Cambridge MedChem Consulting

Covalent Inhibitors

Whilst much of drug discovery deals with non-covalent, reversible interactions with the target protein there are also a class of therapeutic agents that bind covalently to the target protein, these are distinct from ligands that have very slow off rates. Depending on the type of covalent interaction the binding can range from readily reversible to effectively irreversible, where restoration of activity requires new protein synthesis. Whilst covalent binding has many attractions, long duration of action, impervious to build-up of natural substate, potential for much lower prolonged systemic exposure, they do raise concerns over potential toxicity caused by non-specific reaction with proteins or DNA etc. Idiosyncratic toxicity has long been studied and in most instances, the liver injury is initiated by the bioactivation of drugs to chemically reactive metabolites, which have the ability to covalently react with cellular macromolecules such as proteins, lipids, and nucleic acids, leading to protein dysfunction, lipid peroxidation, DNA damage, and oxidative stress.

There is an increased interest in covalent therapeutics in the literature as shown Figure 1, using data from PubChem searches.


It is now apparent that an increasing number of marketed drugs target enzymes act as covalent inhibitors, a publication in 2005, Mechanistic Basis of Enzyme-Targeted Drugs DOI many of which were not originally designed as covalent inhibitors.

From an analysis of the FDA Orange Book, there are 317 marketed drugs that work by inhibiting an enzyme. These drugs inhibit 71 enzymes, including 48 human, 13 bacterial, five viral, four fungal, and one protozoal enzyme. Among the 317 drugs, 65% either undergo reactive chemistry in the active site of the target enzyme or contain a structural motif related to the substrate. Among the 71 enzyme targets, 25 are irreversibly inhibited by drugs, and 19 of the 25 irreversibly inhibited enzymes are covalently modified by the drug.

A number of enzyme classes contain nucleophilic residues within the active site including serine proteases, cysteine proteases which also have a histidine as part of the catalytic triad. Other enzymes contain histidine or lysine in the active site so it is perhaps not surprising that most of the covalent ligands contain an electrophile.

Penicillin Mechanism

The beta lactam antibiotics (Penicillin and Cephalosporins) are a very well studied class of therapeutic agent, the mechanism of action is the inhibition of cell wall synthesis. Penicillin inhibits the formation of peptidoglycan cross-links in the bacterial cell wall; this is achieved through reaction of the β-lactam ring of penicillin to the enzyme DD-transpeptidase. As a consequence, DD-transpeptidase cannot catalyze formation of the cell wall cross-links.


One of the mechanisms for beta lactam antibiotic resistance is through Penicillinase a specific type of β-lactamase, showing specificity for penicillins, by hydrolysing the β-lactam ring. This lead to another class of compounds designed to inhibit Penicillinase. Like beta-lactam antibiotics, they are processed by beta-lactamases to form an initial covalent intermediate, however the oxygen acts as a leaving group revealing an electrophilic imine that can react with the beta lactamase. This class of compounds have little intrinsic antibiotic activity but enhance the activity of other beta-lactam antibiotics.


This is an example where activation is required to reveal the electrophilic species.


Clopidogrel is used to reduce the risk of heart disease and stroke in those at high risk, it acts by inhibiting the P2Y12 receptor on platelet cell membranes. It is a prodrug, which requires CYP2C19 oxidation for its activation. Oxidation of the thiophene ring followed by tautomerisation and ring opening reveals a free thiol group which forms a disulphide bond with a cysteine on the P2Y12 receptor.


Covalent Inhibitor Drug Discovery

Covalent therapeutics have particular attraction in some therapeutic areas, for example, cancer and anti-invectives where prolonged inhibition of the molecular target is essential without high systemic exposure. Or cases where the natural ligand has extremely high affinity or is present in high concentrations (e.g. kinases). In addition covalent inhibitors may offer advantages when it does to drug resistance. Whilst mutations may slow the binding of the inhibitor, providing the nucleophilic residue ids still present irreversible inhibition will still occur eventually. With a covalent inhibitor the duration of action is largely governed by resynthesis of the target protein, if a protein is rapidly turned over then covalent inhibition may have little advantage.

A number of enzyme classes contain nucleophilic residues within the active site including cysteine, serine, histidine or lysine so it is perhaps not surprising that most of the covalent ligands contain an electrophile. If the target protein contains a potential nucleophile in or near the active site it may be suitable for covalent inhibition. More recently efforts have been undertaken to screen the entire proteome to identify targets that might be susceptible to covalent inhibition DOI

Here we report a quantitative analysis of cysteine- reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins.

Screening for covalent ligands

High-throughput screening is a popular approach for hit-finding but if the objective is to find covalent ligands then it is likely that any molecules containing potential reactive groups will have been removed from the screening collection. Screening these sort of molecules comes with a number of inherent risks and it is essential to have clear strategy to distinguish between genuine covalent inhibitors and false positives that might be interfering with the assay. It is also important to check for off-target covalent binding early in the screening process.

If the putative target is a cysteine residue in the active site it may be possible to mutate the CYS to SER and reduce covalent binding.

Virtual screening for covalent hits has also been undertaken DOI, identifying submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including covalent inhibitors of ​JAK3.

Design of covalent inhibitors.

If we assume that you don't want to rely on general metabolic activation in the liver then the approach undertaken is to identify a specific substrate and attach a reactive warhead. There are a variety of reactive functional groups, the most reactive will certainly react with the target protein but may also react with a variety of off-target proteins.


The anti-cancer agent Carfilzomib irreversibly binds to and inhibits the chymotrypsin-like activity of the 20S proteasome, an enzyme that degrades unwanted cellular proteins. Inhibition of proteasome-mediated proteolysis results in a build-up of polyubiquinated proteins, which causes cell cycle arrest, apoptosis, and inhibition of tumor growth.


E-64 is an epoxide which can irreversibly inhibit a wide range of cysteine peptidases. The compound was first isolated and identified from Aspergillus japonicus in 1978. It has since been shown to inhibit many cysteine peptidases such as papain, cathepsin B, cathepsin L, calpain and staphopain and is used as a biochemical tool.

Halo ketones

Peptidyl chloromethyl ketones were among the first affinity labels developed for serine proteases, however due to the inherent chemical reactivity of the chloroketone functional group, the major disadvantage is their lack of selectivity. Thienylhalomethylketones (HMK-32), have been reported, as irreversible inhibitors of GSK-3β DOI reacting with Cys199 in the active site. This observation was used to convert the reversible inhibitor (1) into an irreversible inhibitor (2) DOI.


α,β-unsaturated carbonyls

The use of unsaturated carbonyls as Micheal acceptors is particularly popular because by changing the double-bond substitution it is possible to fine tune the reactivity. There is an excellent review of thiol reactivity of Michael acceptors possessing an α,β- unsaturated carbonyl group DOI.


Telaprevir (purple in image below) is a potent HCV protease inhibitor that forms a stabilised (but reversible) semi acetal with the active site Ser139 (shown in green below) [PDB 3SV6]. In contrast, the sulphonamide of compound (3) (in orange below) forms a hydrogen bonding network with Ser139, but the acrylamide reacts with Cys159 (yellow) [PDB3OYP] . Mutation of Cys159 to Ser results in a dramatic reduction in potency.


Gefitinib and Erlotinib are first generation EGRF inhibitors, EGFR is over expressed in the cells of certain types of human carcinomas - for example in lung and breast cancers, and both agents have been used in to treat ling cancer. As with other ATP competitive small molecule kinase inhibitors, patients rapidly develop resistance. In the case of erlotinib this typically occurs 8–12 months from the start of treatment. Over 50% of resistance is caused by a mutation in the ATP binding pocket of the EGFR kinase domain involving substitution of a small polar threonine residue with a large nonpolar methionine residue (T190M). The covalent inhibitor Afatinib covalently targets Cys-797 which is positioned in the solvent channel of the kinase. Afatinib is not only active against EGFR mutations targeted by first generation tyrosine-kinase inhibitors (TKIs) like erlotinib or gefitinib, but also against mutations such as T790M which are not sensitive to these standard therapies. Similar acrylamide functionality is used in the EGFR inhibitors rociletinib, dacomitinib, neratinib.


Whilst the basic dimethylamino in Afatinib is important in improving solubility it has bee suggested that this functionality may also enhance reactivity with glutathione DOI via an intramolecular protonation mechanism shown below. This can be avoided by either ring constraints or by moving the position of the basic amine as in Osimertinib.


Protein kinases are an important class of oncology targets, however kinase selectivity within the over 500 in the human genome is a concern. Since all use the same cofactor (ATP), the pocket is structurally similar among all kinases, therefore selectively targeting a single kinase can be challenging. Designing compounds to covalently bind to poorly conserved residues such as a non-catalytic cysteine adjacent to the active site offers potential gains in selectivity for a target kinase over kinases not containing a cysteine in that position

Vinyl sulphone and Nitriles

Cathepsin K cleaves Type I collagen at multiple positions, the recognition sequence is Gly-Leu-Lys-Gly-His, using an optimised recognition sequence and a vinyl sulphone war head gives an irreversible inhibitor DOI. Type1collagen

Lysine is more abundant than cysteine and is a harder nucleophile, therefore targeting lysine offers alternative routes to covalent inhibition, however only the neutral form of lysine will act as a nucleophile (pKa 10.4). Whilst surface lysine residues are likely to be protonated, buried lysines in hydrophobic environments are less solvent accessible and may favour the neutral form. Model reactions using N-a-acetyl-lysine and glutathione have suggested that lysine is less reactive than cysteine towards soft electrophiles such as acrylamides, but more reactive towards vinyl sulphones or sulphonyl fluorides.

Using a similar strategy Odanacatib has an electrophilic nitrile that acts covalently with a Cys in Cat K .

Odanacatib OdanacatibPDB

It is worth noting that Odanacatib has excellent selectivity over other Cathepsins DOI.

CatK = 0.2 nM, Cat B = 1034 nM, Cat L = 2995 nM, Cat S = 60 nM.

Alkyl Nitro

A nitroalkane has been shown to react with an active-site cysteine residue to yield a thiohydroximate adduct. DOI.



The boron atom in bortezomib binds the catalytic site of the 26S proteasome with high affinity and specificity, the boronic acid ensures high affinity for hard oxygen nucleophiles in contrast to soft cysteine nucleophiles, according to the Lewis hard-soft acid-base principle. [PDB 2F16].

Bortezomib BortezomibPDB

Suicide Substrates

Whilst the beta-lactams are perhaps the best known of the suicide substrate based irreversible inhibitors other mechanism are known. Pargyline is an irreversible selective monoamine oxidase (MAO)-B inhibitor drug that reacts with the redox cofactor flavin after initial oxidation.


The natural product Physostigmine and the synthetic analogue Rivastigmine both act as substrates for acetylcholinesterase, whilst the acylated intermediate resulting from reaction with acetylcholine is rapidly hydrolysed to regenerate the enzyme, the corresponding carbonates resulting from reaction with Physostigmine or Rivastigmine are relatively stable thus inhibiting the enzyme.


Rivastigmine has modest human pharmacokinetics DOI with a half-life of around 1 hour, however the duration of action is 12 h in AD patients due to the very slow hydrolysis of the carbamate.

Mitigating Toxicity Risks

Maximise selectivity of binding to the target protein, this is key in light of emerging data that suggest that off-target binding leads to a greater risk of toxicity DOI.

We find that covalent kinase inhibitors, including approved drugs, have defined, but limited concentration windows across which selective target inhibition can be achieved. Once this selectivity range is breached, substantial off-target protein reactivity and kinase target-independent cytotoxicity is observed. Our results thus indicate that medicinal chemistry efforts aimed at optimizing the selectivity of covalent kinase inhibitors should account for their reactivity across the entire human proteome in order to ensure suitable windows of selectivity for basic pharmacology and drug development initiatives.

Reduce dose as much as possible. Reviews of the characteristics of drugs that have been withdrawn from the market because of an unacceptably high incidence of toxicity, or which have received “black box” warnings for toxicity, have highlighted the role of daily dose.

On an empirical basis, drugs administered at a total dose of 10 mg per day are unlikely to be associated with a high incidence of IDRs

Glutathione conjugation can be used as a surrogate for off-target reactivity.

Minimal formation of reactive metabolites. There is an increasing body of information that implicates reactive metabolites as the mediators of a number of drug-induced toxicities,

In a study of compounds withdrawn due to hepatotoxicity in 5 out 6 cases there was evidence of reactive metabolite

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor designed to target the specific T790M mutation. A recent publication DOI used proteomics to look at off-target activities.

Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug.

So whilst the drug appears selective in in vitro assays, because the drug accumulates in the lysosome the selectivity is eroded.



Despite the highly engineered EGFR mutant inhibition profile achieved by all three third-generation inhibitors and their shared unsubstituted acrylamide reactive group, the inhibitors exhibited strikingly distinct proteome-wide reactivity profiles in human cancer cells.

Worth Reading

Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases, Chemical Reviews, 2002, Vol. 102, No. 12 p 4369 DOI.
Proteome-wide covalent ligand discovery in native biological systems, Nature, 2016, Vol. 534, p 570 DOI
Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β- Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions, DOI
Structure-based design of targeted covalent inhibitors. DOI
Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology DOI

Last Updated 7 January 2019