Identification and preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral and anti-inflammatory therapy

doi:10.21203/rs.3.rs-1533971/v1

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Identification and preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral and anti-inflammatory therapy

https://doi.org/10.21203/rs.3.rs-1533971/v1

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published 13 Jan, 2023

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Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still scarce and the emergence of new variants challenging immunized individuals, suggests that mutant viruses might also emerge because of antiviral pressure. Therefore, beyond the recently alleged positive antiviral clinical results with molnupiravir™ and paxlovid™, the continuous search for drugs against 2019 coronavirus disease (COVID-19) is necessary. Because severe COVID-19 is a virus-triggered immune and inflammatory disfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that N6-furfurylaminopurine (kinetin, MB-905) inhibits the in vitro replication of SARS-CoV-2 at the sub-micromolar range in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. As a pro-drug, MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce an error-prone virus replication. Consistently, co-inhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously placed nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. SARS-CoV-2-infected transgenic mice expressing human ACE2 were treated with MB-905 and decreased viral replication of the gamma variant was observed, along with reduced lung necrosis, hemorrhage and inflammation, together with increasedmice survival. MB-905 showed good oral absorption, its metabolites were stable and achieved long-lasting plasma concentrations exceeding those required for the in vitro inhibition. Besides, MB-905 was neither mutagenic, toxic during chronic treatment, nor cardiotoxic. Because kinetin has already been clinically investigated for a rare genetic disease at regimens that are beyond the predicted concentrations of antiviral/anti-inflammatory inhibition demonstrated here, our investigation strongly suggests the opportunity for a rapid clinical development of a new and orally available antiviral substance for the treatment of COVID-19.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of 2019 coronavirus disease (COVID-19), global health have been confronted by an unprecedented burden that has taken over 200,000 lives/month in almost two years¹, caused deep economic depression and continued uncertainties. Despite previous threats imposed by highly pathogenic coronaviruses in 2002 and 2014, the response to these diseases did not result in clinically approved vaccines or antivirals. Thus, the development of prophylactic and therapeutic responses to COVID-19 had to unfold during the ongoing pandemic².

The unprecedently fast development of vaccines against SARS-CoV-2 have resulted in reduced rates of hospitalization and deaths³. However, SARS-CoV-2 variants of concern (VoC) are constantly emerging endowed with the ability to escape the vaccinal humoral response⁴ thus provoking new pandemic waves⁵. VoC emergence highlights that virus genetic barrier to become resistant may not be insurmountable. In fact, antiviral resistant SARS-CoV-2 strains have been clinically identified with or without a direct drug pressure⁶. This notion reinforces the urgency of antiviral development, as an additional alternative to fight COVID-19. Continued discovery and development are required as antiviral resistant strains to the first generation of antivirals are likely to emerge. Moreover, antivirals are necessary because sustainability of the anti-SARS-CoV-2 vaccinal response may be short and require frequent boosters⁷, especially for individuals with impaired immune system, for whom vaccines may not work properly. Thus, the discovery and development of newer and more effective antivirals continue to be an unmet medical need.

