<p dir="ltr">Human babesiosis is a malaria-like, tick-borne infectious disease of major public health importance with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is primarily caused by Babesia microti and Babesia duncani. Of these parasites, B. duncani infection is lethal to susceptible patients. Current treatment for babesiosis includes either the synergistic use of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects and the resistance posed by these parasites called for alternative approaches for the treatment of human babesiosis. Parasite-derived proteases play several functions in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression and cell migration. We hypothesized that proteases are promising class of drug targets in Babesia parasites. Using the SYBR-Green assay, we screened a protease inhibitor library consists of 160 compounds against B. duncani in vitro culture at 50µM and identified 13 preliminary hits. Additionally, dose response assays of hit compounds against <i>B. duncani</i> and <i>B. microti</i> in vitro cultures identified 5 compounds as effective inhibitors against parasite growth. Of these 5 compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for further studies based on its lower IC50 of 58nM as well as a higher therapeutic index as compared to other hit compounds. We demonstrated that in a mouse model infected with <i>target,</i>, the most effective inhibitor, the prodrug of ixazomib at a low dose of 2.5mg/kg lowers parasite proliferation without causing any adverse effects in animals. Thus, our studies suggest that Babesia proteasome may be an important drug target, and ixazomib may be a potential compound that may be used for the treatment of human babesiosis.</p>

漢厚朴酚是從木蘭科植物中萃取之天然化合物，已知具有抗氧化、抗發炎及神經保護之生理活性功能。先前的研究證明漢厚朴酚可以保護多巴胺神經元對抗6-OHDA所引起的細胞傷害，並且可以減緩6-OHDA 動物模式由apomorphine所誘發的旋轉行為，但漢厚朴酚對於神經保護之分子機制的相關研究尚未釐清。蛋白激酶CK2是具有多功能的絲氨酸/蘇氨酸激酶，高度表現在大腦紋狀體中，先前的研究證實蛋白激酶CK2參與調節神經系統功能和具有神經保護之作用。先前研究也指出轉錄因子Nrf1（Nuclear factor E2-related factor 1）是蛋白激酶CK2下游磷酸化受質，會調控小鼠胚胎纖維細胞中蛋白酶體基因的表現。抗細胞凋亡蛋白Mcl-1 (myeloid cell leukemia 1) 屬於Bcl-2蛋白家族的成員之一，在細胞凋亡的過程中，其蛋白含量減少與細胞凋亡有密切關聯性，抑制Mcl-1蛋白的降解可以延遲細胞死亡。因此本論文主要探討漢厚朴酚的神經細胞保護機制是否透過CK2-Nrf1細胞訊息路徑調控蛋白酶體活性，進而減少Mcl-1的降解速率。實驗結果顯示，轉染CK2α-EGFP DNA質體會增加Nrf1磷酸化並抑制蛋白酶體活性，泛素化之Mcl-1蛋白含量亦伴隨增加；轉染CK2α siRNA則會降低Nrf1磷酸化並促進蛋白酶體活性，導致naive Mcl-1蛋白質含量減少24小時的漢厚朴酚後處理（post-treatment）可以部份恢復因轉染CK2α siRNA所造成之CK2蛋白、Phosphoserine蛋白和Mcl-1蛋白質含量減少，在設計縮短間隔5小時漢厚朴酚後處理（post-treatment）的實驗結果雖然仍無法有效恢復CK2蛋白含量，但對於Phosphoserine和Mcl-1蛋白含量以及蛋白酶體活性則具有部份恢復的功效。利用過氧化氫造成細胞氧化壓力環境下，實驗發現間隔3小時的漢厚朴酚後處理才能有效恢復細胞存活率，間隔5小時的漢厚朴酚後處理則無法恢復細胞存活率。在大白鼠紋狀體腦區給予漢厚朴酚微量注射則對pTH、TH和GAD蛋白質含量皆有促進增加的作用，乙醯化的Histone H3蛋白含量也有顯著增加。綜合以上結果，推測漢厚朴酚對細胞保護作用的其中一個機制是參與調控CK2-Nrf1路徑而抑制蛋白酶體活性，減少Mcl-1蛋白質降解速率和提升氧化壓力下之細胞存活能力；此外，從活體動物的實驗結果顯示漢厚朴酚亦可能參與調控多巴胺和γ-氨基丁酸神經細胞功能的機制之中。 / Honokiol is a natural compound, extracted from the Magnolia officinalis, and is known as its anti-oxidative, anti-inflammatory and neuroprotective effects. The previous study has been demonstrated that the honokiol can protect striatal dopamine neuron against 6-OHDA induced damage and reverse the apomorphine-induced rotational behavior in Parkinson’s disease model of rats. However, the cellular mechanisms for its neuroprotective effects are not fully investigated. Protein kinase Casine kinase 2 (CK2) is a serine/threonine kinase has a highly abundant expression in the striatum compared with other brain areas. Further, CK2 is shown to regulate many neuronal functions including neuroprotection. The nuclear factor E2-related factor 1 (Nrf1) has been identified as one of the substrate proteins for CK2 and is indicated to involve in the induction of proteasome subunits gene expressions in mouse embryonic fibroblasts. The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) is shown to play a critical initiation role during the apoptosis process due to its synthesis blockage and proteasome degradation. The present study is aimed to investigate whether one of protective effects of honokiol is through CK2-mediated Nrf1 signaling pathway to regulate the proteasome activity in the mouse N2a neuroblastoma cell line. In the current results, transfection of the CK2α-EGFP plasmid DNA increased Nrf1 phosphorylation accompanied with the decrease in the proteasome activity but increased the ubiquitinated Mcl-1 protein. Whereas, transfection of CK2α siRNA decreased Nrf1 phosphorylation leading to the increase in proteasome activity and Mcl-1 protein degradation. The 24 hr duration of honokiol post-treatment only slightly reversed the knock-down effect of CK2α siRNA on CK2α and Mcl-1 protein levels. However, 5 hr duration of honokiol post-treatment could partially reverse the Mcl-1 protein level and proteasome activity but no effect on CK2α protein levels. In the H2O2-induced oxidative stress condition, only 3 hr duration of honokiol post-treatment could protect cells against H2O2-induced cell death. In the experiments of in vivo rat animal model, local administration of honokiol was found to increase phospho-TH, naive TH, GAD as well as acetylated Histone H3 protein levels. These above results suggest one of the protective mechanisms of honokiol might be through CK2-mediated Nrf1 signaling to inhibit the proteasome activity. and to promote cell survival under oxidative stress. Beside these functions, honokiol might also involve in the regulation of nurophysiological functions of dopamine and GABA neurons.