An Overview on Anti-COVID-19 Drug Achievements and Challenges Ahead

Abstract

The appearance of several coronavirus pandemics/epidemics during the last two decades (SARS-CoV-1 in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019) indicates that humanity will face increasing challenges from coronaviruses in the future. The emergence of new strains with similar transmission characteristics as SARS-CoV-2 and mortality rates similar to SARS-CoV-1 (∼10% mortality) or MERS-CoV (∼35% mortality) in the future is a terrifying possibility. Therefore, getting enough preparations to face such risks is an inevitable necessity. The present study aims to review the drug achievements and challenges in the fight against SARS-CoV-2 with a combined perspective derived from pharmacology, pharmacotherapy, and medicinal chemistry insights. Appreciating all the efforts made during the past few years, there is strong evidence that the desired results have not yet been achieved and research in this area should still be pursued seriously. By expressing some pessimistic possibilities and concluding that the drug discovery and pharmacotherapy of COVID-19 have not been successful so far, this short essay tries to draw the attention of responsible authorities to be more prepared against future coronavirus epidemics/pandemics.

Undoubtedly, coronavirus disease 2019 (COVID-19) is one of the most pernicious pathological conditions in history that has disrupted all aspects of human life. Unbelievably, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was able to invade all countries at an astonishing speed in just a few months, killing millions of people and causing a loss of billions of dollars in the economy. Although one of the important reasons for this amazing transmissibility is related to the nature of this virus, the development of modern traveling facilities, and vehicles used in the air, sea, and land, has played a key role in the rapid spread of the virus around the world.¹ Despite the fact that modernization has contributed to the rapid spread of viruses, it should not be overlooked that the scientific advances achieved in the modern era (such as advanced genome sequencing for identifying new viruses, developed modern vaccines, and optimized modern methods for producing effective drugs) have increased humanity’s ability to face the challenge of emerging future viral pandemics. Since facing future viral epidemics/pandemics is inevitable, the question raised here is how much countries have been able to understand these threats and try to use their facilities and resources to secure the future of humanity. To correctly answer this question, it is necessary to have a brief overview of viral pandemics in recent decades along with scientific findings and drug achievements against these viruses and their possible dangers.

1. Future Threats Caused by Coronaviruses

During the last two decades, the world has faced six important viral pandemics, whose characteristics are summarized in Table 1. As can be seen, all these pandemics are caused by RNA viruses, which usually have a much higher mutation rate than DNA viruses. This higher rate is because viral RNA-polymerases are not as evolutionarily proofreading as viral DNA-polymerases.² From the data in Table 1, it can also be concluded that the RNA viruses belonging to the coronaviridae family were the most prevalent and approximately triggered one pandemic every ten years. Considering the importance of increasing threats of viral diseases, here we take a brief look at the main mechanisms of the evolution and emergence of RNA viruses.

Table 1. Six Major Viral Pandemics SARS-CoV-1, MERS-CoV, Ebola, Zika, and SARS-CoV-2, Emerged during the Last Two Decades, Along with Some of Their Important Characteristics.

pandemics	virus family	virus type (DNA/RNA)	emergence period	territorial origin	involved countries	number of cases	fatality ratio (%)
SARS-CoV-1	Coronaviridae	RNA virus	2002–2004	China	30	>8000	∼11
Influenza A H1N1	Orthomyxoviridae	RNA virus	2009–2010	Mexico	all	>700 000 000	<0.001
MERS-CoV	Coronaviridae	RNA virus	2012 until now (2023)	Saudi Arabia	27	>2500	34–37
Ebola	Filoviridae	RNA virus	2013–2016	Western Africa	11	>27 000	25–90
Zika Virus (ZIKV)	Flaviviridae	RNA virus	2015–2016	Brazil	87	>3500	N/A
SARS-CoV-2	Coronaviridae	RNA virus	2019 until now (2023)	China	all	>662 000 000	∼1

In general, the five factors including mutation, recombination, natural selection, genetic drift, and migration are mentioned as the main forces for creating evolutionary change to shape the genetic structure of viral populations.³ All these factors play a basic role in the evolution of both human and animal DNA/RNA viruses, but the three factors of mutation, selection, and genetic drift are key for RNA viruses.⁴ A better understanding of evolutionary change processes in RNA viruses requires considering their four important features: First, RNA viruses frequently have highly large population sizes compared to other viruses. Second, these viruses reproduce very quickly and their population grows explosively. Since natural selection leads to the emergence of more upgraded and efficient viruses in the presence of larger populations, the last two features are of fundamental importance in the evolution of RNA viruses. Third, due to the lack of proofreading activity in the polymerase proteins of RNA viruses, these agents have a much higher mutation rate compared to DNA viruses.⁵ Fourth, the genome sizes of RNA viruses are usually smaller (ranging from 3 kb to 30 kb) compared to DNA viruses, because the high mutation rate can limit the size of the genome.⁶ Having these important evolutionary features allows RNA viruses to continuously create effective mutations in a short period of time and thus increase their adaptability to their surrounding environment so that they hardly extinct. In the high evolutionary adaptation of RNA viruses, the large population also plays a key role.⁷ This issue can apply strongly to SARS-CoV-2, which has spread to all countries in the world and infects both humans and animals.⁸ In addition, the subsequently identified substrains of the SARS-CoV-2 (such as Omicron and its derived substrains) have the ability to spread and multiply much more than the original wild type. Apart from the theoretical issues, it has also been observed in practice that SARS-CoV-2 continuously evolves through the occurrence of mutations during the replication of its genome. The occurrence of such changes in the genetic codes of this virus has affected its characteristics (including transmissibility, antigenicity, and infectiousness) and can have more effects in the future. Although coronaviruses mentioned in Table 1 have different infectiousness and transmissibility characteristics, they have a remarkable level of shared genetic similarity (the genomic sequence of SARS-CoV-2 is approximately 79% similar to SARS-CoV-1 and about 50% similar to MERS-CoV).⁹ Furthermore, the genomic sequencing of key proteases PLpro and 3CLpro in viruses SARS-CoV-2 and SARS-CoV-1 have even much higher similarities (PLpro enzyme of the two viruses have about 83% similarity and their 3CLpro enzymes have about 96% similarity, while the amino acid sequence of their active sites is almost the same).^10,11 Despite this high degree of similarity, we should appreciate that SARS-CoV-2 does not have the same mortality rates as SARS-CoV-1 and MERS-CoV with rates of 11% and 34%, respectively. Although this point is fortunate, it has some serious potential risks behind it due to the possibility of the emergence of more lethal new strains in the future. The emergence of a new coronavirus with a fatality rate similar to MERS/SARS-CoV-1 and simultaneously having SARS-CoV-2-transmissibility potential is a pessimistic hypothesis, but it is possible in the future. Such viruses can kill hundreds of millions of people in the world within a few months only in their first round of infection (Figure 1). Indeed, if such viruses appear in the near future, how much knowledge and preparation would be needed to fight them, and how much of our recently gained experience from facing the recent epidemics/pandemics of coronaviruses would be useful to us? To answer these questions, it is necessary to have a brief overview of drug achievements and challenges related to coronavirus diseases.

Figure 1.

Possibility of the emergence of highly infectious coronavirus strains in the future. As schematically shown, societies will face serious problems if new coronaviruses emerge with simultaneous high transmissibility potential of SARS-CoV-2 and high infectious/fatality rate of SARS-CoV-1 (hypothetical virus A) or MERS-CoV (hypothetical virus B).

