Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to α_vβ₃ without Promoting Integrin Allosteric Activation

Abstract

The isoDGR sequence is an integrin-binding motif that has been successfully employed as a tumor-vasculature-homing molecule or for the targeted delivery of drugs and diagnostic agents to tumors. In this context, we previously demonstrated that cyclopeptide 2, the product of the conjugation of c(CGisoDGRG) (1) to 4-( N-maleimidomethyl)cyclohexane-1-carboxamide, can be successfully used as a tumor-homing ligand for nanodrug delivery to neoplastic tissues. Here, combining NMR, computational, and biochemical methods, we show that the succinimide ring contained in 2 contributes to stabilizing interactions with αβ, an integrin overexpressed in the tumor vasculature. Furthermore, we demonstrate that various cyclopeptides containing the isoDGR sequence embedded in different molecular scaffolds do not induce αβ allosteric activation and work as pure integrin antagonists. These results could be profitably exploited for the rational design of novel isoDGR-based ligands and tumor-targeting molecules with improved αβ-binding properties and devoid of adverse integrin-activating effects.

Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations

“…This change was ascribed to the substitution of a Trp residue for a Ser immediately after the RGD sequence because the Trp forms a π-π interaction with β 3 Y122 on the β 1-α1 loop, thus preventing the latter's movement toward the MIDAS, a key element in triggering the conformational change (63). The importance of interacting with Y122 to prevent the conformational change in αVβ3 is also supported by studies demonstrating that nonenzymatic conversion of Asn to isoAsp in the GNGRG sequence in fibronectin repeat 5 results in the repeat developing high affinity for αVβ3; the cyclic peptide CisoDGRC retains this high affinity without inducing the conformational change in αVβ3 (64-66) because the C1 of the peptide interacts via its N-terminus with the Y122 carbonyl in β 3 (65,66).…”

“…62 The importance of interacting with Tyr122 to prevent the conformational change in αVβ3 is also supported by studies demonstrating that non-enzymatic conversion of Asn to isoAsp in the GNGRG sequence in fibronectin repeat 5 results in the repeat developing high affinity for αVβ3; the cyclic peptide CisoDGRC is reported to retain this high affinity without apparently inducing the conformational change in αVβ3 63,64 because the C1 of the peptide interacts via its N-terminus with the Tyr122 carbonyl in β3. 63,64 Based on Arnaout's observations, we synthetically modified the high-affinity RGD-based αVβ3 antagonist MK-429 so as to establish a π-π interaction with β3 Tyr122, guided by a threedimensional molecular model of MK-429's interaction with αVβ3 refined by molecular dynamics (MD) simulations. We searched for compounds that inhibit the adhesion of HEK-293 cells expressing αVβ3 to one of its ligands (fibrinogen), but do not trigger the activating conformational change in the receptor.…”

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin αvβ3

“…Flow cytometry analysis of α v β 3 integrin was carried out as described previously . Briefly, human melanoma MSR3 cells were detached, washed, and re‐suspended with Dulbecco's phosphate‐buffered saline (DPBS with Ca 2+ and Mg 2+ ) containing 1 % FCS (binding buffer) in the presence of an RGD‐ or isoDGR‐containing compound (0, 50, or 500 μ m ) and one of the following antibodies, LM609 (10 μg mL −1 ) or AP5 (10 μg mL −1 ), for 90 min in ice (3×10 5 cells/100 μL in binding buffer).…”

Nanogold Functionalized With Lipoamide-isoDGR: A Simple, Robust and Versatile Nanosystem for αvβ3-Integrin Targeting

“…We have previously shown that peptides containing isoAsp-Gly-Arg (isoDGR), a tripeptide motif that recognizes the αvβ3 integrin overexpressed in tumor vessels and on different tumor cell types (Avraamides et al, 2008;Desgrosellier and Cheresh, 2010), can be exploited as ligands for targeted delivery of various compounds to tumors, including drugs, imaging compounds and nanoparticles (Curnis et al, 2008;Curnis et al, 2013;Corti et al, 2017;Nardelli et al, 2018). In particular, we have shown that a cyclic CGisoDGRG peptide (called iso1) coupled to human serum albumin (iso1-HSA) can be used for the functionalization of nanogold bearing TNF or interleukin-12 (IL12), to enable "active" targeted delivery of nanoparticles to the tumor vasculature (Curnis et al, 2013;Gasparri et al, 2019).…”