TW202302093A - 含有麥角硒因的covid 19治療或預防藥 - Google Patents
含有麥角硒因的covid 19治療或預防藥 Download PDFInfo
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- TW202302093A TW202302093A TW111106368A TW111106368A TW202302093A TW 202302093 A TW202302093 A TW 202302093A TW 111106368 A TW111106368 A TW 111106368A TW 111106368 A TW111106368 A TW 111106368A TW 202302093 A TW202302093 A TW 202302093A
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Abstract
本發明之目的為提供由SARS-CoV-2引起之感染症(COVID19)的治療或預防用組成物。本發明基於確立具有SARS-CoV2之蛋白酶的抑制活性之藥劑的篩選方法,及發現麥角硒因具有SARS-CoV2之蛋白酶的抑制活性,而提供包含麥角硒因的COVID19之治療或預防用組成物。
Description
本發明係關於含有麥角硒因(selenoneine)之COVID19治療或預防的技術領域。
對於2019年秋至2022年間引起廣泛流行之由SARS-CoV-2病毒所引起之COVID19,開發其疫苗或治療藥為當務之急。數種見到效果之疫苗正在開發中且被使用。另一方面,病疫學上顯示亞洲各國,包含日本,COVID19患者之重症化率及死亡率比歐洲各國低,因而思量何種要因賦予COVID19抗性。關於該要因之候選者,正在研究中。已知藉由體內之硒缺損,柯薩奇病毒或流感病毒等RNA病毒的毒性增高。從此種背景,有報導中國之都市間的硒狀態,與COVID19患者之結果的關連性(非專利文獻1:Am J Clin Nutr.2020 Jun 1;111(6):1297-1299.doi:10.1093/ajcn/nqaa095)。
冠狀病毒具有單股RNA作為基因組,若感染宿主細胞,從RNA基因組轉譯為長的多蛋白質。藉由將多蛋白質適當切斷,各片段變成具有作為病毒增殖所需要之構造蛋白質或酵素的功能,而使病毒增殖。就
作為多蛋白質切斷之主要觸媒的蛋白酶而言,可列舉主要蛋白酶(main protease)(Mpro)及木瓜酶樣蛋白酶(papain-like protease,亦稱類木瓜酶蛋白酶)(PLpro),此等蛋白酶,成為有力的藥物創作標的。根據對使用結晶解析之三次元模型之電腦篩選的結果,報導有幾種已存在之藥劑,可具有作為有效之Mpro抑制劑的功能(非專利文獻2:Nature(2020)vol.582(7811):289-293)。其中,為硒化合物之一的依布硒(ebselen),對於觸媒區域顯示優異的親和性,因此可期待被開發作為COVID19之治療藥(非專利文獻3:Sci.Adv.2020 6.eadb0345)。依布硒為分子內含有必需微量元素之一之硒的功能性分子。依布硒為潛在具有呈互變異構物之游離硒醇基的分子。藉由依布硒抑制Mpro之機制,研判係蛋白酶之活性中心的Cys145硫醇基,與依布硒之硒醇基形成共價鍵結。又,在電腦解析中,由於有機硒化合物對SARS-CoV-2之主要蛋白酶(Mpro)呈現高的鍵結親和性,顯示有機硒化合物可成為抗病毒藥的候選分子(非專利文獻4)。
[先前技術文獻]
[非專利文獻]
[非專利文獻1]Am J Clin Nutr. 2020 Jun 1; 111(6): 1297-1299. doi: 10.1093/ajcn/nqaa095
