3.1. Leucoverdazyls Are Potent Inhibitors of EVs at Low Micromolar Concentrations
At the beginning of the study, the virus-inhibition activity of leucoverdazyls in viral yield reduction assay was determined. Cytotoxicity of the compounds for permissive cell lines used was evaluated in MTT assay. The results are summarized in
Table 1 below. Pleconaril was used for comparison as a reference compound due to the fact that CVB3 Nancy is a pleconaril-resistant strain [
37].
As can be seen from the data presented in the table, the least toxic compounds were the following: 1b, 2b, 3b, 3d, and 4d. Two of them (1b, 2b) contain a methoxy group in the aromatic fragment. Halogen containing compounds 2d and 2e showed the greatest toxicity. In viral yield reduction assay, a large majority (57 %) of leucoverdazyls from the library (11 out of 19 tested) demonstrated remarkable anti-enteroviral activity in vitro against pleconaril resistant CVB3 strain in comparison to pleconaril. SI values for these compounds exceeded 10, which is indicative of high anti-enteroviral potential. Compounds 1a-1c exhibited the most pronounced antiviral activity, with IC50 values much lower than that of pleconaril. It should be noted that the structure of these compounds lacks a substituent at position 6 of the tetrazine ring. In addition, the best values of the selectivity index were also noticed for compounds that do not contain a substituent in this position of the tetrazine ring (compounds 1a-1d), as well as for compounds containing the least bulky substituent (Me) – compounds 2a, 2b. Compound 1a with the lowest IC50 value (2.7 µM) and the highest SI (230) was selected as the leader for further study.
In order to trace the effect of the substituent at the position 6 of the tetrazine ring on the antioxidant activity of dihydrotetrazines, a DPPH test was performed in the series of
1a,b, 2a,b, 3a,b and
4a,b. The results are presented in
Figure 2.
As can be seen from the data obtained, the most pronounced antioxidant activity was detected for compounds 1a and 1b, and their antioxidant potentials were even superior to vitamin C used as the reference compound. Therefore, we suppose that addition of substituent in the sixth position of the tetrazine ring has a negative impact on the antioxidant activity of dihydrotetrazines in vitro.
The data on antioxidant activity indicate that compounds without substituent in the sixth position of the tetrazine ring, as well as compounds with a methoxy group in the aromatic fragment at N1, turned out to possess the highest antioxidant activity.
3.2. 1a Possesses Wide-Range Activity against Group A, B and C Enteroviruses
To assess the prospects for further development of the most potent compound (
1a), the spectrum of its anti-enterovirus activity was assessed against a panel of group A, B and C enteroviruses including both Coxsakievirus B4 (strain Powers) and patient isolates in viral yield reduction assay. The following patients isolates of viruses were used: CVA16, CVB5, ECHO30, CVA24. Among them CVA16 is common agent of HFMD, while CVB5 and ECHO30 has been reported to be associated with HFMD also [
38,
39,
40]. The results are presented in the
Table 2 below. Guanidine hydrochloride targeting initiation step of viral RNA synthesis was used as reference drug [
41].
Compound 1a showed high activity towards other strains of group A, B and C enteroviruses superior to reference compound guanidine hydrochloride, though guanidine hydrochloride was less toxic.
We further investigated whether
1a is capable of inhibiting the life cycle of other phylogenetically distinct RNA or DNA viruses. Compound
1a was tested against influenza (ss RNA-negative enveloped virus), HSV1 (dsDNA enveloped virus), Ad5 (dsDNA non-enveloped virus), and SARS-CoV-2 (ssRNA-positive enveloped virus) in viral yield reduction assay (
Table 3).
According to the results, compound 1a showed only modest activity against influenza viruses, Ad5, and HSV1. Surprisingly, however, it inhibited replication of another ssRNA-positive enveloped virus, namely SARS-CoV-2. This leads us suppose that 1a targets some biological entity (protein or process) important to both the enterovirus and coronavirus life cycle.
3.3. 1a Does Not Increase Virion Thermostability, and Inhibits Late Stages of the Enterovirus Life Cycle
The plausible mechanism of action for
1a was studied using in vitro assays. We addressed whether
1a has capsid binding properties using a thermal stability assay. It is known that capsid binders (pleconaril and its derivatives) directly interact with capsid of enteroviruses and stabilize its structure, thereby preventing the virus from entering the host cell. This interaction increases the resistance of the viral capsid to a short-term temperature increase, and the heated virus retains its ability to infect a permissive cell line. The results are presented in the
Figure 3 below.
In the virus control, as well as when test drug 1a was mixed with the virus, no infectious particles were detected when heated above 45 degrees. As expected, the reference capsid binding drug pleconaril had a thermostabilizing effect on Coxsackie B4 virus, ensuring the presence of infectious particles even when heated to 55 degrees (the highest temperature used). Thus, it was concluded that 1a does not belong to the capsid-binding group of inhibitors.
Next, we focused on the stage in the viral cycle when 1a demonstrated the highest inhibitory activity in time-of-addition assay. CVB4 was propagated in Vero cells with addition and removal of
1a at distinct time points before or after the zero point when virus was added. After one cycle of replication (8 hpi), the infectious titer of viral progeny was determined in end-point dilution assay. The titer of viral progeny versus interval of
1a presence in the media is presented in
Figure 4.
The most pronounced inhibitory effect of 1a was demonstrated if it was present in the culture medium starting from -2 to 4 h post-infection (hpi). Inhibition of viral replication was not observed if the substance was added later than 6 hours after the adsorption of the virus. Pleconaril used as a reference compound demonstrated the highest activity between (-2) and 0 hours, as expected for early-stage inhibitors. The results obtained suggest that the substance acts on steps involved in viral replication.
The Coxsackievirus lifecycle is relatively short (6–8 h) and has been extensively studied previously. Briefly, after receptor mediated endocytosis, viral genomic RNA is translated into a polyprotein, which in turn is proteolytically processed by viral 2Apro and 3Cpro to release viral proteins, and viral RNA replication begins. Viral RNA replication is performed via a dsRNA intermediate in specialized replication organelles. Nascent viral (+)RNA is encapsidated by structural proteins to form new virions, which are released either lytically or non-lytically (in autophagic vesicles).
According to previously obtained results, viral RNA replication is initiated 2–3 h after infection, and translation of viral capsid proteins is detectable as early as after 4 hpi [
42]. Therefore, the inhibitory effect of
1a spans the following stages of the viral life cycle: cell attachment, penetration, genomic RNA transcription, proteolytic processing, and RNA replication.
3.5. 1a-Resistant Strain Selection and Its Genomic and Phenotypic Characteristics
In order to assess the genetic barrier to resistance development to
1a, we further passaged CVB3 (Nancy strain) in Vero cells at increasing concentrations of 1a, evaluated the emerging resistance level, and identified amino acid substitutions. Three viral strains were analyzed: o’riginal’ CVB3 from the bank, which was used to generate w’ild-type’ virus (CVB3 WT, passaged without
1a in Vero cells), and r’esistant’ strain (CVB3 R, passaged at increasing concentrations of
1a). After nine subsequent passages of the virus in cell culture, the IC
50 of
1a was determined to be 12.9 μM for the resistant strain, which was 7-fold higher than that of the original virus (IC
50=1.8 µM), and higher than wild type virus (IC
50=0.48 µM). Therefore,
1a stimulates the selection of resistance of CVB3 virus, suggesting its direct antiviral activity and a virus-specific target (
Figure 6). We also investigated the growth characteristics of resistant virus in comparison to the wild type one in vitro (
Figure 7). In the presence of
1a, the resistant strain was able to effectively propagate in contrast to the wild type virus. Nevertheless, without
1a, the growth speed of the resistant strain was significantly lower than that of the wild-type virus during the first 48 hours (p<0.05 by Mann–Whitney U-test).
After the resistant viral variant was obtained, viruses were plaque purified, and full genomes of three clones from each viral type (initial, wild-type, 1a-resistant) were sequenced. Their nucleotide sequences were translated to localize amino acid substitutions. After a comparison of the amino acid sequences, substitution S1209I was identified. Since position 1209 of viral polyprotein corresponds to 2C protein, here and further we use amino acid numeration corresponding to this specific protein (p‘osition 109’, instead of 1209). AlphaFold software was used to generate the complete structural model of 2C protein. Based on the model, the position 109 in 2C was mapped (
Figure 8).
Therefore, with sequential passage of the Coxsackievirus in the presence of 1a, a decrease in the sensitivity of the virus to 1a occurs. This is accompanied by a deterioration in the growth characteristics of the resistant virus and the appearance of mutation in the 2C protein. This indicates an influence of 1a on processes associated with replication of the viral genome.