Commentary Potential Enhancement by 3-Deazauridine of the Antiviral Activity of Molnupiravir in Patients with COVID-19 ()
1. Introduction
The human pandemic coronavirus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has produced substantial worldwide morbidity and mortality [1] . What is urgently needed is effective drug therapy to treat patients with advanced COVID-19 to arrest progression prevent death due to the viral infection. An interesting agent to test for therapeutic activity against COVID-19 is molnupiravir, a prodrug of the cytosine ribonucleoside analog, beta-D-N4-hydroxycytidine (NHC) that contains isopropylester at its 5’ end [2] . After removal of the isopropylester by esterases, NHC is phosphorylated by host kinases to its 5’-triphosphate form (NHC-TP) and incorporated into RNA by the viral RNA-dependent RNA polymerase. The presence of NHC in the viral RNA results in mutation ns during viral replication that leads to a non-infective virus [3] [4] . NHC exhibits in vitro antiviral activity against all coronaviruses tested, including SARS-CoV-2 [5] . Treatment of animals with NHC significantly inhibited SARS-CoV-2 [6] . Therapeutic treatment of infected animals with NHC significantly reduced upper respiratory tract SARS-CoV-2 load and completely suppressed spread to untreated animals [6] . These interesting preclinical results on the antiviral activity of NHC lead to the design of a clinical trial where patients diagnosed with advanced COVID-19 were treated with oral NHC to determine if this antiviral therapy can prevent progression to severe illness, and block transmission of severe acute respiratory syndrome [7] . The formulation of NHC for oral administration was accomplished by the insertion of isopropylester to the 5’end of NHC [8] . This chemical modification facilitated the oral administration of molnupiravir to patients with COVID-19.
The preliminary results of the oral treatment with molnupiravir exhibited positive responses in many COVID-19 patients treated with NHC [7] . This novel therapy in patients with COVID-19 was well tolerated and associated with potent antiviral efficacy as shown by reduced infectious virus isolation, reduction in the time to elimination of SARS-CoV-2 RNA and a greater reduction in SARS-CoV-2 viral RNA from baseline compared to placebo treatment [7] [8] . These remarkable preliminary results suggest that NHC has the potential to prevent the progression of patients with advanced COVID-19 to a fatal outcome.
An interesting question is whether this promising antiviral therapy with NHC to arrest and prevent COVID-19 progression can be enhanced by the addition of another drug. Studies on the pharmacology of cytosine nucleoside analogues suggest that it may be possible to enhance the antiviral action of NHC by its use in combination with an agent that modulates its metabolism.
3-Deazauridine (3DU) is a cytosine ribonucleoside analogue that after its phosphorylation to its triphosphate and acts as a competitive inhibitor of CTP synthetase [9] (Figure 1). Inhibition of this enzyme reduces the level of CTP in the cells by the reduction of its competition with cytosine nucleoside analogues for incorporation into RNA. A good example of this interaction is shown by the remarkable enhancement by 3DU of the antileukemic activity of the cytosine ribonucleoside analogue, 5-azacytidine [10] . Treatment of leukemic cells with 3DU increased the incorporation of 5-azacytidine into RNA by 80%. By this similar mechanism, 3DU has the potential to enhance the incorporation of NHC-TP into viral RNA, which should increase significantly its antiviral action against SARS-CoV-2 (Figure 2). The antiviral activity of NHC is most likely dependent on the number of molecules of NHC-TP incorporated into the viral RNA. If the number of incorporated molecules of NHC-TP falls below the threshold for complete viral inactivation, some viruses may survive the treatment. Treatment with 3DU in combination with HMC can prevent these possibilities.
The reduction in the level of CTP in cells by 3DU also leads to the reduction
![]()
Figure 1. Molecular mechanism of action of 3-deazauridine (3-DU). The phosphorylation of 3-DU is catalyzed by uridine-cytidine (UC) kinase to its monophosphate (3-DUMP) and by cellular kinases to its diphosphate (3-DUDP) and triphosphate (3-DUTP).
![]()
Figure 2. The metabolism of molnupiravir (5’-isopropylester-NHC). After removal of the 5’-isopropylester, NHC is phosphorylated by cellular kinases to its monophosphate (NHC-MP), diphosphate (NHC-DP) and triphosphate (NHC-TP). The viral RNA-dependent RNA polymerase incorporates NHC-TP into viral RNA. The reduced level of CTP (shown by smaller size) permits a greater incorporation of NHC-TP Into viral RNA by the viral RNA polymerase.
in dCTP, since CTP is its precursor. This is the mechanism by which 3DU enhanced the anti-HIV activity of the cytosine deoxyribonucleoside analogues, zalcitabine and lamivudine, which have to compete with dCTP for incorporation into the viral DNA in the reverse transcriptase reaction [11] . The reduction of dCTP by 3DU is also responsible for the enhancement of the antileukemic action of 5-aza-2’-deoxycytiine due to its increased incorporation into DNA [12] [13] . In these latter studies, 3DU exhibited remarkable reproducible inhibitory activity against leukemic cells in cell cultures, animal models and in patients. These observations indicate that the antineoplastic action of 3DU is very reproducible both in vitro and in vivo. The clinical studies on 3DU in patients with leukemia indicate that it exhibits minimal side effects and would be safe to use in combination with molnupiravir [13] . The preclinical and clinical studies on 3DU show that the effective plasma concentration of 3DU to modulate the metabolism of cytosine nucleoside analogues is in the range of 50 µM [13] .
3DU in combination with NHC merits preclinical investigation to confirm its enhancement of the in vitro antiviral activity of NHC against SARS-CoV-2 in cell culture and its in vivo antiviral activity in animal models. Positive results in these preclinical models will provide a very good rationale for the investigation of 3DU plus NHC in patients with advanced COVID-19. Initial studies on patients with advanced COVID-19 can start with an oral dose of molnupiravir of 200 mg twice per day for 2 days [8] . After oral administration of molnupiravir, NHC achieves its maximum serum concentration with a median time of 1.00 to 1.75 h [8] . The serum concentration of NHC declines slowly with a half-life of approximately 1 h. Therapeutic concentrations of NHC against SARS-CoV-2 after oral administration are estimated to have a duration of about 6 h. Therefore, after the oral dose of molnupiravir, the duration of the infusion of 3DU should be about 6 h to obtain maximum incorporation of NHC into viral RNA. In patients with leukemia, 3DU was infused at a rate of 5 mg/kg/h for up to 72 h without any sign of adverse events [13] . Pharmacokinetic studies in patients administered an i.v. infusion of 3DU the estimated plasma half-life of 3DU was 4 h [14] .
The objective is to find the optimal dose schedule of molnupiravir and 3DU that will arrest the progression of viral disease. The rapid development of SARS-CoV-2 variants that are resistant to standard COVID vaccines provides a very good rationale to improve the effectiveness of antiviral drug therapy to rescue patients with progressive disease due to the infection with vaccine-resistant variants.
2. Conclusion
Molnupiravir after its metabolic conversion to the active inhibitor, NHC-TP, and incorporation into viral RNA, exhibits remarkable antiviral against COVID-19 [15] [16] . The pharmacology of 3-DU predicts that it has the potential to enhance the incorporation of NHC-TP into the SARS-CoV-2 RNA leading to a non-functional viral RNA. This action predicts that 3-DU will increase the antiviral potency of molnupiravir in patients with COVID-19. This novel antiviral therapy merits preclinical and clinical investigation.
Financial Support
This work received financial support from the Dean of Medicine, Université de Montréal.