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Proteomic Ligandability Assessment

Tractable Sites for Targeting Proteins with Small Molecules

Ligandability describes the propensity of a protein target to bind a small molecule with high affinity. It is a precursor to evaluating druggability, which requires more advanced translational pharmacological effects and drug-like properties in vivo. In a drug discovery context, ligandability assessments may help justify further investments into a given target or lead molecule.1 Small-molecule drug discovery has traditionally focused on ligandable sites of enzymes, receptors, or ion channels that have “druggable” cavities, typically a deep binding pocket or active site. However, ~80% of the proteome is considered “undruggable” (or difficult-to-target) due to flatter surfaces with shallow grooves or non-catalytic sites that make binding and modulation by small molecules significantly more challenging (Figure 1). However, due to their implications in myriad disease pathways, these proteins – such as transcription factors, scaffolding and non-enzymatic proteins, or those that operate through protein–protein interactions – are attractive targets should the tools exist to study and exploit them for therapeutic benefit or chemical probe development.1–3 Recent chemoproteomic advances in fragment-based ligand discovery (FBLD) are facilitating the identification of hot spots on proteins, especially for undruggables.4–6 Hot spots are residues functionally susceptible to small-molecule interaction where its mutation to alanine results in a significant decrease in free energy of binding.2, 7

Ligandability of “druggable” (left) and “undruggable” (right) proteins. While discerning where to target undruggables with small molecules may be more challenging, chemoproteomics is facilitating the identification of “hot spots” to pursue in lead discovery efforts.

Figure 1.Ligandability of “druggable” (left) and “undruggable” (right) proteins. While discerning where to target undruggables with small molecules may be more challenging, chemoproteomics is facilitating the identification of “hot spots” to pursue in lead discovery efforts.

Advantages of Chemoproteomic FBLD for Assessing Ligandability

  • Target scope beyond traditional “druggable” protein targets where sites of interest may be non-catalytic, allosteric, or at the interface with another protein
  • Covalent modification of target reduces reliance on high-affinity binding
  • Fragments provide starting points for further elaboration into lead compounds
  • Can be performed with lysates or intact cells for assessing whole-proteome ligandability

General Chemoproteomic Workflow

Quantitative mass spectrometry (MS)-based proteomics workflow for identification of fragment–protein interactions in cells

Figure 2.Quantitative mass spectrometry (MS)-based proteomics workflow for identification of fragment–protein interactions in cells. A) Electrophilic scout fragments, typically α-chloroacetamides and acrylamides. isoTOP-ABPP is isotopic tandem orthogonal proteolysis-activity-based protein profiling; IA-alkyne is iodoacetamide-alkyne, a cysteine-reactive probe.4 B) Fully functionalized (enantiomeric) fragments. SILAC is stable isotope labeling by amino acids in cell culture; ReDiMe is reductive demethylation.5–6 Could also apply to lysates; method amenable for TMT-based multiplexing experiments.

Scout Fragments

Covalent fragments provide a means to identify hot spots as well as starting points for chemical probes or drug lead elaboration. The Cravatt Lab has used such fragment electrophiles, primarily α-chloroacetamides and acrylamides, on a global scale to quantitatively assess targetable cysteine residues in human proteomes and cells. The ability to analyze ligandability at the proteome level is an advancement from conventional FBLD that requires purified proteins.4 Termed “scout fragments,” electrophiles 912131, 912654, and 911798 (Figure 3) were shown to have broad reactivity with cysteines, including at protein–protein interfaces and were leveraged as electrophilic PROTAC® degraders for E3 ligase discovery.4, 8–9 Other fragments shown below also successfully identified ligandable cysteines.4,8

Electrophilic scout fragments

Figure 3.Electrophilic scout fragments


In addition to electrophilic fragments, the Cravatt Lab has pioneered the development of fully functionalized fragments (FFFs) that bear photoreactive and biorthogonal reporter groups to map thousands of reversible small-molecule interactions with proteins in human cells. This was a significant extension from electrophilic fragments that may only target a subset of the proteome.5 Because molecules often interact with proteins stereoselectively, they designed a set of eight enantiomeric probe pairs differing only in absolute stereochemistry to help assure specific protein labeling (Figure 4). Using the enantioprobes, the lab identified >170 stereochemistry-dependent protein-fragment interactions in human cells.6 Follow-up studies would map these sites of binding and optimize the fragments into higher affinity, selective ligands for probe or drug development.

Enantioprobe library

Figure 4.Enantioprobe library. Each enantioprobe possesses a photoactivatable diazirine that produces a covalent bond with the target protein upon irradiation and an alkyne handle for click-mediated affinity enrichment. Each pair differs only in its absolute stereochemistry.For reproducible delivery of UV light in cells, check out the new LightOx PhotoReact 365.



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