As it has been the case with other viral infections, the possibility to inhibit the RNA polymerase using nucleoside/nucleotide analogs have aroused special interest^8,9. Structural and enzymatic studies indicate that SARS-CoV-2 RNA polymerase [composed of the nonstructural protein 12 (nsp12) and the co-factors nsp7/nsp8] is a key enzymatic system in the virus life cycle¹⁰. Nsp12 is relatively promiscuous to incorporate the activated forms of various antiviral nucleotides¹⁰: i) non-obligate chain terminators, such as remdesivir™ (RDV), AT-527 and sofosbuvir (SFV); ii) obligate chain terminators, like tenofovir (TFV); and iii) the error-prone purine analog favipiravir (FPV) and molnupiravir (N4-hydroxycytidine, EIDD-2801, MK-4482). Certain factors may have jeopardized the unequivocal success of these antivirals against COVID-19. The phosphoroamidates¹¹ RDV, AT-527 and SFV seem to be highly vectorized to the liver, limiting their bioavailability in the lungs. SFV¹² and FPV¹³ require higher plasma concentrations against SARS-CoV-2, than their plasma exposures for treating hepatitis C and influenza virus, respectively. The required higher doses of these drugs may be because nsp12 RNA-dependent RNA-polymerase (RdRp) activity is coupled with a viral exonuclease (nsp14/10) that proofreads the newly synthetized viral genome and excises atypical nucleotides^14,15. Molnupiravir, on the other hand, delivers a tautomeric N4-hydroxycytidine in the viral RNA, leading to an error-prone catastrophic replication beyond the excision capacity of the exonuclease¹⁶. Despite relevant concerns that N4-hydroxycytidine may lead to mutagenesis in the host cells^17,18, clinical trials with molnupiravir have demonstrated that treated patients undergo faster SARS-CoV-2 RNA clearance¹⁹ and reduced hospitalization rates²⁰. Molnupiravir was not particularly effective in hospitalized individuals²⁰. Nevertheless, it has been authorized, by Food and Drug Administration (FDA) with a narrow difference of votes (https://www.youtube.com/watch?v=fR9FNSJT64M), and other regulatory agencies in different countries, for early antiviral intervention.

Indeed, in hospitalized COVID-19 patients, and in especial those critically ill, SARS-CoV-2 triggers an unbalanced host-dependent response, involving pro-inflammatory cytokine storm and coagulopathy^21,22. The progression from mild to severe COVID-19 is characterized by intense viral replication and tissue necrosis, with release of lactate dehydrogenase (LDH) in the respiratory tract²¹, involvement of possible neurogenic spread of the virus^23–25 and leukopenia^21,22. In this harmful scenario, the increased levels of IL-6, TNFα and C-reactive protein, among other pro-inflammatory substances, result in a virus-triggered sepsis-like disease. Anti-inflammatory and anti-clotting agents enhance the survival likelihood of COVID-19 patients at these late stages of disease^26,27. Therefore, antiviral compounds endowed with anti-inflammatory activity and broad tissue bioavailability would be highly desirable as ideal anti-COVID-19 drug candidates. Up to now, this has only been experimentally accomplished by the complex administration of dexamethasone and antibodies to SARS-CoV-2²⁸.

To discover potential leads against SARS-CoV-2, we tested a library of nitrogenous bases, nucleoside and nucleotide analogs in virus-infected human hepatoma (huh-7) cells, type II pneumocytes (calu-3) and in primary monocytes. N6-Furfurylaminopurine (kinetin) hereby designated MB-905 (Extended Figure 1A) inhibited SARS-CoV-2 replication in a concentration-dependent manner in huh-7 and calu-3 cells (Extended Figure 2, green line). By blocking N9 position (MB-906, Extended Figure 1B) or adding an unphysiological excess of adenine, the antiviral activity of MB-905 was impaired (Extended Figure 2, black lines), suggesting that MB-905 may require activation by the host-cell through the adenine phosphoribosyl transferase pathway (APRT), similarly to FPV²⁹. Considering that APRT could convert MB-905 to its riboside 5’-monophosphate and that 5’-nucleotidases (which are common in the liver³⁰, the port of entry of orally available drugs) would convert it to the corresponding nucleoside, these two potential pro-drugs were respectively synthesized, as the phosphoroamidate MB-711 and the nucleoside MB-801 (Extended Figure 1C and D). MB-905, MB-801 and MB-711 showed similar in vitro potencies to inhibit SARS-CoV-2 replication in calu-3 cells (Figure 1A and Extended Table 1). We further demonstrated that their efficacies were improved by daily administration (Figure 1B and Extended Table 1). Although adenine-related MBs were less potent than RDV, MB-905, MB-801 and MB-711 had 6- to 40-fold higher selective indexes than molnupiravir. These adenine-derived compounds displayed one order of magnitude lower cytotoxicity when compared to molnupiravir. (Extended Table 1).

Next, we tested if MB-905, its related compounds, as well as the controls RDV and molnupiravir, could reduce SARS-CoV-2 RNA levels and virus-induced inflammatory markers in human primary monocytes. In contrast with molnupiravir, which showed relatively low activity in SARS-CoV-2-infected monocytes, MB-905, MB-801, MB-711 and RDV reduced cell-associated virus RNA levels in a concentration-dependent manner (Figure 1C). Additionally, we observed that molnupiravir was highly cytotoxic to these myeloid cells. With molnupiravir at 10 µM, monocyte viability had been drastically reduced (Figure 1C). Further, at 10 µM, MB-905 also diminished SARS-CoV-2-induced TNF-α and IL-6 levels (Figure 1D and E), a highly desired feature for antivirals against COVID-19.

Considering that MB-905 is most likely a pro-drug of its corresponding nucleotide triphosphate, we further tested whether inhibition of virus replication could be related to a direct action on SARS-CoV-2 RNA polymerase (Figure 2A). Kinetin riboside 5’-triphosphate inhibits the viral RNA polymerase, but with an IC₅₀ 3-fold higher when compared to the active triphosphate form of RDV (GS-443902) (Figure 2A). Additionally, by measuring cell-associated genomic and sub-genomic viral RNA in SARS-CoV-2-infected calu-3 cells, we found that MBs are active to impair viral RNA synthesis (Figure 2B). Whereas RDV inhibited both the markers of replication (genomic RNA) and transcription (subgenomic RNA) at similar magnitudes, MBs were more effective in reducing replication rather than transcription (Figure 2B). Considering that MB-905 and related prodrugs deliver the riboside 5’-triphosphate, which could be a substrate for SARS-CoV-2 RNA polymerase (Figure 2A), N6-furfuryladenine would be incorporated in the virus genome from MB-905-treated SARS-CoV-2-infected calu-3 cells. To test this hypothesis, a high affinity anti-N6-furfuryadenine IgG was used to immunoprecipitate (IP) virus RNA, follow by quantification by RT-PCR. Nil-treated cells already presented a basal level of kinetin in the viral RNA compared to control isotype immunoprecipitation (Figure 2C), suggesting a natural occurrence of N6-furfuryladenine in the viral genome – which could be due to a natural oxidation of RNA components during sample preparation. Remarkably, viral RNA obtained from anti-N6-furfuryladenine IP of MB-905-treated SARS-CoV-2-infected calu-3 cells enriched by more than 10-fold viral RNA levels, compared to isotype control and nil-treated cells (Figure 2C). Thus, N6-furfuryladenine seems to be incorporated in the viral genome upon treatment with MB-905. We next sequenced the full-length virus genome from MB-905- and nil-treated SARS-CoV-2-infected cells and observed increased changes at the first base level, especially T(U) → A and C → A (Figure 2D). The error-prone replication made the virus populations grown in the presence and absence of MB-905 phylogenetically distinguishable (Extended Figure 3A). Cumulative effects of four SARS-CoV-2 passages of in calu-3 cells in the presence of MB-905 and molnupiravir are comparable (Extended Figure 3B). Modeling the presence of N6-furfuryladenine in a double-stranded nucleic acid highlights the N6-furfuryl moiety bumping into neighboring adenine (Extended Figure 4A); thus, widening the strands distance and likely impairing polymerase activity. This non-canonical base paring (Extended Figure 4B) could lead to an error-prone replication.

