Selective Targeting of Distinct Active Site Nucleophiles by Irreversible Src-Family Kinase Inhibitors

Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5′-electrophile. A 5′-fluorosulfonylbenzoate (<b>1</b>) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5′-vinylsulfonate (<b>2</b>) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both <b>1</b> and <b>2</b> bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using <b>1</b> as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on endogenous Fyn or Src, it potently blocked the critical T-cell kinase, Lck. Probes <b>1</b> and <b>2</b> thus enable competitive profiling versus distinct kinase subsets in living cells

Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors

Fragment-based ligand design and covalent targeting of noncatalytic cysteines have been employed to develop potent and selective kinase inhibitors. Here, we combine these approaches, starting with a panel of low-molecular-weight, heteroaryl-susbstituted cyanoacrylamides, which we have previously shown to form reversible covalent bonds with cysteine thiols. Using this strategy, we identify electrophilic fragments with sufficient ligand efficiency and selectivity to serve as starting points for the first reported inhibitors of the MSK1 C-terminal kinase domain. Guided by X-ray co-crystal structures, indazole fragment <b>1</b> was elaborated to afford <b>12</b> (RMM-46), a reversible covalent inhibitor that exhibits high ligand efficiency and selectivity for MSK/RSK-family kinases. At nanomolar concentrations, <b>12</b> blocked activation of cellular MSK and RSK, as well as downstream phosphorylation of the critical transcription factor, CREB

Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles

Data_Sheet_1_Pathological Insights From Quantitative Susceptibility Mapping and Diffusion Tensor Imaging in Ice Hockey Players Pre and Post-concussion.DOCX

<p>Myelin sensitive MRI techniques, such as diffusion tensor imaging and myelin water imaging, have previously been used to reveal changes in myelin after sports-related concussions. What is not clear from these studies, however, is how myelin is affected: whether it becomes degraded and possibly removed, or whether the myelin sheath loosens and becomes “decompacted”. Previously, our team revealed myelin specific changes in ice hockey players 2 weeks post-concussion using myelin water imaging. In that study, 45 subjects underwent a pre-season baseline scan, 11 of which sustained a concussion during play and received follow-up scans: eight were scanned within 3 days, 10 were scanned at 14 days, and nine were scanned at 60 days. In the current retrospective analysis, we used quantitative susceptibility mapping, along with the diffusion tensor imaging measures axial diffusivity and radial diffusivity, to investigate this myelin disruption. If sports-related concussive hits lead to myelin fragmentation in regions of lowered MWF, this should result in a measurable increase in magnetic susceptibility, due to the anisotropic myelin fragmenting into isotropic myelin debris, and the diamagnetic myelin tissue being removed, while no such changes should be expected if the myelin sheath simply loosens and becomes decompacted. An increase in radial diffusivity would likewise reveal myelin fragmentation, as myelin sheaths block water diffusion out of the axon, with little to no changes expected for myelin sheath loosening. Statistical analysis of the same voxels-of-interest that were found to have reduced myelin water fraction 2 weeks post-concussion, revealed no statistically significant changes in magnetic susceptibility, axial diffusivity, or radial diffusivity at any time-point post-concussion. This suggests that myelin water fraction changes are likely due to a loosening of the myelin sheath structure, as opposed to fragmentation and removal of myelin debris.</p

Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes

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 <b>2</b> (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe <b>2</b> 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

Role of histone kinases in Tat transactivation.

<p>(A) Chromatin immunoprecipitation analysis of Jurkat T cells containing an integrated HIV promoter in the absence or presence of Tat. Immunoprecipitations were performed with α-phospho-histone H3 antibodies (serine 10) followed by radioactive PCR with primers specific for the HIV LTR, the c-fos, or the β-globin genes. (B) Jurkat 1G5 cells containing an integrated HIV LTR luciferase construct were transiently transfected with Tat/FLAG (25 ng) and kinase-deficient (KD) kinase expression vectors (200 ng). (C) Western blot analysis of cellular lysates from 293 cells cotransfected with the indicated expression plasmids. (D) Transfection of CMV luciferase (25 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. (E) Transfection of 5xUAS luciferase and Gal4-CDK9 (20 ng) with the KD RSK2 expression plasmid (200 ng) in Jurkat cells. Values are means±SEM of three experiments.</p

Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes

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 <b>2</b> (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe <b>2</b> 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

Superinduction of Tat activity in CLS fibroblasts.

<p>(A) Western blot analysis of cellular extracts of fibroblasts from a patient with CLS and control human fibroblasts. (B) Nuclear microinjection of CLS fibroblasts with synthetic Tat (amino acids 1–72), the HIV LTR luciferase reporter, a CMV-GFP expression plasmid, and either the empty vector, an RSK2 expression construct, or a plasmid expressing kinase-deficient RSK2. Values are means±SEM of five experiments. (C) Coinjection of the 5xUAS luciferase reporter, a plasmid expressing the Gal4-VP16 transactivator and CMV-GFP with either the RSK2-expressing plasmid or the vector alone. Values are means±SEM of three experiments.</p

Relative myelin water fraction change post-injury.

<p>Change scores for myelin water fraction, relative to baseline, plotted against time for each subject with a mild traumatic brain injury in all significant voxels A) across the whole brain; B) in the splenium of the corpus callosum (a structure most commonly affected in mild TBI). Dots represent data points for each injured athlete (mean ± standard error plotted in grey). Note: time zero refers to baseline.</p

Brain areas with significantly reduced myelin water fraction.

<p>Areas of significantly reduced myelin water fraction in athletes with concussion at two weeks post-injury, superimposed on a standard brain. These areas include the splenium of the corpus callosum, right posterior thalamic radiation, left superior corona radiata, left superior longitudinal fasciculus, and left posterior limb of the internal capsule.</p