[非專利文獻2]Nature (2020) vol. 582(7811): 289-293
[非專利文獻3]Sci. Adv. 2020 6. eadb0345
[非專利文獻4]Chemrexiv (2020-07-02) DOI: 10.26434/chemrxiv. 12594134
本發明之目的為提供作為藥物創製標的之具有SARS-CoV-2之蛋白酶抑制活性的藥劑。
本發明人等著眼於SARS-CoV-2之主要蛋白酶(Mpro)與木瓜酶樣蛋白酶(PLpro)同為半胱胺酸蛋白酶,確立以木瓜酶抑制活性作為指標的篩選系統。藉由該篩選,篩選出具有比被特定為Mpro抑制劑的依布硒更高抑制活性的物質,即麥角硒因。再者,調製SARS-CoV-2之Mpro,確認藉由麥角硒因可抑制該蛋白酶活性,於是完成本發明。其中,本發明係關於下述事項:
[1]一種冠狀病毒之蛋白酶抑制劑,其包含麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽。
[2]如項目1記載之蛋白酶抑制劑,其中前述蛋白酶為主要蛋白酶或木瓜酶樣蛋白酶。
[3]如項目1記載之蛋白酶抑制劑,其中前述冠狀病毒為SARS-CoV2。
[4-1]一種冠狀病毒感染症之治療或預防用的組成物,其包含麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽。
[4-2]一種麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽,其係為了使用於冠狀病毒感染症之治療或預防。
[4-3]一種方法,於因冠狀病毒之感染之需要治療或預防之對象中用於治療或預防冠狀病毒感染症的方法,其中包含對前述對象,投與麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽。
[4-4]一種麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽的使用,其係用於冠狀病毒之治療藥或預防藥的製造。
[5]如項目[4-1]至[4-4]中任一項記載之發明,其中前述冠狀病毒感染症為COVID19。
[6]一種冠狀病毒感染症之治療或預防藥的篩選方法,其係以對木瓜酶之抑制活性作為指標。
[7]如項目6記載之方法,其中前述冠狀病毒感染症為COVID19。
[8]如項目6或7記載之方法,其中前述治療藥或預防藥,係抑制冠狀病毒之主要蛋白酶或木瓜酶樣蛋白酶。
麥角硒因發揮比依布硒高的Mpro抑制活性。又,麥角硒因與其他含硒化合物比較,發揮高Mpro抑制活性。
圖1為將依布硒對Mpro之活性中心之作用,以三次元模型表示的圖。依布硒互變異化所出現之硒醇基與半胱胺酸145進行共價鍵結。
圖2係展示為半胱胺酸蛋白酶之(1)Mpro之序列及活性中心之立體構造,與(2)木瓜酶之序列及活性中心之立體構造的比較。
圖3係展示為半胱胺酸蛋白酶之(1)PLpro之序列及活性中心之立體構造,與(2)木瓜酶之序列及活性中心之立體構造的比較。
圖4展示於依布硒或麥角硒因存在下或不存在下的木瓜酶酵素活性。藉由木瓜酶酵素活性,螢光基質分解,隨著時間經過,螢光強度變高。
圖5展示依布硒或麥角硒因對木瓜酶酵素活性之抑制曲線。
圖6展示搭載Mpro之基因之質體的示意圖。
圖7展示將藉由大腸菌表現,以His標籤精製之Mpro供於SDS-PAGE的結果。33.8kDa之帶域相當於Mpro之帶域。
圖8展示依布硒、麥角硫鹼(ergothioneine)或麥角硒因之存在下或不存在下的Mpro之蛋白酶活性。藉由Mpro,螢光基質分解,隨著時間經過而螢光強度變高。
圖9展示藉由依布硒、麥角硫鹼或麥角硒因的Mpro之蛋白酶活性的抑制曲線。
