Proteomic characterization of post-translational modifications in drug discovery

Abstract

Protein post-translational modifications (PTMs), which are usually enzymatically catalyzed, are major regulators of protein activity and involved in almost all celluar processes. Dysregulation of PTMs is associated with various types of diseases. Therefore, PTM regulatory enzymes represent as an attractive and important class of targets in drug research and development. Inhibitors against kinases, methyltransferases, deacetyltransferases, ubiquitin ligases have achieved remarkable success in clinical application. Mass spectrometry-based proteomics technologies serve as a powerful approach for system-wide characterization of PTMs, which facilitates the identification of drug targets, elucidation of the mechanisms of action of drugs, and discovery of biomakers in personalized therapy. In this review, we summarize recent advances of proteomics-based studies on PTM targeting drugs and discuss how proteomics strategies facilicate drug target identification, mechanism elucidation, and new therapy development in precision medicine.

Keywords: drug mechanism; drug target; off-target; precision medicine; protein post-translational modification; proteomics.

Fig. 1. Different mass-spectrometry-based proteomic technologies in drug research.

a Proteomics-based PTM profiling supplies the systematic comparison study of the PTM substrates and PTM sites in cells with and without drug treatment; The globally protein and PTM profiling of diseases or drug treatment samples provide accurate molecular subtyping for precision medicine, screening potential biomarkers, revealing drug targets and uncovering systematic mechanisms of drug. b Different drug screening strategies such as TPP, DARTS, LiP are developed based on the protein stability under drug binding condition. The stability-based proteomics approach supplies the high efficiency and high throughput for drug target screening. c Affinity proteomics technology provides the high-efficiency and high throughput approach to directly screen the drug targets. This affinity-based proteome profiling supplies a high-efficiency technology for directly drug target screening.

Fig. 2. Schematic diagram of proteomics strategies in study on drugs targeting phosphorylaton.

Phosphoproteome profiling is widely used in kinase inhibitor study. Combined phospho-peptide enrichment and LC-MS/MS analysis, the landscapes of altered phosphorylation substrates, phosphorylation sites and kinase network are provided for understanding of signal pathway activity, drug action, and side effect mechanism. Affinity-based proteome profiling provides a holistic view of binding proteins of kinase inhibitors. The targeted proteins would be captured directly by affinity beads or with abundance altered under competitive condition, and then detected by the quantitative proteomics. Both competitive and noncompetitive ABPP can benefit identification of drug targets and off-target effects.

Fig. 3. Schematic diagram of proteomics strategies in studying drugs targeting acetylation and methylation.

Acetylation and methylation take place on both histones and non-histone proteins, and are regulated by three classes of enzymes: writers that add the modification, erasers that remove the modification and readers that recognize the modification. For histone substrates, regulators function in complex with other proteins, regulating gene transcription through histone markers. For non-histone substrates, they play a role in various cellular processes by regulating DNA, RNA binding ability, stability, and activity of substrates. Those three classes are all potential drug targets and several proteomics strategies are of benefit to studying mechanisms of drugs targeting acetylation and methylation. Interactome profiling can identify partners in protein complexes. Histone PTM profiling can reveal global histone marker changes. Acetylome and methylome profiling can uncover those non-histone modifications.

Fig. 4. Proteomics strategies to explore protein turnover, E3 ligase complex dynamics and degraded substrates regulated by drugs targeting ubiquitin-proteasome system.

a Cells are treated with MLN4924 for incremental time. Samples from different treatment time are quantified to explore cumulative curve of MLN4924-regulated proteins. In these samples, cullin-interacting proteins were pulled down by bait protein cullin to study the dynamics of CRL E3 ligase complex. b Molecular glue/PROTAC-induced degraded substrates were identified by integrating proteome, ubiquitinome, and interactome after drug treatment by quantitative proteomics coupled with affinity enrichment approach.