Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways

Photosensitization of pancreatic cancer cells by cationic alkyl-porphyrins in free form or engrafted into POPC liposomes: The relationship between delivery mode and mechanism of cell death

Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode

Effect of DNA Glycosylases OGG1 and Neil1 on Oxidized G-Rich Motif in the KRAS Promoter

The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription

hnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression

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Photodynamic Therapy for ras-Driven Cancers: Targeting G-Quadruplex RNA Structures with Bifunctional Alkyl-Modified Porphyrins

Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers