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首页> 外文期刊>Journal of Pharmacy and Pharmaceutical Sciences >Optimisation and Validation of a High Throughput Screening Compatible Assay to Identify Inhibitors of the Plasma Membrane Calcium ATPase Pump - a Novel Therapeutic Target for Contraception and Malaria
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Optimisation and Validation of a High Throughput Screening Compatible Assay to Identify Inhibitors of the Plasma Membrane Calcium ATPase Pump - a Novel Therapeutic Target for Contraception and Malaria

机译：高通量筛选兼容测定法的优化和验证，以鉴定血浆膜钙ATPase泵的抑制剂-避孕和疟疾的新型治疗靶标

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Purpose. ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF? Transcreener? ADP (TR-FRET) assay to screen a drug library. Methods and Results. PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/μl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1μM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. Conclusions. We have optimised the HTRF? Transcreener? ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on on the issue’s contents page.
机译：目的。 ATP酶是人体离子转运的主要类别，具有多种重要的生物学和病理学作用。但是，缺乏用于ATPase的高通量检测方法已大大限制了该领域的药物发现。我们最近发现，ATP依赖性钙泵PMCA4（质膜钙/钙调蛋白依赖性ATPase，亚型4）的基因缺失会导致雄性小鼠的不育，这是由于精子活力的选择性缺陷所致。另外，人类的最新发现表明，PMCA4基因中的单核苷酸多态性（SNP）决定了对疟原虫感染的易感性。因此，迫切需要开发特定的PMCA4抑制剂。在当前研究中，我们旨在优化和验证使用重组表达的PMCA4和HTRF？的高通量筛选兼容测定法。转写器？ ADP（TR-FRET）分析以筛选药物库。方法和结果。由过表达PMCA4的HEK293细胞制备PMCA4膜微粒体。蛋白质印迹定量显示，与感染LacZ（对照病毒）的细胞相比，PMCA4的表达增加了近9倍。在TR-FRET分析中，以15ng /μl微粒体浓度，在37°C下与化合物进行30分钟预孵育以及在1mM EGTA钙缓冲液中提供1μM游离钙的条件下，可实现最大的PMCA4微粒体活性。最后，在最佳条件下，羧曙红（一种非特异性PMCA抑制剂）的剂量反应曲线显示出显着的PMCA4抑制作用。在确认该测定法适合进行高通量筛选后，我们针对PMCA4测定法筛选了ChemBioNet小分子文库（约21,000种化合物），以鉴定那些明显的抑制剂。这次筛选产生了1,494个主要匹配。结论。我们已经优化了HTRF？转写器？ ADP分析用于高通量筛选，以鉴定PMCA4抑制剂。筛查活动的结果提供了初步的化学起点，可以将其进一步开发为用于非激素避孕或抗疟疾治疗的特定PMCA4抑制剂。本文对发布后审查开放。已注册的读者（请参阅“针对读者”）可以通过单击问题的内容页面来发表评论。

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