The Potential Protective Role of GS-441524, a Metabolite of the Prodrug Remdesivir, in Vaccine Breakthrough SARS-CoV-2 Infections

Cases of vaccine breakthrough, especially in variants of concern (VOCs) infections, are emerging in coronavirus disease (COVID-19). Due to mutations of structural proteins (SPs) (e.g., Spike proteins), increased transmissibility and risk of escaping from vaccine-induced immunity have been reported amongst the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Remdesivir was the first to be granted emergency use authorization but showed little impact on survival in patients with severe COVID-19. Remdesivir is a prodrug of the nucleoside analogue GS-441524 which is converted into the active nucleotide triphosphate to disrupt viral genome of the conserved non-structural proteins (NSPs) and thus block viral replication. GS-441524 exerts a number of pharmacological advantages over Remdesivir: (1) it needs fewer conversions for bioactivation to nucleotide triphosphate; (2) it requires only nucleoside kinase, while Remdesivir requires several hepato-renal enzymes, for bioactivation; (3) it is a smaller molecule and has a potency for aerosol and oral administration; (4) it is less toxic allowing higher pulmonary concentrations; (5) it is easier to be synthesized. The current article will focus on the discussion of interactions between GS-441524 and NSPs of VOCs to suggest potential application of GS-441524 in breakthrough SARS-CoV-2 infections. Supplementary Information The online version contains supplementary material available at 10.1007/s44231-022-00021-4.

There are three different groups of proteins that are encoded in the SARS-CoV-2 genome, namely structural proteins (SPs), non-structural proteins (NSPs), and accessory proteins (APs) [24]. Spike, Nucleocapsid, Envelope, and Membrane proteins are the major SPs ( Fig. 1) [24]. With respect to SPs, mutations mainly take place on the receptor-binding domain (RBD) in Spike proteins resulting in changes of its binding affinity to the membrane receptor angiotensin-converting enzyme 2 (ACE2) on host cells [25,26]. NSPs and APs are more conserved than SPs, yet whose mutational changes may facilitate new pathways involving viral replication and release [24].

The Non-phosphoramidate GS-441524 Could be Superior Over Remdesivir (Contains the Functional Group Phosphoramidate) Against SARS-CoV-and VOCs
Although the catalytic NTP that interacts and interferes with RdRp can be derived from both GS-441524 and Remdesivir, the complicated bioactivation pathway, preferential expression of Remdesivir prodrug bioactivating enzymes in the liver, and short half-life of Remdesivir (~ 1 h) render GS-441524 (~ 3-5 h) a better therapeutic candidate [33,34,43]. GS-441524 is bioactivated by nucleoside kinases, which are expressed more evenly across all tissues in the body [33]. Considering that it is quite common to observe comorbidities in patients with severe SARS-CoV-2 infections, GS-441524 could be potentially more potent than Remdesivir and could be  an antiviral therapeutic option against SARS-CoV-2 variants with greater patient tolerability.

Nucleoside Kinase is the Only Enzyme Required for GS-441524 Conversion to Active NTP
The molecular basis of bioactivation demonstrates differences in enzymatic requirement between GS-441524 and Remdesivir. GS-441524 requires only nucleoside kinase for bioactivation [33]. In contrast, Remdesivir requires carboxylesterase 1 (CES1), cathepsin A (CTSA), and histidine triad nucleotide-binding protein 1 (HINT1) that are expressed in kidney and liver tissues to be metabolised involving esterase and phosphoramidase pathways for bioactivation [33,44,45]. In severe COVID-19 patients with underlying comorbidities, the liver and kidney are likely to malfunction for the conversion of Remdesivir to bioactive NTP against SARS-CoV-2 ( Fig. 2).

Orally Bioavailable Prodrug of GS-441524
In in vitro studies, GS-621763, an orally bioavailable prodrug of GS-441524, has been shown to have low cytotoxicity and a similar EC 50 to GS-441524 [46,47]. Recent studies suggested that administration of GS-621763 is efficacious against SARS-CoV-2 in ferrets and mice [45,48]. A pharmacokinetic study revealed higher and more consistent plasma concentrations of GS-441524 in ferrets receiving oral GS-621763 compared to those receiving intravenous administration of Remdesivir or GS-441524 [48]. GS-621763 supports the exploration of GS-441524 oral prodrug in the management of breakthrough COVID-19.

Safety of GS-441524 Over Remdesivir
Data in both cell culture and in animal models indicate that GS-441524 is much less cytotoxic in cells and better tolerated in animals compared to Remdesivir [33,43,49,50]. The latter has shown to induce adverse effects in rhesus macaques (e.g., renal tubular atrophy) and patients (e.g., liver and kidney inflammation) [33,44]. The nonphosphoramidate GS-441524 may minimise liver and kidney adverse events, enables the drug to be administered in higher doses. The first human study of orally administered GS-441524 for COVID-19 (Trial ID: NCT04859244) in a healthy woman has also shown sustained plasma concentrations and excellent safety profile [51,52].

Socio-economic Benefits of GS-4414524 Over Remdesivir
The structural complexity of Remdesivir makes drug production costly and difficult. The minimum production cost of Remdesivir is USD$9.30 for a 10-day treatment course (100 mg two times on Day 1 and 100 mg one time on Days 2-9), which is much more expensive than many other repurposed antiviral drugs for COVID-19 such as fluvoxamine [53][54][55]. In contrast, GS-441524, with its simpler structure (3 functional groups less than Remdesivir) and as the prodrug of Remdesivir during the production procedure and in tissue metabolism, would be significantly less expensive than Remdesivir to produce [33].

Combination Therapy of GS-441524
As SARS-CoV-2 variants continue to emerge, there have been increasing interests in developing combination therapies (both virus-and host-targeted) through repurposed drugs, with the goal of better inhibiting viral infections by targeting different mechanistic pathways [56,57]. As each drug has different yet specific mechanism of action, one advantage of utilizing combination therapies is the potential of achieving drug synergy, offering a treatment that performs better than when administering the individual drugs alone. This also raises the possibility of using lower effective concentrations of each drug in a combination therapy to minimize drug toxicity, side effects, and costs.

Combination Therapy with Functional Inhibitors of Acid Sphingomyelinase (FIASMA)
FIASMA (e.g., fluoxetine, amiodarone, and imipramine) is a group of psychotropic medications that inhibits the lysosomal enzyme acid sphingomyelinase and regulates the homeostasis of the endolysosomal host-pathogen interface [58,59]. In in vitro models, FIASMA has been found to efficiently inhibit SARS-CoV-2 entry and propagation via mechanisms such as impairing endolysosomal acidification and inducing cholesterol accumulation within the endosomes [59,60]. The antiviral potency of FIASMA is further supported by recent clinical studies [55,58,61,62]. For example, in patients with psychiatric disorders hospitalized for severe COVID-19, those receiving FIASMA medications at baseline had significantly reduced risk of intubation or death as compared to those receiving non-FIASMA antidepressants (p < 0.01) [58]. Together, these data suggest the potential antiviral potency of FIASMA in treating SARS-CoV-2 infections.
Recently, many papers revealed the synergistic antiviral potential of therapies that combine remdesivir and/or GS-441524 with FIASMA for treating SARS-CoV-2 variants [56,57,59]. For example, in in vitro models of SARS-CoV-2, combined therapy of GS-441524 with fluoxetine showed a more superior viral titer reduction (over 99% reduction) than using fluoxetine (60-70% reduction) or GS-441524 treatment alone (90% reduction) [57]. More importantly, the synergistic antiviral effects are not only observed in the SARS-CoV-2 parental strain, but also in the Alpha and Beta variants, providing support for the applicability of such combination treatments for the currently prevailing Omicron variant infections [57]. Omicron variants have also been found to rely heavily on the endocytic pathways for viral entry, which further supports the use of host endolysosome-directed FIASMA with viral replication-directed remdesivir/GS-441524 for treating SARS-CoV-2 infections [63].

Combination Therapy with Other Drugs
Additionally, combining remdesivir with many non-FIASMA drugs, including itraconazole, baricitinib, and MEK1/2 Inhibitor ATR-002 (Zapnometinib) have also shown synergistic antiviral effects against SARS-CoV-2 [56,64,65]. In polarized Calu-3 cell culture model, itraconazoleremdesivir combination inhibits the production of infectious SARS-CoV-2 particles by over 90% and shows synergistic effects [56]. In a double-blind, randomized, placebocontrolled trial, baricitinib-remdesivir treatment reduces recovery time of patients with COVID-19 and accelerates their improvement in clinical status as compared to using remdesivir alone [64]. Treatment combinations of ATR-002 with remdesivir have also been found to display synergistic antiviral effects [65]. The above drugs are promising targets to be used in conjunction with the direct-acting antiviral remdesivir against SARS-CoV-2 and in vivo and clinical studies are critical in further validating their potency.

Limitations of Combination Therapy
Patients with severe SARS-CoV-2 infections often hold other comorbidities that may be exacerbated when giving additional repurposed drugs or combination therapies [66]. While both GS-441524 and FIASMA have been reported with little adverse effects on organs, careful evaluations need to be taken about the suitability and safety of GS-441524-FIASMA treatments before making a treatment decision [57,66,67].

Inhibition of RdRp (NSP12, 7, 8) Polymerization
RdRp is a multi-unit transcription complex consisting of NSP12, NSP7, and NSP8, which is essential for the replication of the SARS-CoV-2 genome (Table 1) [35,68]. NTP is a high-affinity substrate for RdRp through interaction with NSP12 [35,69] and inhibits viral replication through incorporation by RdRp into nascent viral RNA, predominately resulting in chain termination at the i + 3 position with a steric clash of the NTP 1′-CN group with residue R858 of RdRp [70]. The efficient incorporation of NTP into the newly synthesized viral RNA chain is due to the superior selectivity of NTP compared to ATP and other nucleoside analogues [71,72]. The effective inhibition of RdRp is dependent on the complementarity between NTP and RdRp. When mutations in amino acid residues that interact with NTP are present, the binding properties may be altered to reconstruct the interactions between NTP and RdRp [73].

Inhibition of NSP5
NSP5 mediates the processing of NSPs at 11 cleavage sites (NSP4-11, NSP12-15) ( Table 1) [24,79]. In conjunction with NSP3, the two proteases cleave SARS-CoV-2 encode precursor polyproteins pp1a and pp1b into 16 NSPs to assemble the viral replicase complex [24]. Among crucial NSP5 residues, C145 and H164 exhibit strong hydrogen bonding with Remdesivir [80,81]. Current data suggest H164 as an essential active site for NSP5 function, which when disrupted, may potentially halt its proteolytic activity [80]. As the structure of the interaction site in Remdesivir is conserved in GS-441524, such interactions between GS-441524 and the NSP5 active site are highly probable [81]. Thus, it is important to investigate whether mutations in the active site are observed that could potentially disrupt GS-441524-NSP5 interactions. Similar to RdRp, since no mutation of NTP-interacting residues in NSP5 had been found as of March 20th, 2022, the interactions between GS-441524 and NSP5 of VOCs (Alpha, Beta, Gamma, Delta, and Omicron variants) would appear similar to that between GS-441524 and Wuhan wildtype NSP5 (Table 2) (https:// nexts train. org/ ncov/ global) [4,5].

Blockage of the Active Site in NSP14
As a 3′-to-5′ exoribonuclease and a guanine-N7methyltransferase, NSP14 is a crucial component securing the replication of SARS-CoV-2 (Table 1) [82,83]. The exoribonuclease domain of NSP14 is critical for viral replication given that mutant exoribonuclease knockout SARS-CoV-2 results in interruption of viral replication [84]. NSP10, the replicative cofactor of NSP14, stabilizes and stimulates enzymatic activities through interaction with exoribonuclease [85]. Furthermore, NSP10 has been proposed to interact with NSP12 to undergo RNA repair processes that may arise during RNA synthesis, indicating the possibility of interactions between NSP10, NSP12, and NSP14 [83]. Studies provided evidence suggesting that NSP14 interacts with NTP, where the cyano group at the 1′-ribose position of NTP fit complementarily with the active site of NSP14 exoribonuclease [40,83]. The distorted base of NTP is predicted to prevent the proper distances for efficient two-metal ion catalysis, thus disrupting the function of exoribonuclease [40]. Due to the importance of complementarity in ensuring the effect of NTP on NSP14 activities, it is worth investigating the NSP14 mutations in VOCs to assess their influences on the potency of NTP.
As of March 20th, 2022, one mutated NSP14 residue that interacts with GS-441524 is observed on Nextstrain in a sample of the Omicron variant sub-lineage BA.1 (https:// nexts train. org/ ncov/ global) [4,5]. The H95Y mutation is not characteristic for the BA.1 lineage, implying that it would not be present in all samples of the sub-lineage (Table 2). However, it is attention-worthy due to the high global prevalence (12%) of the BA.1 sub-lineage (Supplementary  Table S2). Therefore, a more conservative conclusion is that the interactions of GS-441524 with NSP14 would be changed in the Omicron variant but would remain relatively conserved across the other VOCs.

Preclinical and Clinical Studies Using GS-441524 in SARS-CoV-2 Infection
GS-441524 has shown potency in lowering SARS-CoV-2 replication in in vitro human lung Calu-3 cell infection [34], in mice with an increased viral clearance 2 days postinfection and reduced weight loss [43], and has demonstrated exceptional safety, tolerability, and pharmacokinetics in one human (case report; Cmax: 12·01 μM, surpassing the concentration required to eradicate SARS-CoV-2 in vitro [51,52]) and several preclinical species [50]. A human study of orally administered GS-441524 for COVID-19 is underway [51,52]. Taken together, clinical studies of GS-441524 on VOCs are of great interest, given its antiviral potentials.

Limitations
The data for the NSP amino acid residues that interact with GS-441524 are based largely on in silico studies, and continual vigorous analysis are needed for further verification [40,71,77,86]. The effect of mutations on the binding affinity of NSPs to GS-441524 remains to be monitored in emerging VOCs. The potential discrepancy may exist between the microscopic effect of the mutated amino acids and their macroscopic influence on NSP structure and protein-protein interactions. Nevertheless, our analysis provides a foundation for future clinical trial testing of GS-441524 in breakthrough VOCs. The promising results of combination therapies in recent literature also suggest that combining virus-directed and host-directed drugs may partially help to counteract the possible reduction in potency of antiviral drugs against the emerging SARS-CoV-2 variants.

Conclusion
Given the recent rise of breakthrough SARS-CoV-2 cases and the emerging Alpha, Beta, Gamma, Delta, and Omicron variants that have shown spike protein mutations, there is an urgent need to examine antiviral candidates that could contain these VOCs from escaping vaccines. The major amino acid sites of NSPs (NSP3, 5, 12, and 14) that interact with the parental nucleotide GS-441524 are not altered in the emerging VOCs. As such, we believe that the ready-touse GS-441524 is a potential antiviral approach against the breakthrough VOCs (Fig. 3). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.