Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.
Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.
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DOI：
10.1021/acschembio.3c00437
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发表时间：
2023-11-17
影响因子：
4
通讯作者：
Conway, Stuart J.
中科院分区：
文献类型：
--
作者：
Thomas, Adam M.;Serafini, Marta;Grant, Emma K.;Coombs, Edward A. J.;Bluck, Joseph P.;Schiedel, Matthias;Mcdonough, Michael A.;Reynolds, Jessica K.;Lee, Bernadette;Platt, Michael;Sharlandjieva, Vassilena;Biggin, Philip C.;Duarte, Fernanda;Milne, Thomas A.;Bush, Jacob T.;Conway, Stuart J.
靶标验证仍然是药物发现中的一个挑战，这导致药物发现过程中的高损耗率，特别是在II期临床试验中。因此，新的方法，以加强目标验证是有价值的工具，以改善药物发现过程。在这里，我们报告了定点诱变和亲电子片段的组合，使选择性抑制突变蛋白的小分子的快速鉴定。以含溴结构域蛋白BRD4为例，我们采用了基于结构的方法来鉴定BRD4第一个溴结构域[BRD4（1）]中的L94C突变对BRD4（1）功能的影响最小。然后，我们筛选了一个集中的，KAc模拟含有片段集和一个不同的片段库对突变体和野生型蛋白质，并确定了一系列的片段，表现出高选择性的突变蛋白。这些化合物被精心制作以包括炔点击标签以使得能够附着荧光染料。然后在瞬时表达BRD4（1）WT或BRD4（1）L94C的HEK293 T细胞中评估这些可点击化合物，以确定它们对BRD4（1）L94C的选择性超过其他可能的细胞靶标。鉴定了一种化合物，其对BRD4（1）L94C的选择性非常高，超过所有其他蛋白质。这项工作提供了一个概念验证，即定点诱变和亲电子片段的组合，在突变和缀合物的方法，可以快速识别小分子抑制剂的适当突变的蛋白质的兴趣。该技术可用于评估抑制目标蛋白的细胞表型，并且亲电配体为非共价配体开发提供了起点。
Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
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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
DOI：
10.1126/science.1249830
发表时间：
2014-10-31
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Baud MGJ;Lin-Shiao E;Cardote T;Tallant C;Pschibul A;Chan KH;Zengerle M;Garcia JR;Kwan TT;Ferguson FM;Ciulli A
通讯作者：
Ciulli A
DOI：
10.1126/science.aaz8455
发表时间：
2020-04-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gilan O;Rioja I;Knezevic K;Bell MJ;Yeung MM;Harker NR;Lam EYN;Chung CW;Bamborough P;Petretich M;Urh M;Atkinson SJ;Bassil AK;Roberts EJ;Vassiliadis D;Burr ML;Preston AGS;Wellaway C;Werner T;Gray JR;Michon AM;Gobbetti T;Kumar V;Soden PE;Haynes A;Vappiani J;Tough DF;Taylor S;Dawson SJ;Bantscheff M;Lindon M;Drewes G;Demont EH;Daniels DL;Grandi P;Prinjha RK;Dawson MA
通讯作者：
Dawson MA
选择性抑制BET溴结构域。
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
7.3
作者：
Hewings, David S.;Fedorov, Oleg;Filippakopoulos, Panagis;Martin, Sarah;Picaud, Sarah;Tumber, Anthony;Wells, Christopher;Olcina, Monica M.;Freeman, Katherine;Gill, Andrew;Ritchie, Alison J.;Sheppard, David W.;Russell, Angela J.;Hammond, Ester M.;Knapp, Stefan;Brennan, Paul E.;Conway, Stuart J.
通讯作者：
Conway, Stuart J.