The Role of Pyrazolopyridine Derivatives on Different Steps of Herpes Simplex Virus Type-1 In Vitro Replicative Cycle

Herpes simplex virus type-1 (HSV-1) infection causes several disorders, and acyclovir is used as a reference compound. However, resistant strains are commonly observed. Herein, we investigate the effects of N-heterocyclic compounds (pyrazolopyridine derivatives), named ARA-04, ARA-05, and AM-57, on HSV-1 in vitro replication. We show that the 50% effective concentration (EC50) values of the compounds ARA-04, ARA-05, and AM-57 were 1.00 ± 0.10, 1.00 ± 0.05, and 0.70 ± 0.10 µM, respectively. These compounds presented high 50% cytotoxic concentration (CC50) values, which resulted in a selective index (SI) of 1000, 1000, and 857.1 for ARA-04, ARA-05, and AM-57, respectively. To gain insight into which step of the HSV-1 replication cycle these molecules would impair, we performed adsorption and penetration inhibition assays and time-of-addition experiments. Our results indicated that ARA-04 and ARA-05 affected viral adsorption, while AM-57 interfered with the virus replication during its α- and γ-phases and decreased ICP27 content during initial and late events of HSV-1 replication. In addition, we also observed that AM-57 caused a strong decrease in viral gD content, which was reinforced by in silico calculations that suggested AM-57 interacts preferentially with the viral complex between a general transcription factor and virion protein (TFIIBc-VP16). In contrast, ARA-04 and ARA-05 interact preferentially in the proteins responsible for the viral adsorption process (nectin-1 and glycoprotein). Thus, our results suggest that the 1H-pyrazolo[3,4-b]pyridine derivatives inhibit the HSV-1 replicative cycle with a novel mechanism of action, and its scaffold can be used as a template for the synthesis of promising new molecules with antiviral effects, including to reinforce the presented data herein for a limited number of molecules.


Introduction
Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus, whose virions consist of a large, double-stranded DNA genome packaged within an icosahedral capsid encased by a proteinaceous tegument and a lipidic envelope composed of various glycoproteins [1,2]. Most people have their first contact with HSV-1 during childhood, and if HSV-1 succeeds in invading the orolabial mucosa, they acquire a lifelong persistent infection, in which the viruses can establish latency in nervous system cells and even reactivate depending on the immune status of the human host. Recent estimates indicate 63-90% seropositivity among adults worldwide [3,4].
First, IE gene expression occurs [11]. HSV-1 immediate-early proteins, such as infectedcell protein 0 (ICP0), ICP27, and ICP4, regulate the subsequent steps of viral replication and affect normal host cell function [12,13]. ICP0 is known to be an E3-ubiquitin ligase that counteracts host repression of the viral genome, DNA damage responses, and host-driven chromatin repression of the viral genome [14]. ICP27 modulates 3 processing, splicing, and exportation of mRNA [12,15,16]. ICP4 is the main viral transcriptional factor, stimulating the transcription of early and late genes, while ICP22 promotes transcription elongation of viral genes [17]. Second, delayed-early (DE; β) gene transcripts, such as viral helicase (UL-8), DNA polymerase (UL- 30), and thymidine kinase, are expressed, and viral DNA is synthesized. Finally, after viral DNA replication, late (L; γ) genes, which encode HSV-1 structural proteins, such as gD, gB, gC, and ICP5, are expressed [5,6]. Among the most important HSV-1 proteins, the immediate-early protein ICP27 plays a key role during viral replication. It has been shown that this viral protein affects both viral and host cell gene expression, being crucial for the efficient expression of DE and L genes [16,18,19]. Another HSV-1 protein vital for viral replication is glycoprotein D (gD) [5,9]. This glycoprotein has structural roles and serves as a binding point to anchor interactions with three alternative receptors for virus entry: nectin-1, herpesvirus entry mediator (HVEM), and modified heparan sulfate [20,21]. Viral gD is also one of the most conserved proteins among the Herpesviridae family and constitutes the core apparatus of the viral fusion protein [7,8,22].
The treatment of HSV-1-infected individuals and various antiherpetic compounds have been widely investigated, including acyclovir (ACV), ganciclovir, and penciclovir, and also their prodrugs valacyclovir, valganciclovir, and famciclovir [23]. Nonetheless, the selective emergence of drug-resistant HSV-1 strains may result from prolonged administration of these agents to immunocompromised patients. Therefore, drug repurposing and the search for new antiviral compounds, particularly with novel mechanisms of action, is a critical aim [24,25].
Our group demonstrated the inhibition of HSV-1 cytopathic effects by 1H-pyrazolo [3,4b]pyridine derivatives [38]. However, the mechanism of action towards this virus replication remained to be characterized. Thus, to understand the main active derivatives against the mode of action of HSV-1 replication (Figure 1), we performed a set of in vitro studies to determine the 50% effective concentration (EC 50 ) and 50% cytotoxic concentration (CC 50 ) of these compounds, as well as their effects on HSV-1 adsorption, penetration, and virus replicative cycle. To offer a reasonable, molecular-level explanation of the mechanism of the assayed compounds, in silico calculations via molecular docking were carried out with the main targets identified in the experimental assays. We found that ARA-04, ARA-05, and AM-57 were strong inhibitors of HSV-1 replication and fully capable of diminishing the content of some important viral proteins. pyrazolo [3,4-b]pyridine and thieno[2,3-b]pyridine derivative systems was described by our group [35][36][37][38][39]. Our group demonstrated the inhibition of HSV-1 cytopathic effects by 1Hpyrazolo [3,4-b]pyridine derivatives [38]. However, the mechanism of action towards this virus replication remained to be characterized. Thus, to understand the main active derivatives against the mode of action of HSV-1 replication (Figure 1), we performed a set of in vitro studies to determine the 50% effective concentration (EC50) and 50% cytotoxic concentration (CC50) of these compounds, as well as their effects on HSV-1 adsorption, penetration, and virus replicative cycle. To offer a reasonable, molecular-level explanation of the mechanism of the assayed compounds, in silico calculations via molecular docking were carried out with the main targets identified in the experimental assays. We found that ARA-04, ARA-05, and AM-57 were strong inhibitors of HSV-1 replication and fully capable of diminishing the content of some important viral proteins.

