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Lem3p对于烷基磷酸胆碱类药物(依地福新和米替福新)的摄取和效力至关重要。

Lem3p is essential for the uptake and potency of alkylphosphocholine drugs, edelfosine and miltefosine.

作者信息

Hanson Pamela K, Malone Lynn, Birchmore Jennifer L, Nichols J Wylie

机构信息

Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

出版信息

J Biol Chem. 2003 Sep 19;278(38):36041-50. doi: 10.1074/jbc.M305263200. Epub 2003 Jul 3.

Abstract

The alkylphosphocholine class of drugs, including edelfosine and miltefosine, has recently shown promise in the treatment of protozoal and fungal diseases, most notably, leishmaniasis. One of the major barriers to successful treatment of these infections is the development of drug resistance. To understand better the mechanisms underlying the development of drug resistance, we performed a combined mutant selection and screen in Saccharomyces cerevisiae, designed to identify genes that confer resistance to the alkylphosphocholine drugs by inhibiting their transport across the plasma membrane. Mutagenized cells were first selected for resistance to edelfosine, and the initial collection of mutants was screened a second time for defects in internalization of a short chain, fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD))-labeled phosphatidylcholine reporter. This approach identified mutations in a single gene, YNL323W/LEM3, that conferred resistance to alkylphosphocholine drugs and inhibited internalization of NBD-labeled phosphatidylcholine. Loss of YNL323W/LEM3 does not confer resistance to N-nitroquinilone N-oxide or ketoconazole and actually increases sensitivity to cycloheximide. The defect in internalization is specific to NBD-labeled phosphatidylcholine and phosphatidylethanolamine. Labeled phosphatidylserine is internalized at normal levels in lem3 strains. LEM3 is a member of an evolutionarily conserved family and has two homologues in S. cerevisiae. Single point mutations that produce resistance to alkylphosphocholine drugs and inhibition of NBD-labeled phosphatidylcholine internalization were identified in several highly conserved domains. These data demonstrate a requirement for Lem3p expression for normal phosphatidylcholine and alkylphosphocholine drug transport across the plasma membrane of yeast.

摘要

包括依地福新和米替福新在内的烷基磷胆碱类药物最近在治疗原生动物和真菌疾病,尤其是利什曼病方面显示出前景。成功治疗这些感染的主要障碍之一是耐药性的产生。为了更好地理解耐药性产生的潜在机制,我们在酿酒酵母中进行了联合突变体选择和筛选,旨在鉴定通过抑制烷基磷胆碱类药物跨质膜转运而赋予耐药性的基因。首先选择诱变细胞使其对依地福新产生耐药性,然后对最初收集的突变体进行第二次筛选,以检测短链、荧光(7-硝基苯并-2-恶唑-1,3-二氮杂-4-基(NBD))标记的磷脂酰胆碱报告分子内化缺陷。这种方法鉴定出一个单一基因YNL323W/LEM3中的突变,该突变赋予对烷基磷胆碱类药物的耐药性并抑制NBD标记的磷脂酰胆碱的内化。YNL323W/LEM3的缺失不会赋予对N-硝基喹啉N-氧化物或酮康唑的耐药性,实际上还会增加对环己酰亚胺的敏感性。内化缺陷特异性针对NBD标记的磷脂酰胆碱和磷脂酰乙醇胺。标记的磷脂酰丝氨酸在lem3菌株中以正常水平内化。LEM3是一个进化上保守的家族成员,在酿酒酵母中有两个同源物。在几个高度保守的结构域中鉴定出了产生对烷基磷胆碱类药物耐药性并抑制NBD标记的磷脂酰胆碱内化的单点突变。这些数据表明Lem3p表达对于酵母质膜上正常的磷脂酰胆碱和烷基磷胆碱类药物转运是必需的。

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