Dihydrocoumarin (DHC) is a compound found in Melilotus officinalis (sweet clover) that has been found to inhibit the NAD-dependent histone deacetylase (HDAC) Sir2 in yeast. Because HDAC inhibitors inhibit the DNA damage response (DDR), DHC might suppress the DNA repair machinery by inhibiting HDAC activity. However, these activities have not been identified due to the sequence-independent nature of radiation or chemotherapy drug-induced damage.

Dihydrocoumarin as an HDAC Inhibitor

Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC), a flavoring agent, causes deficiencies in double-stand break (DSB) repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC), was found to mimic dihydrocoumarin -induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of dihydrocoumarin in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.[1]

Targeting DNA repair pathways is an increasingly popular strategy for improving the efficacy of DNA damage-based cancer therapy. Although yeasts are not a good model for cancer research, we exploited the functional conservation of the DNA repair pathways between yeast and humans to mechanistically identify inhibitors of DNA repair proteins in yeast that could be extended to human use in the future. Our results show that DHC increases DNA damage sensitivity by suppressing DNA repair pathways, which suggests that dihydrocoumarin might have potential uses as a chemo- or radiosensitizer. Targeting human RAD52 represents a selective therapeutic approach for BRCA2-deficient cancers due to the synthetic lethality of RAD52 and BRCA2. In this study, we provide the first demonstration that the inhibitory activity of DHC toward the recombination protein Rad52 is critical for dihydrocoumarin -mediated sensitivity to DNA damage. Therefore, dihydrocoumarin can potentially be used for sensitizing BRCA2-deficient cancer cells. Our research supports the preclinical relevance of identifying molecular targets for DNA damage repair proteins, which will be of paramount importance in devising future therapeutic interventions. Scientists noticed that wild-type cells are extremely sensitive to DHC combined with MMS but only slightly sensitive to dihydrocoumarin in combination with 4NQO or UV.

It is known that MMS generates single- and double-strand breaks that can be repaired by HR, whereas 4NQO or UV generates cross-linked DNA that can be repaired by nucleotide excision repair (NER). Therefore, this phenomenon indicates that dihydrocoumarin might inhibit DNA repair pathways mainly by regulating HR-related protein(s). HDAC inhibitors, such as valproic acid (VPA) and curcumin, have been reported, and these can effectively inhibit HR repair and sensitize cells to DNA damage. In this study, we also revealed that dihydrocoumarin, an effective HDAC inhibitor, inhibits DNA repair and increases DNA damage sensitivity in yeast. Reportedly, DHC inhibits NAD-dependent HDAC Sir2 in yeast. However, the expression of Rad52 recombinase is not suppressed in sir2 mutants; therefore, it is unlikely that DHC downregulates homologous recombination by Sir2 inhibition. Whether DHC Influences the DNA damage checkpoint through a Sir2-dependent pathway needs to be further tested. Surprisingly, our results showed that dihydrocoumarin attenuates DNA repair through Rad52 recombinase inhibition, which is a Rpd3-dependent process. Robert et al. reported that VPA, an HDAC inhibitor with a different mechanism than dihydrocoumarin, inhibits both Rpd3 and Hda1 and triggers Sae2 degradation to suppress DNA end resection by increasing the acetylation of Sae2. In this study, we found that rpd3 deletion alone mimics the effect of DHC treatment in Rad52 regulation, which suggests that DHC might inhibit Rad52 through its Rpd3 inhibitor activity. HDAC inhibition often results in the activation of gene transcription by hyperacetylating chromatin and loosening DNA structure, but the mechanism through which rpd3 mutants and DHC treatment downregulate Rad52 needs further investigation.

