The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 microM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from re-purposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.

The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 µM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) and ∼100-fold reductions in Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 100 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus.

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

Since its discovery about 40 years ago, the transcription factor p53 has turned into the most extensively studied protein in the context of human cancers due to its essential role in promoting tumor suppression. P53 regulates central processes such as the induction of cell cycle arrest, senescence, or apoptosis in response to cellular stresses, thus preventing tumorigenesis in mammals. Being the major negative regulator of p53, the E3 ubiquitin ligase MDM2 is as much of interest to cancer researchers as p53 itself. Down to the present day, its major function is assigned to antagonizing p53. However, evidence of additional, p53independent, functions is accumulating.One of these functions is the role of MDM2 as a p53-independent regulator of transcription.Previous studies have proven that MDM2 can interact with the general transcription machinery.Additionally, it acts as a chromatin-modifying co-factor promoting the formation of the repressive histone modifications H3K27me3 and H2AK119ub1 by Polycomb repressor complexes. However, since most of these studies were either conducted in vitro or in the absence of p53 in vivo, a comprehensive analysis of the MDM2 chromatin association in the presence of its major interaction partner p53 is still missing.In this thesis, we have investigated the global chromatin-binding pattern of endogenous MDM2 protein in various cell systems with diverse p53 status. Strikingly, comparative analyses of MDM2 binding sites identified in p53 wild-type, deleted and mutated systems revealed that MDM2 associates with more than 50 % of all CpG islands identified in human cells. This targeted binding of MDM2 to CpG islands is mediated through its direct interaction with the histone demethylase and CpG island-binding protein KDM2B, a known component of a variant Polycomb repressor complex.Preliminary results addressing the function of this KDM2B-directed chromatin recruitment of MDM2 indicate that both proteins cooperate in the repression of CpG island-associated genes, potentially through affecting the recruitment of RNA Polymerase II to those sites. This hypothesis is further strengthened by gene expression studies conducted in p53 mutated cells.In these studies, we found that MDM2 and Polycomb repressor complexes cooperatively repress target genes of the inducible TNF signaling pathway.Since CpG islands associate with the transcriptional start sites of about 50-60 % of all human genes, it is highly possible that this newly identified MDM2-KDM2B axis is central to the regulation of a multitude of physiological processes in the cell.(PIC) is formed at the core promoters of genes, starting with the sequence-specific binding of the general transcription factor TFIID followed by the sequential recruitment of TFIIA, TFIIB,