Explore the vast range of titles available.

Oral outpatient treatment for Covid-19 is needed. In this phase 3, double-blind, randomized, controlled trial, molnupiravir, a small-molecule antiviral, was studied in unvaccinated patients with less than 5 days of Covid-19 illness. By day 29, hospitalization for progression of Covid-19 was lower with molnupiravir (6.8%) than with placebo (9.7%).

Molnupiravir, an orally administered drug for the treatment of mild‐to‐moderate COVID‐19, is a prodrug of the ribonucleoside β‐D‐N4‐hydroxycytidine (NHC). NHC incorporation in the SARS‐CoV‐2 RNA strand causes an accumulation of deleterious errors in the genome, resulting in reduced viral infectivity and replication. Exposure‐response (E‐R) analyses for viral RNA mutation rate and virologic outcomes were conducted using data from three phase 2/3 studies of molnupiravir (P006, MOVe‐IN, and MOVe‐OUT). Three dose levels (200, 400, and 800 mg every 12 hours [Q12H]) and placebo were evaluated. E‐R datasets were generated for SARS‐CoV‐2 RNA mutation and longitudinal SARS‐CoV‐2 RNA viral load. E‐R models were defined for RNA mutation rate and viral load change from baseline at days 5 and 10. The models supported plasma NHC AUC0‐12 as the appropriate pharmacokinetic driver for assessing E‐R relationships. The highest percentage of participants with > 20 low‐frequency nucleotide substitutions (LNS) per 10,000 bases, a measure of likely meaningful drug effect, was predicted in the 800 mg Q12H treatment group. A strong drug effect on the reduction of viral load was observed on days 5 and 10. E‐R relationships were best represented by an Emax structural model with reasonable consistency in the estimated AUC50s (~2.3‐fold), across the models, of 10,260 and 4390 nM*hr. for day 5 viral load change from baseline and LNS error rate, respectively. These biomarker E‐R curves support the choice of 800 mg Q12H as providing near‐maximal drug effect, consistent with findings from the previously published molnupiravir E‐R model of clinical outcomes.

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β- d - N 4 -hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir. Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques 1 , 2 . Because previously described cytidine deaminases operate on single-stranded nucleic acids 3 , their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria 4 . As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases 9 , 10 .Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders. An interbacterial toxin that catalyses the deamination of cytidines within double-stranded DNA forms part of a CRISPR-free, RNA-free base editing system that enables manipulation of human mitochondrial DNA.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified 1 – 4 , the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy. Induction of APOBEC3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that its suppression may represent a potential therapeutic strategy in the prevention of acquired resistance to lung cancer targeted therapy.

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications. Improved cytosine base editors are created by repurposing an adenine deaminase.

Background RNA–DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. Results Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2′-O-methylation (2′-OMe). Moreover, we find that 2′-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2′-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2′-OMe, R-loop stability and CSR. Surprisingly, FBL, AID’s interaction partner and aSNORD1C promoted AID targeting to the IgH locus. Conclusion Taken together, our results suggest that 2′-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

CDD plays a pivotal role within the pyrimidine salvage pathway. In this study, a novel, rapid method for the identification of cell lines lacking functional cytidine deaminase was developed. This innovative method utilizes immunocytochemical detection of the product of 5-fluorocytidine deamination, 5-fluorouridine in cellular RNA, enabling the identification of these cells within two hours. The approach employs an anti-bromodeoxyuridine antibody that also specifically binds to 5-fluorouridine and its subsequent detection by a fluorescently labeled antibody. Our results also revealed a strong correlation between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and cytidine deaminase content. On the other hand, no correlation was observed between the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio and deoxycytidine monophosphate deaminase content. Similarly, no correlation was observed between this ratio and equilibrative nucleoside transporters 1 or 2. Finally, concentrative nucleoside transporters 1, 2, or 3 also do not correlate with the 5-fluorouridine/5-fluorocytidine cytotoxicity ratio.

Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells, macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR), while decreasing at higher doses over a broad physiological range, AID and Blimp1 expression, CSR, somatic hypermutation and plasma cell differentiation. In human and mouse B cells, butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3′UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors, not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes, these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models. Dietary fiber-derived short-chain fatty acids (SCFA) act as histone deacetylase (HDAC) inhibitors on Tregs and innate immune cells, promoting immune tolerance by altering gene expression. Here the authors show that SCFA HDAC inhibitor activity impacts B cell differentiation, antibody responses and antibody-driven autoimmunity.

Enzymes of the nucleotide salvage pathway are shown to have substrate selectivity that protects newly synthesized DNA from random incorporation of epigenetically modified forms of cytosine; a subset of cancer cell lines that overexpress cytidine deaminase (CDA) are sensitive to treatment with 5hmdC or 5fdC (oxidized forms of 5-methyl-cytosine), which leads to DNA damage and cell death, indicating the chemotherapeutic potential of these nucleoside variants for CDA-overexpressing cancers. Specificity in nucleotide recycling As well as synthesizing DNA nucleotides de novo , cells utilize nucleotides recycled from dying cells. It is unclear how the nucleotide salvage pathway deals with the various oxidized forms of 5-methyl-cytosine such as 5hmdC and 5fdC. Here Skirmantas Kriaucionis and colleagues demonstrate that the nucleotide salvage pathway has a substrate selectivity that protects newly synthesized DNA from random incorporation of epigenetically modified forms of cytosine. However, some cancer cells that overexpress cytidine deaminase (CDA) are sensitive to overexpression of 5hmdC or 5fdC, which leads to DNA damage and cell death. The authors speculate that drugs based on these nucleoside variants may have chemotherapeutic potential in CDA-overexpressing cancers. Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands 1 , 2 , 3 , random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2′deoxycytidine (5mdC) has been investigated before 4 , 5 , 6 , it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2′deoxycytidine (5hmdC), 5-formy-2′deoxycytidine (5fdC) and 5-carboxyl-2′deoxycytidine (5cadC) 7 , 8 , 9 , 10 . Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues 11 .