Covalent Inhibition of SARS-CoV-2 RBD-ACE2 Interaction by Aptamers with Multiple Sulfur(VI) Fluoride Exchange Modifications

Abstract

The SARS-CoV-2 spike protein uses its receptor-binding domain (RBD) to interact with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells, establishing the first step of SARS-CoV-2 infection. Inhibitors of RBD-ACE2 interaction, therefore, have shown great promise in preventing SARS-CoV-2 infection. Currently known RBD-ACE2 inhibitors are all based on reversible binding and must compete with ACE2 or RBD at the equilibrium. On the other hand, covalent inhibitors, such as those based on sulfur(VI) fluoride exchange (SuFEx) chemistry, can form irreversible chemical bonds with target proteins and offer advantages including higher potency and longer duration of inhibition. Here, we report covalent aptamer inhibitors that can block RBD-ACE2 by forming covalent bonds with RBD. These covalent aptamer inhibitors were developed by equipping known RBD aptamers with multiple SuFEx (mSuFEx) modifications. The mSuFEx-aptamer 6C3-7SF underwent strong covalent bonding with RBD and some of its variants at fast rates (t1/2 = 20 ~ 29 min−1) and induced more efficient RBD-ACE2 inhibition (IC50 = 26 ~ 37 nM) than the original aptamer (IC50 > 200 nM) according to an in vitro enzyme-linked immunosorbent assay (ELISA). The covalent bond formation was highly selective to RBD over human serum albumin (HSA) and ACE2, and could occur efficiently in diluted human serum. Peptide fragmentation analyses of the RBD-6C3-7SF adducts revealed multiple sites of covalent bonding on RBD, including K378, R408, Y422, Y424, Y453, and K458. The surprising R408 suggests that context-specific non-N-terminal arginine could be a new type of targetable residue by SuFEx-based covalent inhibitors, which were never reported as reactive with any non-N-terminal arginine in target proteins. In addition, RBD R408 is responsible for binding with ACE2 N90 glycan, and this arginine is conserved in SARS-CoV-2 variants of concern or interest, suggesting that R408 could be the potential site of interest for developing SuFEx-based covalent inhibitors against threatening SARS-CoV-2 variants. Although the compatibility of mSuFEx-based covalent aptamers in cellular and in vivo systems should be further investigated, our study demonstrated the promise of mSuFEx chemistry in constructing potent covalent aptamers to inhibit important protein-protein interactions (PPIs).

bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements

“…The drug effect of the covalently bound bioTCI should far outlast even after the clearance of the unbound drug in the serum. An excellent theoretical treatment of TCI kinetics can be found in [ 64 ], and experimental data for small molecule TCI [ 67 ] and aptamer TCI [ 33 , 34 , 68 ] have been reported.…”

“…Aptamers have been touted as potential antibody replacements given their high specificity and affinity [ 83 , 85 ] but the limitation of a very short in vivo half-life has prevented their practical application [ 81 ]. In theory, these already identified aptamers can be rendered potential TCIs with a long drug half-life just by incorporating a warhead, as recently demonstrated for the thrombin binding aptamer [ 28 , 34 ] and SARS-CoV-2 spike protein binding aptamer [ 42 , 68 ]. Whether other warhead-introduced aptamers will conjugate with the target protein depends on the orientation of the aptamer to the target dictating the proper positioning of the warhead on the aptamer, and the availability of the interaction-capable amino acids on the target protein.…”

“…The second-generation nucleotidic bioTCI is based on a pre-identified DNA aptamer showing high affinity for the target, and a warhead directly conjugated at a specific position of the DNA ( Table 2 ) [ 28 , 34 , 68 ]. It should be noted that TCI aptamers created in such a fashion are better described as a tethered-TCI (TeTCI) ( Figure 7 ) since the protein domain forming the covalent bond is outside [ 14 , 88 , 89 ] the actual aptamer docking domain.…”

“…Structural information is usually unavailable for most aptamers bound to its target, and the determination of the position of warhead introduction becomes a labor-intensive trial-and-error process where every T residue is replaced with an OctdU and screened for efficient covalent binding to the target. Qin et al [ 68 ] reported a potential docking-structure-independent method of warhead introduction by simply extending the 3′ end of an aptamer with a phosphorothioate (PS)-linked nucleic acid tail, and subsequent introduction of a warhead through a simple nucleophilic reaction between a Br-warhead and the S-atom of the PS linker. The authors chose two SARS-CoV-2 S-protein binding aptamers previously selected by the conventional SELEX [ 85 , 93 ] and tailed the 3′ end with 7 T residues possessing 1, 3, 5, or 7 PS bonds.…”

“…For example, DNA aptamers targeting the SARS-CoV-2 spike protein have been reported to inhibit its binding to the angiotensin-converting enzyme 2 receptor necessary for viral particle infection of cells [ 93 , 98 ]. Although not tested in vivo, a recently reported covalently binding anti-spike-protein nucleotidic TCI [ 68 ] should be effective as a long-lasting antibody equivalent neutralizing viral infection. Several aptamers targeting the check point proteins and their ligands have been reported, and a similar warhead introduction into these aptamers could create bioTCIs disrupting the immune check point mechanism used by cancer cells to evade elimination by the immune system.…”