Modular Approaches to Synthesize Activity- and Affinity-Based Chemical Probes

Abstract

Combinatorial and modular methods to synthesize small molecule modulators of protein activity have proven to be powerful tools in the development of new drug-like molecules. Over the past decade, these methodologies have been adapted toward utilization in the development of activity- and affinity-based chemical probes, as well as in chemoproteomic profiling. In this review, we will discuss how methods like multicomponent reactions, DNA-encoded libraries, phage displays, and others provide new ways to rapidly screen novel chemical probes against proteins of interest.

Keywords: DNA-templated; SuFEx; activity-based probe; affinity-based probe; chemical probe design; iminoboronate chemistry; protein profiling.

Figure 1

(A) Schematic representations of the components of chemical probes. (B) Schematic representation of activity-based chemical probe design. The reactive group is leading in the design and commonly reactive groups that exploit the mechanism of the target enzyme are used as starting point. (C) Schematic representation of affinity-based chemical probe design. The ligand is leading in the design of affinity-based probes and often known bioactive molecules are used as starting point. (D) Schematic representation of a chemoproteomic fragment library. Small molecular fragments (ligands) are equipped with the identical reactive groups and reporter groups and are screened against complex protein mixtures using chemoproteomic approaches. To visualize the differences between the fragments, the proteins that bind to different fragments are plotted in an interaction map. The color correlates with the enrichment fold. Dark colored proteins are enriched the most. The resulting selectivity profiles enable the identification of new probe-protein combinations.

Figure 2

(A) Schematic representation of probe synthesis by multicomponent reactions (MCRs). Ligands can be combined with reactive groups and reporter groups to generate probes in a single step. (B) Examples of probes discovered by screening probe libraries prepared via MCRs. (C) Structure of the isocyanide building block (8), containing both a diazirine reactive group and an alkyne reporter group, developed by Jackson and Lapinsky, that can be used for the synthesis of MCR probes.

Figure 3

(A) Schematic representation of fluorosulfate probe synthesis with SuFEx. (B) An example of a fluorosulfate probe (9) prepared from a phenolic compound via SuFEx chemistry. (C) Schematic representation of sulfuramidimidoyl fluoride probe synthesis with SuFEx. (D) An example of a sulfuramidimidoyl fluoride probe (10) prepared from a primary and a secondary amine.

Figure 4

Schematic representation of the modular synthesis of chemical probes by DNA-technology. A binding strand, functionalized with a ligand that binds selectively to the protein of interest, is hybridized with a capture strand that contains a protein reactive group. In DPAL, the 3′ of the capture strand is functionalized with one of the depicted photocrosslinkers and the 5′-end of the capture strand contains a reporter group. In DTPC, the 3′ of the capture strand is functionalized with one of the depicted chemical crosslinkers and the 5′-end of the capture strand contains a reporter group. Upon binding of the ligand, the reactive group on the capture strand will covalently modify the protein of interest. The labeled proteins can be visualized via the reporter group on the capture strand.

Figure 5

Schematic representation of chemical probes prepared with a combination of DPAL/DTPC and DEL. A DEL library is functionalized with a general ssDNA linked to a dsDNA barcode. The general strand is hybridized with the capture strand. After covalent labeling of the target protein, the target protein is enriched by affinity enrichment. The unbound probes are washed away and the bound probes are analyzed by DNA sequencing.

Figure 6

(A) Traditional synthesis of cyclic peptide probes. (B) Schematic representation of simultaneous peptide cyclisation with a clickable benzophenone photocrosslinker (17). (C) Specific alkylation of a cysteine residue and a methionine residue to prepare cyclic probes. The sulfonium reactive group reacts with cysteine residues within the proximity of the probe binding site.

Figure 7

Schematic representation of the phage-display cyclic peptide probe synthesis and screening approach. Different peptides containing two cysteine residues on fixed positions are expressed on the PIII protein of phages. The phage peptide library is cyclized with a dichloroacetone-functionalized vinylsulfone (18) or fluorophosphonate (19) reactive group. The library is incubated with the immobilized target protein. After the labeling step, all the unbound phage is washed away. The bound phage is liberated by denaturing or by cleavage with a protease. The enriched phage is amplified in E. coli cells. After three cycles, the consensus sequence is determined by PCR.

Figure 8

(A) Schematic representation of a trifunctional reagent. (B) Schematic representation of the synthesis of a probe using a trifunctional reagent. (C) Structures of a first-generation trifunctional reagent. (D) Examples of minimalistic trifunctional photocrosslinker reagents. (E) Examples of minimalistic trifunctional chemical crosslinking reagents. (F) Examples of reactive groups that contain dual-purpose elements.

Figure 9

Schematic representation of in situ probe formation with hydrazone chemistry. A hydrazide ligand is reacted with an aldehyde-functionalized reactive group, for example a sulfonyl fluoride (40), to form a hydrazone probe. The resulting probe is used without further purification. Probe labeling is visualized via a copper-catalyzed click reaction with a reporter group.

Figure 10

Schematic representation of in situ probe formation with iminoboronate chemistry. A hydrazide ligand is reacted with a 2-FPBA-functionalized reactive group, for example a sulfonyl fluoride (41), to form an iminoboronate probe. The resulting probe is used without further purification. Probe labeling is visualized via a transimination exchange reaction with an α-amino hydrazide reporter group (42). This figure has been derived from van der Zouwen et al. (2020, Figure 1) and is used under a CC-BY 4.0 license.

Figure 11

Schematic representation of in situ probe formation with a pentafluorophenyl (PFP) ester-functionalized trifunctional reagent (43). An amine-containing ligand reacts with the PFP ester to introduce a cysteine reactive chloroacetamide and an alkyne bioorthogonal tag. The resulting probe is used without further purification. Probe labeling is visualized via a copper-catalyzed click reaction with a reporter group.