Post-translational site-selective protein backbone α-deuteration

Galan, Sébastien R. G.; Wickens, James; Daďová, Jitka; Ng, Wai-Lung; Zhang, Xinglong; Simion, Robert; Quinlan, Robert B. A.; Pires, Elisabete; Paton, Robert S.; Caddick, Stephen; Chudasama, Vijay; Davis, Benjamin G.

doi:10.1038/s41589-018-0128-y

Cited by 31 publications

(28 citation statements)

References 57 publications

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“…15,16 To obviate this issue and to leverage the fact that the precursor peptide of 1 (MroA1) lacked Cys, we hypothesized that replacement of Ser1 and Ser6 residues with Cys would enable a convenient desulfurization using dibromohexanediamide (3, DBHDA). [20][21][22][23] The product of this reaction would be the putative substrate for an MroD-catalyzed [4+2]-cycloaddition ( Figure 2B). (Figure S1).…”

Section: Resultsmentioning

confidence: 99%

Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

hudson¹,

Hooper²,

DiCaprio³

et al. 2020

Preprint

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<p>Owing to advances in genomic sequencing and bioinformatics, the breadth of natural product biosynthetic gene clusters (BGCs) has meteorically risen. This remains true for ribosomally synthesized and post-translationally modified peptides (RiPPs), where the rate of bioinformatically identifying clusters vastly outpaces characterization efforts. Uniting bioinformatics and enzymological knowledge to predict the chemical product(s) of a RiPP BGC with total chemical synthesis to obtain the natural compound is an effective platform for investigating cryptic gene clusters. Herein, we report the bioinformatic identification of a biosynthetically divergent class of RiPP bearing a subset of enzymes involved in thiopeptide biosynthesis. These natural products were predicted based on BGC architecture to undergo a formal, enzymatic [4+2]-cycloaddition with subsequent elimination of the leader peptide and water to produce a tri-substituted pyridine-based macrocycle. Bearing a pyridine similar to thiopeptides but lacking the ubiquitous thiazole heterocycles, these new RiPPs were termed pyritides. One of the predicted natural products was chemically synthesized using an 11-step synthesis. This structure was verified to be chemically identical by an orthogonal chemoenzymatic synthesis utilizing the precursor peptide and the cognate [4+2]-cycloaddition enzyme. The chemoenzymatic platform was used to synthesize a second in-cluster pyritide product as well as analogs from other bioinformatically identified pyritide BGCs. This work exemplifies complementary bioinformatic, enzymological, and synthetic techniques to characterize a structurally distinct class of RiPP natural product.</p>

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Section: Resultsmentioning

confidence: 99%

Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

hudson¹,

Hooper²,

DiCaprio³

et al. 2020

Preprint

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show abstract

“…46,47 Similar to the often-targeted chemistry that links peptidoglycan in the bacterial cell wall, FlgE Lal formation, also in the periplasm, has no known parallel in mammals and thus, crosslinking inhibitors are well positioned to avoid cross-reactivity with host metabolism. 48 Finally, DHA is increasingly being used for protein engineering and proteomic studies; 39,40,49,50 therefore the understanding of its formation and reactivity in the context of FlgE may advance its utility for such applications.…”

Section: Discussionmentioning

confidence: 99%

“…3a, top inset). The DHA intermediate is fleeting in the course of normal Lal crosslinking; thus, to produce the intermediate in high yield, WT Td FlgE D2 was treated with the cysteine alkylating agent 2,5-dibromohexanediamide (DBA) 39,40 and DHA formation was confirmed by LC-MS ( Supplementary Fig. 7a).…”

Section: Structural Characterization Of Wt Dha and Lal Crosslinked mentioning

confidence: 99%

Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook

et al. 2019

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The flagellar hook protein FlgE from spirochaete bacteria self-catalyzes the formation of an unusual inter-subunit lysinoalanine (Lal) crosslink that is critical for cell motility. Unlike other known examples of Lal biosynthesis, conserved cysteine and lysine residues in FlgE spontaneously react to form Lal without the involvement of additional enzymes. Oligomerization of FlgE via its D0 and Dc domains drives assembly of the crosslinking site at the D1-D2 domain interface. Structures of the FlgE D2 domain, dehydroalanine (DHA) intermediate, and Lal crosslinked FlgE subunits reveal successive snapshots of the reaction. Cys178 flips from a buried configuration to release hydrogen sulfide (H 2 S/HS −) and produce DHA. Interface residues provide hydrogen bonds to anchor the active site, facilitate β-elimination of Cys178, and polarize the peptide backbone to activate DHA for reaction with Lys165. Cysteine-reactive molecules accelerate DHA formation, whereas nucleophiles can intercept the DHA intermediate, thereby indicating a potential for Lal crosslink inhibitors to combat spirochaetal diseases.

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“…Posttranslational mutagenesis displays excellent site selectivity and regioselectivity (>98% β γ), time efficiency, compatible with a board spectrum of macromolecules (all α, α/β folds, all β, receptor, enzyme, antibody) and untouching biological functional groups 199,207,208 . Now, these methods have been used in a chemical biology way to understand the biological functions of PTM isoforms 209 through direct protein‐engineering 210 . A widely used case is the installation of fluorescent dyes on proteins, allowing the measurements of physiological parameters with optical techniques 184 .…”

Section: Targeting Ptm Protein Isoforms In Drug Designmentioning

confidence: 99%

Drug design targeting active posttranslational modification protein isoforms

Meng

Liang

Zhao

et al. 2020

Medicinal Research Reviews

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Modern drug design aims to discover novel lead compounds with attractable chemical profiles to enable further exploration of the intersection of chemical space and biological space. Identification of small molecules with good ligand efficiency, high activity, and selectivity is crucial toward developing effective and safe drugs. However, the intersection is one of the most challenging tasks in the pharmaceutical industry, as chemical space is almost infinity and continuous, whereas the biological space is very limited and discrete. This bottleneck potentially limits the discovery of molecules with desirable properties for lead optimization. Herein, we present a new direction leveraging posttranslational modification (PTM) protein isoforms target space to inspire drug design termed as “Post‐translational Modification Inspired Drug Design (PTMI‐DD).” PTMI‐DD aims to extend the intersections of chemical space and biological space. We further rationalized and highlighted the importance of PTM protein isoforms and their roles in various diseases and biological functions. We then laid out a few directions to elaborate the PTMI‐DD in drug design including discovering covalent binding inhibitors mimicking PTMs, targeting PTM protein isoforms with distinctive binding sites from that of wild‐type counterpart, targeting protein‐protein interactions involving PTMs, and hijacking protein degeneration by ubiquitination for PTM protein isoforms. These directions will lead to a significant expansion of the biological space and/or increase the tractability of compounds, primarily due to precisely targeting PTM protein isoforms or complexes which are highly relevant to biological functions. Importantly, this new avenue will further enrich the personalized treatment opportunity through precision medicine targeting PTM isoforms.

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Post-translational site-selective protein backbone α-deuteration

Cited by 31 publications

References 57 publications

Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook

Drug design targeting active posttranslational modification protein isoforms

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