Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development.
Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development.
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DOI：
10.1128/spectrum.00439-22
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发表时间：
2022-04-27
影响因子：
3.7
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中科院分区：
文献类型：
--
作者：
新生隐球菌是一种主要的真菌病原体，在免疫功能低下的人群中经常会导致危及生命的脑膜炎。这种酵母菌对棘球绦虫药物卡泊芬净具有高度抗药性。以往的研究表明，隐球菌脂转位酶(Flippase)是该真菌对卡泊芬净产生抗性所必需的。携带脂质翻转酶亚单位CDC50缺失的突变体对卡泊芬净的敏感度增加。在这里，我们设计了一种针对P4-ATPase功能的抗菌肽。我们合成了基于CDC50环区的稳定多肽，通过阻断Flippase功能来鉴定能够对卡泊芬净增敏的多肽，发现基于AS15序列的肉豆蔻化多肽在高浓度下是有效的。修饰多肽“AW9-mA”对H99野生型的最低抑菌浓度为 μg/mL，与卡泊芬净合用时的分数抑菌浓度指数为0.5。最值得注意的是，在aw9-mA多肽存在下，新生弧菌野生型对卡泊芬净高度敏感，其最低抑菌浓度为4 μg/m L，与cdc50Δ突变体相同。进一步的流式细胞仪检测显示，脂质翻转酶活性受到抑制，磷脂酰丝氨酸在细胞膜表面显著积累。利用荧光标记的多肽，我们证实该多肽与mCherry标记的P4-ATPase蛋白APT1在新生梭菌中是共定位的。AW9序列的构效关系研究表明，多肽上的两个赖氨酸残基可能是与P4-ATPase相互作用的原因，因此对其抗真菌活性至关重要。重要性作者开发了一种先导化合物多肽抗真菌药物，靶向是来自生物新生隐球菌的一种蛋白质。药物与目标真菌蛋白的结合使带电的脂类分子保留在表面。这种多肽与现有的抗真菌药物卡泊芬净有协同作用。棘球菌素类药物，如卡泊芬净，是现有的为数不多的抗真菌药物之一。由于所需的高浓度，卡泊芬净很少用于治疗新生葡萄球菌感染。作者认为，他们的新化合物提供了一种降低治疗此类感染所需卡泊芬净浓度的方法，从而为这些抗真菌药物的更大用途打开了可能性。
Cryptococcus neoformans is a major fungal pathogen that often causes life-threatening meningitis in immunocompromised populations. This yeast pathogen is highly resistant to the echinocandin drug caspofungin. Previous studies showed that Cryptococcus lipid translocase (flippase) is required for the caspofungin resistance of that fungus. Mutants with a deleted subunit of lipid flippase, Cdc50, showed increased sensitivity to caspofungin. Here we designed an antifungal peptide targeting the P4-ATPase function. We synthesized stable peptides based on the Cdc50 loop region to identify peptides that can sensitize caspofungin by blocking flippase function and found that myristylated peptides based on the “AS15 sequence” was effective at high concentrations. A modified peptide, “AW9-Ma” showed a MIC of 64 μg/mL against H99 wild type and a fractional inhibitory concentration (FIC) index value of 0.5 when used in combination with caspofungin. Most notably, in the presence of the AW9-Ma peptide, C. neoformans wild type was highly sensitive to caspofungin with a MIC of 4 μg/mL, the same as the cdc50Δ mutant. Further assays with flow cytometry showed inhibition of the lipid flippase enzyme activity and significant accumulation of phosphatidylserine on the cell membrane surface. Using a fluorescently labeled peptide, we confirmed that the peptide co-localized with mCherry-tagged P4-ATPase protein Apt1 in C. neoformans. Structure-activity relationship studies of the AW9 sequence showed that two lysine residues on the peptide are likely responsible for the interaction with the P4-ATPase, hence critical for its antifungal activity. IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal.
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影响因子：
3.3
作者：
Bahn, YS;Kojima, K;Heitman, J
通讯作者：
Heitman, J
影响因子：
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作者：
Konarzewska, Paulina;Wang, Yina;Xue, Chaoyang
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影响因子：
2.9
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影响因子：
4.9
作者：
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Selitrennikoff, CP
影响因子：
56.9
作者：
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通讯作者：
Nureki, Osamu