Special Issue on RNA-Based Catalysts that Revolutionized the Discovery of Bioactive Peptides

IF 2.3 4区 化学 Q3 CHEMISTRY, MULTIDISCIPLINARY Israel Journal of Chemistry Pub Date : 2024-09-23 DOI:10.1002/ijch.202400064
Assoc. Prof. Seino A. K. Jongkees, Assoc. Prof. Joseph M. Rogers, Dr. Louise J. Walport
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Focusing on his development of RNA-based aminoacylation catalysts, his share of the prize <i>“For developing RNA-based catalysts that revolutionized the discovery of bioactive peptides</i>” is emphasised here in a collection of nine review articles and one research article that span aspects of oligonucleotide acylation catalysis, reprogramming of the genetic code, and applications of this in peptide drug discovery.</p><p>The RNA-based catalysts developed by Suga are called flexizymes. These were initially developed using the oligonucleotide selection platform ‘SELEX’ (systematic evolution of ligands by exponential enrichment). The <i>in vitro</i> selection scheme was designed to select for self-acylating activity by evolving a 5’ extension on tRNA, mimicking a key step in a potential transition between RNA-based life and modern protein-dominant life. RNA molecules that were able to attach a biotinylated amino acid to their own 3’ end were enriched by streptavidin pull-down, connecting survival to catalytic activity. After several more selection campaigns aiming to improve the level of activity and scope of the 5’ extension to independently aminoacylate added tRNA in <i>trans</i>, the modern flexizymes eFx, dFx, and aFx were born (discussed in detail in several reviews elsewhere).<span><sup>1, 2</sup></span> While this work began in fundamental biochemistry, the practical applications of a catalyst that is largely agnostic about which amino acid it attaches to which tRNA soon became apparent. However, as outlined in the contribution by <b>Cho, Lee, and coworkers</b>,<span><sup>3</sup></span> an understanding of the precise working of flexizymes at the molecular level is still lacking. While a crystal structure of the oligonucleotide has been available for some time,<span><sup>4</sup></span> the amino acid component is poorly resolved and so this leaves open questions about the positioning of the substrate ester and contributions of nucleotides in the catalytic pocket. They describe attempts to profile the rules for design of good flexizyme substrates, and how this has expanded research into novel bio-based polymers. One consequence of having relatively easy access to tRNA acylated with amino acid-like non-canonical building blocks is the ease with which translational space could be explored, and this fed back into further fundamental insights into the translation process itself.</p><p>Flexizymes have wide-ranging applications in ‘genetic code reprogramming’ of <i>in vitro</i> translation by providing tRNA pre-loaded with amino acids. However, the full potential of engineering translation in a test tube is yet to be reached. <b>Jones and Hartman</b><span><sup>5</sup></span> describe efforts to reduce the degeneracy of the genetic code, with a long-term goal of increasing the number of assignable codons and thereby the total number of building blocks that can be translated at once. In their work they give a detailed break down per codon box of the decoding process and how tRNA modifications influence this, as well as how evolved ribosomes can shift this balance. Further approaches on how to shift the balance between native decoding and reprogramming is discussed in work by <b>Hopstaken, Große-Wichtrup, and Jongkees</b>,<span><sup>6</sup></span> where they cover this challenge from the perspective of the competing factors throughout the translation process (beyond tRNA base pairing with mRNA in the ribosome). Included here are options for removing competition by targeting aaRS enzymes, amino acids, tRNA, release factors, and initiation elements. Finally, within the set of articles describing aminoacylation and genetic code reprogramming approaches, <b>Watkins, Kavoor, and Musier-Forsyth</b><span><sup>7</sup></span> profile <i>in vitro</i> and cellular strategies for detecting enzymatic aminoacylation. Covered here are radioisotope assays, coupled enzyme assays for detecting pyrophosphate release or AMP production, ATP consumption, and acylation-dependent biotinylation for application <i>in vitro</i>. However, these assays typically use <i>in vitro</i> transcribed tRNA that lack modifications. Also described are cell-based assays that use mature tRNA, profiling isoacceptor specificity arising from aaRS-related diseases through tRNA capture and analysis of the released amino acid or by oxidation of unacylated tRNA and poly-A extension of the remaining pool. These approaches can further be combined with high-throughput sequencing of the tRNA pool to correlate acylation levels with modification profiling.</p><p>Combining acylation of tRNA with non-canonical amino acids (using flexizymes, chemoenzymatic synthesis or endogenous/evolved aaRS enzymes) with approaches to remove competing factors (vacating sense or stop codons) allows the reassignment of the genetic code. As a result, a vast pool of non-standard building blocks can be incorporated into peptides and proteins by the ribosome. <b>Zhou and Obexer</b><span><sup>8</sup></span> profile this breadth of possibilities in their contribution. They outline the functionality accessible with this diverse building block pool, including contributions from artificial enzymes with novel catalytic cores and natural product-like peptides. Next to genetic code reprogramming, further diversification in the latter can be accessed using chemical and/or enzymatic modification following translation (which can synergise with reprogramming by building on novel reaction handles). In a research contribution from <b>Zhang, Goto, and Suga</b><span><sup>9</sup></span> such an approach is described, showing engineering of a geranyltransferase to broaden its peptide specificity. Using their engineered enzyme, C-lipidation of histidine can be achieved in an efficient manner that is more tolerant of a broad peptide context, an important consideration in library building where sequence context is difficult to control.</p><p>The application of genetic code reprogramming and (chemo)enzymatic diversification into display approaches for drug discovery is profiled by <b>Yin and Hipolito</b>.<span><sup>10</sup></span> The combination of genetic code reprogramming using flexizymes in the context of <i>in vitro</i> translation (coined flexible <i>in vitro</i> translation or the FIT system) together with mRNA display of the resulting peptides, together termed the Random non-standard Peptides Integrated Discovery (RaPID) system, has proven highly effective at generating hits against a diverse array of targets that are difficult to target using traditional modalities. In this review, a number of representative hits, targets, and structures are showcased. Also discussed are approaches for macrocyclisation of the libraries used to generate such hits, a critical consideration in allowing application of small peptides in a biological setting. Further development of initial hits is also discussed, covering two prominent cases wherein such hits have been developed further from the initial hit to a clinical candidate with oral bioavailability. An alternate but complementary modality, the highly knotted cysteine-rich plant-derived peptides ‘knottins’ (exemplified by the cyclotides), are discussed in the contribution by <b>Xie, Craik, and coworkers</b>.<span><sup>11</sup></span> These are composed of canonical amino acids, but their highly complex cyclic and disulfide cross-linked structure makes them highly stable in biological settings, while the loops between these critical cysteines are amenable to engineering or replacement with target-binding sequences (‘grafting’). Further discussed are approaches to display of such molecules in genetically encoded libraries, including suitable locations to break the head-to-tail macroycle for display and which loops are most amenable to grafting. This closes with a case study wherein this scaffold was engineered by mRNA display, giving 350-fold improved affinity.</p><p>Rounding out the collection are two articles focusing on specific applications of several of the above technologies in drug discovery efforts. In the contribution by <b>Ullrich and Nitsche</b>,<span><sup>12</sup></span> they describe drug discovery campaigns against targets related to the SARS-CoV-2 pandemic. These focused on the main protease and the spike protein as targets, and 8 hits are discussed in more detail. These include some with particularly challenging building blocks such as gamma amino acids targeting the main protease and with novel binding sites targeting the spike protein. <b>Sakai, Sato, and Matsumoto</b><span><sup>13</sup></span> present efforts to modulate activity of cytokine receptors using diverse modalities, including small molecules, oligonucleotide aptamers, antibody fragments, DARPins, and peptides. For this last category, an important contribution from the RaPID system was highlighted, wherein a potent binder of the Met receptor was dimerised to be able to induce receptor dimerisation, thereby converting it into a novel agonist. These same peptides could also be grafted into loops on recombinant proteins while maintaining binding of the peptide motif and function of the host protein (similar to the approach described above for cyclotides). These grafted proteins can host multiple loops, allowing tuned avidity and combinatorial binding, while at the same time expanding serum half life based on host protein re-uptake (when based on an antibody or Fc domain). 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引用次数: 0

