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Publication Date

5-2024

Abstract

Background: The investigation of p53 mutations, prevalent in osteosarcoma (OS), has led to the identification of potential anti-cancer compounds—KU0171032, KU-D2, and KU-D2-F. These compounds exhibit cytotoxic effects in p53-deficient OS cells. We hypothesize that the KU017032 compound series induces reactive oxygen species (ROS) production and inhibits DNA double-stranded break repair by interacting with protein targets, yet the specific proteins binding to KU0171032 remain unclear. Previous targeted experiments including Cellular Thermal Shift Assays (CETSA) suggest a potential interaction between KU0171032 and NBS1, which is crucial for DNA repair. Here, we describe the application of untargeted proteomic methods to identify protein targets of the KU0171032 compound series. Stability Proteomic Methods (SPMs) measure changes to protein stability due to compound binding on a proteome-wide scale using mass spectrometry. Recently these methods have been used to identify the protein targets of small molecules.

Methods: SPMs implemented include Thermal Proteomic Profiling (TPP), Stability of Proteins from Rates of Oxidation (SPROX), Limited Proteolysis (LiP), and Solvent Proteome Integral Solubility Alteration (solvent-PISA). These SMPs quantify compound stabilization/destabilization of protein targets from thermal denaturation, methionine oxidation, proteolysis, and solvent-induced precipitation. The pilot studies involve treating p53-deficient U2OS cell lysates with staurosporine, a pan-kinase inhibitor, and analyzing samples by quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS), revealing thermodynamically stabilized proteins.

Results: OnePot TPP and LiP workflows successfully quantified kinase targets of staurosporine binding. We quantified 2,460 proteins including 6 kinase targets and 3,319 proteins including 27 kinase targets of staurosporine using a OnePot TPP workflow and a LiP workflow, respectively. SPROX, while potentially able to provide good resolution for peptide-level binding events, failed to provide sufficient protein coverage. Thus far we’ve shown that OnePot TPP and LiP are feasible approaches to identify small molecule-protein interactions in OS cells. Analysis of KU-D2-F treated p53-deficient U2OS cell lysates using OnePot TPP and LiP workflows displayed good protein coverage and interesting putative interactions. Ongoing work is necessary to reproduce findings and will be aided by using higher concentrations and reducing batch effects.

Conclusions: This work presents a systematic approach, using staurosporine as a benchmark in piloting SPMs. This allows for the pre-assessment of technique feasibility, emphasizing the potential of OnePot TPP and LiP methodologies in uncovering novel drug targets. Future directions include piloting orthogonal SPMs such as solvent-PISA and further experiments to identify protein binders and better understand the mechanism of the KU0171032 compound series.

Document Type

Poster

Stability Proteomic Methods To Detect Novel Drug Targets

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