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Ligand‐based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand‐based modulation of integrin functions. Inhibitors of integrin αvβ3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg‐Gly‐Asp (RGD) or isoAsp‐Gly‐Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo‐RGDf(NMe)V (Cilengitide), cyclo[DKP3‐RGD], cyclo[DKP3‐isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand‐controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site. [ABSTRACT FROM AUTHOR]

This work takes advantage of one of the hallmarks of cancer, that is, the presence of tumor infiltrating cells of the immune system and leukocyte‐secreted enzymes, to promote the activation of an anticancer drug at the tumor site. The peptidomimetic integrin ligand cyclo(DKP‐RGD) was found to accumulate on the surface of αvβ3 integrin‐expressing human renal cell carcinoma 786‐O cells. The ligand was conjugated to the anticancer drug paclitaxel through a Asn‐Pro‐Val (NPV) tripeptide linker, which is a substrate of neutrophil‐secreted elastase. In vitro linker cleavage assays and cell antiproliferative experiments demonstrate the efficacy of this tumor‐targeting conjugate, opening the way to potential therapeutic applications. Exploiting inflammation in cancer: A RGD‐drug conjugate accumulates on the surface of integrin‐expressing human renal cell carcinoma cells and releases paclitaxel upon linker cleavage mediated by neutrophil‐secreted elastase. [ABSTRACT FROM AUTHOR]

This work reports the synthesis of three multimeric RGD peptidomimetic-paclitaxel conjugates featuring a number of αVβ3 integrin ligands ranging from 2 to 4. These constructs were assembled by conjugation of the integrin αVβ3 ligand cyclo[DKP-RGD]-CH2NH2 with paclitaxel via a 2′-carbamate with a self-immolative spacer, the lysosomally cleavable Val-Ala dipeptide linker, a multimeric scaffold, a triazole linkage, and finally a PEG spacer. Two monomeric conjugates were also synthesized as reference compounds. Remarkably, the new multimeric conjugates showed a binding affinity for the purified integrin αVβ3 receptor that increased with the number of integrin ligands (reaching a minimum IC50 value of 1.2 n m for the trimeric), thus demonstrating that multivalency is an effective strategy to strengthen the ligand-target interactions. [ABSTRACT FROM AUTHOR]

Herein we report the first example of an isoDGR-drug conjugate ( 2), designed to release paclitaxel selectively within cancer cells expressing integrin αVβ3. Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin αVβ3 receptor (IC50=11.0 n m). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin αVβ3 expression: human glioblastoma U87 (αVβ3+) and U87 β3-KO (αVβ3−). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4). [ABSTRACT FROM AUTHOR]

The cyclo[DKP- isoDGR] peptidomimetics 2- 5, containing bifunctional diketopiperazine (DKP) scaffolds that differ in the configuration of the two DKP stereocenters and in the substitution at the DKP nitrogen atoms, were prepared and examined in vitro in competitive binding assays with purified αvβ3 and αvβ5 integrin receptors. IC50 values ranged from low nanomolar (ligand 3) to submicromolar with αvβ3 integrin. The biological activities of ligands cyclo[DKP3-RGD] 1 and cyclo[DKP3- isoDGR] 3, bearing the same bifunctional DKP scaffold and showing similar αVβ3 integrin binding values, were compared in terms of their cellular effects in human U373 glioblastoma cells. Compounds 1 and 3 displayed overlapping inhibitory effects on the FAK/Akt integrin activated transduction pathway and on integrin-mediated cell infiltration processes, and qualify therefore, despite the different RGD and isoDGR sequences, as integrin antagonists. Both compounds induced apoptosis in glioma cells after 72 hour treatment. [ABSTRACT FROM AUTHOR]

A perfect fit: Shown here is the docking best pose of a low ‐ nanomolar αvβ3 integrin ligand cyclo[DKP ‐ isoDGR] (atom color tube representation; see figure) into the receptor binding pocket, overlaid on the bound conformation of Cilengitide (green tube representation). The extended isoDGR conformation permits all the important interactions with the extracellular domain of the αvβ3 integrin. The metal ion in the MIDAS region is represented by a magenta sphere. [ABSTRACT FROM AUTHOR]

Multivalent binding is often used to increase the strength of ligand–receptor interactions. In oncology, the development of multimeric conjugates aims at promoting a more efficient recognition of target antigens, possibly leading to better therapeutic performance. The affinity for integrin αVβ3 of multimeric conjugates (cyclo[DKP‐RGD])n‐Val‐Ala‐PTX (n=1–4) increases with the number of cyclo[DKP‐RGD] ligands up to n=3. For more information, see the Communication by L. Pignataro, C. Gennari et al. on page 14410 ff. [ABSTRACT FROM AUTHOR]

Ligand‐based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand‐based modulation of integrin functions. Inhibitors of integrin αvβ3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg‐Gly‐Asp (RGD) or isoAsp‐Gly‐Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo‐RGDf(NMe)V (Cilengitide), cyclo[DKP3‐RGD], cyclo[DKP3‐isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand‐controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site. [ABSTRACT FROM AUTHOR]

This work reports the synthesis of three multimeric RGD peptidomimetic-paclitaxel conjugates featuring a number of αVβ3 integrin ligands ranging from 2 to 4. These constructs were assembled by conjugation of the integrin αVβ3 ligand cyclo[DKP-RGD]-CH2NH2 with paclitaxel via a 2′-carbamate with a self-immolative spacer, the lysosomally cleavable Val-Ala dipeptide linker, a multimeric scaffold, a triazole linkage, and finally a PEG spacer. Two monomeric conjugates were also synthesized as reference compounds. Remarkably, the new multimeric conjugates showed a binding affinity for the purified integrin αVβ3 receptor that increased with the number of integrin ligands (reaching a minimum IC50 value of 1.2 n m for the trimeric), thus demonstrating that multivalency is an effective strategy to strengthen the ligand-target interactions. [ABSTRACT FROM AUTHOR]

Multivalent binding is often used to increase the strength of ligand–receptor interactions. In oncology, the development of multimeric conjugates aims at promoting a more efficient recognition of target antigens, possibly leading to better therapeutic performance. The affinity for integrin αVβ3 of multimeric conjugates (cyclo[DKP‐RGD])n‐Val‐Ala‐PTX (n=1–4) increases with the number of cyclo[DKP‐RGD] ligands up to n=3. For more information, see the Communication by L. Pignataro, C. Gennari et al. on page 14410 ff. [ABSTRACT FROM AUTHOR]