The positioning of the 13-mer within the landscape of the spike proprotein is schematically represented in Fig. 1. To gain insight into the possible function of the 13-mer, Blastp analysis was performed against non-viral taxa, revealing significant identity and coverage with a segment derived from prolyl tRNA synthetase and its bifunctional analogue, glutamyl-prolyl tRNA synthetase (ligase) derived from diverse taxa including a bacterium, a protozoan parasite, and a boney fish (Fig. 2). This enzyme catalyzes the covalent attachment of proline to its cognate tRNA molecule and therefore plays a key role in protein translation. These blastp hits in turn revealed highly conserved identity with the key drug binding site of human glutamyl-prolyl tRNA synthetase1 (15) as delineated in Fig. 2, implying that the drug might also bind to the 13-mer region of the spike protein. Structural analysis of the SARS-CoV-2 Spike trimer-ACE2 complex revealed that the 13-mer forms a projection from S1 RBD that allows for asymmetric pairing with ACE2, forming the primary docking site in the co-complex (Fig. 3a), (10, 12). Comparatively, the 13-mer sequence from SARS-CoV-1, Bat RaTG13 CoV, and endemic hCoV NL63 demonstrated less relatedness to the SARS-CoV-2 13-mer, implying possible ectopic acquisition of this 13-mer sequence. Corresponding analysis of the 13-mer region of the spike protein from the South African variant of concern (VOC), Pango lineage B.1.351, revealed the presence of a single amino acid substitution, E484K, which is predicted to impair drug binding based on the conversion of a negatively charged residue to a positively charged residue at a critical drug contact surface (Fig. 3.b). In comparison, the delta variant, which is currently circulating world-wide, retains a persevered 13-mer sequence relative to the reference strain (data not shown), indicating that this VOC has retained the genetic ability to theoretical respond to halofuginone treatment and can be considered as a potential candidate for intervention of severe COVID-19.
The human orthologue, glutamyl-prolyl tRNA synthetase 1 (EPRS), plays an additional regulatory role in Stat and Smad3 signal transduction in response to various cytokines. Specifically, phosphorylation of EPRS allows for the enzyme to combine and form the Gamma-interferon Activated Inhibitor of Translation (GAIT) complex that regulates translation in immune cells in response to IFN-γ stimulation (16) and can be pharmacologically targeted using the inhibitor halofuginone (17). In particular, halofuginone has been shown to exert pleotropic effects on the immune system (18) and has received orphan drug designation by the FDA in March 2020 for the treatment of scleroderma. More recently, halofuginone has been shown to be a potent inhibitor of SARS-CoV-2 infection in vitro, through two distinct putative mechanisms: down regulation of TMPRSS2 or through the effects of amino acid starvation on genomic biosynthesis and heparin sulfate decoration of proteoglycans on the cell surface (19, 20).
Due to the degree of shared similarity between the EPRS drug binding domain and the SARS-CoV-2 13-mer, we pursued in silico docking analysis of the drug on the RBD of spike protein. Halofuginone docked flawlessly in an internal cavity formed inside the 13-mer projection, with a theoretical affinity of interaction of -3.9 kcal/mol. We posit that the apparent effect of drug binding to the 13-mer cavity is to lock it in the down configuration such that the S1 subunit is unable to transition upwards and facilitate the jack-knife-like movement of the S2 subunit to facilitate the ensuing fusion event (Fig. 3b). In comparison, cladosporin, a structural analogue with specificity for lysyl tRNA synthase bound slightly below and outside the cavity formed by the 13-mer region, with a theoretical affinity of -4.9 kcal/mol (Supplemental Fig. 1). Structural analogues (+)-deoxyhalofuginone and febrifugine were also docked. Deoxyhalofuginone docked within the 13-mer cavity, although not as precisely as halofuginone, with an apparent affinity of -4.5 kcal/mol (Supplemental Fig. 2), whereas febrifugine did not exhibit any binding proximal to the 13-mer (data not shown). The structure of these four drugs is represented in Supplemental Fig. 3 and underscores the specificity of halofuginone docking to the 13-mer concavity. Lastly, the drug was docked against the spike protein of SARS-CoV (supplemental Fig. 4). Halofuginone failed to bind within the concavity formed by the 13-mer, though it did bind below the 13-mer with a theoretical binding affinity of -4.8 kcal/mol, indicating that the drug was specific for the 13-mer of SARS-CoV-2.
We subsequently undertook in vitro enzymatic experiments to see if the drug could inhibit binding of the spike protein to the ACE2 receptor, thereby restoring the enzymatic activity of ACE2. In this analysis, the receptor binding domain (RBD) of spike protein (consisting of amino acids 330–530) was used rather than complete spike protein so that we could restrict our findings as tightly as possible to a minimal structure containing the 13-mer region (amino acids 482–494) and therefore definitively conclude the mechanism by which the drug might be inhibiting infection as reported by Chen et al., 2020, and Sandoval et al., 2021. Indeed, at the 0.01–0.1 nM range, halofuginone blocked the ability of the RBD to bind to ACE2, as evidenced by restoration of ACE2 activity in the context of spike protein RBD.