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Bicyclic RGD Peptides with Exquisite Selectivity for the Integrin αvβ3 Receptor Using a “Random Design” Approach
journal contribution
posted on 2019-01-09, 00:00 authored by Dominik Bernhagen, Vanessa Jungbluth, Nestor Gisbert Quilis, Jakub Dostalek, Paul B. White, Kees Jalink, Peter TimmermanWe
describe the identification of bicyclic RGD peptides with high
affinity and selectivity for integrin αvβ3 via high-throughput screening of partially randomized libraries.
Peptide libraries (672 different compounds) comprising the universal
integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop and
a randomized sequence XXX (X being one of 18 canonical l-amino
acids) in the second loop, both enclosed by either an l-
or d-Cys residue, were converted to bicyclic peptides via
reaction with 1,3,5-tris(bromomethyl)benzene (T3). Screening
of first-generation libraries yielded lead bicyclic inhibitors displaying
submicromolar affinities for integrin αvβ3 (e.g., CT3HEQcT3RGDcT3, IC50 = 195 nM).
Next generation (second and third) libraries were obtained by partially
varying the structure of the strongest lead inhibitors and screening
for improved affinities and selectivities. In this way, we identified
the highly selective bicyclic αvβ3-binders CT3HPQcT3RGDcT3 (IC50 = 30 nM), CT3HPQCT3RGDcT3 (IC50 = 31 nM), and CT3HSQCT3RGDcT3 (IC50 = 42 nM) with affinities comparable to that of a knottin-RGD-type
peptide (32 amino acids, IC50 = 38 nM) and outstanding
selectivities over integrins αvβ5 (IC50 > 10000 nM) and α5β1 (IC50 > 10000 nM). Affinity measurements using
surface plasmon-enhanced fluorescence spectroscopy (SPFS) yielded Kd values of 0.4 and 0.6 nM for the Cy5-labeled
bicycle CT3HPQcT3RGDcT3 and RGD “knottin” peptide, respectively.
In vitro staining of HT29 cells with Cy5-labeled bicycles using confocal
microscopy revealed strong binding to integrins in their natural environment,
which highlights the high potential of these peptides as markers of
integrin expression.
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10000 nMintegrin-binding sequence Arg-Gly-AspaffinityHT 29 cellsC T 3 HSQ C T 3 RGD c T 3Bicyclic RGD PeptidesIntegrin α v β 3 Receptorbicyclic α v β 3Cy 5-labeled bicycle C T 3 HPQ c T 3 RGD c T 3surface plasmon-enhanced fluorescence spectroscopyCy 5-labeled bicyclesα 5 β 1integrins α v β 5binders C T 3 HPQ c T 3 RGD c T 3HEQintegrin α v β 3IC 50SPFS18 canonical l-amino acidsbicyclic RGD peptidesrandomized sequence XXX
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