SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA, USA.
Int J Parasitol. 2017 Oct;47(12):811-821. doi: 10.1016/j.ijpara.2017.08.005. Epub 2017 Sep 9.
Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.
贝氏巴贝斯虫是一种顶复门寄生虫，可引起牛贝氏巴贝斯虫病，这是一种慢性且使人虚弱的疾病，可导致公牛出现全身和皮肤症状以及不育。目前尚无治疗方法或疫苗。在寻找治疗候选药物的过程中，钙依赖性蛋白激酶已成为其他顶复门寄生虫（例如，新孢子虫、刚地弓形虫、疟原虫和艾美耳球虫）有希望的药物靶点，并且被 bumped 激酶抑制剂有效地靶向。在这项研究中，我们鉴定并克隆了编码 BbCDPK1 的基因。通过荧光素酶测定法评估了九种 bumped 激酶抑制剂类似物文库对重组 BbCDPK1 活性的影响。之后，进一步筛选了这些化合物对在 Marc-145 细胞中生长的贝氏巴贝斯虫速殖子的疗效。在 5µM 的初步测试中，有 8 种化合物显示出超过 90%的入侵和增殖抑制作用。进一步对化合物 BKI 1294、1517、1553 和 1571 进行了表征，通过 3 天增殖测定的实时定量聚合酶链反应确定了 EC 值（1294：2.38µM；1517：2.20µM；1553：3.34µM；1571：2.78µM）。在高达 48 小时的时间内，用 EC 浓度的这些药物暴露于受感染的培养物中并没有杀寄生虫作用。电镜观察证实了 bumped 激酶抑制剂处理干扰了速殖子的细胞周期调节和非分离，导致形成大的多核复合物，这些复合物与寄生空泡内的存活寄生虫共存，从而证实了没有杀寄生虫作用。然而，在面对主动免疫反应的情况下，寄生虫清除可能会发生。总之，bumped 激酶抑制剂可能是控制贝氏巴贝斯虫感染的有效候选药物。应计划进一步进行体内实验，因为已经证明 BKIs 1294、1517 和 1553 在小牛中可以达到并维持治疗性血浆水平，而没有毒性。
