Shaping Development of Autophagy Inhibitors with the Structure of the Lipid Kinase Vps34

Abstract

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases with diverse roles in health and disease. The primordial PI3K, Vps34, is present in all eukaryotes and has essential roles in autophagy, membrane trafficking, and cell signaling. We solved the crystal structure of Vps34 at 2.9 angstrom resolution, which revealed a constricted adenine-binding pocket, suggesting the reason that specific inhibitors of this class of PI3K have proven elusive. Both the phosphoinositide-binding loop and the carboxyl-terminal helix of Vps34 mediate catalysis on membranes and suppress futile adenosine triphosphatase cycles. Vps34 appears to alternate between a closed cytosolic form and an open form on the membrane. Structures of Vps34 complexes with a series of inhibitors reveal the reason that an autophagy inhibitor preferentially inhibits Vps34 and underpin the development of new potent and specific Vps34 inhibitors.

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

“…This compound has been shown to be specific over PI3K kinases (10 fold more potent against PI4KIIIβ relative to p110α, and >100 fold more potent against PI4KIIIβ compared with all other class I and class III PI3K isoforms). Comparing the structure of PI4KIIIβ bound to BQR695 compared with structures of different PI3K inhibitor structures33, 34, 35, 36 reveals the molecular basis for this specificity. PI4KIIIβ has a larger binding pocket to accommodate the glycyl methyl amide group compared to the class I and class III PI3Ks.…”

The machinery of macroautophagy

“…The C2 domain does not affect the catalytic activity of the kinase and is thought to be involved in protein-protein interactions. The crystal structure of Vps34 lacking the C2 domain has been determined [172]. Vps34 has a highly ordered phosphatidylinositol-binding loop located in its catalytic domain, while the corresponding activation loop that is critical in substrate recognition in phosphoinositide 3-kinases is largely disordered.…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…[183][184][185][186] An early study resolved the molecular structure of PIK3C3, and revealed that PIK3C3 has a smaller and more rigid ATP binding pocket than that of PIK3CG, which restricts the binding of classical PI3K inhibitors. 187 This molecular dissection of PIK3C3 paved the way for the subsequent development of novel inhibitors that fit well within the PIK3C3 ATP cavity. A number of recent studies reported new PIK3C3 inhibitors, including VPS34-IN1, 184 Compound 31 (a tetrahydropyrimidopyrimidinone derivative), 186 PIK-III, 185 and SAR405; 183 among them, Compound 31 and SAR405 exhibit the highest potency (IC 50 PIK3C3 equals 2 nM and 1.2 nM, respectively) and favorable selectivity toward PIK3C3.…”