RNA Display Methods for the Discovery of Bioactive Macrocycles

Huang, Yichao; Wiedmann, Mareike M.; Suga, Hiroaki

doi:10.1021/acs.chemrev.8b00430

Cited by 184 publications

(182 citation statements)

References 272 publications

(448 reference statements)

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“…1–5 Effective exploitation of ncAAs enhances our understanding of basic biology 6–8 and provides opportunities for engineering new classes of materials 9 and biological therapeutics. 2, 10–12 Many of these applications require high efficiency, high fidelity ncAA incorporation and subsequent careful evaluation of such events. The use of mass spectrometry-based characterizations offers the highest level of rigor, 9, 13 but lacks the throughput needed for initial screening and characterization.…”

Section: Introductionmentioning

confidence: 99%

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Potts

Stieglitz

Lei

et al. 2019

Preprint

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The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N-and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a “drop-in” version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems—and protein translation apparatuses—to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.Design, System, Application ParagraphOn earth, the genetic code provides nearly invariant instructions for generating the proteins present in all organisms using 20 primary amino acid building blocks. Scientists and engineers have long recognized the potential power of altering the genetic code to introduce amino acids that enhance the chemical versatility of proteins. Proteins containing such “noncanonical amino acids” (ncAAs) can be used to elucidate basic biological phenomena, discover new therapeutics, or engineer new materials. However, tools for measuring ncAA incorporation during protein translation (reporters) exhibit highly variable properties, severely limiting our ability to engineer improved ncAA incorporation systems. In this work, we sought to understand what properties of these reporters affect measurements of ncAA incorporation events. Using a series of ncAA incorporation systems in yeast, we evaluated reporter architecture, measurement techniques, and alternative data analysis methods. We identified key factors contributing to quantification of ncAA incorporation in all of these categories and demonstrated the immediate utility of our approach in identifying genomic knockouts that enhance ncAA incorporation efficiency. Our findings have important implications for how to evolve cells to better accommodate alternative genetic codes.

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Section: Introductionmentioning

confidence: 99%

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Potts

Stieglitz

Lei

et al. 2019

Preprint

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“…These studies resulted in the discovery of lanthipeptide inhibitors of HIV budding process, 15 urokinase plasminogen activator 16 and α v β 3 integrin binders. 13 Similarly, we anticipate that the integration of the FIT-Laz system with powerful in vitro screening techniques such as mRNA display 66 will provide access to artificial thiopeptides with desirable pharmacological profiles for drug discovery purposes.…”

Section: Discussionmentioning

confidence: 99%

Minimal lactazole scaffold for in vitro production of pseudo-natural thiopeptides

Vinogradov

Shimomura

Ozaki

et al. 2019

Preprint

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26Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb 27 biosynthetic gene cluster. Here, we established a platform for in vitro biosynthesis of lactazole A, 28 referred to as the FIT -Laz system, via a combination of the flexible in vitro translation (FIT) system 29 with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole 30 biosynthesis revealed remarkable substrate tolerance of the biosynthetic enzymes, and led to the 31 development of the "minimal lactazole scaffold", a construct requiring only 6 post-translational 32 modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz 33 enabled access to diverse lactazole analogs with 10 consecutive mutations, 14-to 62-membered 34 macrocycles, and up to 18 amino acid-long tail regions. Moreover, utilizing genetic code 35 reprogramming, we demonstrated synthesis of pseudo-natural lactazoles containing 4 non-36 proteinogenic amino acids. This work opens possibilities in exploring novel sequence space of pseudo-37 natural thiopeptides. 38 39 595

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“…Messenger RNA (mRNA) display is another powerful method for in vitro peptide‐screening . Depending on different in vitro translation systems, like rabbit reticulocyte lysates, E. coli S30, and the PURE (protein synthesis using recombinant elements) system, mRNA display can synthesize ribosomal cyclopeptides effectively, especially some uncommon cyclopeptides which are difficult to be expressed in a cellular system.…”

Section: Strategies To Develop Cyclic Peptides Into Therapeutic Agentsmentioning

confidence: 99%

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

Jing

Jin

2019

Medicinal Research Reviews

114

123

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As a versatile therapeutic modality, peptides attract much attention because of their great binding affinity, low toxicity, and the capability of targeting traditionally “undruggable” protein surfaces. However, the deficiency of cell permeability and metabolic stability always limits the success of in vitro bioactive peptides as drug candidates. Peptide macrocyclization is one of the most established strategies to overcome these limitations. Over the past decades, more than 40 cyclic peptide drugs have been clinically approved, the vast majority of which are derived from natural products. The de novo discovered cyclic peptides on the basis of rational design and in vitro evolution, have also enabled the binding with targets for which nature provides no solutions. The current review summarizes different classes of cyclic peptides with diverse biological activities, and presents an overview of various approaches to develop cyclic peptide‐based drug candidates, drawing upon series of examples to illustrate each strategy.

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RNA Display Methods for the Discovery of Bioactive Macrocycles

Cited by 184 publications

References 272 publications

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Minimal lactazole scaffold for in vitro production of pseudo-natural thiopeptides

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

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