Inhibition Profile of HSV-1 by Pyrazolopyridine Derivatives
We have already described the synthesis and antiviral activity of some 1Hpyrazolo [3,4-b]pyridine derivatives [38]. Here, we named the studied compounds ARA-04, ARA-05, and AM-57 ( Figure 1). First, we determined the potency of the studied compounds. As shown in Table 1, the 50% effective concentration (EC50) values for ARA-04 and ARA-05 were about 1 µM, while for AM-57, it was 0.70 ± 0.10 µM. These values were comparable to the EC50 value for acyclovir (ACV, positive control). Importantly, the CC50 values for ARA-04 and ARA-05 were 1000 µM, while for AM-57, it was 600 µM. We calculated the selective index (SI) values based on the ratio of CC50 and EC50, which were equal to 1000 for ARA-04 and ARA-05 and 857.01 for AM-57, again comparable to the SI value for ACV [40].

Inhibition Profile of HSV-1 by Pyrazolopyridine Derivatives
We have already described the synthesis and antiviral activity of some 1H-pyrazolo [3,4b]pyridine derivatives [38]. Here, we named the studied compounds ARA-04, ARA-05, and AM-57 ( Figure 1). First, we determined the potency of the studied compounds. As shown in Table 1, the 50% effective concentration (EC 50 ) values for ARA-04 and ARA-05 were about 1 µM, while for AM-57, it was 0.70 ± 0.10 µM. These values were comparable to the EC 50 value for acyclovir (ACV, positive control). Importantly, the CC 50 values for ARA-04 and ARA-05 were 1000 µM, while for AM-57, it was 600 µM. We calculated the selective index (SI) values based on the ratio of CC 50 and EC 50 , which were equal to 1000 for ARA-04 and ARA-05 and 857.01 for AM-57, again comparable to the SI value for ACV [40]. Considering that pyrazolo [3,4-b]pyridine derivatives inhibit HSV-1 replication, and it has been shown that these compounds are non-nucleoside analog inhibitors of the HIV-1 enzyme reverse transcriptase [34,41], the structure of these derivatives could be an interesting model to develop novel drugs that could simultaneously target HIV-1 and HSV-1 replication since HSV-1 is known to be an opportunistic pathogen often found in HIV-infected patients [42].

