Remdesivir, previously known as GS-5734, is a phosphoramidate prodrug of GS-441524, a 1′-cyano-substituted adenine nucleoside analogue. As a phosphoramidate prodrug, intravenous remdesivir is rapidly hydrolyzed in the serum by extracellular kinases to a nucleoside monophosphate (GS-441524), which then undergoes intracellular conversion to the antiviral, pharmacologically active nucleoside triphosphate metabolite (GS-443902). Remdesivir and GS-441524 are bioisosteres of monophosphates, meaning that they can more quickly be activated and undergo intracellular conversion to the active metabolite. By competing with endogenous nucleosides, GS-443902 can incorporate into viral RNA via inhibition of RNA-dependent RNA polymerase (8–12).

All remdesivir metabolites confer greater selectivity to RNA-dependent RNA polymerase in comparison to human polymerases (8). The presence of exoribonuclease (ExoN) within virus cells acts to correct RNA chain errors to assist in preventing antiviral activity (13). The activity of remdesivir is only minimally affected by ExoNs, as it can more effectively incorporate into viral RNA than other nucleotide analogs (14, 15). This effective incorporation into RNA allows for improved antiviral activity against single-stranded RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory distress syndrome coronavirus (SARS-CoV) and SARS-CoV-2 (9, 11).

Upon intravenous administration, remdesivir concentrations decline quickly (half-life ∼1 h) as extracellular kinases work to actively metabolize it to GS-441524 (half-life ∼27 h (11, 16). Remdesivir is highly protein bound (80%–90%), though GS-441524 is not (<5%) (16). It acts as a substrate to several cytochrome (CYP) P450 enzymes in vitro, including CYP2C8, CYP2D6, and CYP3A4. However, the specifics of these pathways are yet to be quantified but are thought to be minor given the rapid metabolism of the prodrug. GS-441524 is not a substrate of major CYP enzymes, suggesting that GS-441524 does not undergo extensive hepatic metabolism (8, 11, 16, 17). Remdesivir byproducts are predominantly excreted via urine, primarily as GS-441524 (49%), followed by remdesivir (10%) and GS-704277 (2.9%). Excretion via feces is negligible (16).

Adenosine is an endogenous purine nucleoside. It is composed of an adenine molecule with a ribose sugar moiety and is an essential component of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) (18, 19). Adenosine activity in the body is widespread, including activity to reduce blood pressure and heart rate, regulation of the sympathetic nervous system, and induction of vasodilation (18).

Adenosine binds to four receptor subtypes that are found on the surfaces of most cells within the body: A1, A2A, A2B, and A3. A1 receptor activation leads to negative chronotropic and dromotropic effect, A2 receptor activation leads to inotropic effects and vasodilation, and A3 receptors are minimally expressed in the myocardium. The adenosine A1 receptors are G_i protein-coupled receptors on cell surfaces with inhibitory functions when activated, leading to negative chronotropic and dromotropic effects (18, 20, 21). A1 receptors are expressed in the brain, spinal cord, kidney, spleen, and heart and have a strong affinity for adenosine (20).

In the heart, adenosine binds to A1 receptors primarily expressed in the atria, leading to decreased cAMP production, inhibition of protein kinase A and voltage-gated calcium channels, and subsequent opening of ATP-sensitive potassium gated channels. The inhibition of this calcium influx and increased potassium current mediates the inhibition of atrioventricular (AV) node conduction, causing a shortened action potential duration with refractoriness (18, 20, 21).

The human adenosine A1 receptor-Gi2-protein complex (PDB ID: 6D9H) (22) was used as the target receptor. The 3D structural coordinates for both adenosine (compound CID 60961) and GS-441524,(2R,3R,4S, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile (Compound CID 44468216) structures were acquired from the PubChem database at the NIH NCBI (23). GS-441524 was the only metabolite tested due to the lack of structural plausibility for the other metabolites. Prior to modeling, both ligand structures were minimized using WebMO (24). Ligand docking was carried out using UCSF Chimera (25) and AutoDock Vina (26). The AutoDock Vina search volume was centered on the A1 adenosine binding site, as described previously (22). The search volume dimensions were set to encompass both the putative binding site and the adjacent trans-membrane helical protein regions. Maximum binding modes was set at ten, with a maximum energy difference of 2 kcal/mol. Models were chosen on the basis of greatest binding affinity and minimum root mean square deviation. Figures of resulting structural models were prepared using UCSF Chimera or PyMOL 2.3.5. Datasets are available upon request: The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

Our team simulated the interaction between both adenosine and GS-441524 with the A1 receptor. Figure 2 shows the adenosine molecule (purple) binding within the G protein complex.

Figure 3 demonstrates GS-441524 (green) in its pre-docking state with the A1 G protein complex. The adenosine docking site is highlighted in this figure by a green square.

Figures 4, 5 demonstrate GS-441524 (green) in its docking state within the A1 G protein complex.

By modeling the interaction between the A1 G protein complex and both adenosine and GS-441524, we found that the proposed mechanism of exogenous A1 receptor activation is feasible based on docking compatibility.