Demir Ahu, Oguariri Raphael M, Magis Andrew, Ostrov David A, Imamichi Tomozumi, Dunn Ben M
University of Florida College of Medicine Biochemistry and Molecular Biology, Gainesville, FL, USA.
Antivir Ther. 2012;17(5):883-92. doi: 10.3851/IMP2090. Epub 2012 Mar 21.
Human T-cell leukemia virus-1 (HTLV-1) was the first identified human retrovirus and was shown to be associated with diseases such as adult T-cell leukemia lymphoma and tropical spastic paraparesis/HTLV-1 associated myelopathy. Retroviral proteases (PRs) are essential for viral replication by processing viral Gag and Gag-(Pro)-Pol polyproteins during maturation. Full-length HTLV-1 PR is 125 residues long; whether the C-terminal region is required for catalytic activity is still controversial. In this study, we characterized the effect of C-terminal amino acids of HTLV-1 PR for PR activity and examined the binding of compounds identified by in silico screening. One compound showed inhibition against the virus in infected cells.
Truncated (116-, 121- and 122-residue) forms of HTLV-1 PR were prepared and proteins from expression of the genes were purified. In silico screening was performed by docking small molecules into the active site of HTLV-1 PR. The kinetic constants k(cat), K(m), k(cat)/K(m) and inhibition constants K(i) for inhibitors identified by the computational screening were determined. Western blot and ELISA analyses were used to determine the effect of the most potent PR inhibitors on HTLV-1 protein processing in infected cells.
The constructs showed similar catalytic efficiency constants (k(cat)/K(m)); thus HTLV-1 PR C-terminal amino acids are not essential for full activity. Computational screening revealed new PR inhibitors and some were shown to be inhibitory in enzyme assays. In HTLV-1-infected cells, one of the small molecules inhibited HTLV-1 gag cleavage and decreased the amount of HTLV-1 p19 produced in the cells.
We have identified an HTLV-1 PR inhibitor that is biologically functional. Inhibitor screening will continue to develop possible drugs for therapy of HTLV-1 infection.
人类T细胞白血病病毒1型（HTLV-1）是首个被鉴定出的人类逆转录病毒，被证明与成人T细胞白血病淋巴瘤以及热带痉挛性截瘫/HTLV-1相关脊髓病等疾病有关。逆转录病毒蛋白酶（PRs）在病毒成熟过程中通过加工病毒Gag和Gag-(Pro)-Pol多蛋白对病毒复制至关重要。全长HTLV-1 PR有125个氨基酸残基；其C末端区域对于催化活性是否必需仍存在争议。在本研究中，我们对HTLV-1 PR的C末端氨基酸对PR活性的影响进行了表征，并检测了通过计算机筛选鉴定出的化合物的结合情况。一种化合物在感染细胞中显示出对该病毒的抑制作用。
制备了截短形式（116、121和122个氨基酸残基）的HTLV-1 PR，并对基因表达产生的蛋白质进行了纯化。通过将小分子对接至HTLV-1 PR的活性位点进行计算机筛选。测定了通过计算机筛选鉴定出的抑制剂的动力学常数k(cat)、K(m)、k(cat)/K(m)和抑制常数K(i)。采用蛋白质印迹法和酶联免疫吸附测定法分析最有效的PR抑制剂对感染细胞中HTLV-1蛋白加工的影响。
构建体显示出相似的催化效率常数（k(cat)/K(m)）；因此HTLV-1 PR的C末端氨基酸对于其全部活性并非必需。计算机筛选揭示了新的PR抑制剂，其中一些在酶分析中显示出抑制作用。在HTLV-1感染的细胞中，一种小分子抑制了HTLV-1 gag的切割，并减少了细胞中产生的HTLV-1 p19的量。
我们鉴定出了一种具有生物学功能的HTLV-1 PR抑制剂。抑制剂筛选将继续开发可能用于治疗HTLV-1感染的药物。