2. Anti-COVID-19 Drugs

During the last three years and after the emergence of COVID-19, many related studies were carried out. As a result, several natural-based molecules, synthetic compounds, and monoclonal antibodies with significant therapeutic potentials have been identified to fight SARS-CoV-2, some of which have even been licensed for the treatment of COVID-19 patients (Figure 2).¹² In addition, some molecules with high abilities to inhibit SARS-CoV-2 have been withdrawn or have not received approval due to health concerns and serious side effects on vital organs such as the cardiovascular system.¹³ Although the amount of effort that has been made so far to fight SARS-CoV-2 is remarkable, the introduced medications were not able to thoroughly treat those suffering from COVID-19. The question that arises here is how much is the therapeutic effect of these medications compared to collective/herd immunity.

Figure 2.

Chemical structure of anti-COVID-19 drugs licensed for emergency use or fully approved by the FDA. Among the drugs shown above, baricitinib and remdesivir have been fully approved by the FDA for the treatment of COVID-19 patients, but molnupiravir and paxlovid (nirmatrelvir + ritonavir) are licensed for emergency use and have not yet been fully approved for this purpose.

So far, more than ten drugs have been authorized by the United States Food and Drug Administration (FDA) under Emergency Use Authorization (EUA) for the treatment of COVID-19 patients, and dozens of vaccines have been approved worldwide. Since the rules for granting licenses for the use of therapeutic agents are different in every country, we focus here on the anti-COVID-19 medications licensed by the FDA as one of the most respected authorities in the world. Table 3 provides a list of FDA-recommended anti-COVID-19 drugs, along with some of their characteristics including mechanism of action, approval/withdrawal history, and clinical indications for COVID-19 patients. As can be seen, these agents are divided into two major categories, monoclonal antibodies, and synthetic small molecules. These two categories are each divided into two subcategories, which include direct-acting anti-SARS-CoV-2 agents and agents with anti-inflammatory effects. Here, we briefly evaluate the success or failure of each of these agents in treating COVID-19.

2.1. Monoclonal Antibodies

As pointed out in recent studies, the binding of SARS-CoV-2 through the spike (S) glycoprotein to the human angiotensin-converting enzyme 2 (ACE2) receptor is the main route to enter the host cells and start the virus life cycle.¹⁴ For most monoclonal antibodies designed to directly damage the SARS-CoV-2 replication, the S protein is the main target.¹⁵ Since many mutations occur in the S protein-coding gene which causes changes in the receptor-binding domain (RBD) of the S protein,¹⁶ the binding affinity of the virus with hACE2 changes in the mutated substrains. It is expected that in mutants in which the amino acid sequence of the RBD region is changed in a such way that increases the binding affinity of RBD with hACE2, the transmissibility increases and dominates in the viral population. These events pose serious challenges in combating mutated substrains of SARS-CoV-2 and to the efficacy of drugs previously designated to inhibit the binding of the S protein of older substrains to host receptors. Among the agents listed in Table 3, eight of them are monoclonal antibodies, the vast majority of which have either been withdrawn from treatment processes (including bamlanivimab, bebtelovimab, copackaged tixagevimab plus cilgavimab, and sotrovimab, all of which are designed to inhibit the SARS-CoV-2 spike protein binding to the host ACE2 receptor), or have not yet gained fully approval (anakinra, etesevimab, and combination of the casirivimab plus imdevimab). Tocilizumab (with the brand name actemra), which previously received FDA approval in 2010 for the treatment of inflammatory and autoimmune disorders such as cytokine release syndrome, is the only recombinant human monoclonal antibody that recently received full approval for the use in the treatment of some hospitalized COVID-19 patients receiving systemic corticosteroids and requires supplemental oxygen, extracorporeal membrane oxygenation, and mechanical ventilation.

In general, it can be concluded that the use of direct-acting anti-SARS-CoV-2 monoclonal antibodies has not been successful so far, which is due to the continuous and numerous mutations in the spike protein of newer SARS-CoV-2 substrains.

2.2. Small Molecules

In the case of small molecule anti-COVID-19 drugs, the situation seems somewhat better. In total, baricitinib (brand name: olumiant), paxlovid (nirmatrelvir plus ritonavir), remdesivir (brand name: veklury), and molnupiravir (brand name: lagevrio) have been able to get EUA for the treatment of COVID-19. Among them, just baricitinib and remdesivir have been fully approved by the FDA. Both these drugs (baricitinib and remdesivir) were previously developed for other diseases and later repurposed to treat COVID-19, so they cannot be considered entirely as the result of the efforts made during the battle against SARS-CoV-2. As can be seen from the data in Table 3, except for baricitinib which is a JAK1/2 inhibitor with anti-inflammatory activity, the rest of these molecules have direct antiviral effects and act by disrupting the activity of critical SARS-CoV-2 enzymes including 3CLpro and RdRp. Since these antiviral molecules target highly conserved regions of SARS-CoV-2 enzymes, some studies have shown that their antiviral effect persists also against newly emerged Omicron subvariants.¹⁷Table 2 shows the reported antiviral efficacy (IC₅₀) of the drugs remdesivir, molnupiravir, and nirmatrelvir against SARS-CoV-2 variants. In order to compare the antiviral effects of these drugs, we used here only data from studies in which IC₅₀ values were reported for all three drugs. As can be seen from the data presented in Table 2, all three of these drugs strongly inhibit the proliferation of different SARS-CoV-2 strains including wild-type, Alpha, Beta, Gamma, Delta, and Omicron subvariants at mostly comparable single-digit IC₅₀ values (the average IC50 values for the drugs remdesivir, molnupiravir, and nirmatrelvir presented in Table 2 are 2.09, 5.42, and 2.33, respectively). Here we give a brief overview of the characteristics and capabilities of these molecules in the fight against SARS-CoV-2 and the treatment of COVID-19.

Table 2. IC₅₀ Values (μM) of Small Molecules Remdesivir, Molnupiravir, and Nirmatrelvir against Different SARS-CoV-2 Variants Including Wild-Type, Alpha, Beta, Gamma, Delta, and Omicron Sub-variants^a.

viral strain	Remdesivir (or GS-441524)b	Molnupiravir (or EIDD-1931)b	Nirmatrelvir	ref.
hCoV/Korea/KCDC03/2020	2.24	11.08	1.51	(Cho et al., 2023)17
hCoV/Wuhan/Hu-1/2019	1.7	2.8	2.7	(Takashita, Yamayoshi, Simon et al., 2022)18
hCoV/UT-NC002–1T/2020/Tokyo	0.98	0.59	1.71	(Takashita, Yamayoshi, Fukushi et al., 2022)19
hCoV/UT-NC002–1T/2020/Tokyo	1.23	1.46	1.08	(Takashita, Yamayoshi, Halfmann et al., 2022)20
hCoV/UT-NC002–1T/2020/Tokyo	1.8	2.5	2.2	(Imai et al., 2023)21
B.1 wild-type	6.90	1.1	5.8	(Fiaschi et al., 2022)22
Alpha (B.1.1.7)	1.75	6.19	1.02	(Cho et al., 2023)17
Beta (B.1.351)	1.84	7.97	0.98	(Cho et al., 2023)17
Gamma (P.1)	2.73	11.62	1.371	(Cho et al., 2023)17
Delta (B.1.627.2)	3.70	12.96	1.01	(Cho et al., 2023)17
Delta (USA/WI-UW-5250/2021)	0.61	1.85	3.29	(Takashita, Yamayoshi, Halfmann et al., 2022)20
Delta (Japan/TKYTK1734/202)	1.43	0.88	2.75	(Takashita et al., 2023)23
Delta (USA/WI-UW-5250/2021)	1.09	1.20	5.43	(Takashita et al., 2023)23
BA.1	1.42	4.66	1.11	(Cho et al., 2023)17
BA.1	1.9	7.5	4.8	(Takashita, Yamayoshi, Simon et al., 2022)18
BA.1 (hCoV-19/Japan/NC928-2N/2021)	0.99	0.51	3.92	(Takashita et al., 2023)23
BA.1.1	2.0	6.0	3.9	(Takashita, Yamayoshi, Simon et al., 2022)18
BA.1.1 (Japan/NC929-1N/2021)	0.67	1.11	1.71	(Takashita et al., 2023)23
BA.2.2	1.95	4.63	0.84	(Cho et al., 2023)17
BA.2	2.77	8.09	2.07	(Cho et al., 2023)17
BA.2 (Japan/UT-NCD1288-2N/2022)	2.68	6.60	3.69	(Takashita, Yamayoshi, Halfmann et al., 2022)20
BA.2 (Japan/UT-NCD1288-2 N/2022)	5.9	8.7	6.9	(Takashita, Yamayoshi, Simon et al., 2022)18
BA.2 (Japan/UTNCD1288-2N/2022)	1.32	0.25	1.69	(Takashita, Yamayoshi, Fukushi et al., 2022)19
BA.2 (Japan/UT-NCD1288-2N/2022)	2.3	3.5	3.2	(Imai et al., 2023)21
BA.2.12.1	1.28	9.67	1.25	(Cho et al., 2023)17
BA.2.12.1 (USA/NY-MSHSP-PV56475/2022)	0.5	3.2	1.8	(Takashita, Yamayoshi, Simon et al., 2022)18
BA.2.3	1.07	7.53	2.11	(Cho et al., 2023)17
BA.4.1.1	3.34	9.19	1.83	(Cho et al., 2023)17
BA.4 (USA/MD/HP30386/2022)	1.2	3.3	2.9	(Takashita, Yamayoshi, Simon et al., 2022)18
BA.5 (Japan/TY41-702/2022)	2.0	4.1	4.4	(Takashita, Yamayoshi, Simon et al., 2022)18
BA.5 (Japan/TY41-702/2022)	0.78	8.36	2.01	(Takashita, Yamayoshi, Halfmann et al., 2022)20
BA.5 (Japan/TY41-702/2022)	0.45	0.23	1.50	(Takashita, Yamayoshi, Fukushi et al., 2022)19
BA.5 (Japan/TY41-702/2022)	2.3	4.6	2.9	(Imai et al., 2023)21
BA.5.2.1	1.91	1.94	0.51	(Cho et al., 2023)17
BA.4/BA.5	2.67	1.37	6.82	(Takashita et al., 2023)24
BA.2.75	1.59	7.31	0.54	(Cho et al., 2023)17
BA.2.75 (Japan/TY41-716/2022)	1.52	0.90	1.78	(Takashita, Yamayoshi, Fukushi et al., 2022)19
BA.4.6	1.82	4.74	0.92	(Cho et al., 2023)17
BA.4.6 (USA/WI-UW-12757/2022)	1.95	8.38	4.43	(Takashita, Yamayoshi, Halfmann et al., 2022)20
BA.4.6 (USA/WI-UW-12767/2022)	0.54	2.62	1.29	(Takashita, Yamayoshi, Halfmann et al., 2022)20
BA.2.75.2	4.34	2.92	1.27	(Cho et al., 2023)17
BF.7	2.45	9.4	1.32	(Cho et al., 2023)17
BF.7	1.64	18.37	3.16	(Takashita et al., 2023)24
BQ.1.5	3.04	4.43	1.32	(Cho et al., 2023)17
BQ.1.1	2.00	6.62	1.30	(Cho et al., 2023)17
BQ.1.1	4.89	44.85	10.8	(Takashita et al., 2023)24
BQ.1.1 (Japan/TY41-796/2022)	1.0	2.8	2.6	(Imai et al., 2023)21
BJ.1	3.22	1.56	1.18	(Cho et al., 2023)17
XBB.1	2.76	3.87	0.94	(Cho et al., 2023)17
BA.2.3.20	2.94	4.27	1.15	(Cho et al., 2023)17
BA.2.75.5	2.15	1.92	0.83	(Cho et al., 2023)17
XBB	1.90	2.70	0.69	(Cho et al., 2023)17
XBB (Japan/TY41-795/2022)	1.4	1.2	2.9	(Imai et al., 2023)21
XAY.1	3.44	4.48	0.75	(Cho et al., 2023)17
XBC	0.92	1.89	0.73	(Cho et al., 2023)17
BN.1.9	2.19	2.36	0.59	(Cho et al., 2023)17
BN.1.5	1.67	4.21	0.69	(Cho et al., 2023)17
XBB.1.5	2.19	5.18	1.20	(Cho et al., 2023)17
Average (mean ± SD)	2.09 ± 1.24	5.42 ± 6.41	2.33 ± 1.93

^aIn order to compare the antiviral activities of drugs, only studies were selected in which IC₅₀ values were reported for all three drugs.

^bGS-441524 and EIDD-1931 (also known as N4-hydroxycytidine) are the active forms of remdesivir and molnupiravir prodrugs, respectively.

2.2.1. Baricitinib

In many victims of COVID-19, it was observed that the intensification of inflammation caused by the cytokine storm led to a sudden exacerbation of acute respiratory distress syndrome (ARDS) conditions, and eventually they died due to multiorgan failure.²⁵ By collecting the treatment experiences related to these patients for several years, prevention of the cytokine storm as well as the appropriate time to start anti-inflammatory treatment has been considered as key factors to reduce the mortality rate caused by COVID-19. Indeed, the severe phase of COVID-19 is associated with high levels of cytokine signaling and is related to the Janus kinase signal transducer and activator of the transcription (JAK-STAT) signaling pathway.²⁶ Baricitinib is a selective and reversible inhibitor of Janus kinase (JAK) 1 and 2 that intracellularly blocks cytokine storms by suppressing JAK1/JAK2 and attenuates JAK-mediated immune responses.²⁷ These kinases are factors that play an important role in pro-inflammatory signaling pathways and autoimmune disorders. Baricitinib has already been approved by the European Commission (EC) and the FDA for the treatment of rheumatoid arthritis in 2017 and 2018, respectively. With the emergence of COVID-19 and the proof of the key role of severe inflammation complications in the clinical manifestations of the disease, the use of baricitinib was gradually considered. In 2020, Jorgensen and colleagues reviewed the pharmacology, safety, pharmacokinetics, and emerging clinical experiences of baricitinib for the treatment of COVID-19. The authors concluded that baricitinib has a high capability to be used in the treatment of COVID-19 patients through disrupting the signaling of multiple cytokines involved in SARS-CoV-2 immunopathology, targeting host factors that viruses rely on for cell entry, and suppressing type I interferon-driven upregulation of ACE2.²⁸ In another study, Richardson and his colleagues explained that baricitinib can exert its anti-COVID-19 effects through two different mechanisms, including anti-inflammatory effects by inhibiting JAK1/2 and simultaneously inhibiting SARS-CoV-2 endocytosis by regulating enzymes AP2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK).²⁹ In addition to the mentioned studies, the simultaneous anticytokine activity and antiviral effects of baricitinib, which makes it a unique candidate for use in the treatment of COVID-19, have also been noted in some other studies.³⁰ The latest findings indicate that barcitinib treatment for hospitalized patients with COVID-19 causes a 50% reduction in secondary infections.³¹

