SHP2, a non-receptor tyrosine phosphatase belonging to the PTP family, plays a crucial role in cellular signal transduction [1,2]. This enzyme is involved in cell survival, proliferation and migration, modulating a spectrum of cellular signals that encompass metabolism, cell growth, differentiation, transcription, and oncogenic transformation. SHP2 serves as a central node in oncogenic signaling pathways, including but not limited to JAK/STAT, PI3K/AKT, RAS/Raf/MAPK, and the immune checkpoint interactions of PD-1/PD-L1 [[3], [4], [5]]. It is essential for mediating biological responses to growth factors, hormones, cytokines, and cell adhesion molecules, as well as for signal transduction pathways that govern developmental processes and hematopoietic function. Dysregulation of SHP2, manifested as its overexpression or hyperactivation, has been correlated with a spectrum of diseases and is frequently detected in various solid tumors [6,7].

In recent years, SHP2 has emerged as a promising target for cancer therapeutics, owing to its critical roles in cell proliferation, differentiation, and survival, and has gained increasing recognition. Existing detection methods for SHP2, such as western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and substrate-based assays, have substantially enhanced our understanding of SHP2 biology. However, these methods are often constrained by limitations such as low throughput, reduced sensitivity, and inability to monitor SHP2 activity in real time within living cells.

Fluorescence imaging has become increasingly prominent in the field of target visualization, with small-molecule fluorescent probes being a potent analytical tool [8,9]. Many such probes have been developed and are now widely applied for labeling, observation, and detection of proteins [10,11], nucleic acids [12,13], and cellular physical microenvironments [14], such as polarity [15], pH [16], and viscosity [17,18]. Some of these targeted fluorescent probes have been commercialized and play a crucial role in clinical practice, often in conjunction with multimodal imaging techniques [[19], [20], [21]]. Although some fluorescent modulators targeting the catalytic domain of the PTP protein family have the potential to serve as SHP2 tracers, the high conservation of the PTP catalytic domain within the PTP protein family generally makes it difficult for these fluorescent molecules to achieve high selectivity for SHP2 [[22], [23], [24], [25]].

To date, no small-molecule fluorescent probes specific for SHP2 by targeting allosteric site have been reported. The development of such probes could permit real-time tracking of SHP2 cellular dynamics, utilizing imaging techniques that offer high sensitivity and spatial-temporal resolution. This capability would enhance our understanding of SHP2's role in signal transduction and its implications in disease progression, thereby expediting research endeavors and potentially unveiling new diagnostic and therapeutic avenues for conditions such as cancer [26].

SHP099 is a potent and selective allosteric inhibitor of SHP2, exhibiting an IC50 of 70 nmol/L for SHP2 inhibition, with no discernible inhibitory effect on SHP1 without favorable solubility and permeability profiles [[27], [28], [29], [30]]. Drawing inspiration from the structural attributes of SHP099, this study presents the design, synthesis, and efficacy assessment of novel small-molecule fluorescent probes aimed at SHP2. The newly designed SHP-PS series probes feature an environment-sensitive switching mechanism, high specificity for SHP2, robust binding affinity, and the ability to selectively label SHP2 in living cells and tumor sections. This study pioneered the investigation of small-molecule fluorescent probes for SHP2 allosteric site.