Knowing that the proof-reading mechanism in SARS-CoV-2 replication complex could be actively excising the incorporated nucleotide derived from MB-905, we tested whether the co-inhibition of the exonuclease enhanced the in vitro antiviral activity of MBs under investigation. Either the HIV integrase¹⁵ and HCV NS5A³¹ inhibitors have been proposed to target SARS-CoV-2 exonuclease. Thus, MB-905 or control RNA polymerase inhibitors were combined with suboptimal doses of dolutegravir (DTG), raltegravir (RTG), pibrantasvir (PIB), ombitasvir (OMB) or daclatasvir (Extended Table 2). We observed that combination of RNA polymerase and repurposed inhibitors of SARS-CoV-2 nsp14 enhanced the potency of the former, such as MB-905 and the control antivirals (Extended Table 2). In the case of MB-905, efficient inhibition at EC99 level was achieved when combined with the HIV integrase inhibitors (Extended Table 2). To better demonstrate the enhancement achieved for MB-905, MB-801 or MB-711 in combination with 5 µM of DTG or RTG, results are presented as virus productive titers in untreated and treated virus-infected cells (Figure 2E and F). Whereas MBs inhibited virus replication in 1-log₁₀ at 10 µM, combination with 5 µM RTG (Figure 2E) or DTG (Figure 2F) increased antiviral inhibition to 2-log₁₀. Of note, RTG and DTG showed marginal effects on SARS-CoV-2 replication when tested at 5 µM (Figure 2E and F). These results on drug combination not only reinforce the characterization of MB-905 mechanism of action as an error-prone molecule, but also point out that our lead compound could be used together with repurposed drugs. Especially with DTG, which has very favorable pharmacokinetics³².

MB-905 leads to an error-prone virus replication but, in contrast with mutagenic molnupiravir¹⁸, it was negative for both Ames and micronucleus tests (Extended Table 3 and 4). Additional preclinical studies for MB-905 were performed, included pharmacokinetics in mice and rats, maximum tolerable dose, 28-day repeated oral dose, toxicokinetic assay, in vitro and in vivo potential severe cardiac side effects, and plasma albumin binding (Figure 3, Supplementary Figures 1-3, Supplementary Tables 1-2). When assessed in vivo in mice and rats, MB-905 displayed over 50% of oral bioavailability (Figure 3A and B, Supplementary Figures 1A and B, and Supplementary Tables 1 and 2). MB-905 was found in plasma and lungs (Figure 3B and C). MB-905-derived metabolites were detected (Figure 3D and Supplementary Figures 1 and 2), with molecular weights consistent with its nucleoside (equivalent to MB-801, metabolites 1 and 4 from Figure 3D and Supplementary Figures 1 and 2), nucleotide monophosphate (metabolites 2 and 3 from Figure 3D and Supplementary Figures 1 and 2) and triphosphate (metabolite 5 from Figure 3C and Supplementary Figures 1 and 2). In lung extracts, either the nucleoside, nucleotide monophosphate and triphosphate (metabolite 1, 2 and 5, respectively, from Figure 3C) were more abundant, consistently with the notion that the nucleosides undergo phosphorylation in the target tissue for COVID-19 antiviral activity. Moreover, incubation of MB-711 with mice liver extracts, followed by measurement of 5’-nucleotidase activity, reveals that this molecule could be used as a substrate in substitution of AMP (Supplementary Figure 4). We interpret that upon APRT activation, MB-905 riboside monophosphate may be converted to its corresponding nucleoside (MB-801) to be released in the plasma. Altogether these data suggest that oral administration of MB-905 leads the formation of its active metabolite in the respiratory tract to act as a potent inhibitor of the polymerase and thus inhibiting the virus reproduction.

We next evaluated if MB-905 could protect transgenic mice expressing human ACE2 (K18-hACE2) from a lethal challenge (10⁵ PFU of SARS-CoV-2 VoC gamma). To better mimic a clinical situation, in which patients seek for treatment after the disease onset, oral treatments with MB-905 started 12h after intranasal SARS-CoV-2 infection and continued thereafter, once daily. Higher survival rates were observed for MB-905 at 140 mg/kg/day, combined or not with DTG, and at 70 mg/kg/day with the HIV integrase inhibitor (Figure 4A). Besides mortality, weight loss is the major clinical event after SARS-CoV-2 infection. Under treatment with MB-905 at 140 mg/kg/day alone and in combination with DTG, infected mice sustain their masses beyond the threshold of euthanasia (Figure 4B) and improved their overall clinical scores (Figure 4C). Although MB-905 administered with or without DTG did not alter virus RNA levels in the lungs of the infected mice (Figure 4D), the infectious titers decreased significantly upon treatment (Figure 4E). These results are in line with an error-prone inhibition of virus replication.

