摘要
人类T细胞白血病病毒Ⅰ型(HTLV-Ⅰ)的感染可以导致成人T细胞白血病(ATI)和热带痉挛性下肢瘫痪/HTLV-Ⅰ相关脊髓病(TSP/HAM)等其他慢性疾病,目前全世界范围内有2-3千万人为病毒携带者。HTLV-Ⅰ蛋白酶与这一病毒的复制密切相关,抑制蛋白酶的活性能够有效地阻止病毒的复制。因此,HTLV-Ⅰ蛋白酶是一个非常重要的抗HTLV-Ⅰ感染的研究靶点。
天然肽分子易于被体内的酶降解而失去活性,本文设计将含有羟甲羰基(HMC)结构的(2S,3S)-3-氨基-2-羟基-4-苯基丁酸(Apns)作为非天然氨基酸残基,引入底物的结构中,模拟天然底物被蛋白酶水解的过渡态类似物,从而阻断天然底物与酶的结合,有效抑制HTLV-Ⅰ蛋白酶活性。以KNI-10455(IC_(50)=140nM)作为先导化合物,在C端和N端引入不同的取代胺基和取代羧基,并逐步减小分子结构,通过液相合成的方法制备了66个未见文献报道的拟肽类化合物,其中53个为四肽化合物,9个为三肽化合物,4个为二肽化合物。新化合物的结构经核磁共振氢谱和质谱检测确证。
采用蛋白酶抑制剂活性测定方法对新化合物进行了初步抗HTLV-Ⅰ和HIV-1蛋白酶活性筛选。结果显示53个四肽化合物在50nM浓度下显示了较高的HIV-1蛋白酶抑制率(>97%);42个四肽抑制剂具有高于先导化合物的抗HTLV-Ⅰ蛋白酶活性(IC_(50)<137nM);二肽和三肽化合物在600nM浓度下对HTLV-Ⅰ蛋白酶没有显著的抑制作用(<14%);其中活性最强的化合物是(R)-N-(2,2-二甲基)丙基-3-{{(2S,3S)-3-{(2S-2-[(2S)-2-丁酰胺基-2-苯基]乙酰胺基-3,3-二甲基}丁酰胺基-2-羟基-4-苯基}}丁酰基-5,5-二甲基-1,3-噻唑-4-甲酰胺(IC_(50)=74 nM)。
A large number of diseases including adult T-cell leukemia(ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy(TSP/HAM) are caused by the human T-cell leukemia virus typeⅠ(HTLV-I).HTLV-I infection is currently considered as an intractable disease because 20-30 million individuals worldwide are viral carriers.HTLV-I protease is responsible for the proliferation of the retrovirus and then becomes an attractive target for developing inhibitors against HTLV-I infections.
Substrates containing natural amino acid residues are easily digested by proteases. When a non-natural amino acid,(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid(Apns), containing a hydroxymethylcarbonyl(HMC) isostere,that can mimic the tetrahedral transition-state of amino bond hydrolysis but cannot itself be hydrolyzed by the enzyme,is introduced,the protease's pharmacological actions are suspended.Using KNI-10455 as a reference compound(IC_(50)=140 nM),we were interested in modifying the N-and C-terminals and truncation studies to enhance HTLV-I protease inhibition.Sixty-six peptidic compounds containing Apns were prepared by solution phase peptide synthesis.Fifty-three of them were tetrapeptidic inhibitors.Nine of them were tripeptidic compounds.Four of them were dipeptidic inhibitors.The structures were identified by ~1H-NMR and MS spectra.
All novel compounds were evaluated for HTLV-I and HIV-1 protease assays. Fifty-three tetrapeptides exhibited high HIV-1 protease inhibition at 50nM(>97%).Forty-two tetrapeptides showed potent HTLV-I protease activity(IC_(50)<137nM) that were greater than the reference compound.None of the tri-and di-peptides efficiently inhibited HTLV-I protease at 600 nM(<14%).The most potent HTLV-I protease inhibitor was(R)-N-(2,2-dimethyl) propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-butyrylamino-2-phenyl]acetylamino-3,3-dimethyl}butan oylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-l,3-thiazolidine-4-carboxamide(IC_(50)= 74nM).
引文
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