Quantification of a Pharmacodynamic ERK End Point in Melanoma Cell Lysates: Toward Personalized Precision Medicine

Abstract

Protein kinases are mutated or otherwise rendered constitutively active in numerous cancers where they are attractive therapeutic targets with well over a dozen kinase inhibitors now being used in therapy. While fluorescent sensors have capacity to measure changes in kinase activity, surprisingly they have not been utilized for biomarker studies. A first-generation peptide sensor for ERK based on the Sox fluorophore is described. This sensor called ERK-sensor-D1 possesses high activity toward ERK and more than 10-fold discrimination over other MAPKs. The sensor can rapidly quantify ERK activity in cell lysates and monitor ERK pathway engagement by BRAF and MEK inhibitors in cultured melanoma cell lines. The dynamic range of the sensor assay allows ERK activities that have potential for profound clinical consequences to be rapidly distinguished.

Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2

“…Fluorescence-based kinase assays were performed at 28 °C in a buffer containing 25 mM HEPES, pH 7.5, 50 mM KCl, 2 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, 0.5% glycerol, 2.5 mg/mL BSA, 10 mM MgCl 2 , 2 nM ppERK2 with various concentrations of the ERK-sensor-D1 peptide in the absence or the presence of 100 μM Elk 387-399 . The ERK-sensor-D1 peptide 57 (QRKTLQ RR NLKGLN L N L -X 3 -TGPL S PC-Sox-PF) is a highly efficient ERK2 substrate and includes – (1) a D-site sequence (bold) derived from the yeast MAP kinase kinase (MKK) Ste7, (2) a phosphorylatable serine (bold italics) fused to the C-terminus using a flexible linker (X = 6-aminohexanoic acid) and (3) the cysteine at the +2 position modified by a sulfonamido-oxine (Sox) group. The reaction was initiated after 1 hour of pre-incubation at room temperature by adding 10 μL of 5 mM Mg 2+ /ATP to 90 μL of assay mixture and the fluorescence intensity was recorded at each time point for a total of 6 minutes on a Fluorolog fluorimeter ( Horiba Jobin Yvon ) in quartz cuvettes ( Starna Cells ) with a 10 mm path length and a reaction volume of 100 μL.…”

A real-time, fluorescence-based assay for Rho-associated protein kinase activity

“…Another significant advantage of CSox-based activity sensors is the ability to selectively report on the activity of protein kinases in heterogeneous biological samples such as cell lysates and tissue homogenates [37,38,54,55]. However, in these complex samples off-target activity can be observed from enzymes with overlapping sequence specificity.…”

Preclinical assessment of ulixertinib, a novel ERK1/2 inhibitor

“…Most of the resistance mechanisms to BRAF and MEK inhibitors ultimately lead to increase in phosphorylation of ERK1/2 [3]. Therefore, inhibition of ERK1/2 offers a promising strategy to address both innate and acquired resistance to BRAF and MEK inhibitors in various solid tumors.…”

“…RAS/RAF/MEK signaling pathway is frequently activated by mutations in upstream targets such as BRAF, RAS and receptor tyrosine kinases [2]. Most of the resistance mechanisms to BRAF and MEK inhibitors ultimately lead to increase in phosphorylation of ERK1/2 suggesting the importance of this node in the RAS/RAF/MEK pathway even in the resistance setting [3]. RAS activating mutations have been reported in about 90 % of pancreatic carcinomas, followed by colon carcinomas (50 %), lung cancers and myeloid leukemia cases (30 % each) [4].…”