Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes.
Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes.
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DOI：
10.1021/jacs.6b08536
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发表时间：
2017-01-18
影响因子：
15
通讯作者：
Taunton J
中科院分区：
文献类型：
--
作者：
Zhao Q;Ouyang X;Wan X;Gajiwala KS;Kath JC;Jones LH;Burlingame AL;Taunton J
蛋白激酶包括一个大的家族结构相关的酶。激酶抑制剂开发的一个主要目标是选择性地参与所需的激酶，同时避免无数脱靶激酶。然而，量化抑制剂与活细胞中多种内源性激酶的相互作用仍然是一个未满足的挑战。在这里，我们报道了磺酰氟探针的设计，它以高效的共价标记了细胞内kinome的广泛区域。蛋白质晶体学和质谱学证实了在ATP结合位点上磺酰氟和一个保守的赖氨酸之间的化学选择反应。优化后的探针2 （XO44）共价修饰了多达133种内源性激酶，有效地与高浓度的细胞内ATP竞争。我们使用探针2和无标记质谱法来定量批准的药物达沙替尼的细胞内激酶作用。数据显示，在临床相关浓度下，可饱和达沙替尼与一小部分激酶靶点结合，突出了赖氨酸靶向磺酰氟探针在要求苛刻的化学蛋白质组学应用中的实用性。
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.
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影响因子：
14.8
作者：
Arrowsmith CH;Audia JE;Austin C;Baell J;Bennett J;Blagg J;Bountra C;Brennan PE;Brown PJ;Bunnage ME;Buser-Doepner C;Campbell RM;Carter AJ;Cohen P;Copeland RA;Cravatt B;Dahlin JL;Dhanak D;Edwards AM;Frederiksen M;Frye SV;Gray N;Grimshaw CE;Hepworth D;Howe T;Huber KV;Jin J;Knapp S;Kotz JD;Kruger RG;Lowe D;Mader MM;Marsden B;Mueller-Fahrnow A;Müller S;O'Hagan RC;Overington JP;Owen DR;Rosenberg SH;Roth B;Ross R;Schapira M;Schreiber SL;Shoichet B;Sundström M;Superti-Furga G;Taunton J;Toledo-Sherman L;Walpole C;Walters MA;Willson TM;Workman P;Young RN;Zuercher WJ
通讯作者：
Zuercher WJ
影响因子：
15
作者：
Gushwa, Nathan N.;Kang, Sumin;Chen, Jing;Taunton, Jack
通讯作者：
Taunton, Jack
影响因子：
2.9
作者：
Patricelli, Matthew P.;Szardenings, A. Katrin;Kozarich, John W.
通讯作者：
Kozarich, John W.
影响因子：
4.4
作者：
Ku, Xin;Heinzmeir, Stephanie;Kuster, Bernhard
通讯作者：
Kuster, Bernhard
影响因子：
--
作者：
Patricelli MP;Nomanbhoy TK;Wu J;Brown H;Zhou D;Zhang J;Jagannathan S;Aban A;Okerberg E;Herring C;Nordin B;Weissig H;Yang Q;Lee JD;Gray NS;Kozarich JW
通讯作者：
Kozarich JW