Nintedanib-Containing Dual Conjugates Targeting α_Vβ₆ Integrin and Tyrosine Kinase Receptors as Potential Antifibrotic Agents

Abstract

α V β 6 Integrin plays a fundamental role in the activation of transforming growth factor-β (TGF-β), the major profibrotic mediator; for this reason, α V β 6 ligands have recently been forwarded to clinical phases for the therapy of fibrotic diseases. Herein, we report the synthesis and in vitro biological evaluation as antifibrotic agents of three new covalent conjugates, constituted by c (AmpLRGDL), an α V β 6 integrin-recognizing small cyclopeptide, and nintedanib, a tyrosine kinase inhibitor approved for idiopathic pulmonary fibrosis (IPF) treatment. One of these conjugates recapitulates optimal in vitro antifibrotic properties of the two active units. The integrin ligand portion within the conjugate plays a role in inhibiting profibrotic stimuli, potentiating the nintedanib effect and favoring the selective uptake of the conjugate in cells overexpressing α V β 6 integrin. These results may open a new perspective on the development of dual conjugates in the targeted therapy of IPF.

Nintedanib‐αVβ3 Integrin Ligand Dual‐Targeting Conjugates towards Precision Treatment of Melanoma

“…As stated above, our previous experiments on both sunitinib and nintedanib conjugates of type I and II clearly demonstrated that the tyrosine kinase inhibitory (TKI) activity of the drug portion was maintained, as proven by in vitro assays on isolated human recombinant VEGFR [30,33] . Given the structural resemblance between those compounds and conjugates 1 – 3 in this study, we also logically expected preservation of their TKI activity.…”

“…As stated above, our previous experiments on both sunitinib and nintedanib conjugates of type I and II clearly demonstrated that the tyrosine kinase inhibitory (TKI) activity of the drug portion was maintained, as proven by in vitro assays on isolated human recombinant VEGFR. [30,33] Given the structural resemblance between those compounds and conjugates 1-3 in this study, we also logically expected preservation of their TKI activity. Of course, since nintedanib binds to the intracellular region of growth factor receptors, the in-cell TKI activity of compounds 1-3 may be appreciably detected, provided that cell internalization occurs even if only partially.…”

“…[33] In both works, we were able to prove that: 1) the binding competence of both the integrin peptide ligand and the small molecule to their respective targets (either isolated or in-cell) was maintained in the covalent presentations; 2) the constructs were stable in both rat and human plasma, 3) cell internalized conjugates could be quantified via HPLC-MS of cell extracts and also analysed by cytofluorimetry by exploiting the innate Figure 1. General depiction of IL-SM dual conjugates I [30][31][32] and II [33] described in previous works. Structure of the IL-SM conjugates 1-3 of general formula III (integrin ligand in red, nintedanib unit in blue, and linker moiety in black) described in this work.…”

“…As an additional proof, we carried out the cell internalization assays also on human erythroleukemia cells (K562), that were shown to not express integrins α V β 3 , α V β 6 , and α V β 5 (Figure S5A, Supporting Information). [33] Again, nintedanib was able to efficiently cross the cell membrane, whilst conjugates 1-3 did not show the capacity to accumulate in the cytoplasm (Figure S5B, Supporting Information).…”

“…Thus, by following this scheme in the forward sense, the advanced intermediate 5 was prepared from linker1-piperazyl moiety 8 according to previously reported procedures; [33] likewise, pegylated alkyne 10 and bis-alkyne 11 (Scheme 2) were accessed from the respective linker-piperazyl precursors (Scheme 1, dashed box). [33] All that remained was the final click reaction between the reactivity-complementary azide-and alkyne-compounds.…”