Novel chemistry for covalent inhibitors

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The formation of an irreversible covalent bond between inhibitor and target increases the stability of the complex and can have therapeutic advantages such as longer duration of action. However, the exploration of covalent inhibitors has been limited by concerns about off-target reactivity, which may be alleviated by developing irreversible inhibitors based on weak electrophiles that only form covalent bonds after specific binding to a target protein. So far, most such inhibitors have incorporated acrylamides that form thioethers with cysteine residues in the target protein, but two new papers use different chemistry, and one targets residues other than cysteine.

Huang et al. began with a target — the NSD family of histone methyltransferases — and screened a 1,600 fragment library to identify molecules that bound to members of this family. NSDs are altered in numerous cancers, including the NUP98–NSD1 fusion protein, which is found in childhood forms of acute leukaemia. One of the fragments induced a large NMR shift near the autoinhibitory loop of NSD1. Interestingly, because the published NSD1 crystal structure contains no pockets in that region, the authors concluded that the fragment caused large rearrangements in the autoinhibitory loop.

Replacing a bromine in the fragment with a thiocyanate generated a molecule, BT3, that covalently bound to a residue, C2062, in the autoinhibitory loop, as observed by crystallography. BT3 bound in a pocket that is formed after rearrangement of the loop.

To refine the fragment, the authors tried, unsuccessfully, to use a traditional acrylamide moiety, but the bulkiness of this group likely prevented the drug from entering the pocket. So instead they tried aziridine-based derivatives, one of which bound with high affinity to C2062 of NSD1, and with lower affinity to NSD3. This compound also reduced histone methyl transferase activity, and blocked the proliferation of murine bone marrow progenitor cells and a leukaemia patient blood sample bearing NUP98–NSD1.

Brighty and colleagues took an unusual ‘inverse drug discovery’ approach, starting with compounds containing a weakly reactive moiety to probe HEK293T cell lysates. Their small collection of diverse organic compounds all contained sulfuramidimidoyl fluorides (SAFs), which are weak electrophiles that undergo sulfur(VI) fluoride exchange and can form covalent bonds with nucleophiles in the side chains of tyrosine and lysine residues, depending on the local environment of the protein.

From this screening campaign, the group pursued four proteins that bound to and reacted with four different SAFs as identified from tandem-mass tagging: macrophage migration inhibitory factor (MIF), epoxide hydrolase (EPHX2), branched chain amino acid transaminase 1 (BCAT1), and poly(ADP-ribose) polymerase 1 (PARP1).

Recombinant versions of BCAT1 and EPHX2 did not react with their respective SAFs, possibly because key post-translational modifications were absent on the recombinant proteins, highlighting the biological relevance of screening cell lysates.

To map the binding sites of three drug–protein pairs, the authors used competition with inhibitors known to bind to the protein active sites combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). They thereby mapped the likely SAF-reactive sites to an N-terminal proline (in MIF1), or a tyrosine in the active site (in EPHX2 and PARP1).

The PARP SAF inhibited the generation of poly(ADP-ribose) in vitro and in HeLa cells. Unlike olaparib, an FDA-approved reversible PARP inhibitor, the PARP SAF retained nearly all of its activity after wash-out, suggesting that SAF inhibited PARP until the PARP conjugate was turned over.

FDA-approved covalent inhibitors include afatinib (which targets EGFR) and ibrutinib (which targets BTK). RAS inhibitors, currently under consideration by the FDA, have also highlighted the potential for irreversible drugs. These two papers expand the chemistry that could be used to form covalent bonds with cysteines and also demonstrate that other residues could be targeted by covalent inhibitors.


  1. 1.

    Huang, H. et al. Covalent inhibition of NSD1 histone methyltransferase. Nat. Chem. Biol. (2020)

  2. 2.

    Brighty, G.J. et al. Using sulfuramidimidoyl fluorides that undergo sulfur(vi) fluoride exchange for inverse drug discovery. Nat. Chem. (2020)

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