ABSTRACT: Protein kinases are quintessential regulators of cellular function. Numerous pathologies are intimately linked to the dysregulated activity of a particular protein kinase. Herein we report a technology based on a proximity-induced chemical transformation that enables the detection and imaging of specific kinases. Using two probes that target the nucleotide-binding site and substrate binding site of a target kinase respectively, the reagents appended on the probes are brought within reactive distance thereby enabling the chemical transformation. The reaction used for sensing is a ruthenium-photocatalyzed reduction of a pyridinium immolative linker, which uncages a fluorophore (rhodamine). We demonstrate that this technology can be used to discriminate between closely related kinases with a high signal to noise ratio. We further demonstrate that the technology operates within the complexity of a cellular context with a good correlation between the level of kinase activity and fluorescence output.
Project description:Self-immolative linker is a useful building block of molecular probes, with broad applications in the fields of enzyme activity analysis, stimuli-responsive material science, and drug delivery. This manuscript presents N-methyl dimethyl methyl (i.e., trimethyl) carbamate as a new class of self-immolative linker for the fluorescence detection of enzyme reactions. The trimethyl carbamate was shown to spontaneously undergo intramolecular cyclization upon formation of a carboxylate group, to liberate a fluorophore with the second time rapid reaction kinetics. Interestingly, the auto-cleavage reaction of trimethyl carbamate was also induced by the formation of hydroxyl and amino groups. Fluorescent probes with a trimethyl carbamate could be applicable for fluorescence monitoring of the enzyme reactions catalyzed by esterase, ketoreductase, and aminotransferase, and for fluorescence imaging of intracellular esterase activity in living cells, hence demonstrating the utility of this new class of self-immolative linker.
Project description:Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently identified, in a DNA-templated macrocycle library, inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated 'gatekeeper' mutant. We solved two cocrystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bisubstrate-competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance.
Project description:Exosomes are nanoscale vesicles distinguished by characteristic biophysical and biomolecular features; current analytical approaches, however, remain univariate. Here, we develop a dedicated platform for multiparametric exosome analysis-through simultaneous biophysical and biomolecular evaluation of the same vesicles-directly in clinical biofluids. Termed templated plasmonics for exosomes, the technology leverages in situ growth of gold nanoshells on vesicles to achieve multiselectivity. For biophysical selectivity, the nanoshell formation is templated by and tuned to distinguish exosome dimensions. For biomolecular selectivity, the nanoshell plasmonics locally quenches fluorescent probes only if they are target-bound on the same vesicle. The technology thus achieves multiplexed analysis of diverse exosomal biomarkers (e.g., proteins and microRNAs) but remains unresponsive to nonvesicle biomarkers. When implemented on a microfluidic, smartphone-based sensor, the platform is rapid, sensitive, and wash-free. It not only distinguished biomarker organizational states in native clinical samples but also showed that the exosomal subpopulation could more accurately differentiate patient prognosis.
Project description:Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.
Project description:The chromium photocatalyzed cyclopropanation of diazo reagents with electron-rich alkenes is described. The transformation occurs under mild conditions and features specific distinctions from traditional diazo-based cyclopropanations (e.g., avoiding ?-hydride elimination, chemoselectivity considerations, etc.). The reaction appears to work most effectively using chromium catalysis, and a number of decorated cyclopropanes can be accessed in generally good yields.
Project description:A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells,quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 184.108.40.206).
Project description:During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein-protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.
Project description:Kinase cascades, in which enzymes are sequentially activated by phosphorylation, are quintessential signaling pathways. Signal transduction is not always achieved by direct activation, however. Often, kinases activate pathways by deactivation of a negative regulator; this indirect mechanism, pervasive in Akt signaling, has yet to be systematically explored. Here, we show that the indirect mechanism has properties that are distinct from direct activation. With comparable parameters, the indirect mechanism yields a broader range of sensitivity to the input, beyond saturation of regulator phosphorylation, and kinetics that become progressively slower, not faster, with increasing input strength. These properties can be integrated in network motifs to produce desired responses, as in the case of feedforward loops.
Project description:Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.
Project description:DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13,824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC(50) values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.