Current efforts to impair SARS-CoV-2 transmission are geared primarily at disrupting virus anchoring to the human host cell via blockade of recognition of the angiotensin-converting enzyme 2 (hACE2) receptor.(1,2) This may be achieved via administered or vaccine-induced antibodies with selective affinity toward the receptor binding domain (RBD) or overlapping adjacent regions in the S1 subunit of the virion spike (S) protein.(1,2) Such efforts may be only partially successful because the virus–receptor interface has been evolutionarily perfected and hence its therapeutic disruption may prove more arduous than anticipated. This justifies the need for complementary or alternative targeted therapies aimed at deactivating the virus directly. How would such a therapeutic opportunity arise? Here we argue that clues become apparent after examination of hitherto overlooked aspects of the priming mechanism for cell entry and its optimization along the recent (i.e., in last few months) evolutionary history of the virus. As it turns out, activation for cell entry places the virus in a vulnerable position for targeted molecular therapy because it entails cleavage of the S-protein at the S1/S2 junction, a key event that loosens up the protein assemblage.(3,4) This enzymatic cleavage is in fact a barrier to zoonotic coronavirus transmission, and the acquisition of the cleavage site constituted a “gain of function” that enabled the virus to jump to humans. Thus, S1/S2 cleavage endows S1 and S2 with distinctive roles during the cell invasion process: S1 serves as “anchor” of the human cell via RBD-hACE2 association, while S2 acts as “harpoon” of the cell membrane, as it wields a terminal fusion peptide with dual lipid/water solubility.(3,4) However, effective cell penetration requires that anchoring S1 and harpooning S2 remain in close proximity, with some retention of the quaternary assemblage and organization of the S protein. Otherwise, the purported host cell, with its S1-tag indicative of receptor recognition, would not be amenable to virus penetration (Figure 1a). This aspect is typically overlooked in mechanistic accounts of cell penetration and it is pivotal to design our therapeutic strategy. The S1 shedding during the priming for cell entry is obviously a dysfunctional aspect, detrimental to virus transmissibility. Figure 1. Mechanism of SARS-CoV-2 priming for virus-mediated membrane fusion in strain D614 (a) and dominant strain G614(5) (b), unraveling a vulnerability and suggesting a targeted therapeutic strategy to disassemble the virus. (a) The priming of the virus entails enzymatic cleavage at the S1/S2 junction (red line), a step that endows S1 with an anchoring role and S2 with a harpooning role in the cell-entry process.(3,4) The arrow in S2 represents the fusion peptide. The white segment represents the well wrapped Asp614-Ala647 backbone hydrogen bond in strain D614.(6) The marginal stability of the “anchor–harpoon complex” in the D614 strain begets some S1 shedding,(7,8) which renders S2 somewhat inefficient for cell penetration (lower right). Cell penetration is only possible when the anchor–harpoon complex is retained following recognition of receptor hACE2 (upper right). The host cell is represented by a light purple ellipse. (b) Mutation D614G creates a deshielded solvent-exposed backbone hydrogen bond (dehydron, green segment) pairing residues Gly614 and Ala647.(6) The dehydron is stabilized through intermolecular wrapping/protection (thin blue line) contributed by the S2 region (chains A, B stand respectively for S1, S2 in the inset). In the G614 strain, the anchor–harpoon complex is more stable (and the free anchoring S1, more unstable), thereby enhancing cell penetration efficacy while limiting S1 shedding.(6−8) The dehydron location in S1 suggests a complex-disruptive therapeutic intervention in which a peptidomimetic subsuming the dehydron-wrapping region B859–B864 (inset), or an antibody targeting the highly antigenic dehydron region in S1,(9) displaces S2 from its complexation with S1 (both therapeutic ligands are represented by a yellow ellipse). This therapeutic disruption of the anchor–harpoon complex is tantamount to virus deactivation. The dominant mutation D614G in the S protein(5) removes the transmissibility handicap, as it brings up a selective advantage by stabilizing the S1/S2 association,(6) thereby significantly increasing the harpooning efficacy of S2 (Figure 1b). By creating a packing defect in S1 in the form of a solvent-exposed backbone hydrogen bond (dehydron) pairing residues Gly614 and Ala647, S1 becomes more reliant on binding with S2 to maintain its structural integrity and the packing defect in the free S1 unit becomes compensated upon association with S2, thereby stabilizing the S1/S2 complex.(6) This post-cleavage holding together of anchor and harpoon is precisely what is required for effective cell penetration (Figure 1b), as evidenced by the significantly less S1 shedding and higher transmission efficacy experimentally observed in the G614 strain.(7,8) Taken together, the typically overlooked(3) mechanistic constraint of post-cleavage anchor/harpoon association and the structural impact of dominant D614G mutation upon the association unravel an opportunity for therapeutic intervention to disrupt the activated S1/S2 complex. Thus, the goal is to target the S1-region containing dehydron Gly614-Ala647 in the dominant strain G614. Since the S1 region containing this packing defect is verifiably antigenic,(9) it should be possible to generate or induce complex-disruptive antibodies by exploiting the dehydron-containing antigen. Alternatively, the region in S2 that provides intermolecular shielding to the Gly614-Ala647 dehydron has also been identified(6) (Figure 1b). This implies that it should be possible to synthesize a peptidomimetic that improves the intermolecular protection of the dehydron from structure-disruptive backbone hydration, thereby displacing S2 from the association with S1. The discovery of a drug disruptive of the anchor–harpoon complex may not be limited to synthesizing a peptidomimetic of the S2 region that protects and stabilizes the G614-A647 dehydron (Figure 1b, inset). This is because the active complex is not necessarily in perfect alignment with the S1/S2 complex: The clipping of the S1/S2 junction may give rise to some degree of induced folding, with structural adaptation altering the original structure of the S1/S2 complex. There is no reported structure for the activated complex, and an educated guess is the one proposed, but it is possible that other regions of the activated S2 domain contribute to the stabilization of dehydron G614-A647. Artificial intelligence will ultimately need to be deployed(10) to infer the induced folding upon association of the two key players in the cell-entry process. Be the therapeutic agent an antibody or a peptidomimetic, the targeted disassembly of the “anchor/harpoon complex” in activated SARS-CoV-2 may be regarded as an alternative line of attack on the COVID-19 infection. This strategy is expected to complement the vaccine-induced impairment of the anchoring to the host cell, currently under accelerated development. Even partial or incomplete success of the leveraged vaccine-based strategy may have unfathomable consequences for public health. In this scenario, a backup therapeutic strategy such as the one proposed becomes an imperative. This article references 10 other publications.

中文翻译：

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在 SARS-CoV-2 准备好进行细胞渗透时对其进行靶向分解

目前削弱 SARS-CoV-2 传播的努力主要是通过阻断血管紧张素转换酶 2 (hACE2) 受体的识别来破坏锚定在人类宿主细胞上的病毒。 (1,2) 这可以通过给药或疫苗诱导的抗体对受体结合域 (RBD) 或病毒粒子刺突 (S) 蛋白的 S1 亚基中的重叠相邻区域具有选择性亲和力。 (1,2) 这种努力可能只是部分成功，因为病毒 - 受体界面已经进化完善，因此其治疗中断可能比预期的更艰巨。这证明需要旨在直接灭活病毒的补充或替代靶向疗法。这样的治疗机会如何出现？在这里，我们认为，在检查了迄今为止被忽视的细胞进入启动机制及其在病毒最近（即，最近几个月）进化史中的优化后，线索变得明显。事实证明，细胞进入的激活使病毒处于靶向分子治疗的脆弱位置，因为它需要在 S1/S2 连接处裂解 S 蛋白，这是放松蛋白质组合的关键事件。 (3,4 ) 这种酶促裂解实际上是人畜共患冠状病毒传播的障碍，获得裂解位点构成了“功能获得”，使病毒能够跳到人类身上。因此，S1/S2 切割赋予 S1 和 S2 在细胞侵袭过程中独特的作用：S1 通过 RBD-hACE2 结合充当人体细胞的“锚”，而 S2 充当细胞膜的“鱼叉”，因为它具有具有双重脂/水溶解度的末端融合肽。(3,4) 然而，有效细胞渗透需要锚定 S1 和鱼叉 S2 保持紧密接近，并保留一些 S 蛋白的四级组合和组织。否则，具有指示受体识别的 S1 标签的所谓宿主细胞将不适合病毒渗透（图 1a）。这方面通常在细胞渗透的机械解释中被忽视，它是设计我们的治疗策略的关键。细胞进入启动过程中的 S1 脱落显然是一个功能失调的方面，不利于病毒的传播。图1。SARS-CoV-2 在菌株 D614 (a) 和优势菌株 G614(5) (b) 中引发病毒介导的膜融合的机制，揭示了一个漏洞并提出了一种分解病毒的靶向治疗策略。(a) 病毒的引发需要在 S1/S2 连接处（红线）进行酶切，这一步骤使 S1 在细胞进入过程中具有锚定作用，S2 具有鱼叉作用。(3,4) S2中的箭头代表融合肽。白色部分代表菌株 D614 中包裹良好的 Asp614-Ala647 主链氢键。 (6) D614 菌株中“锚-鱼叉复合物”的边缘稳定性导致一些 S1 脱落，(7,8) 这使得 S2 有点低效用于细胞渗透（右下）。只有在识别受体 hACE2 后保留锚定鱼叉复合物时，细胞渗透才有可能（右上）。宿主细胞由浅紫色椭圆表示。(b) 突变 D614G 产生一个去屏蔽的溶剂暴露骨架氢键（脱氢，绿色段）配对残基 Gly614 和 Ala647。（6）脱氢通过由 S2 区域（链A、B分别代表插图中的S1、S2）。在 G614 菌株中，锚定鱼叉复合物更稳定（而游离锚定 S1 更不稳定），从而增强细胞渗透功效，同时限制 S1 脱落。（6-8）S1 中的脱水子位置表明复合物破坏性治疗干预，其中包含脱氢包裹区域 B859-B864（插图）的拟肽，或靶向 S1 中高抗原性脱氢区的抗体，(9) 取代 S2 与 S1 的复合物（两种治疗配体均由黄色椭圆表示）。这种锚定鱼叉复合物的治疗性破坏相当于病毒灭活。S 蛋白 (5) 中的显性突变 D614G 消除了传播障碍，因为它通过稳定 S1/S2 关联带来了选择性优势，(6) 从而显着提高了 S2 的鱼叉效应（图 1b）。通过以溶剂暴露的骨架氢键的形式在 S1 中产生堆积缺陷（这种锚定鱼叉复合物的治疗性破坏相当于病毒灭活。S 蛋白 (5) 中的显性突变 D614G 消除了传播障碍，因为它通过稳定 S1/S2 关联带来了选择性优势，(6) 从而显着提高了 S2 的鱼叉效应（图 1b）。通过以溶剂暴露的骨架氢键的形式在 S1 中产生堆积缺陷（这种锚定鱼叉复合物的治疗性破坏相当于病毒灭活。S 蛋白 (5) 中的显性突变 D614G 消除了传播障碍，因为它通过稳定 S1/S2 关联带来了选择性优势，(6) 从而显着提高了 S2 的鱼叉效应（图 1b）。通过以溶剂暴露的骨架氢键的形式在 S1 中产生堆积缺陷（脱氢) 配对残基 Gly614 和 Ala647，S1 变得更加依赖与 S2 的结合来维持其结构完整性，并且游离 S1 单元中的包装缺陷在与 S2 结合后得到补偿，从而稳定 S1/S2 复合物。 (6)将锚和鱼叉固定在一起的切割正是有效细胞渗透所需要的（图 1b），正如在 G614 菌株中实验观察到的显着更少的 S1 脱落和更高的传输效率所证明的那样。 (7,8) 总之，典型的被忽视的 (3) 切割后锚定/鱼叉关联的机械约束和显性 D614G 突变对关联的结构影响揭示了治疗干预破坏激活的 S1/S2 复合体的机会。因此，目标是针对优势菌株 G614 中含有脱氢 Gly614-Ala647 的 S1 区域。由于含有这种包装缺陷的 S1 区域具有可验证的抗原性，(9) 应该有可能通过利用含有脱氢的抗原来产生或诱导复合物破坏性抗体。或者，S2 中为 Gly614-Ala647 脱氢提供分子间屏蔽的区域也已被确定 (6)（图 1b）。这意味着应该有可能合成一种肽模拟物，以改善脱氢分子间的保护，使其免受结构破坏性骨架水合，从而取代 S2 与 S1 的结合。对锚定鱼叉复合物的药物破坏的发现可能不仅限于合成保护和稳定 G614-A647 脱氢子的 S2 区域的肽模拟物（图 1b，插图）。这是因为活性复合体不一定与 S1/S2 复合体完美对齐：S1/S2 连接处的剪切可能会引起一定程度的诱导折叠，结构适应性改变了 S1/S2 复合体的原始结构. 没有报告激活复合物的结构，并且提出了有根据的猜测，但激活的 S2 域的其他区域可能有助于稳定脱氢 G614-A647。最终需要部署人工智能 (10) 来推断细胞进入过程中两个关键参与者关联时的诱导折叠。作为抗体或肽模拟物的治疗剂，激活的 SARS-CoV-2 中“锚/鱼叉复合物”的靶向分解可被视为对 COVID-19 感染的替代攻击线。该策略有望补充疫苗诱导的宿主细胞锚定损伤，目前正在加速开发。即使是基于杠杆的疫苗战略的部分或不完全成功，也可能对公共卫生产生难以估量的后果。在这种情况下，像所提议的那样的备用治疗策略变得势在必行。本文引用了 10 篇其他出版物。该策略有望补充疫苗诱导的宿主细胞锚定损伤，目前正在加速开发。即使是基于杠杆的疫苗战略的部分或不完全成功，也可能对公共健康产生不可估量的后果。在这种情况下，像所提议的那样的备用治疗策略变得势在必行。本文引用了 10 篇其他出版物。该策略有望补充疫苗诱导的宿主细胞锚定损伤，目前正在加速开发。即使是基于杠杆的疫苗战略的部分或不完全成功，也可能对公共卫生产生难以估量的后果。在这种情况下，像所提议的那样的备用治疗策略变得势在必行。本文引用了 10 篇其他出版物。