Abstract

This special issue of the Israel Journal of Chemistry is in celebration of the Wolf Prize awarded to Hiroaki Suga in 2023 “for pioneering discoveries that illuminate the functions and pathological dysfunctions of RNA and proteins and for creating strategies to harness the capabilities of these biopolymers in new ways to ameliorate human diseases”. In this issue we collect contributions from former trainees, collaborators, and beyond to profile how his work has impacted the fields of bioorganic chemistry, synthetic biology, and drug discovery. Focusing on his development of RNA-based aminoacylation catalysts, his share of the prize “For developing RNA-based catalysts that revolutionized the discovery of bioactive peptides” is emphasised here in a collection of nine review articles and one research article that span aspects of oligonucleotide acylation catalysis, reprogramming of the genetic code, and applications of this in peptide drug discovery.

The RNA-based catalysts developed by Suga are called flexizymes. These were initially developed using the oligonucleotide selection platform ‘SELEX’ (systematic evolution of ligands by exponential enrichment). The in vitro selection scheme was designed to select for self-acylating activity by evolving a 5’ extension on tRNA, mimicking a key step in a potential transition between RNA-based life and modern protein-dominant life. RNA molecules that were able to attach a biotinylated amino acid to their own 3’ end were enriched by streptavidin pull-down, connecting survival to catalytic activity. After several more selection campaigns aiming to improve the level of activity and scope of the 5’ extension to independently aminoacylate added tRNA in trans, the modern flexizymes eFx, dFx, and aFx were born (discussed in detail in several reviews elsewhere).1, 2 While this work began in fundamental biochemistry, the practical applications of a catalyst that is largely agnostic about which amino acid it attaches to which tRNA soon became apparent. However, as outlined in the contribution by Cho, Lee, and coworkers,3 an understanding of the precise working of flexizymes at the molecular level is still lacking. While a crystal structure of the oligonucleotide has been available for some time,4 the amino acid component is poorly resolved and so this leaves open questions about the positioning of the substrate ester and contributions of nucleotides in the catalytic pocket. They describe attempts to profile the rules for design of good flexizyme substrates, and how this has expanded research into novel bio-based polymers. One consequence of having relatively easy access to tRNA acylated with amino acid-like non-canonical building blocks is the ease with which translational space could be explored, and this fed back into further fundamental insights into the translation process itself.

Flexizymes have wide-ranging applications in ‘genetic code reprogramming’ of in vitro translation by providing tRNA pre-loaded with amino acids. However, the full potential of engineering translation in a test tube is yet to be reached. Jones and Hartman5 describe efforts to reduce the degeneracy of the genetic code, with a long-term goal of increasing the number of assignable codons and thereby the total number of building blocks that can be translated at once. In their work they give a detailed break down per codon box of the decoding process and how tRNA modifications influence this, as well as how evolved ribosomes can shift this balance. Further approaches on how to shift the balance between native decoding and reprogramming is discussed in work by Hopstaken, Große-Wichtrup, and Jongkees,6 where they cover this challenge from the perspective of the competing factors throughout the translation process (beyond tRNA base pairing with mRNA in the ribosome). Included here are options for removing competition by targeting aaRS enzymes, amino acids, tRNA, release factors, and initiation elements. Finally, within the set of articles describing aminoacylation and genetic code reprogramming approaches, Watkins, Kavoor, and Musier-Forsyth7 profile in vitro and cellular strategies for detecting enzymatic aminoacylation. Covered here are radioisotope assays, coupled enzyme assays for detecting pyrophosphate release or AMP production, ATP consumption, and acylation-dependent biotinylation for application in vitro. However, these assays typically use in vitro transcribed tRNA that lack modifications. Also described are cell-based assays that use mature tRNA, profiling isoacceptor specificity arising from aaRS-related diseases through tRNA capture and analysis of the released amino acid or by oxidation of unacylated tRNA and poly-A extension of the remaining pool. These approaches can further be combined with high-throughput sequencing of the tRNA pool to correlate acylation levels with modification profiling.

