Proteome-Wide Fragment-Based Ligand and Target Discovery

Abstract

Chemical probes are invaluable tools to investigate biological processes and can serve as lead molecules for the development of new therapies. However, despite their utility, only a fraction of human proteins have selective chemical probes, and more generally, our knowledge of the "chemically-tractable" proteome is limited, leaving many potential therapeutic targets unexploited. To help address these challenges, powerful chemical proteomic approaches have recently been developed to globally survey the ability of proteins to bind small molecules (i. e., ligandability) directly in native systems. In this review, we discuss the utility of such approaches, with a focus on the integration of chemoproteomic methods with fragment-based ligand discovery (FBLD), to facilitate the broad mapping of the ligandable proteome while also providing starting points for progression into lead chemical probes.

Keywords: Activity-Based Protein Profiling; Chemical Proteomics; Fragment-Based Ligand Discovery; Ligandability; Photoaffinity Labeling.

Figure 1.

Chemoproteomic methods for proteome-wide fragment-based target and ligand discovery. Experiments can be performed in a comparative format, in which protein labeling using broad profiling probes is compared across multiple conditions, or in a competitive format, in which samples are treated with competing tag-free ligands that block probe labeling of proteins. These strategies enable global ligandability mapping, ligand binding site determination, and identification of hit binders that can further be optimized into selective leads that modulate protein function.

Figure 2.

Classification of proteome-wide ligand discovery methods, probe examples and corresponding estimated coverages. (A) Activity-based protein profiling (ABPP) utilizes active-site directed probes (B) to report on enzymatic activity (e.g., serine hydrolases, kinases), allowing for their profiling in native systems and the development of inhibitors. (C-F) Examples of broad-profiling chemoproteomic platforms that have been utilized for proteome-wide fragment-based ligand and target discovery. (C-D) Various reactivity-based probes have been developed that target reactive residues (e.g., cysteine, lysine, and tyrosine) to uncover ligandable binding sites, independent of protein function. (E-F) Photoaffinity-based protein profiling allows for broad profiling of any residue through non-covalent interactions, bypassing the requirement of proximal reactive residues or protein activity. Fully-functionalized fragments (FFFs) are photoaffinity probes that enable ligandability profiling of non-covalent interactions.

Figure 3.

Applications of global ligandability studies. In addition to inhibitor discovery, fragment-based proteome-wide ligandability mapping also facilitates bifunctional molecule development (e.g., TPD), as well as the surveyal of state-dependent changes in proteome-wide ligandability.