Enzymatic Macrolactamization of mRNA Display Libraries for Inhibitor Selection.
Enzymatic Macrolactamization of mRNA Display Libraries for Inhibitor Selection.
复制标题
用于抑制剂选择的 mRNA 展示文库的酶促大内酰胺化。
DOI：
10.1021/acschembio.2c00828
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发表时间：
2023
影响因子：
4
通讯作者：
Bowers,AlbertA
中科院分区：
文献类型：
--
作者：
Bowler,MatthewM;Glavatskikh,Marta;Pecot,ChadV;Kireev,Dmitri;Bowers,AlbertA
mRNA展示是一种强大的高通量技术，用于发现蛋白质靶点的新型肽配体。许多方法已被用来扩大mRNA展示文库的化学多样性超过20个典型的氨基酸，包括遗传密码重编程和双正交化学。然而，迄今为止，很少有报道使用酶作为生物相容性试剂用于多样化mRNA展示文库。在这里，我们报告的评价和实施的共同工业酶，微生物转氨酶（mTG），作为一种通用的生物催化剂，通过赖氨酸-谷氨酰胺异肽键环化的mRNA展示肽库。我们建立了两个独立的基于展示的测定来验证mTG与mRNA连接的肽底物的相容性。这些测定表明mTG具有高度的底物耐受性和低的单轮偏倚。为了证明mTG介导的环化在配体发现中的潜在益处，将高多样性mTG修饰的文库用于两个单独的亲和选择：（1）一个针对钙和整联蛋白结合蛋白CIB 1，和（2）第二个针对免疫检查点蛋白和新兴治疗靶标B7-H3。这两种选择导致鉴定出有效的、环状的、低纳摩尔结合剂，并且随后的结构-活性研究证明了环化对所观察到的活性的重要性。值得注意的是，CIB 1结合剂中的环化稳定了α-螺旋构象，而B7-H3抑制剂采用两个桥，一个mTG衍生的内酰胺和第二个二硫化物来实现其效力。总之，这些结果证明了基于酶的生物催化剂在mRNA展示配体选择中的潜在益处，并建立了在mRNA展示中使用mTG的框架。
mRNA display is a powerful, high-throughput technology for discovering novel, peptide ligands for protein targets. A number of methods have been used to expand the chemical diversity of mRNA display libraries beyond the 20 canonical amino acids, including genetic code reprogramming and biorthogonal chemistries. To date, however, there have been few reports using enzymes as biocompatible reagents for diversifying mRNA display libraries. Here, we report the evaluation and implementation of the common industrial enzyme, microbial transglutaminase (mTG), as a versatile biocatalyst for cyclization of mRNA display peptide libraries via lysine-to-glutamine isopeptide bonds. We establish two separate display-based assays to validate the compatibility of mTG with mRNA-linked peptide substrates. These assays indicate that mTG has a high degree of substrate tolerance and low single round bias. To demonstrate the potential benefits of mTG-mediated cyclization in ligand discovery, high diversity mTG-modified libraries were employed in two separate affinity selections: (1) one against the calcium and integrin binding protein, CIB1, and (2) the second against the immune checkpoint protein and emerging therapeutic target, B7–H3. Both selections resulted in the identification of potent, cyclic, low nanomolar binders, and subsequent structure–activity studies demonstrate the importance of the cyclization to the observed activity. Notably, cyclization in the CIB1 binder stabilizes an α-helical conformation, while the B7–H3 inhibitor employs two bridges, one mTG-derived lactam and a second disulfide to achieve its potency. Together, these results demonstrate potential benefits of enzyme-based biocatalysts in mRNA display ligand selections and establish a framework for employing mTG in mRNA display.
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影响因子：
16.6
作者：
Urban JH;Moosmeier MA;Aumüller T;Thein M;Bosma T;Rink R;Groth K;Zulley M;Siegers K;Tissot K;Moll GN;Prassler J
通讯作者：
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影响因子：
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作者：
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影响因子：
3.2
作者：
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通讯作者：
P. Spycher;Christian A Amann;Jöri E. Wehrmüller;D. Hurwitz;Olivier Kreis;D. Messmer;Andreas Ritler;A. Küchler;A. Blanc;M. Béhé;P. Walde;R. Schibli
RiPP 前体肽的一锅饱和诱变
DOI：
--
发表时间：
2019
期刊：
影响因子：
--
作者：
Mao Qiyue;Kawaguchi Akira T.;Mizobata Shun;Motterlini Roberto;Foresti Roberta;Kitagishi Hiroaki;Yuki Goto
通讯作者：
Yuki Goto
影响因子：
3.2
作者：
Jeremy Touati;A. Angelini;Marlon J. Hinner;C. Heinis
通讯作者：
Jeremy Touati;A. Angelini;Marlon J. Hinner;C. Heinis