Abstract
Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2–HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2–HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.
Highlights
Pseudokinases belong to the protein kinase superfamily, but are catalytically compromised
To validate the concept of allosteric inhibitors of pseudokinases, we report here a differential scanning fluorimetry (DSF) screening strategy to identify small molecules capable of blocking HER3 in a conformation that prevents the formation of an active signalling heterodimer with HER2
Valid data were obtained for 25 285 wells, 709 of which induced a shift in HER3 Tm over 3°C, corresponding to 2835 compounds and 2.84% hit rate (Figure 1A,B, Supplementary Figure S2A for proof-of-concept compound AC3573 data)
Summary
Pseudokinases belong to the protein kinase superfamily, but are catalytically compromised. 10% of kinases are pseudokinases, which account for around 60 proteins encoded by the human genome [2]. Despite displaying defective catalytic kinase activity, pseudokinases actively regulate various signalling pathways, playing an important role in many cellular processes. Pseudokinases bear changes in key amino acids essential for catalytic kinase activity, impairing their ability to bind to either divalent metal cations or nucleotides, or to catalyse transfer of the phosphate group onto substrates [3,4]. 50% of these inactive kinases can still bind to ATP, and this appears to be a prerequisite for their function [5,6]. ATP-bound STRADα adopts an ‘active’ closed conformation required for its oligomerisation with MO25 and LKB1 to promote tumour-suppressor activity of LKB1 [7,8]
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