Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

Abstract

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M−1 s−1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Photocatalytic proximity labelling of MCL-1 by a BH3 ligand

“…Hamachi and co-workers have developed a wide range of LDL methods capable of selectively modifying native proteins in a 'traceless' manner, in which the ligand leaves its binding site after the labelling reaction and the protein is able to perform its native function 14 . These methods include exchange/cleavage approaches based on electrophilic phenylsulfonate esters [15][16][17] , acyl imidazoles 18,19 , N-sulfonyl pyridines 20 , or N-acyl-N-alkyl sulfonamides 21 and catalyst tethering approaches based on N,N-dimethylaminopyridine (DMAP) [22][23][24] or oxime reagents 25 . In addition, several groups have developed LDL reagents incorporating transition metal catalysts.…”

Chemical Methods for Selective Labeling of Proteins

“…The ligand‐directed tosyl chemistry (Scheme a), ligand‐tethered‐DMAP derived nucleophilic catalysis, and ligand‐directed acyl imidazole (LDAI) capacitate the installation of biophysical probes to the protein (Scheme b) . Hamachi's group also demonstrated that sulfonamide derivatives could direct an electrophile for precise modification of a lysine residue . Sodeoka and co‐workers also developed the affinity labeling of Lys residue with simple chemical probe bearing small molecule o‐nitrobenzoxadiazole unit (o‐NBD, Scheme c) .…”

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

“…For this reason, affinity‐guided probes have also been developed for transfer of a biotin handle to the protein. In these cases, the biotin handle was used for blotting of the target protein that had been labeled in living systems …”

“…Recently, sulfonating probes have been included in the repertoire of affinity‐guided reactive moieties (Figure A) . An N ‐sulfonyl pyridone reactive moiety was used to transfer a sulfonyl functionality to a protein with the probe displaying superior labeling efficiency and kinetics compared to the benzenesulfonates and imidazole carbamates.…”

“…Another approach is based on the use of N ‐acyl‐ N ‐alkyl sulfonamide known from Kenner's safety‐catch linkers for preparation of thioester‐modified peptides used in native chemical ligation (Figure B) . The kinetics of the conjugation using ligand directed N ‐acyl‐ N ‐alkyl probes was investigated and showed astonishingly fast conjugation at the rate‐orders of an inverse electron‐demand Diels–Alder reaction.…”