A new ER-specific photosensitizer unravels 1O2-driven protein oxidation and inhibition of deubiquitinases as a generic mechanism for cancer PDT

Abstract

Photosensitizers (PS) are ideally devoid of any activity in the absence of photoactivation, and rely on molecular oxygen for the formation of singlet oxygen ((1)O2) to produce cellular damage. Off-targets and tumor hypoxia therefore represent obstacles for the use of PS for cancer photodynamic therapy. Herein, we describe the characterization of OR141, a benzophenazine compound identified through a phenotypic screening for its capacity to be strictly activated by light and to kill a large variety of tumor cells under both normoxia and hypoxia. This new class of PS unraveled an unsuspected common mechanism of action for PS that involves the combined inhibition of the mammalian target of rapamycin (mTOR) signaling pathway and proteasomal deubiquitinases (DUBs) USP14 and UCH37. Oxidation of mTOR and other endoplasmic reticulum (ER)-associated proteins drives the early formation of high molecular weight (MW) complexes of multimeric proteins, the concomitant blockade of DUBs preventing their degradation and precipitating cell death. Furthermore, we validated the antitumor effects of OR141 in vivo and documented its highly selective accumulation in the ER, further increasing the ER stress resulting from (1)O2 generation upon light activation.

Validation of a SPE HPLC–UV method for the quantification of a new ER-specific photosensitizer OR-141 in blood serum using total error concept

“…We can observe that both regio-isomers have a quite similar behavior giving parallel elimination curves. Concerning the asymmetric carbon atom, as both isomers of both regio-isomers were shown to have similar activities [3], the developed method did not intend to separate Rand S-forms, and it is unlikely that isomerization at this position can occur during metabolization, but a difference in metabolizing enzymes affinity is not excluded between isomers.…”

“…1) in blood serum samples using a solid phase extraction (SPE) HPLC-UV method. As our findings showed that both regio-isomers of both OR-141-R and OR-141-S possess similar activities and selectivity [3], we decided to work on the racemic mixture of both regio-isomers and develop a method which would not separate R-and S-isomers. The method has been fully validated according to accuracy profiles based on tolerance intervals [4].…”

“…It combines a quasi-absence of cytotoxicity in the absence of light activation and a low IC50 (≈10-100 nM according to the cell types and the assay) when activated, limiting the off-targets side effects. Furthermore, its capacity to produce 1 O 2 is equivalent in normoxia and hypoxia, allowing this compound to be effective in the hypoxic environment of many tumors [3].…”

Deep analysis of the USP18-dependent ISGylome and proteome unveils important roles for USP18 in tumour cell antigenicity and radiosensitivity

“…HAP1 lysates were used for GlyGly immunoprecipitation using PTMScan Ubiquitin Remnant Motif Kit (Cell Signaling), according to manufacturer's protocol 45,46 . Briefly, 20 mg of extracts were solubilized and denatured in 10 mL lysis buffer (20 mM HEPES, pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate), reduced using dithiothreitol (4.5 mM final) for 30 minutes at 55 °C.…”

“…Protein ISGylation profiles in human cells have been previously reported, but with limited depth and predominantly based on overexpression systems [39][40][41][42][43][44] . To obtain deep coverage and modified residue information in a more physiologically relevant context, we decided to combine advanced proteomic techniques with the immunoaffinity purification of GlyGly tryptic peptides 45,46 in wild-type (WT) and USP18-/-knockout (KO) HAP1 cells, in order to analyse the first USP18-dependent ISGylome of human cancer cells. We uncovered novel ISGylated substrates that are controlled by USP18, further emphasizing its role as a master regulator of the innate immune response.…”