Alone or combined with DTG, MB-905 reduced lung necrosis (Figure 4F and G). Whereas infected/untreated mice lung histology displays collapsed alveoli septum and intense hemorrhage, treated animals displayed a lung parenchyma closer to mock-infected mice (Figure 4G). In line with in vitro results on SARS-CoV-2-infected monocytes, MB-905 is also presented significant anti-inflammatory properties in vivo also. (Figures 4H to K). Levels of the cytokine storm markers TNF-α (Figure 4H), IL-6 (Figure 4I) and KC (Figure 4J), and inflammatory cells counts (Figure 4K) were reduced in the bronchioalveolar lavage (BAL) of SARS-CoV-2-infeced mice treated with MB-905, in comparison with the untreated animals.

We found that MB-905 could impair the viral RNA synthesis and induce an error-prone replication, with increased U è C and C èA changes. Based on viral sequencing and in silico studies, we interpret that MB-905 can act as tautomeric purine, in which C6-N could be either acting as a donor or acceptor for hydrogen bonds, depending on whether hydrogen at position C6-N is interacting or not with oxygen from furfuryl group. The codon bias observed for MB-905 is supported by other independent studies on a rare disease called familial dysautonomia, in which kinetin has been proposed as a treatment. While healthy individuals have the base T at 6^th nucleotide position of 20^th exon of the gene that encodes the protein kinase complex associated with I-κ-B (IKBKAP), patients with familial dysautonomia have base C at this nucleotide position^33,34. This T è C change affects the splicing process of the intron adjacent to that exon^33,34. Kinetin allows the correct splicing to occur, suggesting it is incorporated in the intronic RNA present as an A/G analogue, which are purines able to respectively act as donor and acceptor of hydrogen bonds at position 6 of the nucleobase. Therefore, there is a convergence between the antiviral mechanism and the effect of kinetin to overcome innate genetic errors. Additionally, kinetin has been used in humans as anti-aging, anti-apoptotic and anti-inflammatory drug^35–38. In line with these studies, MB-905 attenuated SARS-CoV-2-induced inflammation.

Previous Phase I/II clinical trials carried out in patients with familial dysautonomia showed that kinetin doses between 11.1 and 23.5 mg/kg/day were well tolerated^39,40. In these trials, kinetin reached human plasma exposures from 8 to 23 µM⁴⁰, which are above our in vitro EC₉₀ values for MB-905, in the range of 2.1 to 6.3 µM. Thus, considering this range of EC₉₀ values for MB-905 and according to the results on kinetin pharmacokinetics in humans⁴⁰, plasma exposures would last for 8h to 12h above the threshold of virus inhibition. The in vivo dose of 140 mg/kg/day, which displayed antiviral, anti-inflammatory and protective effectiveness, is equivalent in humans to 11.38 mg/kg/day, according to standard conversion calculations between species⁴¹. Therefore, our in vitro and in vivo results are consistent with the doses already used in humans, and to our observation of the active ribosyl-triphosphate metabolite in the lung of treated mice.

