RESEARCH The human genome encodes more than 1,600 transcription factors (TFs), along with additional cofactors, chromatin regulators, and structural proteins that collectively execute the regulatory instructions encoded within the nuclear DNA. Dysfunctions of these proteins are known to drive multiple diseases such as cancer, inflammation-related, and neurological conditions. In cancer, these proteins are frequently amplified or overexpressed to drive a gene expression program that facilitates the initiation and progression of various types of leukemia, sarcoma and other tumors. Despite the importance of these proteins, TFs have been considered undruggable due to challenges in modeling their activity in vitro. We have solved these shortcomings by implementing an in-cell functional proteomics drug discovery platform that quantifies the effects of small-molecules on the abundance of TF bound to the genome in a diversity of cell and tissue types. The platform is based on Chromatin Extraction by Salt Separation, coupled to Data Independent Analysis mass spectrometry (ChESS-DIA), which was recently reported. In this proposal, we adapt this technology to enable the discovery of covalent small molecule inhibitors, a type of chemistry that has recently enabled targeting of the previously undruggable KRAS protein. First, we will compare the efficacy of several different covalent screening approaches in combination with ChESS-DIA proteome analysis, determining which strategy is best for compound discovery. Computational tools will also be built to robustly identify functional covalent hit compounds, and to identify the compound:protein adducts that are formed upon compound binding. Several well-characterized covalent inhibitors will be used to validate the accuracy of these methods. With a validated covalent screening TF assay, we will then optimize a secondary assay using intact protein to verify that hit compounds can label target proteins in a stoichiometric, specific way. With these tools in hand, we will then perform a pilot screen to prove the assay's utility in a discovery setting, using a subset of a commercial covalent compound library. These compounds contain a diverse array of reactive warheads, and this will allow us to understand various performance metrics of the optimized assay. This data package will enable us to perform full-scale internal screens for compounds that target Talus' TFs of interest, as well as provide the foundation for business development discussions with biotech and pharmaceutical companies interested in TF inhibition.
Public Health Relevance Statement: PROJECT NARRATIVE This research aims to develop a technology to enable drug development for undruggable transcription factor proteins. Traditional tools to study these proteins have proven ineffective, so we are developing an in-cell system discovery technology that tests a drug's ability to block disease-causing transcription factors.
Project Terms: Algorithms; inhibitor; Biological Assay; Assay; Bioassay; Biologic Assays; Biotechnology; Biotech; Biotin; Vitamin H; coenzyme R; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Cells; Cell Body; Chemistry; Pharmaceutical Chemistry; Medicinal Chemistry; Pharmaceutic Chemistry; Chromatin; Clinical Research; Clinical Study; Clinical Trials; Cysteine; Half-Cystine; L-Cysteine; Cystine; L-Cystine; Disease; Disorder; DNA; Deoxyribonucleic Acid; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Engineering; Foundations; Future; Gene Expression; Regulator Genes; Transcriptional Regulatory Elements; regulatory gene; trans acting element; Genome; Human Genome; human whole genome; Hand; hands; Human; Modern Man; Inflammation; Lead; Pb element; heavy metal Pb; heavy metal lead; leukemia; Libraries; Ligands; Methods; Modernization; Mutation; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Peptides; Proteins; Publishing; Research; Role; social role; Sodium Chloride; salt; Computer software; Software; Mass Spectrum Analysis; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Talus; Astragalus; Astragalus Bone; Technology; Testing; Tissues; Body Tissues; transcription factor; Basal Transcription Factor; Basal transcription factor genes; General Transcription Factor Gene; General Transcription Factors; Transcription Factor Proto-Oncogene; Transcription factor genes; Triage; Work; Measures; Businesses; cofactor; Label; Phase; Neurological; Neurologic; Ligand Binding Protein; Ligand Binding Protein Gene; Protein Binding; bound protein; Binding Proteins; Measurement; Dysfunction; Physiopathology; pathophysiology; Functional disorder; tool; Machine Learning; machine based learning; programs; System; Nuclear; interest; adduct; Performance; Categories; Reporting; Proteome; Transcription Factor Oncogene; Modeling; Sampling; cellular engineering; cell engineering; drug development; Proteomics; drug discovery; Molecular Interaction; Binding; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; pharmaceutical; Pharmacologic Substance; Malignant Soft Tissue Neoplasm; malignant soft tissue tumor; sarcoma; small molecule; KRAS2 gene; C-K-RAS; K-RAS2A; K-RAS2B; K-Ras; K-Ras 2A; K-Ras-2 Oncogene; KRAS; KRAS2; Ki-RAS; Oncogene K-Ras; RASK2; v-Ki-RAS2 Kirsten Rat Sarcoma 2 Viral Oncogene Homolog; Address; Data; Detection; Mutate; Protein Analysis; Nuclear Extract; Validation; validations; Preparation; preparations; Development; developmental; Instruction; computational tools; computerized tools; new approaches; novel approaches; novel strategy; novel strategies; cancer type; Coupled; innovate; innovative; innovation; Oncogenic; tumor; malignant phenotype; overexpress; overexpression; in vitro activity; comparable efficacy; compare efficacy; comparative efficacy; screenings; screening; small molecule inhibitor; Structural Protein; lead optimization; driver lesion; driver mutation
