Retinaldehyde dehydrogenases belong to a superfamily
of enzymes
that regulate cell differentiation and are responsible for detoxification
of anticancer drugs. Chemical tools and methods are of great utility
to visualize and quantify aldehyde dehydrogenase (ALDH) activity in
health and disease. Here, we present the discovery of a first-in-class
chemical probe based on retinal, the endogenous substrate of retinal
ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme
activity in a panel of cancer cells via both fluorescence and chemical
proteomic approaches. We demonstrate that our probe is superior to
the widely used ALDEFLUOR assay to explain the ability of breast cancer
(stem) cells to produce all-trans retinoic acid.
Furthermore, our probe revealed the cellular selectivity profile of
an advanced ALDH1A1 inhibitor, thereby prompting us to investigate
the nature of its cytotoxicity. Our results showcase the application
of substrate-based probes in interrogating pathologically relevant
enzyme activities. They also highlight the general power of chemical
proteomics in driving the discovery of new biological insights and
its utility to guide drug discovery efforts.
To investigate the potential of tumor-targeting photoactivated chemotherapy, a chiral ruthenium-based anticancer warhead, Λ/Δ-[Ru(Ph 2 phen) 2 (OH 2 ) 2 ] 2+ , was conjugated to the RGD-containing Ac-MRGDH-NH 2 peptide by direct coordination of the M and H residues to the metal. This design afforded two diastereoisomers of a cyclic metallopeptide, Λ-[1]Cl 2 and Δ-[1]Cl 2 . In the dark, the ruthenium-chelating peptide had a triple action. First, it prevented other biomolecules from coordinating with the metal center. Second, its hydrophilicity made [1]Cl 2 amphiphilic so that it self-assembled in culture medium into nanoparticles. Third, it acted as a tumor-targeting motif by strongly binding to the integrin (K d = 0.061 μM for the binding of Λ-[1]Cl 2 to α IIb β 3 ), which resulted in the receptor-mediated uptake of the conjugate in vitro. Phototoxicity studies in two-dimensional (2D) monolayers of A549, U87MG, and PC-3 human cancer cell lines and U87MG three-dimensional (3D) tumor spheroids showed that the two isomers of [1]Cl 2 were strongly phototoxic, with photoindexes up to 17. Mechanistic studies indicated that such phototoxicity was due to a combination of photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) effects, resulting from both reactive oxygen species generation and peptide photosubstitution. Finally, in vivo studies in a subcutaneous U87MG glioblastoma mice model showed that [1]Cl 2 efficiently accumulated in the tumor 12 h after injection, where green light irradiation generated a stronger tumoricidal effect than a nontargeted analogue ruthenium complex [2]Cl 2 . Considering the absence of systemic toxicity for the treated mice, these results demonstrate the high potential of light-sensitive integrin-targeted ruthenium-based anticancer compounds for the treatment of brain cancer in vivo.
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