Synthesis and Biological Evaluation of RGD and isoDGR–Monomethyl Auristatin Conjugates Targeting Integrin α_Vβ₃

Abstract

This work reports the synthesis of a series of small‐molecule–drug conjugates containing the αVβ3‐integrin ligand cyclo[DKP‐RGD] or cyclo[DKP‐isoDGR], a lysosomally cleavable Val‐Ala (VA) linker or an “uncleavable” version devoid of this sequence, and monomethyl auristatin E (MMAE) or F (MMAF) as the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper‐catalyzed azide–alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated αvβ3 receptor and were shown to retain nanomolar IC50 values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with αvβ3 integrin overexpressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess markedly lower cytotoxic activity than the free drugs, which is consistent with inefficient integrin‐mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, the cyclo[DKP‐isoDGR]‐VA‐MMAE conjugate has low nanomolar IC50 values in cell viability assays with both cancer cell lines tested (U87: 11.50±0.13 nm; M21: 6.94±0.09 nm) and is therefore a promising candidate for in vivo experiments.

Molecular Delivery of Cytotoxic Agents via Integrin Activation

“…Monomethyl auristatin E (MMAE) and F (MMAF) are cytotoxic agents that bind to microtubules and prevent cell proliferation by inhibiting mitosis ( Figure 24 ) [ 97 ]. Moreover, they are synthetic analogs of Dolastatin 10, extracted from the sea hare Dolabella Auricularia , but they maintain the same so potent cytotoxic activity [ 98 , 99 ] that they cannot be used as drug themselves [ 100 ]; they are in fact used as payloads in a number of state-of-the-art antibody–drug conjugates (ADCs) [ 101 , 102 , 103 , 104 , 105 ]. Targeted MMAE and MMAF have different cell killing mechanisms in vivo depending on potencies of the respective drugs, together with the enhanced retention of MMAF, compared to the more lipophilic MMAE, within the tumor cells thanks to the negative charge on the C-terminal phenylalanine residue [ 106 ].…”

“…Dias et al [ 100 ] conjugated MMAE to cyclo[DKP-isoDGR] using both cleavable and uncleavable linkers, as reported in Figure 25 . The antiproliferative activity was tested against human glioblastoma (U87) and human melanoma (M21) cells overexpressing α v β 3 integrins.…”

Innovative Linker Strategies for Tumor‐Targeted Drug Conjugates

“… Evolution of PAB‐based self‐immolative spacers from the protease‐promoted release of payloads bearing secondary amine (e.g. MMAF in 27 , connected through a carbamate bond, PABC), tertiary amine (e.g. Tubulysin in 28 , connected through a quaternary ammonium group, PABQ), and phenol group (e.g.…”

“…For instance, a large number of ADCs undergoing clinical evaluations, including the marketed Adcetris™ and Polivy™ feature a para ‐aminobenzyl carbamate (PABC) spacer, in which the release of amine‐bearing drugs (e.g. MMAF in compound 27 , Figure ) takes place through the electronic cascade and loss of CO 2 (mechanism shown in Scheme ).…”

Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-β-ACPC) Scaffolds

“…For both sequences, flanking residues combined to the 3D presentation determine the recognition specificity. Since the pioneering work of Kessler and coworkers [ 11 ], many different RGD peptides and peptidomimetics have been developed as integrin ligands and investigated as potential antitumor drugs with antiangiogenic properties [ 12 , 13 , 14 ] or directing ligands in molecular imaging and targeted anticancer therapy, which emerged as powerful weapon for reducing the toxicity of the antitumor treatments and the insurgence of drug resistance [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ].…”