Targeted Disassembling of SARS-CoV-2 as It Gets Ready for Cell Penetration

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 G614⁵ (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 protein⁵ 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 overlooked³ 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 identified⁶ (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 deployed¹⁰ 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.