Kinase Drug R & D: Helping Your Inhibitor Make the Cut

Finding the best inhibitor for your kinase doesn’t have to be a long trip.

A recent paper in Journal of Medicinal Chemistry, “Discovery of GDC-0853: A Potent, Selective and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development” (1) details some elegant work in chemical modification and extensive testing during exploration of inhibitors for BTK. As a warmup to the article, here is a brief BTK backstory.

BTK (Bruton Tyrosine Kinase): Importance in Health and Disease 

Bruton’s tyrosine kinase (BTK) was initially identified as a mediator of B-cell receptor signaling in the development and functioning of adaptive immunity. More recent and growing evidence supports an additional role for BTK in mononuclear cells of the innate immune system, especially dendritic cells and macrophages. For example, BTK functions in receptor-mediated recognition of infectious agents, cellular maturation and recruitment processes, and Fc receptor signaling. BTK has recently been identified as a direct regulator of a key innate inflammatory machinery, the NLRP3 inflammasome (2).

In the J. Med. Chem. paper highlighted here, the role of BTK in signaling through the B-cell antigen receptor and the Fc receptor was of particular interest. The authors note that targeted mutations in knockout mice as well as spontaneous mutations in the BTK gene have resulted in animals with B-cell development and proliferation defects, such as reduced numbers of mature B cells and decreased titers of immunoglobulin classes IgM and IgG3 (1).

BTK and B Cell Development in Humans

Crawford et al. provide the example of X-linked agammaglobulinemia (XLA), a human congenital immunodeficiency associated with low to undetectable levels of BTK mRNA, and no BTK protein expression. B-cell development in XLA affected persons is almost completely blocked (1).

Finding BTK Inhibitors 

BTK inhibition is an attractive therapeutic target, not only for disorders where B cells cause an excessive autoimmune response, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) but also for B-cell malignancies.

There is a currently available, irreversible BTK inhibitor, irbrutinib, that has been successfully used against B-cell malignancies–it’s approved for treatment of CLL (chronic lymphocytic leukemia) and relapsed MCL (mantle-cell lymphoma). However, there are no approved BTK inhibitors for chronic autoimmune diseases. Treatment of chronic conditions requires stringent safety standards. Crawford et al. sought to identify a BTK inhibitor with a safety profile good enough for use in chronic diseases such as RA and SLE.

One major safety concern for current leading BTK inhibitors like irbrutinib, is that they bind covalently at the BTK binding site (1). This binding is not reversible and can result in selectivity issues. At least 10 additional kinases have the same cysteine residue at their ATP binding site, resulting in the risk of off-target inhibition of these other kinases.

Further, due to their ATP-type binding, covalently-bound inhibitors can show noncovalent binding to other kinases, thus lowering their selectivity profile. And reactive groups present on covalent-binding type inhibitors can cause conglomeration of serum proteins, resulting in serious serum-sickness-type illnesses (1).

For these reasons, Crawford et al. set their criteria on development of a noncovalent BTK inhibitor (1).

GDC-0853 was evolved from earlier efforts with BTK inhibitors, some of which showed toxicity in animals during testing.

Their inhibitor compound evolution included optimizing the hydroxymethyl-substituted aryl ring that links the hinge binding motif to the selectivity pocket, and making additional structural modifications to develop a BTK inhibitor with greater selectivity and noncovalent binding.

Testing Performed 

In their examination of BTK inhibitors, Crawford et al. tested compounds across several biochemical and cellular methods including kinase activity & residence time, whole blood potency, kinase selectivity and hepatotoxicity. See their paper for the details.

There is another method that provides kinase-specific cellular potency in an easier format, in addition to enabling cellular residence time analysis.

Another Way: Testing of Kinase-Inhibitor Binding in Live Cells 

Biochemical and other acellular assays can be good preliminary screening methods. However, kinase inhibitor potency in a biochemical format can differ from that in a cellular environment. The authors used a whole blood assay that monitored BTK phosphorylation or anti-IgM CD69 expression in B-cells. But these methods can be labor intensive and require the sourcing of blood samples.

In inhibitor studies where researchers want to simplify their work yet gain understanding of cell permeability, cellular potency and residence time for their compounds using a specific BTK cellular assay, they could employ the NanoBRET™ TE Intracellular Kinase Assay. This method uses a bioluminescence resonance energy transfer (BRET) competitive binding approach to measure inhibitor binding to full-length BTK (or other kinases) in live cells. The assay can be configured for equilibrium analysis of cellular potency and permeability, as well as nonequilibrium residence time analysis of the compound.

The NanoBRET™ TE Kinase assay format has been applied to over 170 kinases and was recently described in this 2017 Cell Chemical Biology paper, “Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement”(3).

In this paper, Vasta et al. took the NanoBRET TE kinase assay method a step further by examining cellular selectivity of inhibitors in live cells. Specifically, they examined crizotinib binding to 178 kinases and comment that “Due to the cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose “ (3).

Want to learn more?

If you’re interested in finding out which kinases the NanoBRET assay can be used with, see the table listing protocols and assays that will help you investigate >125 full-length kinases on the NanoBRET TE Intracellular Kinase Assay web page.

We’d love to hear about your kinase-inhibitor studies. Our products and scientists are available to aid your success. Keep us posted.


    1. Crawford, J.J. et al. (2018) Discovery of GDC-0853: A Potent, Selective and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development. J. Med. Chem. 61, 2227–45. PMID: 29457982.
    2. Weber, A.N.R. et al. (2017) Bruton’s Tyrosine Kinase: An Emerging Key Player in Innate Immunity. Front. Immunol. 8. PMID: 29167667.
    3. Vasta, J. et al. (2017) Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem. Biol. 25(2), 206–14. PMID: 29174542.
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Kari Kenefick

Kari Kenefick

Kari has been a science writer/editor for Promega since 1996. Prior to that she enjoyed working in veterinary microbiology/immunology, and has an M.S. in Bacteriology, U of WI-Madison. Favorite topics include infectious disease, inflammation, aging, exercise, nutrition and personality traits. When not writing, she enjoys training her dogs in agility and obedience. About the practice of writing, as we say for cell-based assays, "add-mix-measure".

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