Commentary  |
Target Based Discovery

Covalent Inhibitors Discovery: Advancements and Developments

Edited by Oliver Picken |
12 October 2021
Covalent inhibitors have progressed significantly since they were discovered in the 18th century and are now experiencing a resurgence.

What Are Covalent Inhibitors? 

Covalent inhibitors are compounds that are designed to create a covalent bond formation with a molecular target. Depending on the chosen warhead, there are two main types of covalent bonds, either reversible or irreversible. A variety of warheads have been used to target specific amino acid residues. Some examples include cysteine, serine, threonine, tyrosine and lysine. 

Why Have Covalent Inhibitors Increased in Popularity? 

The study of covalent inhibitors has progressed significantly since they were discovered in the 18th century. The advantages of covalent inhibitors are negating the initial concerns of potential off-target toxicity. Research has continuously expanded as a result of recent findings and studies and is currently reporting promising results. Werngard Czechtizky, a leading expert on covalent inhibitor discovery, highlighted just how much research is presently being undertaken, stating that in the past few years, “nearly every week there has been a really interesting paper coming out”. 

Traditionally, the selection and design of small molecules has centred around their ability to interact with their biological targets under equilibrium binding states. However, these binding states are typically short-lasting and reversible, which lowers the duration of therapeutic response.  

The length of time a drug interacts with its target can be a determining factor in the prolongation and effectiveness of therapeutic response. Covalent inhibitors have been proven to dramatically improve therapeutic responses by increasing the duration of interaction between a drug and its target. As a result, numerous drug candidates are progressing through clinical trials or are currently in the FDA (Food and Drug Administration) approval pipeline. Today, there are more than 50 FDA-approved drugs that act as covalent inhibitors. They are helpful in a wide range of applications, as the chart below demonstrates.  

Covalent FDA-Approved Therapeutics by Treatment Area

Advantages of Covalent Inhibitors 

Covalent inhibitors bind to their target in two stages. The first is equilibrium bond formation, while the second and final stage is covalent bond formation. This final state is often considered to be irreversible, resulting in a drug-protein complex that is different from that formed with a regular equilibrium bond. With that said, different types of covalent interaction can lead to effects that range from easily reversible to effectively irreversible, where restoration would require new protein synthesis. Covalent inhibitors also enable new approaches to selectivity. They can be designed to specifically target rare nucleophiles, which avoid bond formation with other family members. This approach prevents off-target selectivity as while the inhibitor can bind temporarily to the active sites of other proteins, no effect will be enabled unless the target nucleophile is present. Covalent binding brings many benefits such as longer duration of action, imperviousness to accumulation of natural substrate, shorter prolonged systemic exposure and excellent selectivity if targeting a non-conserved residue. 

Covalent Inhibitors: Discovery Procedures and Challenges 

While much of the discovery process of covalent drugs is similar to non-covalent, there are several important additional factors to consider. These include residence time, nucleophilic reactivity, and covalent bond formation energy. Robert Soliva (Head of Drug Discovery, Oryzon Genomics) provides a helpful summary, explaining that “you will have to apply most of the tools that you use for reversible inhibitors, you will still need to locate a binding site, you will still have to make sure that this mining site is druggable. But then, once you’ve done this, you will have a layer of complexity on top of this because you will need to you know how to locate which residues to somehow target with your inhibitor.” 

Drug Design and Screening 

Werngard Czechtizky (Executive Director, Head Medicinal Chemistry Respiratory and Immunology, and Chair of AZ Global Chemistry Leadership) describes the identification of targets for covalent inhibitors as “a really lively topic over the last few years”. The drug discovery process starts with identifying and validating biological targets and continues to the last optimisation stage of an identified lead. If a target protein contains a nucleophile at the active site, it may be susceptible to covalent inhibition. Several enzyme classes contain nucleophilic residues within the active site. There are several screening approaches for covalent inhibitors and fragments. Recently there has been a move to screening the entire proteome to identify targets that might be susceptible to covalent inhibition. 

High Throughput Screening 

While high throughput screening (HTS) is the go-to for many forms of early drug discovery, it is not optimal for covalent drug discovery. Most of the currently available covalent inhibitors were discovered by happenstance as part of a broader discovery process. There are few studies where libraries of potential covalent compounds were explicitly screened, although this is changing.  

In this context, it is critical to distinguish covalent inhibitors from Pan Assays Interference Compounds (PAINS). Although PAINS are false positives, which must be discarded, many of such compounds are still reported as new promising hits for various targets. Therefore, one strategy is to rely on PAINS alerts to remove compounds that contain common PAINS substructures from HTS screening. However, a small molecule featuring any given substructure alert may not necessarily be a PAINS, and potential covalent inhibitors would also be discarded.  

Kinase selectivity-profiling  

Many previously reported molecules have been derived from screens of archived inhibitors. Several discovery programs are initiated using a lead molecule known to target a certain kinase, only to find that reactions with other new kinases. Compared to expensive high throughput screening, this method provides an enticing alternative with a high probability of identifying novel, patentable analogues to traditional drugs. 

Cutting Edge Developments: RAS and BTK Targeting 

Amongst oncology targets, the RAS protein remains one of the most difficult to target and was long labelled undruggable. In 2018 Amgen developed AMG510, a covalent inhibitor targeting KRASG12C. This was the first compound to undergo clinical trials after more than thirty years of research. Recently, KRAS has also been looked at within the field of proteolysis targeting chimaeras (PROTACs). PROTACs are an exciting new modality of heterobifunctional molecules which degrade the protein of interest instead of inhibiting it. 

In 2019 attempts were made to create a cereblon-based degrader molecule library targeting KRASG12C via acrylamide warheads. Although the lead PROTAC bound to KRASG12C in vitro, pancreatic and lung cancer cells did not show signs of degradation. Further studies are needed to understand the challenges of targeting KRAS with PROTACs. 

Several inhibitors targeting Bruton’s tyrosine kinase (BTK) have been discovered in the last ten years. Examples include ibrutinib and zanubrutinib. These compounds contain an acrylamide warhead, targeting Cys-481 in the ATP binding domain of BTK. Recently, covalent kinase inhibitors have been developed as PROTACs. Recent studies have shown promising results, with higher levels of BTK degradation compared to prior methods.  

Acrylamide Warheads: Passing the Test 

While acrylamide warheads are some of the most commonly used, they are not passing through stage three trials in many cases. During one of our Drug Discovery Panel Discussions, a member of the audience put forward a question to our three panellist seeking insight into the challenges associated with acrylamide warheads. Werngard’s reply highlighted one of the most common difficulties, stating that in the past, they have often had to go back and “modify the acrylamides due to high reactivity”. Robert agreed, saying that “finding the right electrophilic warhead that is not very reactive” often requires an extensive “series of experimental tests.  Gyorgy M Keserű (Unit Head, Medicinal Chemistry, RCNS Hungary) expanded on this, stating that while there is “ample room to tailor the reactivity, it is only one aspect that needs to be considered.” Other factors that need to be considered are “accessibility, the location of the warhead and the non-covenant recognition at the protein binding site”. In essence, refining non-covalent interaction is also essential.  


Covalent inhibitors offer many advantages, including prolonged duration of action, lower dosage and dosing frequency, the potential for reduced off-target effects and a better therapeutic index. Choosing inhibitors with the optimal techniques is vital to decide whether to continue work on a potential drug. Covalent inhibitors offer an enticing alternative for difficult targets where selectivity has proved challenging with non-covalent inhibitors. Additionally, recent advances of using covalent warheads to kick off protein degradation show the promise of exciting new modalities. Industry leaders are working hard to overcome the additional challenges associated with covalent inhibitors, and their market resurgence shows no signs of slowing down. 

Title image credit: pikisuperstar on

Speaker Biographies

Werngard Czechtizky – Executive Director, Head Medicinal Chemistry Respiratory and Immunology, and Chair of AZ Global Chemistry Leadership

Werngard Czechtizky obtained her PhD at ETH Zürich. After a postdoctoral stay at Harvard, she worked on GPCRs in a global Chemical Biology platform at Aventis. In 2005 at Sanofi-Aventis, she moved into a parallel synthesis team working in the fields of CNS and CV diseases. In 2010, she was appointed leader of a medicinal chemistry section focussing on Diabetes targets, and became Head of Medicinal Chemistry at Sanofi R&D Germany in 2014.

György M Keserű - Unit Head, Medicinal Chemistry, RCNS Hungary

György M Keserű obtained his Ph.D. at Budapest University, Hungary. He worked for Sanofi and then moved to Gedeon Richter. In 2007 he was appointed as the Head of Discovery Chemistry. He contributed to the discovery of the antipsychotic Vraylar® (cariprazine) that has been approved and marketed from 2016 in US and EU. He served as a director general of the Research Centre for Natural Sciences (RCNS), Hungary. Since 2015 he is heading the Medicinal Chemistry Research Group at RCNS and is a full professor at the Budapest University of Technology and Economics. His research interests include medicinal chemistry.

Robert Soliva – Head of Drug Discovery, Oryzon Genomics

Robert Soliva is Head of a Drug Discovery unit consisting of 17 scientists and technicians. He coordinates the Biology Department, comprising multiple platforms (Biochemical assays, Cell-based assays, Genomics/Proteomics) and the Medicinal Chemistry Deptartment with outsourced chemistry at several European and Asian CROs. His goal is to generate small molecule clinical candidates for either oncology or CNS indications in the field of epigenetics. Additionally, he provides close support to Development and Clinical Departments in implementing personalised medicine approaches for our two clinical-stage candidates: Iadademstat and Vafidemstat.

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