Mechanistic Insights into Harmine-Mediated Inhibition of Human DNA Methyltransferases and Prostate Cancer Cell Growth.

Mammalian DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, are key DNA methylation enzymes and play important roles in gene expression regulation. Dysregulation of DNMTs is linked to various diseases and carcinogenesis, and therefore except for the two approved anticancer azanucleoside drugs, various non-nucleoside DNMT inhibitors have been identified and reported. However, the underlying mechanisms for the inhibitory activity of these non-nucleoside inhibitors still remain largely unknown. Here, we systematically tested and compared the inhibition activities of five non-nucleoside inhibitors toward the three human DNMTs. We found that harmine and nanaomycin A blocked the methyltransferase activity of DNMT3A and DNMT3B more efficiently than resveratrol, EGCG, and RG108. We further determined the crystal structure of harmine in complex with the catalytic domain of the DNMT3B-DNMT3L tetramer revealing that harmine binds at the adenine cavity of the SAM-binding pocket in DNMT3B. Our kinetics assays confirm that harmine competes with SAM to competitively inhibit DNMT3B-3L activity with a Ki of 6.6 µM. Cell-based studies further show that harmine treatment inhibits castration-resistant prostate cancer cell (CRPC) proliferation with an IC50 of ∼14 µM. The CPRC cells treated with harmine resulted in reactivating silenced hypermethylated genes compared to the untreated cells, and harmine cooperated with an androgen antagonist, bicalutamide, to effectively inhibit the proliferation of CRPC cells. Our study thus reveals, for the first time, the inhibitory mechanism of harmine on DNMTs and highlights new strategies for developing novel DNMT inhibitors for cancer treatment.

Correction to "Synergistic Inhibition of SARS-CoV-2 Replication Using Disulfiram/Ebselen and Remdesivir".

[This corrects the article DOI: 10.1021/acsptsci.1c00022.].

Synergistic Inhibition of SARS-CoV-2 Replication Using Disulfiram/Ebselen and Remdesivir.

The SARS-CoV-2 replication and transcription complex (RTC) comprising nonstructural protein (nsp) 2-16 plays crucial roles in viral replication, reducing the efficacy of broad-spectrum nucleoside analog drugs such as remdesivir and evading innate immune responses. Most studies target a specific viral component of the RTC such as the main protease or the RNA-dependent RNA polymerase. In contrast, our strategy is to target multiple conserved domains of the RTC to prevent SARS-CoV-2 genome replication and to create a high barrier to viral resistance and/or evasion of antiviral drugs. We show that the clinically safe Zn-ejector drugs disulfiram and ebselen can target conserved Zn2+ sites in SARS-CoV-2 nsp13 and nsp14 and inhibit nsp13 ATPase and nsp14 exoribonuclease activities. As the SARS-CoV-2 nsp14 domain targeted by disulfiram/ebselen is involved in RNA fidelity control, our strategy allows coupling of the Zn-ejector drug with a broad-spectrum nucleoside analog that would otherwise be excised by the nsp14 proofreading domain. As proof-of-concept, we show that disulfiram/ebselen, when combined with remdesivir, can synergistically inhibit SARS-CoV-2 replication in Vero E6 cells. We present a mechanism of action and the advantages of our multitargeting strategy, which can be applied to any type of coronavirus with conserved Zn2+ sites.