(J-367) Cancer-specific SRC activity profiling using exogenous artificial peptide probes

Enzymes responsible for post-translational modifications (PTMs) make up around 5% of the proteome, yet are responsible for dynamically regulating the activity of nearly all proteins^1,2. Kinases are critical in many signaling pathways that regulate cell life and death, such as metabolism, proliferation, differentiation, migration, and apoptosis³. Due to their importance in cell regulation, kinases are frequently dysregulated in cancers⁴. Kinase signaling could be an invaluable biomarker-stratification tool for tumors, elucidating the functional consequences of upstream genomic alterations. However, profiling kinase activity in complex and heterogenous clinical specimens is technically challenging. Background activity from non-cancerous cells can result in a low assay signal-to-noise ratio. Current assays that measure enzymatic activity in intact whole cells, such as in-cell ELISA and DELFIA, do not have the sensitivity to analyze samples with low tumor cell purity. In contrast, cell-deliverable artificial peptide probes enable straightforward live-cell measurement of endogenous PTM enzyme activity^5–7. These cell-deliverable peptide probes consist of three modules 1) an enzyme-specific reporter sequence, 2) a bioconjugation handle for detection and readout, and 3) a cell-penetrating peptide (CPP) for rapid non-cytotoxic cellular internalization. By using a tumor-type specific CPP, our goal is to develop a tumor-homing artificial peptide probe. Our approach would enable direct profiling of oncogenic PTM enzyme activity in complex biological specimens. Here we present a new colorectal cancer (CRC)-targeting artificial peptide probe for Src family kinases. Targeting Src kinases is an ideal model system due to their importance in oncogenesis in CRC and other solid tumors⁸.

Probe design: We synthesized two Src-Family Artificial Substrate Peptide (SFAStide) probes. SFAStide-TAT is a well-validated artificial probe⁶ that uses a modified TAT^9–11 CPP for cell-agnostic delivery. SFAStide-CPP2 uses the same enzyme-specific reporter sequence with a novel CPP reported as CRC-specific ¹². Kinase assay: Biochemical performance of each probe was evaluated as previously described¹³. Briefly, kinase assays were performed using purified SRC kinase (SignalChem), peptide probe, and ATP in reaction buffer. Samples of the reaction were quenched at various timepoints. The amount of phosphorylated probe was measured at each time point by ELISA. Cellular uptake/internalization: To evaluate SFAStide-CPP2 peptide uptake specificity, 10 µM FAM-conjugated SFAStide-CPP2 peptides were incubated with LoVo (colorectal cancer), and K562 (leukemia) cells. These samples were then imaged using an IX-83 inverted microscope, and ImageJ was used to measure corrected total region fluorescence for all cells in a representative image. Cells were manually segmented in ImageJ. The ratio of peptide fluorescence to live cell fluorescence was used to quantify peptide uptake.  Cell viability with biosensor: The effect of SFAStide-TAT and SFAStide-CPP2 on viability was evaluated through incubating LoVo cells with increasing concentrations of exogenous probes (5, 10, 20, and 30 µM). Viability was then determined using a Countess 3 Cell Counter.   

For the proposed SFAStide-CPP2 peptide probe to evaluate PTM enzyme activity in heterogeneous samples, it needs to be cell-specific, while maintaining the ability to act as a Src substrate in live cells, as had been demonstrated with SFAStide-TAT.

Changing the cell transduction module from TAT (SFAStide-TAT) to a CRC-specific CPP (SFAStide-CPP2) did not inhibit SFAStide phosphorylation by the kinase. Under matched experimental conditions, measured V₀ was 530.4 pmol/min for SFAS-TAT and 5339 pmol/min for SFAS-CPP2 (p = 0.200, Mann Whitney U Test). The reaction progress curves show SFAStide-CPP2 probe is rapidly phosphorylated with a robust signal that is readily detected by ELISA (Fig 1A). The phosphorylation profile of SFAStide-CPP2 suggests that the peptide probe can be modified for cell-type specificity without compromising its ability to measure Src kinase activity.

The SFAStide-CPP2 peptide probe enables preferential targeting of colorectal cancer cells. When colorectal (LoVo) and leukemia (K562) cells were incubated with FAM-conjugated SFAStide-CPP2, the average normalized FAM fluorescence was two times higher for LoVo compared to K562 (p=0.001, one-way ANOVA, Tukey’s Post-hoc test) (Fig 1C). These preliminary data suggest that introducing a cell-specific transduction element could enhance assay signal-to-noise ratio in complex biological matrices.

While the specific uptake mechanism for the novel CRC-specific CPP is unknown, SFAStide-CPP2 probe internalization has no significant effect on cell viability. Average cell viability remained above 89% for cells incubated with either probe across a range of concentrations (0-30 μM). At highest exogenous probe concentration, cell viability was 95.3% for the SFAStide-TAT group versus 89.7% for the SFAStide-CPP2 group. There is little to no difference in cell viability between untreated LoVo cells (92.7% viability) and cells incubated with SFAStide-TAT (p=0.989) or SFAStide-CPP2 (p=0.136) peptide probes (one-way ANOVA, Tukey’s post-hoc test). (Fig 1C). These findings suggest that the SFAStide-CPP2 probe is a promising candidate for targeted cell-based PTM activity profiling in colorectal cancer. 

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