Effects of Pyrazolopyridine Derivatives on Infected Cells
To gain insight into pyrazolopyridine's inhibitory effect on HSV-1 replication, we analyzed the impact of antiviral treatment during different moments of the virus life cycle. To monitor if the antiviral activity was already present during adsorption, cells were infected and treated simultaneously for 1 h in an ice-cold bath at 4 • C because, at this condition, viral particles can bind but are unable to penetrate the host cells. After this period, cells were washed with ice-cold PBS to remove unbounded viruses, and the temperature was raised to 37 • C. After a temperature shift, the previously bound viral particles may enter the cells and produce plaques after 72 h p.i.
The compounds ARA-04 and ARA-05 significantly reduced the number of plaques when virus infection and treatment were synchronized at 4 • C ( Figure 2A). AM-57 was inactive under these conditions ( Figure 2A). For comparison, we used heparin as a positive control at 1 µg/mL because it competes with heparan sulfate on the cell surface for HSV-1 adsorption ( Figure 2A) [43,44]. To gain insight into whether the HSV-1 replicative cycle would be impaired by the ARA-04, ARA-05, and AM-57 compounds, a time-of-addition assay was performed because the synthesis of most HSV-1 α-, β-, and γ-proteins has been with particular time frames in Vero cells [9]. Vero cells were infected with HSV-1 for 1 h at 37 °C and treated with the compounds at a concentration equivalent to their EC50 concentrations since, at In another set of experiments, cells were infected at 4 • C but without treatment. After 1 h, cells were washed with ice-cold PBS to remove unbounded viruses, and the temperature was shifted to 37 • C to allow virus penetration for an additional 1 h period. During this step, associated with virus penetration, treatments with ARA-04, ARA-05, or AM-57 were performed. After 72 h, PFU were scored. No effect could be attributed to the inhibition of HSV-1 penetration ( Figure 2B).
To gain insight into whether the HSV-1 replicative cycle would be impaired by the ARA-04, ARA-05, and AM-57 compounds, a time-of-addition assay was performed because the synthesis of most HSV-1 α-, β-, and γproteins has been with particular time frames in Vero cells [9]. Vero cells were infected with HSV-1 for 1 h at 37 • C and treated with the compounds at a concentration equivalent to their EC 50 concentrations since, at this level, 50% replication inhibition can be obtained without observing possible nonspecific effects. We found that the antiherpetic activity of AM-57 was preserved when the drug was added to HSV-1-infected cells in the time frames from 0-3 h ( Figure 3A) to 6-20 h after infection ( Figure 3C). These results, along with the absence of anti-HSV-1 activity by AM-57 in Figure 2, could suggest that such a molecule may act both at the initial and late steps of viral replication, which overlaps with the peak of αand γ-protein syntheses. The compounds ARA-05 and ARA-04 showed marginal effects when added post-infection/adsorption ( Figure 3). For comparison, ACV, which inhibits HSV-1 DNA synthesis, a β-phase event, mainly affected the viral life cycle in the time frame from 3 to 6 h ( Figure 3B).  Among the tested compounds, AM-57 seems to have a bimodal effect on the HSV-1 replicative cycle, an activity that is desired among novel compounds in the literature [45][46][47]. Moreover, since there is a gap of time between the initial and late effects of this compound on the HSV-1 replication cycle, these two activities could be independent of each other.
Since the pyrazolopyridine compound blocks HSV-1 replication, considering it has been shown that this virus replication causes an interruption of host cell protein synthesis Among the tested compounds, AM-57 seems to have a bimodal effect on the HSV-1 replicative cycle, an activity that is desired among novel compounds in the literature [45][46][47]. Moreover, since there is a gap of time between the initial and late effects of this compound on the HSV-1 replication cycle, these two activities could be independent of each other.
Since the pyrazolopyridine compound blocks HSV-1 replication, considering it has been shown that this virus replication causes an interruption of host cell protein synthesis [2,6,47,48], and also that AM-57 inhibition on the HSV-1 life cycle is temporally associated with the αand γ-steps of viral replication, we monitored the levels of ICP27 and gD upon AM-57 treatment as proxies of these phases [47,48]. Vero cells were infected for 1 h at 37 • C and then treated with the compounds at their EC 50 values during the time frames of 0-3 or 6-20 h p.i. After that, the cells were harvested, and the samples were submitted to SDS/PAGE electrophoresis and immunoblotting assays. When HSV-1-infected Vero cells were treated with AM-57 during the peak of proteins from the α-phase (0-3 h p.i.) of HSV-1 replication, an 89.6% decrease in ICP27 content was observed ( Figure 4A,B). In addition, when cells were treated with AM-57 from 6 to 20 h p.i., a complete suppression of ICP27 content was observed ( Figure 4C,D). These results indicate that AM-57 may modulate the immediate-early and late events of HSV-1 replication [12,15,18,49]. Interestingly, AM-57 lacks activity from 3 to 6 h p.i. ( Figure 3B) because ICP27 synthesis/content could be turned off during this period, or such a protein would be in association with other cellular/viral molecules that would probably make ICP27 inaccessible to this compound [12,49]. °C and then treated with the compounds at their EC50 values during the time frames of 0-3 or 6-20 h p.i. After that, the cells were harvested, and the samples were submitted to SDS/PAGE electrophoresis and immunoblotting assays. When HSV-1-infected Vero cells were treated with AM-57 during the peak of proteins from the α-phase (0-3 h p.i.) of HSV-1 replication, an 89.6% decrease in ICP27 content was observed ( Figure 4A,B). In addition, when cells were treated with AM-57 from 6 to 20 h p.i., a complete suppression of ICP27 content was observed ( Figure 4C,D). These results indicate that AM-57 may modulate the immediate-early and late events of HSV-1 replication [12,15,18,49]. Interestingly, AM-57 lacks activity from 3 to 6 h p.i. (Figure 3B) because ICP27 synthesis/content could be turned off during this period, or such a protein would be in association with other cellular/viral molecules that would probably make ICP27 inaccessible to this compound [12,49]. The levels of gD from the HSV-1-infected cells treated with AM-57 were completely reduced, reinforcing the notion that the late phase of the HSV-1 life cycle could be affected by this treatment (Figure 4E,F). AM-57 does not necessarily act on ICP27 or gD directly. It is possible that AM-57 more broadly could affect the α-and γ-steps of viral replication.  The levels of gD from the HSV-1-infected cells treated with AM-57 were completely reduced, reinforcing the notion that the late phase of the HSV-1 life cycle could be affected by this treatment (Figure 4E,F). AM-57 does not necessarily act on ICP27 or gD directly. It is possible that AM-57 more broadly could affect the αand γ-steps of viral replication.

