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A fluorescence-based thermal shift assay identifies inhibitors of mitogen activated protein kinase kinase 4
Sankar N Krishna1, Chi-Hao Luan, Rama K Mishra
1Department of Medicine, Northwestern University, Chicago, Illinois, United States of America.
Abstract:
Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors.
Insights
Researchers developed a new screening platform to identify small molecules targeting MAP2K4 (mitogen-activated protein kinase kinase 4) to inhibit prostate cancer (PCa) cell invasion and reduce mortality. This platform successfully identified potent MAP2K4 inhibitors.
Area of Science:
- Oncology
- Biochemistry
- Drug Discovery
Background:
- Prostate cancer (PCa) is a leading cause of cancer death in US males, with metastasis being a key driver of mortality.
- Mitogen-activated protein kinase kinase 4 (MAP2K4) activates signaling pathways that promote invasion in human PCa cells.
- MAP2K4 is a validated therapeutic target for PCa, necessitating the development of effective inhibitors.
Purpose of the Study:
- To develop and characterize an efficient screening process for identifying small molecules that target MAP2K4.
- To discover novel inhibitors of MAP2K4 for potential therapeutic use in prostate cancer.
- To create a robust platform for inhibitor discovery and design.
Main Methods:
- Utilized a fluorescence-based thermal shift assay (FTS) to screen compound libraries against MAP2K4.
- Developed an in vitro kinase assay using JNK1 and p38 MAPK substrates to validate inhibitor efficacy.
- Employed structure-activity relationship data and crystal structure analysis to model ligand binding to MAP2K4's ATP pocket.
Main Results:
- An initial screen of 80 kinase inhibitors identified 8 compounds that stabilized MAP2K4.
- A larger screen of 2000 compounds yielded 7 additional inhibitors, confirmed by the in vitro kinase assay.
- A binding model predicted that identified inhibitors bind to the ATP binding pocket of MAP2K4.
Conclusions:
- A robust inhibitor-screening platform for MAP2K4 was successfully established.
- The platform effectively identified and validated small molecule inhibitors of MAP2K4.
- This work provides a foundation for the discovery and design of potent MAP2K4 inhibitors for prostate cancer treatment.
