Cancer chemotherapy relies on a high ratio of toxicity toward cancer cells vs. nonmalignant cells, making it desirable to protect normal cells. Among the nonmalignant cells, epithelia of the gut belong to the most vulnerable ones toward chemotherapeutics. Here, we use a murine intestinal organoid model to assess a strategy for protecting such epithelia against chemotherapy. Cell cycle progression was first stalled by palbociclib, a clinically established cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Washout of the drug allowed subsequent outgrowth of gut organoids. This transient cell cycle arrest conferred near-complete protection of the cells toward the nucleoside analogue gemcitabine. Moreover, pre-treatment with palbociclib protected the organoids against SN-38, the topoisomerase I-inhibiting metabolite of irinotecan, which is otherwise known for its severe gastrointestinal toxicities. In contrast, RB1-mutated cancer cells were not protected against gemcitabine or SN-38 when pre-treated with palbociclib. Taken together, these results outline a strategy for protecting nonmalignant cells against the toxicities of chemotherapeutics commonly used to treat advanced colorectal and pancreatic cancer. We propose that this strategy is particularly promising to protect the gut when treating RB1-deficient tumors that fail to arrest the cell cycle in response to CDK4/6 inhibitors. [Figure: see text].

Molnupiravir, an orally administered prodrug of β-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.

A bstract: Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for stopping the current COVID-19 pandemic and preventing future outbreaks. The SARS-CoV-2 main protease (M ^pro ), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that M ^pro is subject to redox regulation and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include sulfenylation, disulfide formation between the catalytic cysteine and a proximal cysteine, and generation of an allosteric lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and possess antiviral activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS and SARS-CoV, indicating their potential as common druggable sites.