圖10展示藉由試驗化合物(麥角硒因、依布硒、硒代胱胺酸((SeCys)2)、甲基硒代半胱胺酸(MeSeCys)、硒代甲硫胺酸(SeMet)、二苯基二硒化物(PhSeSePh)、亞硒酸鈉(selenite))對Mpro之抑制活性。
以下,詳細地說明本發明之實施型態(以下,稱為「本實施型態」。),然而本發明不受此等之限定,在不脫逸其要旨的範圍,可進行各種變更。
本發明係關於一種冠狀病毒之蛋白酶抑制劑或者冠狀病毒感染症之治療或預防用組成物,其包含麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽。
在本發明中,麥角硒因為下列之化學名稱:2-氫硒基-Nα,Nα,Nα-三甲基-L-組胺酸(2-selenyl-Nα,Nα,Nα-trimethyl-L-histidine)的化合物。具體而言,意指下列之式(I)所表示的化合物:
分子中之OH基、NH基等可為無氫原子之狀態,亦即,離子化狀態。此化合物能以任何光學異構物、幾何異構物、互變異構物、或二聚體、或者此等之混合物存在。互變異構化及二量化之結果、麥角硒因可採取下述之式(I)至(III)的任何型態:
在本發明中,麥角硒因能以任何比率,含有式(I)至(III)之型態的化合物。二聚化之化合物可依隨周圍環境,還原為單體。又,為了維持單體,包含式(I)或(II)所表示之化合物的組成物可進一步含有還原劑。就此種還原劑而言,可使用任何還原劑,舉一例言之,可使用麩胱甘肽(glutathione)(GSH)、二硫蘇糖醇(DTT)、巰基乙醇。麥角硒因為大量包含於鮪魚類、旗魚類、鯖魚類等之含血肉中的成分,為可於日常攝取之成分。
若為本技術領域人士,可將麥角硒因以適宜方法製造。就麥角硒因之製造方法而言,可依照化學合成方法(Angew.Chem.Int.Ed.2019,58,1-6)製造,亦可藉由從含有麥角硒因之生物組織萃取,或使用微生物發酵而製造。例如,可列舉如日本專利第5669056號公報記載的從魷魚類、魚類、鳥類、哺乳類等之組織萃取的方法;Pluskal T氏等之文獻(Pluskal T et al.,PLoS One 2014 May 14;9(5):e97774)記載的利用導入參與麥角硫鹼生合成系統之基因的分裂酵母之栗酒裂殖酵母(Schizosaccharomyces pombe)的方法;國際公開第2017/026173號記載的
將組胺酸及硒化合物利用過度表現編碼麥角硒因合成酵素之基因的醬油麴菌(Aspergillus sojae)、米麴菌(Aspergillus oryzae)、黑麴菌(Aspergillus niger)等之麴菌屬微生物或大腸菌(Escherichia coli)等之轉形體生產的方法等。其中,在將麥角硒因以工業化規模生產的情況,從以高收量生產麥角硒因的觀點而言,以國際公開第2017/026173號記載之方法為較佳。
再者,國際公開第2017/026173號小冊子記載之使用將麥角硒因合成酵素過度表現之轉形體的方法中,麥角硫鹼與麥角硒因同時生成,由於將此等分離有困難,所得到的含有麥角硒因之轉形體萃取物,除麥角硒因之外,可含有麥角硫鹼。只要能得到,麥角硒因以精製之麥角硒因為較佳。麥角硒因可藉由HPLC等本技術領域人士周知的手法精製。
冠狀病毒之蛋白酶,較佳為關於SARS-CoV2之蛋白酶。就冠狀病毒之蛋白酶而言,可列舉主要蛋白酶(Mpro)、及木瓜酶樣蛋白酶(PLpro)。從藥物創製標的之觀點而言,以SARS-CoV2之主要蛋白酶(Mpro)為較佳。
主要蛋白酶亦稱為3Clpro、非構造蛋白質5(nsp5),其係參與多蛋白質分解的主要蛋白酶。主要蛋白酶為半胱胺酸蛋白酶,具有由相同次單元(subunit)構成之二聚體的功能。SARS-CoV2之主要蛋白酶具有由306個殘基構成的胺基酸序列(序列編號1)。由該序列之Cysl45與His41所形成的活性中心為已知,研判依布硒之硒醇基,對Cys145之硫醇基進行共價鍵結(圖1)。由於為含有硒之化合物的麥角硒因,亦具有硒醇基,所以可與Mpro之活性中心的Cys145進行共價鍵結,而具有Mpro抑制活性。
木瓜酶樣蛋白酶係關於非構造蛋白質3(nsp3)之蛋白酶,為將多蛋白質分解的半胱胺酸蛋白酶。SARS-CoV2之木瓜酶樣蛋白酶,具有由317個殘基構成的胺基酸序列(序列編號3)。木瓜酶樣蛋白酶之活性中心相關的催化三聯體(catalytic triad)構造,為Asp286-His272-Cys111。
木瓜酶為木瓜所含之半胱胺酸蛋白酶的一種,具有由345個殘基構成之胺基酸序列(序列編號2)。半胱胺酸蛋白酶意指於酵素之觸媒區域包含半胱胺酸的蛋白質分解酵素。通常,藉由存在於觸媒區域之半胱胺酸附近的組胺酸,將半胱胺酸之硫醇脫質子化,成為陰離子的硫醇基,攻擊基質肽或蛋白質之羰基碳,將肽鍵水解。因此,蛋白酶抑制劑藉由與觸媒區域之半胱胺酸的硫醇基進行共價鍵結,可抑制半胱胺酸蛋白酶之酵素活性。
木瓜酶及主要蛋白酶或木瓜酶樣蛋白酶皆為半胱胺酸蛋白酶,在活性中心共通之胺基酸殘基形成特徵性催化三聯體(catalytic triad)、或催化二分體(catalytic dyad)。催化三聯體(catalytic triad)意指在數個酵素之活性部位中可見到的3個配位胺基酸。隨著酵素之種類,構成之配位胺基酸相異。半胱胺酸蛋白酶之催化三聯體(catalytic triad)係由半胱胺酸、組胺酸、及為第3個胺基酸的天冬醯胺酸或天冬胺酸構成。其中,半胱胺酸及有助於半胱胺酸之硫醇脫質子化的組胺酸雖然為必需之構成,但在木瓜酶等中可見到之天冬醯胺酸對活性之影響少,在此情況,亦被稱為由半胱胺酸及組胺酸構成之催化二分體(catalytic dyad)。因此,藉由選擇對木瓜酶具有抑制活性的物質,可篩選作為主要蛋白酶或木瓜酶樣蛋白酶之抑制劑。就本發明之其他態樣而言,係關於主要蛋白酶或木瓜酶樣蛋白酶之
抑制劑或冠狀病毒治療或預防藥的篩選方法,其中以對木瓜酶之抑制活性作為指標。該篩選方法,具體而言,包含製備含有候選藥劑、木瓜酶分解性之螢光基質、及木瓜酶的溶液,並測定前述溶液之螢光強度。可經時地測定螢光強度,亦可藉由測定改變候選藥劑之濃度時的螢光強度變化,得到候選藥劑之木瓜酶抑制曲線。從對催化三聯體(catalytic triad)構造或催化二分體(catalytic dyad)構造具有共通點的觀點,顯示木瓜酶之抑制活性的麥角硒因,應具有主要蛋白酶或木瓜酶樣蛋白酶之抑制活性。
本發明之蛋白酶抑制劑,藉由抑制從冠狀病毒生成之多蛋白質的分解,可抑制冠狀病毒之增殖,藉此可使用作為治療藥及預防藥。又,本發明之蛋白酶抑制劑,可包含於食品或食品組成物。
本發明之組成物,包含治療有效量的麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽。再者,亦可包含可被容許作為醫藥之載劑或賦形劑。因此,本發明之組成物亦可稱為醫藥組成物。本發明之蛋白酶抑制劑及組成物可被投與至需要治療或預防的患者。
本發明之其他態樣,包含將麥角硒因或其互變異構物或二聚體或者其可被容許作為醫藥之鹽、根據本發明的蛋白酶抑制劑、或者治療或預防用醫藥組成物投與至需要治療或預防之對象。可藉由經口投與或非經口投與之任一種方式投與。就非經口投與而言,以一例言之,可列舉腹腔內投與、肌肉內投與、靜脈內投與、動脈內投與、經鼻投與、口腔內投與、經肺投與、局部投與。投與量/次數可根據症狀而適宜選擇。
本說明書中所使用的「可被容許作為藥劑之賦形劑」亦包含任何載劑、稀釋劑、輔助劑、或媒介物、保存材料或抗氧化劑、充填劑、
崩散劑、濕潤劑、乳化劑、懸浮化劑、溶劑、分散媒介物、塗覆劑、抗菌劑、殺黴劑、等壓劑及吸收延遲劑等。此等賦形劑對有效成分之使用,為本技術領域中所周知。先前之賦形劑,除非無法與麥角硒因共存,否則均可在本發明之組成物中被用作賦形劑。輔助之有效成分亦可被摻入組成物中,形成適當的治療用組合。
就需要治療或預防之對象而言,可列舉有暴露於冠狀病毒之可能性的對象。
「治療有效量」意指在投與之情況,可抑制參與發病之活性蛋白酶的量,又在投與之情況,意指用於預防或治療COVID19之發病或惡化有效的本發明之化合物/藥劑之量。治療有效量可經由動物實驗或對人類之臨床試驗而決定。另一方面,為本發明之有效成分的麥角硒因,可藉由食魚而攝取,例如只要每一日最多投與1.7mg,將可安全無虞地使用,亦可將參與量納入考慮而決定治療有效量。就一例而言,麥角硒因能以28μg/kg投與,然而非意圖以此等量為限。
本發明之組成物能以任何劑型提供,可形成錠劑、膠囊劑、粉末、點鼻劑、或氣溶膠之型態;注射液、點滴、軟膏、乳膏、噴霧劑、經皮貼片之型態的劑型。
本發明之其他態樣,可為關於包含麥角硒因或其互變異構物或二聚體或者其可被容許作為食品之鹽的食品組成物。就此種食品組成物而言,可為標示「對冠狀病毒,尤其SARS-CoV2之預防或抵抗性」的功能,或「抑制冠狀病毒之蛋白酶,尤其主要蛋白酶」之功能的功能性標示食品、營養功能食品、或特定保健用食品。食品組成物、功能性標示食品、
營養功能食品、或特定保健用食品,就一例而言,可為包含約1至1000ppm,較佳為約10至100,最佳為約30至70ppm之麥角硒因或其互變異構物或二聚體或者其可被容許作為食品之鹽的飲料品、食品、補充料。已知麥角硒因多含於魚類。例如,黑鮪魚中之麥角硒因含量為30mg Se/kg,假設攝食100g之魚肉,在體重60kg之人類體內均勻分布的情況,理論上可推測體內之麥角硒因含量為約1μM,血中濃度為約8μM。
就含有麥角硒因之魚類而言,可列舉鮪魚類、旗魚類、鯖魚類、鰤魚類、鯛魚類、河豚類、鮭魚‧鱒魚類、比目魚‧鰈魚類,尤其在鮪魚類、旗魚類、鯖魚類、鰤魚類中被大量含有。就食品組成物、功能性標示食品、營養功能食品、或特定保健用食品而言,可列舉此等魚類之生食可食部分、或以魚類作為原料的加工食品。此等魚類可為天然產物或養殖產物。由於魚類中之麥角硒因含量隨餌之種類而變動,更佳者以使麥角硒因含量增加的養殖魚為較佳。就以魚類作為原料之加工食品而言,可列舉罐裝、瓶裝、佃煮、乾燥魚、魚乾、魚漿製品、醃魚、補充品等以魚作為原料的任何食品。
就使用於食品組成物、功能性標示食品、營養功能食品、或特定保健用食品之鮪魚類,可列舉鮪魚族及條紋鰹魚族。就鮪魚族而言,可列舉鮪魚屬、花鰹屬、巴鰹屬、鰹屬等,就條紋鰹魚族而言,可列舉磯鮪屬、條紋鰹魚屬等。就鮪魚類而言,例如,可列舉鮪魚屬之長鰭鮪魚、黑鮪魚、南方鮪魚、大西洋鮪魚、大西洋黑鮪魚、黃鰭鮪魚、大眼鮪魚、長尾鮪魚等,鰹魚屬之鰹魚,花鰹屬之平騷多鰹及丸騷多鰹,巴鰹屬之巴鰹等,條紋鰹魚屬之條紋鰹魚,或者,長鰭鮪魚、黑鮪魚、南方鮪魚、大
西洋鮪魚、大西洋黑鮪魚、黃鰭鮪魚、大眼鮪魚、長尾鮪魚、條紋鰹魚或巴鰹。較佳可列舉長鰭鮪魚、黑鮪魚、大西洋鮪魚、大西洋黑鮪魚、黃鰭鮪魚、大眼鮪魚、長尾鮪魚、條紋鰹魚或巴鰹。
在本說明書中所述及之所有文獻,其全文以引用方式被納入本說明書中。
以下說明之本發明的實施例,其目的僅為例示,並非用於限定本發明之技術範圍。本發明之技術的範圍,僅藉由申請專利範圍之記載而限定。以不超脫本發明之旨趣為條件,可進行本發明之變更,例如,本發明之構成要件的追加、刪除及置換。
[實施例]
實施例1:木瓜酶活性之抑制活性
將10μg/ml之木瓜酶(販賣商:Sigma-Aldrich日本)、及5μM之依布硒或麥角硒因,溶解於保持37℃的50mM之Tris-HCl(pH7.4)中,進行預培育(pre-incubation)15分鐘後,在相同反應液中添加10μM之Bz-Arg-MCA,以螢光光度計,隨著時間推移觀察伴隨Bz-Arg-MCA之切斷的螢光強度變化。將結果示於圖4。
將10μg/ml之木瓜酶(販賣商:Sigma-Aldrich日本)、及0至125μM之依布硒或0至5μM之麥角硒因,溶解於保持37℃的50mM之Tris-HCl(pH7.4)中,進行預培育15分鐘後,在相同反應液中添加10μM之Bz-Arg-MCA,以螢光光度計測定伴隨Bz-Arg-MCA之切斷的螢光強度變化,調查抑制活性。將結果示於圖5。從圖5之結果,求得麥角硒因對木瓜
酶之抑制活性(IC50=0.25μM)、及依布硒對木瓜酶之抑制活性(IC50=5.0μM)。
實施例2:SARS-CoV2之Mpro之調製
藉由基因全合成,依照NC_45512之鹼基序列(10055-10972:序列編號4),合成Mpro之DNA。將Mpro導入附GST及組胺酸標籤之質體(圖6),轉形至大腸菌BL21(DE3)株。將大腸菌BL21(DE3)株於37℃培養,使Mpro蛋白質表現。回收細胞體,使用針對組胺酸標籤的結合劑,將Mpro蛋白質精製。藉由Mpro之自己消化及人鼻病毒3C蛋白酶(HRV),將GST及組胺酸標籤除去,使用針對組胺酸標籤之結合劑,將未消化之Mpro及HRV除去,得到Mpro蛋白質的精製標本。將精製之Mpro蛋白質溶解在含20mM Tris-HCl、100mM NaCl、0.01%Triton-X-100、50%甘油、1mM EDTA、1mM DTT之貯藏緩衝液中,供於SDS-PAGE,藉由考馬斯亮藍(coomassie brilliant blue)染色(圖7)。將溶解於含有DTT之貯藏緩衝液的精製Mpro蛋白質,使用微量透析筒匣(cartridge)Xpress Micro/Mini Dialyzer(販賣商:船越股份有限公司)透析,除去DTT,供實施例4的抑制活性試驗使用。
實施例3:SARS-CoV2之PLpro的調製
藉由基因全合成,依照序列編號NC_45512之鹼基序列(4955-5908:序列編號5)合成PLpro之DNA。將PLpro導入附組胺酸標籤之質體,轉形至大腸菌BL21(DE3)株。培養大腸菌BL21(DE3)株,使PLpro蛋白質表現。回收細胞體,使用針對組胺酸標籤之結合劑,將PLpro蛋白質精製。藉由PLpro之自己消化及HRV,將GST及組胺酸標籤除去,使用針對組
胺酸標籤之結合劑,將未消化之PLpro及HRV除去,得到PLpro蛋白質的精製標本。
實施例4:對SARS-CoV2之Mpro的抑制活性
將2μg/ml之Mpro、及5μM之依布硒、麥角硒因、或麥角硫鹼,分別溶解於保持37℃的50mM之Tris-HCl(pH7.4)中,進行預培育5分鐘後,在相同反應液中添加10μM之Ac-Abu-Tle-Leu-Gln-MCA,以螢光光度計測定伴隨Ac-Abu-Tle-Leu-Gln-MCA之切斷的螢光強度變化,調查抑制活性(圖8)。就對照而言,以只有未添加抑制化合物之點不同的條件,測定螢光強度變化。
Ac-Abu-Tle-Leu-Gln-MCA被Mpro分解,如下述生成螢光物質,藉由Mpro抑制化合物之作用,可抑制螢光物質的生成。
在添加依布硒及麥角硫鹼之情況的螢光強度變化,與未添加之對照同等。另一方面,在添加麥角硒因的情況,與未添加之對照比較,螢光強度被壓低,顯示麥角硒因可抑制Mpro的活性。
將2μg/ml之Mpro、及0至25μM之依布硒、0至25μM之麥角硫鹼或0至5μM之麥角硒因,分別溶解在保持37℃的50mM之Tris-HCl(pH7.4)中,進行預培育5分鐘後,在相同反應液中添加10μM之Ac-
Abu-Tle-Leu-Gln-MCA,以螢光光度計測定伴隨Ac-Abu-Tle-Leu-Gln-MCA之切斷的螢光強度變化,求得抑制曲線(圖8)。從回歸曲線算出IC50值,如下述之表所示:
實施例5:SARS-CoV2之對Mpro的抑制活性
將2μg/ml之Mpro、及1μM之試驗化合物,分別溶解在保持37℃的50mM之Tris-HCl(pH7.4)中,進行預培育5分鐘後,在相同反應液中添加10μM之Ac-Abu-Tle-Leu-Gln-MCA,以螢光光度計測定伴隨Ac-Abu-Tle-Leu-Gln-MCA之切斷的螢光強度變化,測定抑制活性(圖10)。就試驗化合物而言,使用麥角硒因、依布硒、硒代胱胺酸((SeCys)2)、甲基硒代半胱胺酸(MeSeCys)、硒代甲硫胺酸(SeMet)、二苯基二硒化物(PhSeSePh)、亞硒酸鈉(sodium selenite)。除亞硒酸鈉外,此等含硒化合物,在電腦之試驗中,為被預測具有Mpro抑制活性的化合物(非專利文獻4)。在以未添加試驗化合物之螢光強度當作100的情況,測定添加試驗化合物1分鐘後之螢光強度時,麥角硒因為約20%,另一方面,依布硒、硒代胱胺酸((SeCys)2)、甲
基硒代半胱胺酸(MeSeCys)、硒代甲硫胺酸(SeMet)、二苯基二硒化物(PhSeSePh)、亞硒酸鈉(selenite)之任一者皆超過90%。因此,麥角硒因與其他含硒化合物相較,就Mpro抑制活性之點而言,顯得特別優良。
實施例6:對SARS-CoV2之增殖抑制活性
依布硒或麥角硒因對於SARS-CoV2感染宿主細胞的抑制,例如,可用斑塊檢定(plaque assay)法測定。將宿主細胞於37℃、5%CO2之培育器中,進行單層培養至成為融合(confluent)。繼而將培養基除去,用PBS(-)將細胞表面洗淨後,添加病毒溶液、及0至100μM之蛋白酶抑制劑溶液,進行30分鐘培育。30分鐘後,除去檢體溶液,疊加瓊脂培養基層。瓊脂固化後,將培養盤反轉,於37℃、5% CO2之培育器中培育2日。然後移除疊層培養基,使培養盤乾燥,藉由結晶紫染色液進行5分鐘染色後,用精製水洗淨並風乾。最後,計算斑塊之數,並與對照群比較,計算蛋白酶抑制劑對病毒的感染抑制率。
<110> 國立大學法人千葉大學(NATIONALUNIVERSITY CORPORATION CHIBA UNIVERSITY) 日商龜甲萬股份有限公司(KIKKOMAN CORPORATION)
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Claims (9)
- 一種冠狀病毒之蛋白酶抑制劑,其包含麥角硒因、或其互變異構物或二聚體、或者其可被容許作為醫藥之鹽。
- 如請求項1所述之蛋白酶抑制劑,其中該蛋白酶為主要蛋白酶或木瓜酶樣蛋白酶。
- 如請求項1或2所述之蛋白酶抑制劑,其中該冠狀病毒為SARS-CoV2。
- 一種冠狀病毒感染症之治療或預防用組成物,其包含麥角硒因、或其互變異構物或二聚體、或者其可被容許作為醫藥之鹽。
- 如請求項4所述之治療或預防用組成物,其中該冠狀病毒感染症為COVID19。
- 一種冠狀病毒感染症之治療或預防用組成物,其包含如請求項1至3中任一項所述之蛋白酶抑制劑。
- 一種冠狀病毒感染症之治療或預防藥的篩選方法,其係以對木瓜酶之抑制活性作為指標。
- 如請求項7所述之篩選方法,其中該冠狀病毒感染症為COVID19。
- 如請求項7或8所述之篩選方法,其中該治療藥或預防藥係抑制冠狀病毒的主要蛋白酶。
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JPWO2022177029A1 (zh) | 2022-08-25 |
WO2022177029A1 (ja) | 2022-08-25 |
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