NADPH-Cytochrome P450 Reductase Ccr1 Is a Target of Tamoxifen and Participates in Its Antifungal Activity via Regulating Cell Wall Integrity in Fission Yeast

Abstract

Invasive fungal diseases are a leading cause of mortality among immune-compromised populations. Treatment is notoriously difficult due to the limited number of antifungal drugs as well as the emergence of drug resistance. Tamoxifen (TAM), a selective estrogen receptor modulator frequently used for treatment of breast cancer, has been found to have antifungal activities, and may be a useful addition to treat fungal infectious diseases. However, its molecular mechanisms underlying the antifungal actions remain obscure. Here, we screened for mutations that conferred sensitivity to antifungal drug azoles by using fission yeast as a model, and isolated the cls1 (ccr1) mutant, an allele of the gene encoding NADPH-cytochrome P450 reductase Ccr1. We found that deletion of ccr1+ gene exhibited hypersensitivities to various drugs, including antifungal drugs azoles, terbinafine, micafungin, immunosuppressor FK506, anticancer drugs TAM and 5-fluorouracil (5-FU). Unexpectedly, overexpression of Ccr1 caused yeast cells resistance to TAM, but not the other drugs tested here. Additionally, deletion of Ccr1 displayed pleiotropic phenotypes, including defects in cell wall integrity and vacuole fusion, enhanced calcineurin activity as well as increased intracellular Ca2+ levels. Overexpression of the constitutively active calcineurin suppressed the drug sensitive phenotypes of the Δccr1 cells. Notably, TAM treatment on wild-type cells resulted in pleiotropic phenotypes, similar to that of Ccr1 lacking. Furthermore, TAM inhibited NADPH-cytochrome P450 reductase activities Ccr1 activity in a dose-dependent manner. Moreover, TAM treatment also inhibited NADPH-cytochrome P450 reductase activities of Candida albicans and resulted in defective cell wall integrity. Collectively, our findings suggest that Ccr1 is a novel target of TAM and is involved in the antifungal activity of TAM through regulating cell wall integrity in fission yeast.

Myriocin enhances the antifungal activity of fluconazole by blocking the membrane localization of the efflux pump Cdr1

“…Therefore, a high concentration of tacrolimus (16 μg/ml) inhibited both calcineurin and TORC1 and decreased the MIC value (from 4 to 1 μg/ml) of FLC in the presence of FNZ ( Figure 2 , Supplementary Figure S2B ). Tamoxifen targets calmodulin ( Dolan et al, 2009 ), then blocks the calcineurin pathway and inhibits NADPH-cytochrome P450 reductase Ccr1 ( Liu et al, 2020 ) and ergosterol biosynthesis in fission yeast. Therefore, tamoxifen could improve the antifungal activity of FLC at a high concentration (16 μg/ml) ( Figure 2 , Supplementary Figure S2C ) due to inhibiting ergosterol biosynthesis and then inactivation Cdr1 ( Pasrija et al, 2008 ).…”

Drug Repurposing in Medical Mycology: Identification of Compounds as Potential Antifungals to Overcome the Emergence of Multidrug-Resistant Fungi

“…In this way, tamoxifen-treated yeasts showed cell lysis and an alteration of fungal development [61,89]. Recently, the interaction between tamoxifen and its target Ccr1 has also been described as causing the disruption of cell wall integrity [62].…”

Phosphoinositide-Dependent Protein Kinases Regulate Cell Cycle Progression Through the SAD Kinase Cdr2 in Fission Yeast

“…The fission yeast Schizosaccharomyces pombe ( S. pombe ) has been known for years to be an excellent model system for the investigation of the fundamental regulations and control mechanisms of various cellular processes, due to its high homology with mammals and advantages in gene manipulation ( Forsburg, 2005 ; Liu et al, 2018 , 2020 ; Jiang G. et al, 2021 ; Vyas et al, 2021 ). Ksg1 ( k inase responsible for s porulation and g rowth 1), which is essential for the cell viability in fission yeast, is a homologous protein of mammalian PDK1 ( Niederberger and Schweingruber, 1999 ).…”