Our preclinical investigation indicates that MB-905 may be clinically developed not only as monotherapy, but also in combination with repurposed drugs that inhibit SARS-CoV-2 nsp14/10, such as HIV integrase inhibitors⁴². Among this class of compound, dolutegravir has the most favorable pharmacokinetics³². When administered at the dose of 50 mg per day, dolutegravir achieves plasma concentrations between 15 and 1.5 µM, at the points of C_max and C_min, respectively³². These concentrations synergized in vitro with MB-905. MB-905 in vitro pharmacological parameters were improved to 5-10 times when combined with dolutegravir at 5 µM. Altogether, our results could motivate clinicals trials with MB-905 at doses of 11.5 mg/kg, twice daily, combined or not with standard dolutegravir posology for patients with COVID-19. The proposed dose and regimen of MB-905 against COVID-19 would be lower and shorter than the 23.5 mg/kg/day used for 28 days in humans in previous studies^39,40. Further, in a more recent clinical trial, kinetin was used orally at a dosage of 30 mg/kg/day by patients with familial dysautonomia during 3 years without interruption⁴³, thus reinforcing that MB-905 is a highly safe and promising candidate against COVID-19. Naturally, to treat a human genetic disease, kinetin would have to be administrated chronically, while its potential use against COVID-19 would be limited to a narrower period.

In summary, our results reconfirm the paramount importance of nucleoside analogs as a rich source of lead molecules capable of inhibiting the replication of biomedically important viruses⁴⁴. We demonstrated that MB-905, a molecule with easy synthetic access and cost effectiveness, is a pro-drug of the corresponding nucleoside tri phosphate which inhibits SARS-CoV-2 RNA synthesis, and therefore its replication in cells relevant to the pathophysiology of COVID-19, such as type II pneumocytes and monocytes. MB-905 Affected the viral pattern of nucleotide recognition and this activity may be potentiated by inhibiting the viral 3’-5’-exonuclease. Oral administration of MB-905 reduced SARS-CoV-2 gamma-induced lung damage and inflammation in vivo. Our preclinical development of MB-905 as a new antiviral compound together with its previous clinical investigation in familial dysautonomia strongly indicates the promising opportunity to advancing MB-905 into clinical trials against Covid-19^8,45,46.

Reagents. The antivirals RDV, tenofovir and molnupiravir were purchased from Selleckhem (https://www.selleckchem.com/). Sofosbuvir and the MBs (Extended Figure 1) were synthetized by Microbiologica Química-Farmacêutica LTDA (Rio de Janeiro, Brazil). ELISA assays were purchased from R&D Bioscience. Recombinant SARS-CoV-2 proteins were purchased by BPS Biosciences (https://bpsbioscience.com/). For in vitro experiments all small molecule inhibitors were dissolved in 100% dimethylsulfoxide (DMSO) and subsequently diluted at least 10⁴-fold in culture or reaction medium before each assay. The final DMSO concentrations showed no cytotoxicity. The materials for cell culture were purchased from Thermo Scientific Life Sciences (Grand Island, NY), unless otherwise mentioned.

Small molecule synthesis. All solvents were used as received from commercial suppliers. All of the other reagents used in the synthesis were purchased from Sigma-Aldrich except the reagent for phosphoramidation reaction ((S)-isopropyl 2-(((S)-(perfluorophenoxy) (phenoxy) phosphoryl) amino) propanoate), purchased from ALCHIMIA LIMITED. All reactions were carried out under an argon atmosphere. Reactions were monitored with analytical TLC on silica gel 60-F254 precoated aluminum plates and visualized under UV (254 nm). Purification was performed by column chromatography on silica gel (35−70 μM) or by crystallization. NMR data were recorded on a Bruker Fourier 80, or Bruker 500 MHz spectrometer in the deuterated solvents indicated, and the spectra were calibrated on TMS. Chemical shifts (δ) are quoted in ppm, and J values are quoted in Hz. In reporting spectral data, the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dddd (doublet of double doublets) and m (multiplet). Mass spectrometry analysis was performed on Shimadzu LCMS-8045. HPLC was carried out on a Shimadzu Prominence chromatographer equipped with a DAD detector (Xterra RP18, 250x4.6 mm 5mm – part n°:186000496 column). Solvents were used as HPLC grade. Structural details are highlighted in Chemistry Supplemental File 1