The Flavoring Agent Dihydrocoumarin

Sirtuins are a family of phylogenetically conserved nicotinamide adenine dinucleotide-dependent deacetylases that have a firmly established role in aging. Using a simple Saccharomyces cerevisiae yeast heterochromatic derepression assay, we tested a number of environmental chemicals to address the possibility that humans are exposed to sirtuin inhibitors. Here we show that dihydrocoumarin (DHC), a compound found in Melilotus officinalis (sweet clover) that is commonly added to food and cosmetics, disrupted heterochromatic silencing and inhibited yeast Sir2p as well as human SIRT1 deacetylase activity. Dihydrocoumarin exposure in the human TK6 lymphoblastoid cell line also caused concentration-dependent increases in p53 acetylation and cytotoxicity. Flow cytometric analysis to detect annexin V binding to phosphatidylserine demonstrated that DHC increased apoptosis more than 3-fold over controls. Thus, dihydrocoumarin inhibits both yeast Sir2p and human SIRT1 deacetylases and increases p53 acetylation and apoptosis, a phenotype associated with senescence and aging. These findings demonstrate that humans are potentially exposed to epigenetic toxicants that inhibit sirtuin deacetylases.

After screening more than 100 environmental chemicals to which humans are exposed, including coumarins, bioflavonoids, benzene metabolites, and arsenic, we identified that DHC disrupted heterochromatic repression. Further analyses demonstrated that dihydrocoumarin -mediated heterochromatic derepression caused yeast colony formation in a concentration-dependent manner that was similar to splitomicin. Splitomicin is an established Sir2p inhibitor with a structure similar to dihydrocoumarin, thus it was likely that DHC-mediated heterochromatic derepression in the mating assay was due to Sir2p inhibition. Further experiments with an overexpressing SIR2-induced death phenotype were conducted to identify if Sir2p was the target of DHC. DHC-mediated reversal of the SIR2 overexpressed death phenotype indicated that DHC is a Sir2p inhibitor and that this inhibition was responsible for the heterochromatic derepression observed in the mating assay. DHC thus joins a short list of established Sir2p inhibitors that includes nicotinamide, splitomicin, and sirtinol.[2]

One-Pot Synthesis of 3,4-Dihydrocoumarins via C-H Oxidation/Conjugate Addition/Cyclization Cascade Reaction

The dihydrocoumarin core is present as a characteristic structural motif to possess a broad range of biological activities. Particularly, 3,4-dihydrocoumarins have attracted considerable attention due to their various pharmacological properties, such as antiviral, anti-inflammatory, and anticancer effects. Therefore, various novel methods for the synthesis of 3,4-dihydrocoumarins have been developed. The most general protocol for the synthesis of 3,4-dihydrocoumarins is the [4 + 2] cycloaddition of ortho-quinone methides (o-QMs). To the best of our knowledge, the single-pot synthesis of dihydrocoumarins from the in situ generation of o-QMs via C–H oxidation of benzyl phenol has not been reported. Therefore, we envisioned a one-pot synthesis of 3,4-dihydrocoumarin derivatives via C–H oxidation and ring closure cascade of 2-benzyl phenol with oxazolone under Br?nsted acid conditions. In connection with our work on conjugate addition reactions, we reported the Michael-type addition/ring closure sequences of o-QM with dipoles. Herein, we describe the one-pot synthesis of 3,4-dihydrocoumarin derivatives from 2-benzyl phenol via C–H oxidation and acid-catalyzed ring closure cascade.[3]

The 3,4-dihydrocoumarin derivatives were obtained from 2-alkyl phenols and oxazolones via C–H oxidation and cyclization cascade in the presence of silver oxide (Ag2O) and p-toluenesulfonic acid as a Br?nsted acid catalyst. This approach provides a one-pot strategy to synthesize the multisubstituted 3,4-dihydrocoumarins with moderate to high yields (64–81%) and excellent diastereoselectivity (>20:1)

References

[1]Chen CC, Huang JS, Wang TH, Kuo CH, Wang CJ, Wang SH, Leu YL. Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52. Int J Mol Sci. 2017 Dec 7;18(12):2655.

[2]Olaharski AJ, Rine J, Marshall BL, Babiarz J, Zhang L, Verdin E, Smith MT. The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases. PLoS Genet. 2005 Dec;1(6):e77.

[3]Kim DY. One-Pot Synthesis of 3,4-Dihydrocoumarins via C-H Oxidation/Conjugate Addition/Cyclization Cascade Reaction. Molecules. 2023 Sep 28;28(19):6853.