formidable challenges. Herein, we present two novel near-infrared (NIR) fluorescent probes, YL1 and YL2, designed by conjugating the PKM2-specific allosteric activator TEPP-46 with a green fluorescent protein (GFP)-chromophore-derived molecular rotor. Operating through a twisted intramolecular charge transfer (TICT) mechanism, these probes exhibit a highly sensitive "turn-on" NIR fluorescence response upon specific binding within the hydrophobic allosteric pocket of PKM2, which restricts their intramolecular rotation. Both probes demonstrate exceptional isoform selectivity over PKM1 and various biological interferents, boasting low detection limits (180 nM for YL1 and 210 nM for YL2) alongside ultra-fast, second-scale binding kinetics. Significantly, YL1 and YL2 function as potent fluorogenic agonists, effectively promoting the allosteric re-assembly of tumor-associated, inactive PKM2 dimers into active tetramers, thereby restoring pyruvate kinase activity. In live-cell imaging, the probes selectively illuminate PKM2-overexpressing cancer cells (HeLa, A549, and 4T1) with high contrast while remaining virtually silent in normal cells (HK-2, MCF-10A). Furthermore, their integration with flow cytometry facilitates rapid, quantitative, and high-throughput screening of malignant cell populations. Finally, in vivo imaging successfully visualizes tumor lesions in mouse models with high signal-to-noise ratios and deep tissue penetration. This multifunctional probe platform seamlessly integrates precision imaging, enzyme activation, and high-throughput cytometric screening, offering a powerful theranostic strategy for tracking metabolic reprogramming and facilitating early cancer diagnosis.