Genetically-Encoded Discovery of Proteolytically Stable Bicyclic Inhibitors of Morphogen NODAL

In this manuscript, we developed a Two-fold Symmetric Linchpin (TSL) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where Xi is any amino acids but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-Tky-nu ovarian carcinoma cells with apparent IC50 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control Tky-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (PronaseTM) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

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Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays

Journal of Immunological Methods ◽

10.1016/j.jim.2011.08.005 ◽

2011 ◽

Vol 373 (1-2) ◽

pp. 79-88 ◽

Cited By ~ 28

Author(s):

Conrad E.Z. Chan ◽

Annie H.Y. Chan ◽

Angeline P.C. Lim ◽

Brendon J. Hanson

Keyword(s):

Phage Display ◽

Rapid Development ◽

Protein Targets ◽

Phage Display Libraries

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Short constrained peptides derived from phage display libraries as epitope models: the case of mAb 2F5

Retrovirology ◽

10.1186/1742-4690-9-s2-p89 ◽

2012 ◽

Vol 9 (S2) ◽

Author(s):

Y Palacios-Rodriguez ◽

T Gazarian ◽

L Huerta ◽

K Gazarian

Keyword(s):

Phage Display ◽

Phage Display Libraries ◽

Constrained Peptides

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Aggregation of Mouse Serum Amyloid A Protein Was Promoted by Amyloid-Enhancing Factors with the More Genetically Homologous Serum Amyloid A

International Journal of Molecular Sciences ◽

10.3390/ijms22031036 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1036

Author(s):

Xuguang Lin ◽

Kenichi Watanabe ◽

Masahiro Kuragano ◽

Kiyotaka Tokuraku

Keyword(s):

Amino Acid ◽

Serum Amyloid A ◽

Animal Species ◽

Amino Acid Sequences ◽

Serum Amyloid ◽

Mouse Serum ◽

Aa Amyloidosis ◽

Basic Residue ◽

Amyloid A ◽

Serum Amyloid A Protein

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-β structure accumulate and are deposited extracellularly in organs, resulting in chronic inflammatory diseases and infections. The incidence of AA amyloidosis is high in humans and several animal species. Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis. Amyloid enhancing factor (AEF) serves as a seed for fibril formation and shortens the onset of AA amyloidosis sharply. In this study, we examined whether AEFs extracted and purified from five animal species (camel, cat, cattle, goat, and mouse) could promote mouse SAA (mSAA) protein aggregation in vitro using quantum-dot (QD) nanoprobes to visualize the aggregation. The results showed that AEFs shortened and promoted mSAA aggregation. In addition, mouse and cat AEFs showed higher mSAA aggregation-promoting activity than the camel, cattle, and goat AEFs. Interestingly, homology analysis of SAA in these five animal species revealed a more similar amino acid sequence homology between mouse and cat than between other animal species. Furthermore, a detailed comparison of amino acid sequences suggested that it was important to mSAA aggregation-promoting activity that the 48th amino acid was a basic residue (Lys) and the 125th amino acid was an acidic residue (Asp or Glu). These data imply that AA amyloidosis exhibits higher transmission activity among animals carrying genetically homologous SAA gene, and may provide a new understanding of the pathogenesis of amyloidosis.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

Cell Discovery ◽

10.1038/s41421-021-00295-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Yongbing Pan ◽

Jianhui Du ◽

Jia Liu ◽

Hai Wu ◽

Fang Gui ◽

...

Keyword(s):

Phage Display ◽

Neutralizing Antibodies ◽

Variable Region ◽

Neutralizing Antibody ◽

Chain Gene ◽

Prophylactic Efficacy ◽

Heavy Chains ◽

Effective Interventions ◽

Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

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Interactions of mouse glycophorin A with the dRTA-related mutant G719D of the mouse Cl–/HCO3– exchanger Ae1This paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o10-147 ◽

2011 ◽

Vol 89 (2) ◽

pp. 224-235 ◽

Cited By ~ 2

Author(s):

Andrew K. Stewart ◽

Fouad T. Chebib ◽

Syed W. Akbar ◽

Maria J. Salas ◽

Rajan A. Sonik ◽

...

Keyword(s):

Amino Acids ◽

Transmembrane Domain ◽

Peer Review Process ◽

Surface Expression ◽

Amino Acid Sequences ◽

Glycophorin A ◽

Specific Expression ◽

Health And Disease ◽

Renal Tubular

The AE1 mutation G701D, associated with recessive distal renal tubular acidosis (dRTA), produces only minimal erythroid phenotype, reflecting erythroid-specific expression of stimulatory AE1 subunit glycophorin A (GPA). GPA transgene expression could theoretically treat recessive dRTA in patients and in mice expressing cognate Ae1 mutation G719D. However, human (h) GPA and mouse (m) Gpa amino acid sequences are widely divergent, and mGpa function in vitro has not been investigated. We therefore studied in Xenopus oocytes the effects of coexpressed mGpa and hGPA on anion transport by erythroid (e) and kidney (k) isoforms of wild-type mAe1 (meAe1, mkAe1) and of mAe1 mutant G719D. Coexpression of hGPA or mGpa enhanced the function of meAe1 and mkAe1 and rescued the nonfunctional meAe1 and mkAe1 G719D mutants through increased surface expression. Progressive N-terminal truncation studies revealed a role for meAe1 amino acids 22–28 in GPA-responsiveness of meAe1 G719D. MouseN-cyto/humanTMD and humanN-cyto/mouseTMD kAE1 chimeras were active and GPA-responsive. In contrast, whereas chimera mkAe1N-cyto/hkAE1 G701DTMD was GPA-responsive, chimera hkAE1N-cyto/mkAe1 G719DTMD was GPA-insensitive. Moreover, whereas the isolated transmembrane domain (TMD) of hAE1 G701D was GPA-responsive, that of mAe1 G719D was GPA-insensitive. Thus, mGpa increases surface expression and activity of meAe1 and mkAe1. However, the G719D mutation renders certain mAe1 mutant constructs GPA-unresponsive and highlights a role for erythroid-specific meAe1 amino acids 22–28 in GPA-responsiveness.

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ChemInform Abstract: General Method for the Synthesis of Enantiomerically Pure β- Hydroxy-α-amino Acids, Containing Fluorine Atoms in the Side Chains. Case of Stereochemical Distinction Between Methyl and Trifluoromethyl Groups. X-Ray Crystal and Molecula

ChemInform ◽

10.1002/chin.199412268 ◽

2010 ◽

Vol 25 (12) ◽

pp. no-no

Author(s):

V. A. SOLOSHONOK ◽

V. P. KUKHAR ◽

S. V. GALUSHKO ◽

N. YU. SVISTUNOVA ◽

D. V. AVILOV ◽

...

Keyword(s):

Amino Acids ◽

Side Chains ◽

Enantiomerically Pure ◽

X Ray ◽

Trifluoromethyl Groups ◽

General Method

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The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

Biochemical Journal ◽

10.1042/bj1310485 ◽

1973 ◽

Vol 131 (3) ◽

pp. 485-498 ◽

Cited By ~ 113

Author(s):

R. P. Ambler ◽

Margaret Wynn

Keyword(s):

Amino Acids ◽

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Cytochrome C ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Mitochondrial Cytochrome ◽

Cytochromes C ◽

Lending Library ◽

Single Peptide

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

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