1,3,5-Triazine as a Modular Scaffold for Covalent Inhibitors with Streamlined Target Identification

Abstract

Small-molecule inhibitors can accelerate the functional annotation and validate the therapeutic potential of proteins implicated in disease. Phenotypic screens provide an effective platform to identify such pharmacological agents but are often hindered by challenges associated with target identification. For many protein targets, these bottlenecks can be overcome by incorporating electrophiles into small molecules to covalently trap interactions in vivo and by employing bioorthogonal handles to enrich the protein targets directly from a complex proteome. Here we present the trifunctionalized 1,3,5-triazine as an ideal modular scaffold for generating libraries of irreversible inhibitors with diverse target specificities. A divergent synthetic scheme was developed to derivatize the triazine with an electrophile for covalent modification of target proteins, an alkyne as a click-chemistry handle for target identification, and a diversity element to direct the compounds toward distinct subsets of the proteome. We specifically targeted our initial library toward cysteine-mediated protein activities through incorporation of thiol-specific electrophiles. From this initial screen we identified two compounds, RB-2-cb and RB-11-ca, which are cell permeable and highly selective covalent modifiers for Cys239 of β-tubulin (TUBB) and Cys53 of protein disulfide isomerase (PDI) respectively. These compounds demonstrate in vitro and cellular potencies that are comparable to currently available modulators of tubulin polymerization and PDI activity. Our studies demonstrate the versatility of the triazine as a modular scaffold to generate potent and selective covalent modifiers of diverse protein families for chemical genetics applications.

Covalent protein modification: the current landscape of residue-specific electrophiles

“…CA has been incorporated into libraries based on peptide [5,37,38], triazine [39], and piperidine [40] scaffolds, leading to the identification of covalent modifiers for nitrilases [37,38], protein disulfide isomerase (PDI) [39], and GSTO1 [40]. The more reactive IA covalently modifies 1000 cysteines in the proteome [7], enabling the global profiling of changes in cysteine reactivity.…”

“…CNB reacts via a nucleophilic aromatic substitution (S N Ar) mechanism, and consequently, relocation of the electron-withdrawing nitro substituent tunes the reactivity in a predictable manner [46 ]. The CNB-based probe, RB-2-cb, covalently modified a reactive cysteine residue on b-tubulin [39], demonstrating the utility of this highly tunable electrophile in ABPP applications.…”

Experimental and theoretical investigations of copper (I/II) complexes with triazine–pyrazole derivatives as ligands and their in situ C–N bond cleavage

“…antiphytovirucide [1][2][3][4]. Especially, some derivatives can be used as potential functional materials in the fields of magnetic, optical, catalyst, and liquid crystal materials [5][6][7][8][9][10].…”

Controlling the unfolded protein response-mediated life and death decisions in cancer

“…Numerous PDI inhibitors have been described in the literature, such as PACMA31 [42], RB-11-ca [43], P1 [44] and 16F16 [45] (Table 3). These molecules possess an electrophilic moiety that reacts covalently with the cysteine residues within the PDI thioredoxin domain.…”