A Panel of Protein Kinase Chemosensors Distinguishes Different Types of Fatty Liver Disease.

The worldwide incidence of fatty liver disease continues to rise, which may account for concurrent increases in the frequencies of more aggressive liver ailments. Given the existence of histologically identical fatty liver disease subtypes, there is a critical need for the identification of methods that can classify disease and potentially predict progression. Herein, we show that a panel of protein kinase chemosensors can distinguish fatty liver disease subtypes.

Design and synthesis of fluorescent activity probes for protein phosphatases.

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this chapter, we provide protocols for repurposing the phosphorylation-sensitive sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phosphatases.

Quantification of Cell Signaling Networks Using Kinase Activity Chemosensors.

The ability to directly determine endogenous kinase activity in tissue homogenates provides valuable insights into signaling aberrations that underlie disease phenotypes. When activity data is collected across a panel of kinases, a unique "signaling fingerprint" is generated that allows for discrimination between diseased and normal tissue. Here we describe the use of peptide-based kinase activity sensors to fingerprint the signaling changes associated with disease states.

Chemoselective Alteration of Fluorophore Scaffolds as a Strategy for the Development of Ratiometric Chemodosimeters.

Ratiometric sensors generally couple binding events or chemical reactions at a distal site to changes in the fluorescence of a core fluorophore scaffold. However, such approaches are often hindered by spectral overlap of the product and reactant species. We provide a strategy to design ratiometric sensors that display dramatic spectral shifts by leveraging the chemoselective reactivity of novel functional groups inserted within fluorophore scaffolds.

Temporal Analysis of PP2A Phosphatase Activity During Insulin Stimulation Using a Direct Activity Probe.

Protein serine/threonine phosphatases (PSPs) are ubiquitously expressed in mammalian cells. In particular, PP2A accounts for up to 1% of the total protein within cells. Despite clear evidence for the role of PP2A in cellular signaling, there is a lack of information concerning the magnitude and temporal dynamics of PP2A catalytic activity during insulin stimulation.

Nebraska Red: a phosphinate-based near-infrared fluorophore scaffold for chemical biology applications.

A series of novel phosphinate-based dyes displaying near-infrared fluorescence (NIR) are reported. These dyes exhibit remarkable photostability and brightness. The phosphinate functionality is leveraged as an additional reactive handle in order to tune cell permeability as well as provide a proof-of-principle for a self-reporting small molecule delivery vehicle.

Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors.

We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylation-sensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases.

Design and evaluation of a real-time activity probe for focal adhesion kinase.

Focal adhesion kinase (FAK) has been identified as a potential therapeutic target for the treatment of metastatic cancers. Herein we describe the design, synthesis and optimization of a direct activity sensor for FAK and its application to screening FAK inhibitors. We find that the position of the sensing moiety, a phosphorylation-sensitive sulfonamido-oxine fluorophore, can dramatically influence the performance of peptide sensors for FAK.

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

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex.

Design, synthesis, and evaluation of a selective chemosensor for leucine-rich repeat kinase 2.

We describe the design, synthesis, and evaluation of a selective activity probe for leucine-rich repeat kinase 2 (LRRK2), a possible molecular target for the treatment of Parkinson's disease. Our optimal chemosensor design, termed Nictide-S2, incorporates a phosphorylation-sensitive sulfonamido-oxine fluorophore at an engineered cysteine within the substrate sequence. This design allows for the direct, real-time analysis of LRRK2 kinase activity with a detection limit of 2.

Quantification of protein kinase enzymatic activity in unfractionated cell lysates using CSox-based sensors.

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox.