Kinase crystal identification and ATP-competitive inhibitor screening using the fluorescent ligand SKF86002

Abstract

The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.

Expression of myeloid Src-family kinases is associated with poor prognosis in AML and influences Flt3-ITD kinase inhibitor acquired resistance

“…To test this idea, we engineered A-419259-resistant mutants of Hck and Fgr. The crystal structure of Hck bound to A-419259 (PDB code: 4LUE) reveals that the kinase domain gatekeeper residue, Thr338, forms a key a hydrogen bond with the 4-amino group on the pyrrolopyrimidine heterocycle [35]. Kinase domain gatekeeper residues are well known to confer resistance to many ATP-site kinase inhibitors and can be substituted with alternative amino acids without loss of kinase activity.…”

“…As a second approach to validate Hck, Fgr and Lyn as relevant targets for A-419259 in AML, we developed kinase mutants with engineered resistance to this inhibitor (Fig 5). Using the crystal structure of Hck bound to A-419259 (PDB 4LUE) as a guide [35], we substituted the gatekeeper residue in each kinase domain (Thr338) with methionine, leucine or phenylalanine. Each mutation conferred resistance to recombinant kinase mutants of Hck and Fgr in vitro, most likely due to loss of a hydrogen bond with the inhibitor and increased steric bulk at the binding site.…”

Identification of Isosteric Replacements of Glycosyl Domain of Ligands by Data Mining

“…The phenol of LI7 [76] sits inside the same pocket (Figure 8B), with its hydroxyl group participating the highly conserved water mediated hydrogen bond network near Lys67, Glu89 and Phe187 in a direction similar to the 3'-OH of AMP. The same pocket also accommodates the catechol of QUE, with its two hydroxyl groups being involved (Figure 8D) hydrogen bond network and the water molecule experiencing negligible shift comparing to reference [74]. Ligand QUE in Table X exhibit activity of IC 50 1.10 μM (Table 1 in reference [77]) toward Pim1, the OH group addition at 6 position of QUE A ring give MYU with IC 50 of 0.34 μM, the highest inhibitory among avonoids analogs; the additional OH group at 5' position of B ring result in MYC with the IC 50 of 0.78 μM, a slight activity increase; the addition of OH group at 5' position of B ring and the deletion of OH group at 3 position of C ring assign MYF IC 50 of 0.65 μM, nearly double the activity of QUE.…”

“…Structurally, ribose is regarded as the transition between purine/pyrimidine and phosphate, its structural replacement may also take the same role. Kinase inhibitor SK8 (SKF86002 in reference [74]) binds to the ATP binding sites of kinase Pim1, makes many hydrophobic interactions typical of other ATP mimetic inhibitors with imidazothiazole shouldering both pyridine and uoro-substituted phenyl ring (Figure 10 E). SK8 does not form direct hydrogen-bonding interactions with protein, hence has no kinase inhibition detected even at 100 mM.…”

Toward an Understanding of Pan-Assay Interference Compounds and Promiscuity: A Structural Perspective on Binding Modes

“…On the other hand, a low protein sequence identity (7.6%) was calculated for the proteins death-associated protein kinase 1 and serine/threonine-protein kinase pim-1, while the binding mode similarity was high (PDB IDs 5AUW 49 and 4LMU 50 ). Although the protein sequence identity of the protein pair was detected to be low, the binding site is mainly conserved.…”