Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay

Lucet, Isabelle S; Hildebrand, Joanne M.; Czabotar, P.E.; Zhang, Jianguo; Nicola, Nicos A.; Babon, Jeffrey J.; Murphy, James M.

doi:10.21769/bioprotoc.1135

Cited by 3 publications

(1 citation statement)

References 2 publications

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“…Thus, protein denaturation can be monitored by the fluorescence change induced by protein–dye interaction, which generates a thermal melting curve measured by the real-time PCR machine. Then the T m value denoted as the midpoint of melting curve can be calculated using the previously reported protocol 29 . Δ T m values are correlated to the protein conformational stability.…”

Section: Resultsmentioning

confidence: 99%

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

Song

Zhao

et al. 2021

Acta Pharmaceutica Sinica B

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The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC 50 = 0.32 μmol/L; WS-635: IC 50 = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC 50 = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.

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Section: Resultsmentioning

confidence: 99%

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

Song

Zhao

et al. 2021

Acta Pharmaceutica Sinica B

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Impact and Evolution of Biophysics in Medicinal Chemistry

Eggen

Schindler

2017

Biophysical Techniques in Drug Discovery

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The complexity of targets in disease biology coupled with increased diversity within the chemical matter and chemical technologies leveraged by the chemist has driven the expansion of biophysical screen approaches for lead matter. Ligand identification begins by leveraging data from techniques such as AS-MS, TDF, SPR and ligand- and protein-detected NMR to identify and validate screening hits. The mechanism of action (MOA) is assessed through enzymology coupled with biophysical and structural biology techniques. While finalizing selections, teams seek to understand the fundamental nature of the thermodynamics and kinetics of the interactions through ITC and SPR. Molecular design iterations couple computational tools built upon structural data, and in turn biophysical methods are leveraged in selecting the best opportunities for x-ray crystallography and dynamic protein solutions through protein NMR. Further, biophysics has been embraced across the drug discovery and development process and into the clinic, where technologies once used to identify screening actives, understand the structure of compounds and proteins, design molecules and deliver protein structure information are now being leveraged to understand their cellular context and mutational status within the tissues of patients. Non-invasive imaging technologies continue the interplay of drug design, target potency, and in vivo kinetics. This chapter will provide a background from the first biophysical approach, through evolving drug discovery strategies, the expansion of chemical diversity and modalities, and increasing target complexity that have driven the integration of biophysics and medicinal chemistry. Examples will demonstrate the power of utilizing orthogonal or combined methods.

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Molecular dissection of the pseudokinase ZED1 expands effector recognition to the tomato immune receptor ZAR1

Diplock,

Baudin,

Xiang

et al. 2024

Plant Physiology

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The highly conserved angiosperm immune receptor HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) is a bacterial pathogen recognition hub that mediates resistance by guarding host kinases for modification by pathogen effectors. The pseudokinase HOPZ-ETI DEFICIENT 1 (ZED1) is the only known ZAR1-guarded protein that interacts directly with a pathogen effector, HopZ1a, from the bacterial pathogen Pseudomonas syringae, making it a promising system for rational design of effector recognition for plant immunity. Here, we conducted an in-depth molecular analysis of ZED1. We generated a library of 164 random ZED1 mutants and identified 50 mutants that could not recognize the effector HopZ1a when transiently expressed in Nicotiana benthamiana. Based on our random mutants, we generated a library of 27 point mutants and found evidence of minor functional divergence between Arabidopsis (Arabidopsis thaliana) and N. benthamiana ZAR1 orthologs. We leveraged our point mutant library to identify regions in ZED1 critical for ZAR1 and HopZ1a interactions and identified two likely ZED1-HopZ1a binding conformations. We explored ZED1 nucleotide and cation binding activity and showed that ZED1 is a catalytically dead pseudokinase, functioning solely as an allosteric regulator upon effector recognition. We used our library of ZED1 point mutants to identify the ZED1 activation loop regions as the most likely cause of interspecies ZAR1-ZED1 incompatibility. Finally, we identified a mutation that abolished ZAR1-ZED1 interspecies incompatibility while retaining the ability to mediate HopZ1a recognition, which enabled recognition of HopZ1a through tomato (Solanum lycopersicum) ZAR1. This provides an example of expanded effector recognition through a ZAR1 ortholog from a non-model species.

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Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay

Cited by 3 publications

References 2 publications

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

Impact and Evolution of Biophysics in Medicinal Chemistry

Molecular dissection of the pseudokinase ZED1 expands effector recognition to the tomato immune receptor ZAR1

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