The development of mechanically interlocked molecular systems programmed to operate autonomously in biological environments is an emerging field of research with potential medicinal applications. Within this framework, functional rotaxane- and pseudorotaxane-based architectures are starting to attract interest for the delivery of anticancer drugs, with the ultimate goal to improve the efficiency of cancer chemotherapy. Here, we report an enzyme-sensitive [2]-rotaxane designed to release a potent anticancer drug within tumor cells. The molecular device includes a protective ring that prevents the premature liberation of the drug in plasma. However, once located inside cancer cells the [2]-rotaxane leads to the release of the drug through the controlled disassembly of the mechanically interlocked components, in response to a determined sequence of two distinct enzymatic activations. Furthermore, in vitro biological evaluations reveal that this biocompatible functional system exhibits a noticeable level of selectivity for cancer cells overexpressing β-galactosidase.

Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3′ hydroxyl of the adenosine diphosphate (ADP)–ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.