Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.
Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds.
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DOI：
10.1128/jvi.00928-23
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发表时间：
2023-12-21
影响因子：
5.4
通讯作者：
Kirkegaard, Karla
中科院分区：
文献类型：
--
作者：
Doherty, Jennifer S.;Kirkegaard, Karla
关键词：
肠道病毒编码两个蛋白水解酶活性位点，分别位于2A和3C编码区。虽然它们以许多宿主蛋白为靶标，但首先需要从嵌入它们的病毒多蛋白中去除它们。多蛋白切割既可以发生在分子内(顺式)，也可以发生在分子间(反式)。以前的工作表明，靶向分子内切割的抗病毒药物可以产生抑制前体，从而抑制耐药变异体的生长。因此，我们希望评估肠道病毒的裂解模式，以确定这种专性的分子内裂解作为药物靶点选择的方法。利用翻译提取物，我们发现2 A蛋白酶N末端连接的顺式切割在三个肠道病毒分支中是保守的，而3C的N末端连接的机制不同，肠道病毒D68 3C裂解发生在顺式，脊髓灰质炎病毒3C裂解发生在反式。以蛋白水解酶为靶点的抗病毒药物通常是通过阻止人工合成的多肽底物的裂解来鉴定的。在这里，我们表明，抗病毒药物被认为具有阻止分子间切割的能力，有时可以阻止蛋白水解酶的分子内切割，但其效果差异很大。此外，对于三种肠道病毒物种，我们证明了在混合种群中，A蛋白水解酶活性缺陷的基因组抑制了野生型病毒的生长，支持了这样的假设，即阻止VP1·2A连接处的分子内切割可以产生主要的抑制前体。这些数据认为，为了减少耐药性的可能性，应评估以蛋白酶为靶标的抗病毒药物除了阻止其他底物的分子间切割外，还能阻止分子内多蛋白切割的能力。大多数以蛋白酶为靶点的抗病毒开发评估小分子抑制人工底物切割的能力。然而，在它们可以切割任何其他底物之前，病毒蛋白酶需要将自己从它们被翻译的病毒多蛋白中切割出来。这可以发生在分子内，也可以发生在分子间。无论这一过程发生在分子内还是分子间，都与前体积累的潜力和抗病毒药物的有效性有关。我们认为，评估候选抗病毒药物阻止这些裂解的能力对药物开发至关重要，因为未裂解前体的积聚可以抑制病毒，并可能抑制耐药变异体的选择。
Enteroviruses encode two protease active sites, in the 2A and 3C coding regions. While they target many host proteins, they first need to be excised from the viral polyprotein in which they are embedded. Polyprotein cleavage can occur either intra-molecularly (in cis) or inter-molecularly (in trans). Previous work suggested that antivirals targeting intra-molecular cleavages could generate inhibitory precursors that can suppress the outgrowth of drug-resistant variants. Therefore, we wanted to evaluate enteroviral cleavage patterns to identify such obligate intra-molecular cleavages as a method of drug target selection. Using translation extracts, we show that cis cleavage of the 2A protease N-terminal junction is conserved across three enterovirus clades, while the mechanism for the N-terminal junction of 3C varies, with enterovirus D68 3C cleavage occurring in cis and poliovirus 3C cleavage occurring in trans. Antiviral agents targeting proteases are generally identified via their ability to block the cleavage of artificial peptide substrates. Here, we show that antivirals identified for their ability to block inter-molecular cleavage can sometimes block intra-molecular cleavage of the protease from its polyprotein but with widely varying efficacy. Additionally, we demonstrate that for three enteroviral species, genomes defective in 2A protease activity suppress the growth of wild-type virus in mixed populations, supporting the hypothesis that preventing intra-molecular cleavage at the VP1·2A junction can create dominantly inhibitory precursors. These data argue that, to reduce the likelihood of drug resistance, protease-targeted antivirals should be evaluated for their ability to block intra-molecular polyprotein cleavages in addition to inter-molecular cleavage of other substrates. Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.
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