Mapping the protein interaction landscape for fully functionalized small-molecule probes in human cells

Area of Science:

• Chemical Biology
• Proteomics
• Drug Discovery

Background:

• Phenotypic screening identifies molecules affecting cell processes but struggles with target identification.
• Fully functionalized probes with photoreactive groups and affinity handles offer a solution for integrated screening and target identification.
• The breadth of protein interactions for such probes in cells is largely unknown.

Purpose of the Study:

• To synthesize and characterize a library of fully functionalized chemical probes.
• To explore the scope of protein interactions of these probes in human cells.
• To assess the utility of these probes for target identification and pharmacological studies.

Main Methods:

• Synthesis of an approximately 60-member probe library using Ugi-azide condensation reactions.
• Incorporation of diazirine for photoreactive cross-linking and alkyne for affinity enrichment.
• Mass spectrometry-based proteomic analysis to identify probe-bound proteins in human cells.
• Structure-activity relationship studies and competition assays with small-molecule ligands.

Main Results:

• A diverse set of cellular proteins were identified as targets, including enzymes, channels, adaptor/scaffolding proteins, and proteins with unknown functions.
• Many identified protein targets lacked previously described small-molecule ligands.
• Well-defined structure-activity relationships were observed across the probe library for most interactions.
• Probe-protein interactions were confirmed to be specific and could be blocked by competing small molecules.

Conclusions:

• Fully functionalized small-molecule probes can effectively interrogate diverse segments of the human proteome.
• This probe library provides a valuable tool for target identification and understanding drug mechanisms of action.
• These findings support the potential of such probes as pharmacological tools in cell biology research.