From a clinical perspective, several studies have investigated the therapeutic potential of this drug for the treatment of COVID-19 patients, and some systematic reviews have analyzed the results of these studies. In a recent systematic review and meta-analysis, Manoharan and his colleague Ying evaluated the results of 15 eligible studies and concluded that baricitinib significantly reduced mortality and disease progression in COVID-19 patients.³² In a more recent and extensive systematic review and meta-analysis study including 27 randomized controlled trials (RCTs) with 13 549 patients, Albuquerque and colleagues reported a similar result for baricitinib in the treatment of hospitalized COVID-19 patients treated with corticosteroids.³³ Almost a year after the emergence of COVID-19, this drug was able to receive an emergency use license (November 2020), and about 18 months later, it was fully approved by the FDA for the treatment of hospitalized COVID-19 patients requiring supplemental oxygen (May 2022).

From the point of view of medicinal chemistry and based on molecular insights obtained from crystallographic studies, it was discovered that baricitinib creates strong interactions in the active site of targets due to its unique structure. So far, three X-ray crystallographic structures for this compound in the active site of kinases have been released with the protein data bank (PDB) codes 4W9X, 6VN8, and 6WTO, which evaluation of them provides useful information regarding its medicinal capabilities. The first cocrystal structure with the PDB code 4W9X was released in 2014 by Sorrell and his colleagues,³⁴ and it is related to the target bone morphogenetic protein 2 (BMP-2)-inducible kinase in which baricitinib is bound in its active site, but is not the target to be discussed in the present study. The next two PDB codes, 6VN8 and 6WTO, are both related to the cocrystal structure of baricitinib in the active site of the JAK2 JH1 domain, whose parent studies were conducted in the post-COVID-19 era (2021).³⁵ As briefly mentioned above, today JAK2 is considered one of the most important targets in the progression of the inflammation-thrombosis phenotype in COVID-19 patients.³⁶

Figure 3 shows the interactions of baricitinib in the active site of the JAK2 target (PDB code: 6VN8). What is striking at first glance is that this small molecule makes a large number of strong interactions with the target amino acids (baricitinib makes 19 interactions with 12 amino acids including Leu855, Lys857, Ser862, Val863, Ala880, Lys882, Met929, Val911, Glu930, Leu932, Leu983, and Asp994, the details of which can be seen in Figure 3). Evaluation of the ligand-target interactions identified in the X-ray crystal structure with the PDB code 6WTO obtained in a separate study shows almost the same interactions as in 6VN8. Importantly, analyzing both crystal structures indicated that there is a highly strong interaction with a short length of 2.14 Å (6VN8) and 2.16 Å (6WTO) which is generated by the interaction of the nitrogen of the pyrimidine ring with the backbone – NH– of Leu932. Undoubtedly, the existence of this strong interaction plays a key role in the high affinity and selectivity of baricitinib toward JAK2. Another noteworthy point is the role of the bicyclic pyrrolo-pyrimidine moiety in creating several interactions in the active site of JAK2, which accounts for more than half of all interactions.

Figure 3.

Schematic representation (2D and 3D) of the binding mode of baricitinib in the active site of JAK2 JH1 domain (PDB code: 6VN8). As highlighted, the most important interacting sequences in the active site of the target are Leu855, Lys857, Ser862, Val863, Ala880, Lys882, Met929, Val911, Glu930, Leu932, Leu983, and Asp994. All interactions are shown with a red dashed line, and for each of them, the type of interaction along with the bond distance is also specified in Angstrom (Å) scale. The graphic images were prepared using ChemDraw Ultra 11.0 (Cambridge Soft Corporation, MA, U.S.A.), and interactions and distances were analyzed by Discovery Studio Visualizer v4.5.

Although baricitinib potently exerts its anti-inflammatory effects through the inhibition of the targets JAK1/2 and has important therapeutic benefits for COVID-19 patients, its disadvantages should not be overlooked. This drug is only available in tablet form, has not been studied in severe renal failure, and is also contraindicated in pregnancy.³³ In addition, baricitinib carries the risk of increased thromboembolic complications, which is a concern in the treatment of COVID-19 patients.³⁷

2.2.2. Remdesivir

SARS-CoV-2 is a positive-sense single-strand RNA virus that shares a similar replication mechanism with other known coronaviruses, all of which depend on RdRp. In the case of SARS-CoV-2, the enzyme is the most conserved part of the RNA virus-encoded proteins encoded by the nonstructural protein 12 (nsp12) and coordinates viral RNA synthesis as part of an assembly called replication–transcription complex (RTC).³⁸ RdRp is one of the most important targets for clinically approved antiviral nucleoside analogues for the inhibition of RNA viruses because it is highly error-prone and modified/synthetic nucleotide analogues can be used as substrates to disrupt its function. Today, nucleotide analogues that inhibit viral polymerases have been developed as a large group of effective antiviral agents.³⁹

Remdesivir is a broad-spectrum antiviral drug that was originally developed to treat hepatitis C and subsequently tested for Ebola virus (EBOV) and Marburg virus infections before being approved for the treatment of COVID-19.⁴⁰ Structurally, remdesivir is a phosphoramidate prodrug containing a 1′-cyano modification on the sugar which first converts to an adenosine nucleoside analog named GS-441524 in vivo. After GS-441524 enters virus-infected cells, this metabolite is phosphorylated by kinases in three steps to finally turn into remdesivir triphosphate.⁴¹ Structurally, remdesivir triphosphate has many similarities to natural nucleoside triphosphates, especially adenosine triphosphate. This structural similarity is the origin of the mechanism of action of remdesivir’s antiviral activity, where RdRp mistakenly uses it to make viral RNA (Figures 4 and 5).⁴² Interestingly, some studies show that remdesivir triphosphate has a higher selectivity for integration into viral RNA than its structural analog adenosine triphosphate (and other nucleoside triphosphates).⁴³ In a recent study by Malone et al., the structures of the SARS-CoV-2 replication–transcription complex bound to remdesivir triphosphate (PDB: 7UO4) and four natural nucleoside analogues including adenosine triphosphate (ATP) (PDB: 7UO4), guanosine triphosphate (GTP) (PDB: 7UOB), cytidine triphosphate (CTP) (PDB: 7UOE), and uridine triphosphate (UTP) (PDB: 7UO9) were released using cryogenic-electron microscopy.⁴⁴ The availability of these costructures and comparing them with each other can significantly increase the structural insight of medicinal chemists in designing more effective nucleotide analogues through structure-based drug design strategies aimed at better targeting the coronaviruses RdRp in the future.

Figure 4.

Schematic representation of the mechanism of remdesivir effect in inhibiting SARS-CoV-2 replication by disrupting RdRp activity. Briefly, remdesivir (which is structurally a prodrug) is first converted to an adenosine nucleoside analog (GS-441524) in vivo. After GS-441524 enters virus-infected cells and is phosphorylated by kinases, it will finally be converted into remdesivir triphosphate (RTP). Due to the structural similarity of RTP to ATP, SARS-CoV-2 RdRp mistakenly uses it to make viral RNA, thereby disrupting the viral replication cycle.

Figure 5.

Schematic representation of the replication–transcription complex bound to the remdesivir triphosphate and natural nucleotide triphosphates including ATP (PDB: 7UO4), GTP (PDB: 7UOB), CTP (PDB: 7UOE), and UTP (PDB: 7UO9) in states poised for incorporation. The high structural similarity of remdesivir triphosphate to each of these nucleotide triphosphates (especially ATP) causes SARS-CoV-2 RdRp to mistakenly insert it into the viral nucleic acid structure, so the replication of the virus is disrupted. In the lower part, the interacting amino acids in the active site of RdRp for each of the mentioned molecules are shown. More cases related to remdesivir triphosphate can potentially be one of the reasons for the success of this compound in competition with other nucleotide triphosphates to integrate into the structure of nucleic acid strands of the virus.

From the perspective of pharmacological activity, remdesivir has shown broad antiviral activity in vitro against several viral families such as Coronaviridae, Flaviviridae, Pneumoviridae, Filoviridae, and Renaviridae.(45) Among the important advantages of this drug which has been considered by researchers is that viral resistance to remdesivir is rare.⁴⁶ From a clinical point of view, the opinions are not the same and sometimes conflicting results are reported about the effectiveness of this drug in the treatment of COVID-19 patients. For example, in a large clinical study conducted in 52 Canadian hospitals on more than 1,260 patients with COVID-19, the results showed that remdesivir in combination with standard therapy improves the need for mechanical ventilation in patients who were not ventilated on arrival.⁴⁷ Also, some new systematic reviews state with high confidence that remdesivir reduces the mortality rate in COVID-19 patients who need oxygen but are not yet in critical condition.⁴⁸ However, in a clinical study conducted in 48 sites in Europe on about 850 patients with COVID-19, the results showed that no significant clinical advantage was observed by the use of remdesivir in hospitalized patients who required oxygen support.⁴⁹ In a multicenter clinical trial conducted in ten hospitals in Hubei, China, the therapeutic effects of remdesivir were evaluated in 237 hospitalized adult patients with severe COVID-19. In this study, it was also reported that remdesivir treatment is not associated with statistically significant clinical benefits.⁵⁰ In another study, it was observed that the mortality rate in hospitalized patients with COVID-19 receiving remdesivir (2743 patients; 301 deaths) is not different from the mortality rate in patients receiving control (2708 patients; 303 deaths).⁵¹ In addition to the mentioned studies, the lack of clinical benefit of using remdesivir in hospitalized COVID-19 patients has also been reported in some newer studies.⁵² Summarizing the various clinical results regarding the effectiveness of remdesivir in the treatment of COVID-19 patients, the American College of Physicians announced that although remdesivir may shorten the time to clinical improvement and slightly reduce side effects, it probably just makes little difference in mortality.⁵³ However, remdesivir is currently one of the few drugs that the National Institutes of Health (NIH) and Infectious Diseases Society of America (IDSA) guidelines recommend for use in COVID-19 patients who do not require mechanical ventilation.

2.2.3. Molnupiravir

The isopropylester prodrug of N4-hydroxycytidine, molnupiravir, is an orally bioavailable broad-spectrum ribonucleoside analog that was designed in 2013 with the goal of finding an oral antiviral agent with direct activity against infection by the encephalitic New World alphavirus Venezuelan equine encephalitis virus (VEEV). In 2019, when the COVID-19 pandemic began, the drug was in preclinical development for the treatment of seasonal influenza.⁵⁴ After the spread of COVID-19 and reporting of clinical evidence that molnupiravir is effective in treating these patients, FDA granted emergency use authorization for its use on December 23, 2021. However, it has not yet been fully approved.

In terms of mechanism of action, molnupiravir works similarly to remdesivir. This prodrug is first hydrolyzed to N4-hydroxycytidine (N4-HCT) in vivo. After N4-HCT enters the SARS-CoV-2 infected cells, it is phosphorylated by kinase enzymes in three steps and converts into its active form N4-hydroxycytidine triphosphate (N4-HCTP) which is also called molnupiravir triphosphate (Figure 6).⁵⁵ Here, the high structural similarity of molnupiravir triphosphate to nucleotide triphosphates (especially CTP) causes SARS-CoV-2 RdRp to mistakenly insert it into the genome of new virions. This eventually causes the accumulation of inactivating mutations, so the replication of the virus is disrupted.⁵⁶

Figure 6.

After the hydrolysis of the molnupiravir prodrug in the in vivo environment and the entry of its primary metabolite, N4-hydroxycytidine, into SARS-CoV-2 infected cells, kinase enzymes convert this metabolite into molnupiravir triphosphate (MTP) in three steps. The high structural similarity of MTP to the natural nucleotides of ATP, GTP, CTP, and UTP (especially CTP) causes the SARS-CoV-2 RdRp to mistakenly insert this molecule into the structure of nucleic acids being produced for new virions, ultimately leading to disruption of virus replication.

One of the most important studies that confirmed the remarkable therapeutic capabilities of molnupiravir for the treatment of COVID-19 was a phase 3 clinical trial conducted by Butler and his colleagues on 1433 nonhospitalized and unvaccinated adults with mild-to-moderate laboratory-confirmed COVID-19 who had at least one risk factor for severe COVID-19. The results of this study showed that early treatment with molnupiravir significantly reduced the risk of hospitalization (approximate 30% reduction) in at-risk and unvaccinated adults with COVID-19.⁵⁷ Since the results of this study are related to unvaccinated patients in 2021 (that is, in the era before Omicron), it cannot explain the effectiveness of this drug against low-risk Omicron strains and in populations that have already been vaccinated and received the primary immunization course.⁵⁸ To find out more, an extensive clinical study with 26 411 participants (the mean age of the population was near to 60 years) was recently conducted that aimed at the evaluation of the effect of molnupiravir on reducing hospitalizations and deaths related to COVID-19 compared to usual care.

Although initial results suggest that early treatment with molnupiravir significantly shortens recovery time in treated COVID-19 patients, results in total indicated that molnupiravir could not reduce the frequency of hospitalization or death due to COVID-19 among high-risk vaccinated adults in the community.⁵⁹ These results can be one of the reasons for not granting a full license for the use of molnupiravir, or it may even lead to the withdrawal of this drug from the treatment regimen of COVID-19 patients in the near future.

2.2.4. Paxlovid (Nirmatrelvir Plus Ritonavir)

Paxlovid is the brand name for a copackaged oral drug containing two peptidomimetic molecules, nirmatrelvir (PF-07321332) and ritonavir, introduced by Pfizer for the treatment of COVID-19.⁶⁰ Because of its promising therapeutic effects reported in clinical trials that can significantly reduce the risk of hospitalization or death from COVID-19 (up to 88%), the FDA granted the emergency use for Paxlovid in December 2021. Pharmacologically, the therapeutic effects of Paxlovid in COVID-19 are mostly related to nirmatrelvir which inhibits the activity of SARS-CoV-2 3CLpro enzyme, while ritonavir which originally is an HIV protease inhibitor acts as a pharmacokinetic enhancer and increases the duration of nirmatrelvir’s activity by inhibiting cytochrome P450 3A4, the enzyme responsible for nirmatrelvir’s metabolism.⁶¹ Nirmatrelvir is an interesting peptidomimetic molecule that was recently discovered by Owen et al. as an orally bioavailable drug with pan-human coronavirus antiviral activity and excellent off-target selectivity and safety profiles, which can achieve oral plasma concentrations exceeding the in vitro antiviral cell potency.⁶² Recently, we also emphasized the importance of the inhibition of SARS-CoV-2 3CLpro and PLpro proteases involved in the maturation of two raw polyproteins pp1a and pp1ab to mature proteins in viral replication cycle processes.¹⁰ Since the amino acid sequence of these viral proteins is highly conserved throughout the subfamily Coronavirinae and these enzymes have different substrate specificity (compared to human enzymes), the discovery of effective SARS-CoV-2 3CLpro/PLpro inhibitors could potentially be accompanied by providing a good safety profile and low adverse effects. However, the examination of sequences in known types of SARS-CoV-2 showed a low mutation frequency, which makes these enzymes attractive targets for the drug development against SARS-CoV-2.^63,64

Nirmatrelvir is the first SARS-CoV-2 protease inhibitor that was able to pass all the necessary preclinical and clinical standards and get a license for clinical use by the competent authorities, which is promising. Moreover, it is interesting that this drug has been able to show good capabilities in clinical investigations. One of the most important studies investigating the therapeutic capabilities of nirmatrelvir as an oral drug against COVID-19 is a phase 2–3 clinical trial conducted in 2022 by Hammond and his colleagues on high-risk, nonhospitalized adults.⁶¹ In this study, about 2200 symptomatic, unvaccinated, nonhospitalized adult patients who were at high risk for progression to severe COVID-19 were examined. The treatment group was given 300 mg of nirmatrelvir plus 100 mg of ritonavir every 12 h for 5 days, and all results finally were compared with the control group receiving a placebo. The results reported for this study were remarkable, where symptomatic treatment with nirmatrelvir plus ritonavir had an 89% lower risk of progression to severe COVID-19 compared to placebo, while side effects observed were similar for both groups. In another important clinical study conducted by Arbel et al., the therapeutic capabilities of nirmatrelvir against newer strains of type B.1.1.529 (Omicron) were investigated. The results of the study also showed the effectiveness of this drug, because the rate of hospitalization and death due to COVID-19 among patients 65 years of age or older receiving the nirmatrelvir was almost a quarter of the corresponding rate for patients in the control group, which was highly significant.⁶⁵

Although nirmatrelvir works by inhibiting SARS-CoV-2 3CLpro which has a conserved active site against mutations, there is concern that drug resistance may develop with increasing use of this drug, especially if used as monotherapy.⁶⁶ A better understanding of the mechanisms of resistance to this drug could provide valuable insight into the development of next-generations of SARS-CoV-2 3CLpro inhibitors. In an in vitro study by Iketani and colleagues, it was reported that SARS-CoV-2 can be resistant to nirmatrelvir in several ways.⁶⁷ Therefore, it is important to maintain the effectiveness of this drug through its use in combined drug regimens.

Structurally, nirmatrelvir is a tripeptide compound designed by considering the structural parts of drugs lufotrelvir and boceprevir.⁶⁸ So far, several important studies have evaluated and released X-ray crystallographic structures of this drug bound in the active site of 3CLpro in various SARS-CoV-2 variants,^62,63,69,70 including PDB codes 7RFS, 7RFW, 7SI9, 7TEO, 7TLL (Omicron P132H), 7U28 (Lambda; G15S), 7U29 (K90R mutant), 7UJU, 7VH8, 7VLO, 7VLP, 7XB4 (D48N mutant), 8DCZ (M165Y Mutant), 8DZ2, 8DZ6 (Q189 K mutant), 8DZ9 (G143S mutant), 8DZA (A193T mutant), 8E1Y (A193S mutant), 8E25 (M49I mutant), 8E26 (N142S mutant), and 8EYJ (C145S mutant). In addition, it has been reported that this drug has the ability to inhibit 3CLpro of other coronaviruses (such as SARS-CoV-1 and MERS-CoV), and now the crystal structure of nirmatrelvir bound to the active site of MERS 3CLpro was released with PDB code 7VTC and is available at the protein data bank Web site.^62,70 The large number of X-ray crystallographic studies on the binding mode of an anti-SARS-CoV-2 drug is unprecedented which indicates the scientist’s interest for evaluating this drug in the treatment of COVID-19. However, it can provide a deep insight into the structure-based understanding of the action of this drug on the mentioned target in different variants. In a concise report by Zhao et al., the inhibitory mechanism of nirmatrelvir against SARS-CoV-2 3CLpro was described well using the X-ray crystallography method at 1.6 Å resolution.⁶³Figure 7 schematically shows the inhibitory mechanism of nirmatrelvir in the active site of SARS-CoV-2 3CLpro. Structurally, nirmatrelvir is a potential covalent inhibitor due to its nitrile warhead, where it can interact with the thiol group of the catalytic Cys145 residue to block the SARS-CoV-2 3CLpro catalytic action through the formation of a reversible covalent bond. The existence of an intramolecular interaction between the two amino acids His41 and Cys145 in the active site of SARS-CoV-2 3CLpro has led to the activation of the thiol group of Cys145 for nucleophilic attack on molecules containing covalent structural warheads. This point has provided a good chance to medicinal chemists for the structure-based design of effective molecules.⁷¹ It should be noted that in addition to the S–C covalent bond connecting the nitrile carbon of nirmatrelvir with the Cys145 thiol group, the drug binds tightly to the active site of SARS-CoV-2 3CLpro through a series of hydrogen bonds and hydrophobic interactions.

Figure 7.

Schematic representation (2D and 3D) of the inhibitory mechanism of nirmatrelvir at the active site of SARS-CoV-2 3CLpro (PDB: 7VH8). Due to having a nitrile warhead, nirmatrelvir is a reversible covalent inhibitor that targets the Cys145 residue and blocks the SARS-CoV-2 3CLpro active site.

In terms of toxicity and side effects, it can be expressed that paxlovid is a drug with an acceptable safety profile (especially in the case of the nirmatrelvir molecule, for which no concerning side effects have been reported).⁷² The major safety concern of paxlovid is related to the ritonavir molecule, which increases significantly the concentration of drugs metabolized by CYP3A in patients with underlying disorders.⁷³ This concern is more critical for transplant patients who are receiving substrates of CYP3A such as tacrolimus and cyclosporine.⁷⁴

3. Combination Therapy: The Latest Strategy for the Treatment of COVID-19

Regarding the mechanism of action of the approved/authorized anti-COVID-19 drugs (as seen in the third column in Table 3), all of them have either anti-inflammatory or direct-acting antiviral effects that are related to the infectious nature of COVID-19. Attention to the targets of these drugs reveals that antiviral agents often target the critical SARS-CoV-2 proteins including spike (S) glycoprotein, RdRp, and 3CLpro, but anti-inflammatory agents have an effect on preventing the release of inflammatory factors such as interleukins. Since direct action strategies against SARS-CoV-2 targets are facing serious challenges due to the possibility of more mutations and also drug resistance,⁷⁵ some scientists highlighted targeting host-based targets (including those involved in inflammatory processes and viral replication) that are not subject to these challenges.⁷⁶ Continuous mutations of SARS-CoV-2, which occur particularly in the spike (S) glycoprotein (and favor evasion of the host’s immune response) have caused most of the spike protein-based monoclonal antibodies and vaccines to lose their effectiveness against newer strains such as Omicron. One serious question is whether small molecules identified as effective drugs against COVID-19, including molnupiravir, remdesivir, and nirmatrelvir, can maintain their effectiveness against a variety of newer mutant strains. To answer this question, some studies have been conducted recently. In an in vitro study by Vangeel et al., the antiviral activity of remdesivir (with its parent nucleoside GS-441524), molnupiravir (with its parent nucleoside N4-hydroxycytidine), and nirmatrelvir against wild-type SARS-CoV2 and five variants of concern (VOCs) including Omicron was evaluated using VeroE6-Green fluorescent protein (GFP) cells.⁷⁷ The results of this study showed that all molecules retained their antiviral activity and effectiveness against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta, and Omicron. These results are in accordance with the previous reports stating that RdRp and 3CLpro enzymes are highly conserved against mutations.^63,78

Table 3. List of Fully FDA-Approved and FDA-Issued Emergency Use Authorization (EUA) Anti-COVID-19 Drugs, along with Some of Their Characteristics Including Mechanism of Action, Approval/Withdrawal History, and Clinical Indications.

no.	drug name	medication type	mechanism of action	FDA-approval or withdrawal date	indication
monoclonal antibodies
1	Anakinra (Kineret)	recombinant human interleukin-1 (IL-1) receptor antagonist	competitive binding to the IL-1 receptor and subsequently reducing the inflammatory responses	EUA in November 2022	Treatment of hospitalized COVID-19 patients with pneumonia requiring supplemental oxygen who are at risk of progression to severe respiratory failure.
2	Bamlanivimab	neutralizing human IgG1κ monoclonal antibody	disrupting the surface spike (S) protein of SARS-CoV-2 by binding to the receptor binding domain (RBD) of the S protein at a position that overlaps the ACE2 binding site	EUA in November 2020.	Bamlanivimab is used in combination with etesevimab to treat mild to moderate COVID-19 in adults and pediatric patients and also in younger children (including newborns), who are at high risk for progressing to severe COVID-19.
				FDA revokes EUA for Bamlanivimab when administered alone to treat COVID-19 in April 2021.
				EUA in February 2021 for the administration of bamlanivimab in combination with etesevimab.
3	Bebtelovimab	human IgG1κ monoclonal antibody	inhibition of spike (S) protein interaction with ACE2 and disrupting the viral entry into human cells	EUA in February 2022	mild-to-moderate COVID-19 patients
				drug withdrawal: November 2022	The EUA was officially withdrawn due to a lack of efficacy against Omicron subvariants.
5	Etesevimab	human and recombinant monoclonal antibody	disrupting the surface spike (S) protein of SARS-CoV-2 and neutralizing the virus by specifically binding to the S protein receptor binding domain	EUA in February 2021, in combination with bamlanivimab	Etesevimab is used in combination with bamlanivimab for postexposure prophylaxis of COVID-19 and to treat mild to moderate COVID-19 in adults and pediatric patients and also in younger children (including newborns), who are at high risk for progressing to severe COVID-19.
6	Evusheld (Tixagevimab + Cilgavimab)	co-packaged the two human monoclonal antibodies tixagevimab and cilgavimab	targeted against the surface SARS-CoV-2 spike (S) protein and disrupting the viral entry into human cells	EUA in December 2021	For the pre-exposure prophylaxis of COVID-19 in adult and pediatric patients aged 12 years and older weighing at least 40 kg, and at increased risk for whom vaccination is not recommended
				drug withdrawal: January 2023	The FDA revised and withdrew the EUA for Evusheld against SARS-CoV-2 in January 2023
9	REGN-COV2	a combination of the antibodies casirivimab and imdevimab	REGN-COV2 was obtained from some humanized mice as well as blood samples from recovered COVID-19 patients and was formulated to bind to different sites on the SARS-COV-2 spike protein.	EUA in November 2020	Treatment of patients with mild to moderate COVID-19 aged 12 years or older and weighing at least 40 kg, and who are at high risk of progression to severe COVID-19.
10	Sotrovimab	recombinant human IgG1κ monoclonal antibody	attaching to the SARS-CoV-2 spike (S) protein and disrupting the endocytosis process	EUA in May 2021	For the treatment of mild-to-moderate COVID-19 (especially in patients at increased risk for death or hospitalization).
				drug withdrawal: April 2022	The FDA withdrew the EUA of sotrovimab against SARS-CoV-2 due to lack of efficacy against the Omicron variant.
11	Tocilizumab (Actemra)	recombinant humanized monoclonal antibody	binding to soluble and membrane-bound IL-6 receptors and then inhibiting IL-6-induced inflammation	fully approved in December 2022	Treatment of adults COVID-19 patients receiving systemic corticosteroids and supplemental oxygen or mechanical ventilation.
small molecules
4	Baricitinib	pyrrolopyrimidine-based small molecule	suppressing the activity of Janus kinase (JAK) proteins (especially JAK1 and JAK2) and modulating the signaling pathway of interleukins, interferons and various growth factors	EUA in combination with remdesivir in November 2020	Treatment of adults hospitalized COVID-19 patients requiring supplemental oxygen, invasive/noninvasive mechanical ventilation, or extracorporeal membrane oxygenation.
				fully approved in May 2022
7	Paxlovid	a copackaged medication containing two peptidomimetic molecules nirmatrelvir and ritonavir	Nirmatrelvir inhibits the SARS-CoV-2 3C-like protease (3CLpro), but ritonavir acts as a pharmacokinetic enhancer and increases the duration of Nirmatrelvir’s activity by inhibiting cytochrome P450 3A4	EUA in December 2021	Treatment of adults and pediatric patients (12 years of age and older weighing at least 40 kg) COVID-19 patients with the mild-to-moderate condition, and who are at high risk for progression to severe disease.
8	Remdesivir	nucleoside-based molecule	impairing the function of viral RdRp enzyme	EUA in May 2020	Treatment of adult and pediatric (28 days of age and older and weighing at least 3 kg) COVID-19 patients requiring hospitalization, and also for nonhospitalized patients who are at high risk for progression to severe COVID-19.
				fully approved in October 2020
12	Molnupiravir	isopropylester cytidine analog (prodrug of N4-hydroxycytidine)	in the physiological environment, this compound is hydrolyzed to N4-hydroxycytidine and then becomes the phosphorylated 5′-triphosphate (active form), increasing the frequency of viral RNA mutations and impairs SARS-CoV-2 replication by disrupting RdRp activity.	EUA in December 2021	Reducing the risk of hospitalization and death and treatment of mild to moderate adult COVID-19 patients with an increased risk of severe disease.

Combination therapy is one of the newest antiviral treatment strategies for COVID-19 and some preclinical studies emphasize it, but it has not yet been well evaluated in clinical trials. Since two or more viral targets are simultaneously targeted in such strategies, the chance of damaging the virus increases significantly.⁷⁹ Another key advantage of this strategy is preventing the emergence of drug resistance against these hard-earned anti-COVID-19 drugs.⁸⁰ Of course, it should be noted that the success rate of such methods is affected by the collective safety of the drug regimen, patient tolerance, and the absence of serious side effects and drug interactions. In an in vivo study conducted by Jeong et al., the therapeutic potential of nirmatrelvir, remdesivir, and molnupiravir and their combination were evaluated in K18-hACE2 transgenic mice infected with SARS-CoV-2. The results of this study showed that combined treatment with nirmatrelvir and molnupiravir (with doses equal to 20 mg/kg) in infected mice led to a significant reduction of virus-induced tissue damage. Correspondingly, inhibition of SARS-CoV-2 replication in both the lung and brain of the tested animals was observed, as well as synergistically increased survival rate of up to 80% compared to mice administered alone with nirmatrelvir (36%) or molnupiravir (43%).⁸¹ In a concise report, Li and his colleagues evaluated the effectiveness of the drugs molnupiravir, nirmatrelvir and their combination on Omicron type in relevant experimental models. The results of their evaluations also showed that both of these drugs strongly inhibit SARS-CoV-2 Omicron infection, but the combination of molnupiravir and nirmatrelvir has a synergistic effect in inhibiting antiviral activity.⁸² Impressed by the remarkable results of this study, these researchers proposed the initiation of clinical studies to evaluate the combination of molnupiravir and nirmatrelvir for the treatment of COVID-19. Interestingly, the significant synergistic effects of the combination of molnupiravir and nirmatrelvir in inhibiting the replication of SARS-CoV-2 were also confirmed in vitro by Gidari and his co-workers.⁸³

In general, it can be said that the results of preclinical studies related to the combined use of nirmatrelvir and molnupiravir have been more promising than other combined regimens tested. Recently, a case report study by Marangoni et al., reported that a 73-year-old man with follicular lymphoma with persistent SARS-CoV-2 infection was successfully treated with 10-day coadministration oral antivirals molnupiravir and nirmatrelvir plus ritonavir. Importantly, the study’s therapists stated that this combination therapy was well tolerated both clinically and biochemically, and no signs of toxicity were observed.⁸⁴

Although there is both preclinical and clinical evidence to support the usefulness of combination therapy, current knowledge is still limited and it needs to be evaluated by conducting reliable clinical studies in the future. The important point is that this strategy has been used so far just for anti-COVID-19 drugs with direct-acting antiviral effects. Research oriented to the combination drug regimens in which anti-COVID-19 drugs with direct antiviral effects coadministered with anti-inflammatory drugs (such as baricitinib) could be beneficial. In addition, agents with simultaneous antiviral and anti-inflammatory activities could be good candidates for the treatment of COVID-19, but there are no such cases among the available FDA-approved/authorized drugs.

4. Conclusions

Among the most important achievements during the post-COVID-19 era is the identification/characterization of SARS-CoV-2-based targets including spike (S) glycoprotein, RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin-like protease (3CLpro), and papain-like protease (PLpro) as key targets which their high-resolution X-ray crystal structures are now available in precise detail. By targeting these structural/nonstructural viral proteins, a number of medications including vaccines, small molecules, and monoclonal antibodies have been designed (or repurposed) which are currently licensed for the treatment of COVID-19 patients. Despite significant progress in the field of new anti-COVID-19 vaccines, there is still an urgent need for safe/effective oral anti-COVID-19 treatments that can prevent the progression of infection and death, and also reduce the transmission rate.⁸⁵ Clinical applications of nucleotide/nucleoside antiviral drugs remdesivir and molnupiravir as well as peptidomimetic drug nirmatrelvir in the treatment of COVID-19 patients emphasize the importance of SARS-CoV-2 RdRp and 3CLpro enzymes as promising drug targets for the design and development of more effective analogues in the future. In addition, due to the highly conserved nature of the active site of their targets (RdRp and 3CLpro), such drugs can be considered candidates as pan-anticoronavirus drugs that may be effective against emerging strains and could be repurposed in the future to fight new coronaviruses. PLpro is another important enzyme of SARS-CoV-2, whose key role in virus replication and regulation of the host antiviral immune responses has been identified, but drugs that work by the mechanism of inhibiting this enzyme have not yet been approved. In comparison, it can be stated that targeting the SARS-CoV-2 spike (S) glycoprotein is a failed strategy because the occurrence of continuous/abundant mutations in the structure of this protein (especially in its receptor-binding domain) causes the designed medications to lose their effectiveness against newer substrains.

Although the results of clinical trials have suggested the effectiveness of the mentioned anti-COVID-19 medications, the news of thousands of deaths and hospitalizations in the first 4 years after the start of the pandemic in wealthy countries is deeply worrying. The official authorities of China recently announced that they had nearly 60 000 deaths due to SARS-CoV-2 in just one month (from December 8, 2022, to January 12, 2023), while this country has prescribed nearly 3.5 billion doses of anti-COVID-19 vaccines before these events and has access to all recommended drugs for the treatment of the disease. In addition, this country has a famous traditional medicine with a history of several thousand years, in which many natural compounds with known therapeutic activity can be used to treat viral diseases such as COVID-19.⁸⁶ In Japan, whose health care system is one of the most leading ones in the world and benefits from the advanced pharmaceutical and vaccine companies, a similar situation has been recently reported, where their Minister of Health reported daily new COVID-19 cases in the first week of January 2023 at around 200 000 to 300 000 and daily deaths at 400 to 500 (a high record of 520 deaths on January 11, 2023). According to the information provided on the Web site of the World Health Organization, this country is one of the few ones where the ratio of prescribed vaccine doses against COVID-19 (370 400 000 doses) to its population (125 700 000) is almost 3, which indicates a high medical index in a healthcare system, but it has faced such a high death rate. All these tragic events that happened in the presence of many approved anti-COVID-19 drugs and vaccines remind us that we have not yet succeeded in defeating coronavirus diseases. Of course, it should not be ignored that during the recent few years, we have gained unprecedented experiences in the field of research and international joint cooperation, which can be a beacon of the future to fight such diseases. Also, we are still in the middle of the road, and the efficacy of promising strategies such as combination therapy in which multiple anti-COVID-19 drugs simultaneously attack different SARS-CoV-2 targets has not been well studied in clinical trials.

Glossary

Abbreviation

AAK1

AP2-associated protein kinase 1

ACE2

Angiotensin-converting enzyme 2

ATP

Adenosine triphosphate

BMP-2

Bone morphogenetic protein 2

COVID-19

Coronavirus disease of 2019

CC₅₀

Cytotoxicity concentration 50%

CTP

Cytidinee triphosphate

CYP

Cytochromes P450

EC₅₀

Half maximal effective concentration

EBOV

Ebola virus

EUA

Emergency Use Authorization

EC

European Commission

FDA

U.S. Food and Drug Administration

GAK

Cyclin G-associated kinase

GFP

Green fluorescent protein

GTP

Guanosine triphosphate

HIV

Human Immunodeficiency Virus

IDSA

Infectious Diseases Society of America

IL

Interleukin

IC₅₀

Half maximal inhibitory concentration

JAK

Janus kinase

JAK-STAT

Janus kinase signal transducer and activator of the transcription

MERS-CoV

Middle East respiratory syndrome coronavirus

MTP

Molnupiravir triphosphate

N4-HCT

N4-hydroxycytidine

N4-HCTP

N4-hydroxycytidine triphosphate

NSP

Nonstructural protein

NIH

National Institutes of Health

VEEV

Venezuelan equine encephalitis virus

PLpro

Papain-like protease

PDB

Protein Data Bank

RCTs

Randomized controlled trials

RdRp

RNA dependent RNA polymerase

RBD

Receptor-binding domain

RTP

Remdesivir triphosphate

RTC

Replication–transcription complex

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

SARS-CoV-1

Severe acute respiratory syndrome coronavirus 1

SI

Selective index

UTP

Uridine triphosphate

VOCs

Variants of concern

ZIKV

Zika virus

3CLPro

3-Chymotrypsin like protease

2D

Two-dimensional

3D

Three-dimensional

Author Contributions

M.V. contributed in the conceptualization and writing the original draft. H.I. contributed in editing the manuscript. H.K. contributed in the supervision of virology topics.

The authors declare no competing financial interest.