Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

• Klíčová slova
• PI4K2A, PtdIns4P, SNARE, Sorting, VAMP3, Vesicle fusion,
• MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endozomy metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• fúze membrán fyziologie   MeSH
• lidé MeSH
• membránový protein 3 asociovaný s vezikuly metabolismus   MeSH
• proteiny SNARE metabolismus   MeSH
• receptory transferinu metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• vedlejší histokompatibilní antigeny MeSH
• vezikulární transportní proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránový protein 3 asociovaný s vezikuly MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• proteiny SNARE MeSH
• receptory transferinu MeSH
• vedlejší histokompatibilní antigeny MeSH
• vezikulární transportní proteiny MeSH

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.

• Klíčová slova
• ATP-binding site, PI4K2A inhibitor, Phosphatidylinositol 4-kinase class II, Quinazoline derivative, SAR investigation,
• MeSH
• 1-fosfatidylinositol-4-kinasa * chemie   metabolismus   MeSH
• adenin MeSH
• adenosintrifosfát * metabolismus   MeSH
• ligandy MeSH
• racionální návrh léčiv MeSH
• simulace molekulového dockingu MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-fosfatidylinositol-4-kinasa * MeSH
• 4-aminoquinazoline MeSH Prohlížeč
• adenin MeSH
• adenosintrifosfát * MeSH
• ligandy MeSH

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

• Klíčová slova
• endosome, phosphoinositide, vesicular traffic,
• MeSH
• 1-fosfatidylinositol-4-kinasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• biologický transport MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endozomy účinky léků   metabolismus   MeSH
• Golgiho aparát účinky léků   metabolismus   MeSH
• HEK293 buňky MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• preklinické hodnocení léčiv MeSH
• rychlé screeningové testy * MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 1-fosfatidylinositol-4-kinasa MeSH
• inhibitory enzymů MeSH