Technologies for Direct Detection of Covalent Protein–Drug Adducts

Abstract

In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein–drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein–drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.

Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: An Update

“…Finally, it is important to emphasize the relevance of proper characterization of the covalent mechanism, which should be done via two or more orthogonal methods. The latter may include indirect methods like time course, jump dilution or washout experiments, mutation of the target amino acid, and testing of non-reactive close analogs, and direct methods like MS and X-ray crystallography. ,, …”

N‐Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1

“…Covalently acting small molecules equipped with electrophilic moieties are invaluable tools in chemical biology to explore the modulation of a wide variety of enzymes [1–5] . However, their intrinsic chemical reactivity can lead to promiscuous targeting of diverse sets of proteins in cells, requiring thorough characterisation [1,6] . Mild electrophiles as warheads such as nitriles (in the form of alkyl or aryl nitriles and cyanamides) offer an attractive avenue for achieving greater selectivity within a range of potential binding sites.…”

N‐Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1

“…[1][2][3][4][5] However, their intrinsic chemical reactivity can lead to promiscuous targeting of diverse sets of proteins in cells, requiring thorough characterisation. [1,6] Mild electrophiles as warheads such as nitriles (in the form of alkyl or aryl nitriles and cyanamides) offer an attractive avenue for achieving greater selectivity within a range of potential binding sites. Among those, cyanamides have recently garnered attention as privileged scaffolds for the selective targeting of active site cysteines of different classes of deubiquitinases (DUBs).…”