Supporting data for thesis "Chemoprotomics-driven discovery of covalent inhibitors as novel cancer therapeutics"

<p dir="ltr">The unmet demand for novel oncology therapeutics drives the resurgence of covalent inhibitors. Traditionally, the characterization of their target profiles lag behind the approval due to challenges in target identifications. Hence, this comeback of covalent drugs requires advanced methodologies to identify and validate therapeutic targets with high precision and efficiency. Hence, this thesis investigates the potential of chemoproteomics to assist the development of targeted therapeutics for complex diseases such as cancer. By leveraging activity-based protein profiling (ABPP) and novel chemical probes, this work identifies selective anti-cancer inhibitors as well as providing insights into cancer progression and assessing inhibitors’ safety profiles, thereby contributing to the paradigm of covalent drug development.</p><p dir="ltr">The first research story explores the application of protein-centric drug discovery. It applies ABPP in gel-based screening to identify small-molecule inhibitors CL16 and CL26 against two different oncogenic targets, RhoA in colorectal cancer (CRC) and AGPAT4 in hepatocellular carcinoma (HCC) respectively. Coupled with mass-spectrometry based detection by alkyne-functionalized probes, the chemoproteomics approach enable the discovery of hits as well as evaluation of their selectivity. Both lead compounds CL16 and CL26 resulted in potent anti-cancer effect in cell models (e.g., cell death, inhibition on cell mobility) and shrinkage of tumor in vivo models. These cases highlight the ability of ABPP to streamline the identification of druggable proteins, and novel therapeutic leads in complex biological systems.</p><p dir="ltr">In the second project, the focus extends to the design and application of novel cysteine-reactive probes for comprehensive cysteine profiling to identify leads in a top-down approach. This session includes: 1) the use of NAIA-5 in identifying acrylamide-based inhibitor CL1 with cell cycle modulating ability in HCC; 2) applying NAI-DTB to uncover HCC metastasis-associated cysteines/proteins deciphering the molecular basics behind metastasis; 3) developing NAIA-C5-Amide, with altered protein engagement preferences to facilitate the unique discovery of a proteasome inhibitor; and 4) developing KTY42 which feature an internal acrylamide warhead to profile off-target interactions of FDA-approved inhibitors such as Selinexor. These probes collectively expand the chemoproteomic toolkit by offering versatile molecules for precise mapping of cysteine reactivity over different proteins. They demonstrate the utility of cysteine profiling in uncovering novel lead molecules as well as enhancing safety of drug development by detecting off-target interactions.</p><p dir="ltr">The final chapter integrates computational and experimental strategies through virtual screening to develop a covalent inhibitor targeting the RhoA Y42C mutation in diffuse gastric cancer. By combining in silico modelling with chemoproteomic validation, this approach yielded a selective inhibitor that covalently engages the mutant protein, offering a promising therapeutic candidate for precision oncology. This work exemplifies the cooperation between computational design and chemoproteomic profiling in accelerating the discovery of mutation-specific therapeutics.</p><p dir="ltr">Collectively, this thesis underscores the power of chemoproteomics as a cornerstone of covalent drug discovery. By developing innovative probes and integrating virtual screening, it addresses key bottlenecks in target identification. These advancements pave the way for more effective and selective treatments for cancer, demonstrating the potential of chemoproteomics to accelerate drug discovery and improve patient outcomes.</p>