Bicyclic RGD Peptides with Exquisite Selectivity for the Integrin α_vβ₃ Receptor Using a “Random Design” Approach

Abstract

We describe the identification of bicyclic RGD peptides with high affinity and selectivity for integrin α v β 3 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 α v β 3 (e.g., C T3 HEQc T3 RGDc T3 , IC 50 = 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 α v β 3 -binders C T3 HPQc T3 RGDc T3 (IC 50 = 30 nM), C T3 HPQC T3 RGDc T3 (IC 50 = 31 nM), and C T3 HSQC T3 RGDc T3 (IC 50 = 42 nM) with affinities comparable to that of a knottin-RGD-type peptide (32 amino acids, IC 50 = 38 nM) and outstanding selectivities over integrins α v β 5 (IC 50 > 10000 nM) and α 5 β 1 (IC 50 > 10000 nM). Affinity measurements using surface plasmon-enhanced fluorescence spectroscopy (SPFS) yielded K d values of 0.4 and 0.6 nM for the Cy5-labeled bicycle C T3 HPQc T3 RGDc T3 and RGD "knottin" peptide, respectively. In vitro staining of HT29 cells with Cy5labeled 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.

Radiosynthesis, Biological Evaluation, and Preclinical Study of a 68Ga-Labeled Cyclic RGD Peptide as an Early Diagnostic Agent for Overexpressed αvβ3 Integrin Receptors in Non-Small-Cell Lung Cancer

“…erefore, in recent years, many studies have focused on describing novel radiolabeled RGD peptides for molecularbased imaging [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36].…”

“…As α v β 3 integrin has high expression on tumor cells and α v integrins contribute to angiogenesis and tumor progression, α v β 3 integrin can be considered as an excellent target for early detection, treatment, and prognostic marker for lung cancer. e α and β subunits of α v β 3 integrin have different structural domains, and the extracellular domains from α and β subunits provide the binding site for the ligand [21][22][23][24]. e tripeptide RGD sequence binds to the interface of the β-propeller domain from the α subunit and the βα-domain from the β subunit ( Figure S1) [25,26].…”

Size‐Dependent Cellular Uptake of RGD Peptides

“…As M21L cells do not present integrin α v β 3 , uptake of peptide 1 proceeds in an integrin‐independent fashion . The large shift between the negative control (no peptide) and the 0 min sample is caused by ionic interactions of positively charged Arg side chains with negatively charged phospholipids of the membrane resulting in an accumulation of RGD peptides on the cell surface . Therefore, the localization of peptides was further studied by live cell imaging.…”

SARS-CoV-2 attachment to host cells is possibly mediated via RGD-integrin interaction in a calcium-dependent manner and suggests pulmonary EDTA chelation therapy as a novel treatment for COVID 19

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