Combining acylation of tRNA with non-canonical amino acids (using flexizymes, chemoenzymatic synthesis or endogenous/evolved aaRS enzymes) with approaches to remove competing factors (vacating sense or stop codons) allows the reassignment of the genetic code. As a result, a vast pool of non-standard building blocks can be incorporated into peptides and proteins by the ribosome. Zhou and Obexer8 profile this breadth of possibilities in their contribution. They outline the functionality accessible with this diverse building block pool, including contributions from artificial enzymes with novel catalytic cores and natural product-like peptides. Next to genetic code reprogramming, further diversification in the latter can be accessed using chemical and/or enzymatic modification following translation (which can synergise with reprogramming by building on novel reaction handles). In a research contribution from Zhang, Goto, and Suga9 such an approach is described, showing engineering of a geranyltransferase to broaden its peptide specificity. Using their engineered enzyme, C-lipidation of histidine can be achieved in an efficient manner that is more tolerant of a broad peptide context, an important consideration in library building where sequence context is difficult to control.

The application of genetic code reprogramming and (chemo)enzymatic diversification into display approaches for drug discovery is profiled by Yin and Hipolito.10 The combination of genetic code reprogramming using flexizymes in the context of in vitro translation (coined flexible in vitro translation or the FIT system) together with mRNA display of the resulting peptides, together termed the Random non-standard Peptides Integrated Discovery (RaPID) system, has proven highly effective at generating hits against a diverse array of targets that are difficult to target using traditional modalities. In this review, a number of representative hits, targets, and structures are showcased. Also discussed are approaches for macrocyclisation of the libraries used to generate such hits, a critical consideration in allowing application of small peptides in a biological setting. Further development of initial hits is also discussed, covering two prominent cases wherein such hits have been developed further from the initial hit to a clinical candidate with oral bioavailability. An alternate but complementary modality, the highly knotted cysteine-rich plant-derived peptides ‘knottins’ (exemplified by the cyclotides), are discussed in the contribution by Xie, Craik, and coworkers.11 These are composed of canonical amino acids, but their highly complex cyclic and disulfide cross-linked structure makes them highly stable in biological settings, while the loops between these critical cysteines are amenable to engineering or replacement with target-binding sequences (‘grafting’). Further discussed are approaches to display of such molecules in genetically encoded libraries, including suitable locations to break the head-to-tail macroycle for display and which loops are most amenable to grafting. This closes with a case study wherein this scaffold was engineered by mRNA display, giving 350-fold improved affinity.

Rounding out the collection are two articles focusing on specific applications of several of the above technologies in drug discovery efforts. In the contribution by Ullrich and Nitsche,12 they describe drug discovery campaigns against targets related to the SARS-CoV-2 pandemic. These focused on the main protease and the spike protein as targets, and 8 hits are discussed in more detail. These include some with particularly challenging building blocks such as gamma amino acids targeting the main protease and with novel binding sites targeting the spike protein. Sakai, Sato, and Matsumoto13 present efforts to modulate activity of cytokine receptors using diverse modalities, including small molecules, oligonucleotide aptamers, antibody fragments, DARPins, and peptides. For this last category, an important contribution from the RaPID system was highlighted, wherein a potent binder of the Met receptor was dimerised to be able to induce receptor dimerisation, thereby converting it into a novel agonist. These same peptides could also be grafted into loops on recombinant proteins while maintaining binding of the peptide motif and function of the host protein (similar to the approach described above for cyclotides). These grafted proteins can host multiple loops, allowing tuned avidity and combinatorial binding, while at the same time expanding serum half life based on host protein re-uptake (when based on an antibody or Fc domain). This new approach to generation of a unique class of biologicals may again open new directions in therapeutic development.

