Catalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation. Thus, assays that easily monitor kinase activity are critical in both academic and pharmaceutical settings. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a β-turn focused (BTF) design for the continuous assay of kinase activity in vitro and in cell lysates. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg2+ and undergoes chelation-enhanced fluorescence (CHEF). While the design was applied successfully to the development of several kinase sensors, an intrinsic limitation was that only residues C- or N-terminal to the phosphorylated residue could be used to derive specificity for the target kinase. To address this limitation, a new, recognition-domain focused (RDF) strategy has been developed that also relies on CHEF. In this approach, the requirement for the constrained β-turn motif is obviated by alkylation of a cysteine residue with a Sox-based derivative to afford an amino acid termed C-Sox. The RDF design allows inclusion of extended binding determinants to maximize recognition by the cognate kinase, which has now permitted the construction of chemosensors for a variety of representative Ser/Thr (PKCα, PKCβI, PKCδ, Pim2, Akt1, MK2 and PKA), as well as receptor (IRK) and non-receptor (Src, Abl) Tyr kinases with greatly enhanced selectivity. The new sensors have up to 28-fold improved catalytic efficiency and up to 66-fold lower KM when compared to the corresponding BTF probes. The improved generality of the strategy is exemplified with the synthesis and analysis of Sox-based probes for PKCβI and PKCδ, which were previously unattainable using the BTF approach.
Recent efforts have identified the p38α Ser/Thr kinase as a potential target for the treatment of inflammatory diseases as well as non-small cell lung carcinoma. Despite the significance of p38α, no direct activity probe compatible with cell lysate analysis exists. Instead, proxies for kinase activation, such as phosphospecific antibodies, which do not distinguish between p38 isoforms, are often used. Our laboratory has recently developed a sulfonamido-oxine (Sox) fluorophore that undergoes a significant increase in fluorescence in response to phosphorylation at a proximal residue, allowing for real-time activity measurements. Herein we report the rational design of a p38α-selective chemosensor using this approach. We have validated the selectivity of this sensor using specific inhibitors and immunodepletions and show that p38α activity can be monitored in crude lysates from a variety of cell lines, allowing for the potential use of this sensor in both clinical and basic science research applications.Methods for assessing kinase activities have relied on the transfer of a radioactive γ-phosphoryl moiety from ATP to substrate and have been very useful for in vitro studies of kinases. However, beyond being discontinuous, this assay is incompatible with unfractionated cell lysates since ATP is a common substrate for most kinases. This has prompted the development of protein-based fluorescence resonance energy transfer (FRET) sensors for probing kinase activity (1-3). While useful, these FRET-based sensors produce modest changes in fluorescence upon phosphorylation. Alternatively, the development of methods based on small organic fluorophores has provided sensitive probes for interrogating biological functions (4,5). Recently our laboratory has introduced a direct kinase assay strategy based on chelation-enhanced fluorescence of a cysteine derivative of a sulfonamidooxine fluorophore (6) which we term CSox (Figure 1, panel a). Placed (−)2 or (+)2 relative to the phosphorylation site in an optimized kinase substrate, the CSox amino acid provides a readily observable increase in fluorescence signal in response to phosphorylation due to chelation of Mg 2+ between the newly installed phosphoryl group and CSox. These probes afford sensitive real-time fluorescence readouts of kinase activity in unfractionated cell lysates (7,8), provided that selective substrate sequences for the kinase of interest can be identified (5,9,10).Recently p38α, a member of the Mitogen-Activated Protein Kinase (MAPK) family, has been the target of a variety of drug development efforts (11,12) since inhibitors of this kinase may provide treatments for inflammatory diseases (13). Additionally, increased activation of p38α in tumor tissue derived from patients with non-small cell lung carcinoma has been observed (14,15 In the case of MAPKs the development of selective probes has proved more challenging due to the minimal local consensus phosphorylation sequence, S/T-P. This class of enzymes (including the ERK, JNK, and p38 family members) ...
Summary Protein kinases catalyze protein phosphorylation and thereby control the flow of information through signaling cascades. Currently available methods for concomitant assessment of the enzymatic activities of multiple kinases in complex biological samples rely upon indirect proxies for enzymatic activity, such as posttranslational modifications to protein kinases. Our laboratories have recently described a method for directly quantifying the enzymatic activity of kinases in unfractionated cell lysates using substrates containing a phosphorylation-sensitive unnatural amino acid termed CSox, which can be monitored using fluorescence. Herein, we demonstrate the utility of this methodology using a probe set encompassing p38α, MK2, ERK1/2, Akt, and PKA. This panel of chemosensors provides activity measurements of individual kinases in a model of skeletal muscle differentiation and can be readily used to generate individualized kinase activity profiles for tissue samples from clinical cancer patients.
Since the first recognition of a cluster of novel respiratory viral infections in China in late December 2019, intensivists in the United States have watched with growing concern as infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus―now named coronavirus disease of 2019 (COVID-19)―have spread to hospitals in the United States. Because COVID-19 is extremely transmissible and can progress to a severe form of respiratory failure, the potential to overwhelm available critical care resources is high and critical care management of COVID-19 patients has been thrust into the spotlight. COVID-19 arrived in the United States in January and, as anticipated, has dramatically increased the usage of critical care resources. Three of the hardest-hit cities have been Seattle, New York City, and Chicago with a combined total of over 14,000 cases as of March 23, 2020. In this special article, we describe initial clinical impressions of critical care of COVID-19 in these areas, with attention to clinical presentation, laboratory values, organ system effects, treatment strategies, and resource management. We highlight clinical observations that align with or differ from already published reports. These impressions represent only the early empiric experience of the authors and are not intended to serve as recommendations or guidelines for practice, but rather as a starting point for intensivists preparing to address COVID-19 when it arrives in their community.
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