In Silico Calculations for the Main Targets of Pyrazolopyridine Derivatives
We conducted in silico calculations, via molecular docking, to obtain insights into the molecular level of the ARA-04, ARA-05, and AM-57 antiviral mechanism by evaluating the interaction mode and binding capacity between each pyrazolopyridine derivative with the proteins responsible for the viral adsorption process (glycoprotein and nectin-1 (gD-nectin-1)) [10] and one of the main proteins responsible for the ICP27 content (isolated proteins and complex composed by a general transcription factor and virion protein, TFIIBc-VP16) [50,51]. Table 2 summarizes the docking score values for each case. For GOLD 2020.2 software, more positive score values indicate better interactions [52]. Thus, in silico results suggested that ARA-04 and ARA-05 interact preferentially with the proteins responsible for the viral adsorption process, mainly gD, e.g., 55.8 and 46.8 for ARA-04 into gD and nectin-1, respectively. On the other hand, AM-57 interacts preferentially with the proteins responsible for the ICP27 content, mainly in the complex TFIIBc-VP16 (in this case, the docking score value for AM-57 is about 5 times higher than that for ARA-04 and ARA-05). These in silico results corroborate the experimental data that described different mechanisms between ARA compounds (inhibit the viral adsorption process) and AM-57 (decrease ICP27 content). A combination of biochemical and biophysical assays, e.g., thermal shift surface plasmon resonance and, most importantly, structural experiments, e.g., X-ray-diffraction and nuclear magnetic resonance, should be performed to further clarify how ARA-04 and ARA-05 could target gD or nectin-1, as well as how AM-57 could target TFIIBc-VP16, TFIIBc, or VP16.   Figure 5B,C), directly impacting the number of amino acid residues that stabilize the interactive profile of each compound. In this case, interactions via van der Waals forces were detected.
In line with the inhibition of the αand γ-steps of viral replication, by means of in silico assays, AM-57 impairs the system TFIIBc-VP16. AM-57 strongly interacts with the key amino acid residues of the TFIIBc/VP16 interface ( Figure 6B-D), e.g., for AM-57, t-stacking interactions with Tyr-165 (1.9 Å), van der Waals forces with Phe-195 (3.4 Å), and hydrogen bonding with Lys-196, Arg-286, and Arg-290 (2.9, 2.3, and 3.7 Å, respectively) were detected, while for ARA-04 and ARA-05, interactions via van der Waals forces were detected (Tyr-165, Phe-195, and Arg-286 within a distance of 2.7, 2.1, and 3.4 Å, respectively), and only one hydrogen bonding interaction was detected with Tyr-293 (1.9 Å). Therefore, the mechanistic hypothesis for AM-57 is that this pyrazolopyridine compound might competitively interact with TFIIBc, inhibiting its interaction with the substrate VP16 to not form the complex TFIIBc-VP16. As a consequence, the subsequent steps of the virus life cycle would also be impaired.

Cells and Virus
Vero cells (African green monkey kidney cells, ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Life Technologies, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and incubated at 37 • C in 5% CO 2 . For virus stock preparation, Vero cells were infected with HSV-1 (KOS strain) [57] at a multiplicity of infection (MOI) of 0.1. Twenty-four hours post-infection (h.p.i.), the cells were lysed after three rounds of freezing and thawing, centrifuged at 1500× g at 4 • C for 20 min, and the resulting supernatant was pooled and stored at -70 • C for further studies [58].

Cytotoxic Assays
Vero cell monolayers (10 4 cells/well) in 96-well plates were treated for 72 h with increasing amounts (312.5-2500 µM) of the tested compounds. Then, 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) diluted in 1 × PBS was added to the cells, following the manufacturer's protocol. After 4 h at 37 • C, 10% SDS was added. Following incubation for 2 h at 37 • C, the plates were read in a spectrophotometer at 570 nm. All the compounds were resuspended in DMSO 100% for in vitro testing. In the assays, the final DMSO concentrations were equal to or lower than 1% (v/v), diluted in DMEM, and not toxic to the cells [59]. The 50% cytotoxic concentration (CC 50 ) was calculated through a nonlinear regression analysis of the dose-response curves created from the data with GraphPad Prism 8 software.

Yield-Reduction Assays and Virus Titration
Vero cell monolayers in 24-well plates (4 × 10 4 cells/well) were infected with HSV-1 at an MOI of 1 for 1 h at 37 • C. Then, the cells were washed, and the compounds were added at semilog concentrations (0.1-10 µM) in DMEM with 5% FBS. In every assay, we chose to use acyclovir (ACV; Sigma-Aldrich/Merck, St. Louis, MO, USA, >99% pure) as a positive control. After 24 h, the cells were lysed, centrifuged for clarification, and virus titers in the supernatant were determined by plaque-forming assays in Vero cell monolayers using 6-well plates (3 × 10 5 cells/well). In this assay, Vero cells were exposed to supernatants from yield-reduction assays for 1 h at 37 • C. Next, residual viruses were discarded, and carboxymethylcellulose solution (DMEM-HG 1×, 1.0% carboxymethylcellulose, and 2% fetal bovine serum) was added. After 72 h at 37 • C, the cytopathic effect (CPE) was analyzed under an optical microscope, and 10% formalin was added to fix the cells. After 3 h, the solution was harvested, plaques were colored with 0.4% bromophenol blue, and the virus titers were calculated by scoring the plaque-forming units (PFU) [38,39,60]. The 50% effective concentration (EC 50 ) was obtained through a nonlinear regression analysis of the dose-response curves generated from the data using GraphPrism 8 software. The selectivity indexes (SIs) for each assayed compound were calculated through the ratio between the CC 50 and EC 50 values.

Adsorption and Penetration Inhibition Assays
To test whether ARA-04, ARA-05, or AM-57 could inhibit virus adsorption, Vero cells (3 × 10 5 cells/well) grown in 6-well plates were infected with 200 PFU/well of HSV-1 and treated with each compound EC 50 at 4 • C for 1 h. Then, cells were washed and covered with overlay medium, and the temperature was increased to 37 • C for 72 h [61]. After that, plaques were analyzed, as described in the plaque-forming assay. For this assay, heparin at 1 µg/mL (Sigma-Aldrich/Merck, St. Louis, MO, USA, >99% pure) was used as a positive control.
HSV-1 penetration assays were also performed, in which the same protocol mentioned above was used with slight modifications. In brief, Vero cells (3 × 10 5 cells/well) were infected with 200 PFU/well of HSV-1 at 4 • C for 1 h. Next, the temperature was raised to 37 • C in the presence of ARA-04, ARA-05, or AM-57; 1 h after this step, cells were washed with PBS supplemented with glycine at pH 2.2. After this step, cells were covered with overlay medium, and at 72 h p.i., the PFUs were counted as described [62].

Time-Of-Addition Assays
To evaluate whether the addition of the compounds could be delayed without losing their capacity to inhibit HSV-1 replication and thus identify which period of the HSV-1 replication cycle is affected by these compounds, time-of-addition assays were performed. For this purpose, Vero cells in 6-well plates were infected with 200 PFU/well for 1 h at 37 • C. Then, monolayers were washed, and the derivatives were added at their respective EC 50 at different hours p.i. [61]. After that, the medium containing the compounds was removed, and cells were covered with overlay medium. At 72 h p.i., the plaques were counted. As a control, the reference compound (ACV) at 1.1 µM (EC 50 ) was analyzed in parallel.

In Silico Procedure
The 3D chemical structure of ARA-04, ARA-05, and AM-57 was built and energyminimized by density functional theory (DFT) via Spartan'18 software (Wavefunction Inc., Irvine, CA, USA). The crystallographic structure for gD-nectin-1, TFIIBc-VP16, and VP16 was obtained from the Protein Data Bank (PDB): 3SKU, 2PHG, and 16VP, respectively [50,51,53]. GOLD 2020.2 software (Cambridge Crystallographic Data Center Software Ltd., CCDC, Cambridge CB2 1EZ, UK) was applied for molecular docking calculations. Hydrogen atoms were added to the biomacromolecules following the ionization and tautomeric states at pH 7.4. Since there is no crystallographic structure for drugs associated with gD-nectin-1 and TFIIBc-VP16, the standard function ChemPLP was used for the molecular docking calculations by selecting a spherical cavity with a 10 Å radius around the active binding site. PyMOL Delano Scientific LLC software (Schrödinger, New York, NY, USA) was used to build the figures.

Statistical Analysis
All data presented in this paper represent the means ± standard deviation (SD) (SEM) of at least three independent experiments. The inhibition percentages are related to control assays with virus infection without treatment. All statistical analyses were performed using the software GraphPrism 8. Values of p ≤ 0.001 were considered statistically significant by Dunnett's multiple comparisons test, one-way ANOVA (Figures 2 and 3), or paired t-test (Figure 4).

Conclusions
The pyrazolopyridine derivatives evaluated here exhibited a new and different mechanism of inhibition when compared with standard anti-HSV-1 agents typically found in clinical practice, such as ACV, which inhibit viral DNA polymerase. Therefore, even mutations in DNA polymerase of HSV-1, which could produce resistance to the reference compounds, could not affect the action of the studied pyrazolopyridine derivatives. Moreover, we describe for the first time that the compound AM-57 could inhibit different steps of viral replication, affecting critical viral proteins, such as ICP27 and gD, by targeting the viral complex between TFIIBc and VP16. We conclude that the constant design and study of novel antiviral compounds may provide a future basis for novel antiviral therapeutic molecules with unique structures and alternative mechanisms of action. Thus, we propose that the chemical structures of ARA-04, ARA-05, and AM-57, both derivatives of the 1Hpyrazolo [3,4-b]pyridine system, are promising for future bioisosterically broad-spectrum analysis for novel antivirals, with a good perspective for structure-activity relationship (SAR) studies.