Discovery and Characterization of Allosteric WNK Kinase Inhibitors.

Area of Science:

• Biochemistry
• Pharmacology
• Molecular Biology

Background:

• Protein kinases possess a conserved ATP-binding site, complicating the development of selective inhibitors.
• Allosteric inhibitors offer a promising strategy for achieving high kinase selectivity by targeting alternative binding regions.
• Screening or hit triaging at high ATP concentrations can identify inhibitors binding outside the ATP pocket.

Purpose of the Study:

• To discover selective allosteric inhibitors for With-No-Lysine (K) (WNK) kinases.
• To identify lead compounds for a next-generation antihypertensive drug with a stringent safety profile.
• To elucidate the binding mode and specificity of novel WNK kinase inhibitors.

Main Methods:

• Applied stringent hit triaging at high adenosine triphosphate (ATP) concentration to screen for WNK kinase inhibitors.
• Optimized identified lead compounds and performed cocrystallization with WNK1.
• Assessed the functional activity of optimized compounds by measuring rubidium uptake via sodium chloride cotransporter 1 (NKCC1) in HT29 cells.

Main Results:

• Identified several ATP noncompetitive inhibitors targeting WNK1-4 kinases.
• Cocrystallization revealed an allosteric binding mode for an optimized WNK1 inhibitor, confirming high specificity.
• The optimized compound demonstrated inhibition of NKCC1 activity, consistent with WNK kinase physiology in renal electrolyte handling.

Conclusions:

• High ATP concentration screening is an effective strategy for discovering selective, allosteric kinase inhibitors.
• The identified allosteric inhibitors represent promising leads for developing novel antihypertensive therapeutics targeting WNK kinases.
• The study provides structural and functional insights into WNK kinase inhibition and its physiological relevance.