False Friends in Fluorescence - Simple HPLC Evaluation of False Positive Fragment Hits in a DiFMUP Assay for Protein...

Despite their essential role in the reversible protein phosphorylation cycle governing cellular signaling and pathogenesis, phosphatases are frequently overlooked in drug development compared to protein kinases. Protein tyrosine phosphatase 1B (PTP1B) is a prominent therapeutic target due to its crucial role in the etiology of diabetes, obesity, and cancer. Consequently, the goal of this study was to identify fragments inhibiting PTP1B via the fluorogenic DiFMUP assay. Furthermore, we wanted to develop a method to evaluate our primary hits and identify optical artifacts associated with these standard fluorogenic screening assays.

Utilizing the DiFMUP assay, we screened four fragment libraries against PTP1B. To identify false positives caused by spectral interference, the preliminary hits were evaluated through a fluorescence control experiment. Furthermore, we developed and validated a rapid HPLC method to directly quantify the substrate (DiFMUP) and product (DiFMU) for orthogonal verification independent of optical artifacts.

Our initial screening filtered 515 compounds down to 56 preliminary hits. The subsequent fluorescence control experiment revealed a high count of optically interfering fragments. Eventually, the direct quantification of DiFMUP and DiFMU by HPLC yielded three compounds demonstrating inhibition independent of optical interference.

Our fragment screening identified three verified hits. Furthermore, we developed and validated a simple and rapid HPLC-based evaluation method. Although this orthogonal approach does not account for non-specific mechanisms like protein aggregation or oxidation, it serves as a highly useful tool to verify hits from fluorogenic assays. We propose the described screening and verification workflow as an efficient bridge between high-throughput optical assays and resource-intensive downstream testing.