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基于 RNA 的催化剂彻底改变了生物活性肽的发现》特刊
本期《以色列化学杂志》特刊旨在庆祝 2023 年颁发给菅谷宏明的沃尔夫奖,该奖项 "表彰他在阐明 RNA 和蛋白质的功能和病理功能障碍方面的开创性发现,以及为以新方法利用这些生物聚合物的能力来改善人类疾病而制定的战略"。在本期杂志中,我们收集了来自前学员、合作者以及其他人士的文章,介绍他的工作如何影响了生物有机化学、合成生物学和药物发现领域。本期我们收集了九篇评论文章和一篇研究文章,内容涉及寡核苷酸酰化催化、遗传密码重编程以及在肽类药物发现中的应用等方面,重点介绍了他开发的基于 RNA 的氨基酰化催化剂。这些催化剂最初是利用寡核苷酸选择平台 "SELEX"(通过指数富集配体的系统进化)开发的。体外选择方案旨在通过进化 tRNA 上的 5' 延伸部分来选择自酰化活性,模仿 RNA 生命与现代蛋白质主导生命之间潜在过渡的关键步骤。通过链霉亲和素下拉法,能够在自身 3' 端连接生物素化氨基酸的 RNA 分子被富集,从而将存活与催化活性联系起来。为了提高 5' 延伸部分的活性水平和范围,以独立地反式氨基酰化添加的 tRNA,又进行了几次筛选,现代柔性酶 eFx、dFx 和 aFx 应运而生(在其他地方的几篇综述中有详细论述)。然而,正如 Cho、Lee 和同事3 的文章所述,人们对柔性酶在分子水平上的精确工作原理仍缺乏了解。虽然寡核苷酸的晶体结构已经问世了一段时间4 ,但氨基酸成分的解析度却很低,因此在底物酯的定位和核苷酸在催化袋中的贡献方面还存在问题。他们描述了设计良好柔性酶底物的规则的尝试,以及这如何扩展了对新型生物基聚合物的研究。通过提供预装氨基酸的 tRNA,柔性酶在体外翻译的 "遗传密码重编程 "方面有着广泛的应用。然而,试管中翻译工程的全部潜力仍有待开发。琼斯和哈特曼5 描述了减少遗传密码退化的努力,其长期目标是增加可分配密码子的数量,从而增加可一次性翻译的构件总数。他们在工作中详细分析了每个密码子盒的解码过程、tRNA修饰如何影响这一过程,以及进化的核糖体如何改变这一平衡。Hopstaken、Große-Wichtrup 和 Jongkees6 的研究从整个翻译过程(核糖体中 tRNA 与 mRNA 碱基配对之外)的竞争因素角度探讨了如何改变原生解码与重编程之间平衡的进一步方法。其中包括通过针对 aaRS 酶、氨基酸、tRNA、释放因子和启动元件来消除竞争的方案。最后,在一组介绍氨基酰化和遗传密码重编程方法的文章中,Watkins、Kavoor 和 Musier-Forsyth7 介绍了体外和细胞检测酶促氨基酰化的策略。其中包括放射性同位素测定法、用于检测焦磷酸释放或 AMP 产生的耦合酶测定法、ATP 消耗以及应用于体外的酰化依赖性生物素化。不过,这些检测方法通常使用缺乏修饰的体外转录 tRNA。还介绍了使用成熟 tRNA 的基于细胞的检测方法,通过 tRNA 捕获和分析释放的氨基酸,或通过氧化未酰化的 tRNA 和剩余池的 poly-A 延伸,分析 aaRS 相关疾病引起的等位受体特异性。这些方法可进一步与 tRNA 池的高通量测序相结合,从而将酰化水平与修饰谱分析联系起来。
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来源期刊
Israel Journal of Chemistry
Israel Journal of Chemistry 化学-化学综合
CiteScore
6.20
自引率
0.00%
发文量
62
审稿时长
6-12 weeks
期刊介绍: The fledgling State of Israel began to publish its scientific activity in 1951 under the general heading of Bulletin of the Research Council of Israel, which quickly split into sections to accommodate various fields in the growing academic community. In 1963, the Bulletin ceased publication and independent journals were born, with Section A becoming the new Israel Journal of Chemistry. The Israel Journal of Chemistry is the official journal of the Israel Chemical Society. Effective from Volume 50 (2010) it is published by Wiley-VCH. The Israel Journal of Chemistry is an international and peer-reviewed publication forum for Special Issues on timely research topics in all fields of chemistry: from biochemistry through organic and inorganic chemistry to polymer, physical and theoretical chemistry, including all interdisciplinary topics. Each topical issue is edited by one or several Guest Editors and primarily contains invited Review articles. Communications and Full Papers may be published occasionally, if they fit with the quality standards of the journal. The publication language is English and the journal is published twelve times a year.
期刊最新文献
Cover Picture: (Isr. J. Chem. 8-9/2024) Special Issue on RNA-Based Catalysts that Revolutionized the Discovery of Bioactive Peptides Hexagonal and Trigonal Quasiperiodic Tilings Breaking the Degeneracy of Sense Codons – How Far Can We Go? Cover Picture: (Isr. J. Chem. 6-7/2024)
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