Abstract
SummaryDiphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a “catch-and-pass” reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.
Highlights
The process of signal transduction that governs many cellular activities frequently relies upon evolutionarily conserved families of small, regulatory molecules
PPIP5Ks synthesize the 1-diphosphate (Wang et al, 2012); there are two isoforms in mammals (Thomas and Potter, 2014). Interest in this field has recently been heightened by demonstrations that diphosphoinositol polyphosphates operate at the interface of cell signaling and organismic homeostasis (Choi et al, 2005; Szijgyarto et al, 2011; Shears, 2009; Illies et al, 2007; Chakraborty et al, 2010; Pulloor et al, 2014)
We investigated the significance of E192 by preparing E192G and E192Q mutations that we posited would eliminate electrostatic repulsion between the amino acid side chain and phosphorylated ligands, enhancing ligand binding to the substrate capture site
Summary
The process of signal transduction that governs many cellular activities frequently relies upon evolutionarily conserved families of small, regulatory molecules. The synthesis of 5-PP-InsP5 by IP6Ks inhibits the PtdIns(3,4,5)P3/ PDK1/AKT/mechanistic target of rapamycin (mTOR) cascade (Chakraborty et al, 2010) that controls cell growth and metabolism in response to changes in levels of nutrients, growth factors, and bioenergetic status (Benjamin et al, 2011) This inhibitory action of 5-PP-InsP5 is reversed through its further phosphorylation by the PPIP5Ks (Gokhale et al, 2013). We recently published proof-of-principle of the latter idea by demonstrating that AKT phosphorylation in myoblasts is inhibited when PPIP5K1 expression is ‘‘knocked-down’’ (Gokhale et al, 2013) It is just such therapeutic motives that frequently drive the development of drugs that can target kinases such as PPIP5Ks. Candidate molecules may be rationally designed when information on protein structure is available. The architecture of the active site exhibits substantial geometric and electrostatic constraints that raise challenges for the design of an effective yet specific inhibitor
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