在白色念珠菌中,Cdr1将唑类药物从细胞中泵出,以减少有害浓度的细胞内积累,导致唑类药物耐药性。米伯霉素肟,一种兽用抗寄生虫药,强烈且特异性地抑制Cdr1。然而,Cdr1如何识别和出口唑类药物,以及米尔贝霉素肟如何抑制Cdr1仍不清楚。这里,我们报道了Cdr1在不同状态下的三种低温EM结构:apo态(Cdr1Apo),氟康唑结合态(Cdr1Flu),和米尔贝霉素肟抑制状态(Cdr1Mil)。氟康唑底物和米尔贝霉素肟抑制剂都主要通过疏水相互作用在Cdr1的中心腔内被识别。建议氟康唑通过TM2,TM5,TM8和TM11驱动的横向途径从结合位点输出到环境中。我们的发现揭示了米尔贝霉素肟的抑制机制,通过竞争抑制了Cdr1,阻碍出口,并阻碍基材进入。这些发现促进了我们对白色念珠菌中Cdr1介导的唑类耐药性的理解,并为开发针对Cdr1的创新抗真菌药物以对抗唑类耐药性奠定了基础。
In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1Apo), fluconazole-bound state (Cdr1Flu), and milbemycin oxime-inhibited state (Cdr1Mil). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance.