BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3297

Peptides:

Sequences	Affinity Values
ACNTWNPWCPWDAPLCA ACNNFPFRCVYYPDICA	[2.3 ± 2.7][0.40 ± 0.24] [0.44 ± 0.07][NT]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 29517911

Target Name:

Plasma kallikrein (EC:3.4.21.34)

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

CX6CX6C phage display library

Affinity Measurement Method:

Inhibition assay

Affinity Measurement Description:

NaCl, 1.5 μM bovine serum albumin, pH 7.5. PKal (typical nominal concentration 1−2 nM) was preincubated in 96-well plates with different concentrations of the tested peptide (or of aprotinin, used as a control molecule) for 15 min. The maximum final concentration of a given peptide was chosen based on its potency and was followed by a 1.25- to 3-fold serial dilution, plus a control with no peptide. The reactions were initiated by the addition of H-Pro-Phe-Arg-AMC at a final concentration of 10 to 25 μM, and initial rates of substrate hydrolysis were determined by linear fit of the raw fluorescence versus time traces. In the case of H-Pro-Phe-Arg-AMC however, the Km for PKal is large, and all experiments were performed under conditions where [S]≪ Km. The affinity of peptide binding to human and rat PKal are showed respectively.

Brief Description:

Bicyclic peptides with inhibitory activity against PKal were identified using Bicycle Therapeutics proprietary phage display platform. Briefly, linear peptide libraries containing randomized AA between 3 cysteine residues were displayed on the surface of filamentous phage and cyclized on the phage with a thiol-reactive molecular scaffold (TBMB). The libraries formats used were 5 × 5 and 6 × 6, such that either 5 or 6 randomized AA were between the first and second and second and third cysteine, respectively. Following cyclization of the phage libraries, repeated selections were performed against human and rat PKal. Selections and subsequent affinity maturation selections were performed against PKal using methodology described previously. Rat and human PKal orthologous enzymes were alternated as baits during each selection round in order to identify bicyclic peptides with good cross-reactivity.

BiopanningDataSet Comments:

peptides were chemically synthesized for characterization.

Curator:

Lixu Jiang

Curation Date:

2018-04-19

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