Synthesis of Kinetin, MB-905 (A): In a three-neck round-bottom flask quipped with a reflux condenser, a thermometer, an addition funnel and mechanic stirring, a suspension of 6-chloropurine (50 g, 0.323 mol) in ethanol (500 mL) was cooled to 5° C. A solution of furfurylamine (57 mL, 0.647 mol) and 4-Dimethylaminopyridine (0,4g, 3.2 mmol) in ethanol (50 mL) was slowly added. Then, the system was heated to reflux for 4 hours. After this time the system was cooled down to 5°C and the crystalline solid was filtered and washed with 150 mL of a 1:1 ethanol/water solution. The crude product was recrystallized from ethanol. The resulting white crystalline solid was washed with cold ethanol, and dried under vacuum. Yield: 66 g, 95 %, m.p. 265-272 °C dec, mass spectra LC-MS (m/z): [M]⁺ = 216.10, ¹H NMR (500 MHz, DMSO-d6) δ 12.99 (s, 1H), 8.20 (s, 1H), 8.11 (s, 2H), 7.54 (s, 1H), 6.35 (s, 1H), 6.22 (s, 1H), 4.69 (s, 2H) ppm, ¹³C NMR (126 MHz, DMSO-d6) δ 154.0, 153.2, 152.3, 149.7, 141.8, 139.1, 119.0, 110.5, 106.6, 36.5 ppm, HPLC (X Terra RP18, 250x4.6x5mm, isocratic 10% fase B [MeOH:MeCN (1:1)] : 90% fase A [3,4g/L KH₂PO₄ pH 2,54 (H₃PO₄)], 1.0 mL/min, 210 nm, 30 °C) t_r= 8.768 min, λmax 272 nm.

Synthesis of 6-furfurylamino-9-(tetrahydropyran-2-yl)-9H-purine, MB-906 (B): To a suspension of kinetin (10 g, 0.046 mol) in ethyl acetate (150 mL), 3,4-Dihydropyrane (8,5 mL, 0.092 mol) was added followed to addition of 8 mL of formic acid. The mixture was refluxed for 5 hours. After this time TLC analysis confirmed total consumption of starting material. The reaction mixture was cooled and basified with saturated aqueous solution of NaHCO₃. The organic phase was washed with water (2 X 50 ml), dried with anhydrous sodium sulphate, and evaporated to a yellow residue that was crystallized from ethanol (85 ml). The resulting white crystalline solid was washed with cold ethanol, and dried under vacuum. Yield: 12 g, 86 %, m.p. 138 – 140 °C, mass spectra LC-MS (m/z): [M]⁺ = 300.15, ¹H NMR (80 MHz, DMSO-d₆) δ (ppm): 8.43 (s, 1H), 7.93 (s, 1H), 7.55 – 7.19 (m, 2H), 6.27 (d, J = 1.4 Hz, 2H), 5.70 (dd, J = 7.5, 4.3 Hz, 1H), 4.90 (d, J = 5.8 Hz, 2H), 4.15 (dd, J = 11.2, 2.8 Hz, 1H), 3.98 – 3.39 (m, 1H), 2.14 – 1.50 (m, 6H).¹³C NMR (20 MHz, DMSO-d₆) δ (ppm): 154.55, 153.11, 152.00, 148.75, 141.95, 137.72, 119.61, 110.33, 107.29, 81.67, 68.64, 37.63, 31.74, 24.84, 22.78, HPLC (X Terra RP18, 250x4.6x5mm, gradient 5-100% phase B [90% ACN/10% H2O (0,1% TFA)] : phase A [90% H2O (0,1% TFA)/10% ACN], 1.2 mL/min, 210 nm, 30 °C) t_r= 14.443 min, λmax 264 nm.

Synthesis of Kinetin riboside, MB-801 (D): To a suspension of 6-chloropurine riboside (10g, 0,034 mol) in EtOH (100 mL), furfurylamine (6 mL, 0.069 mmol) was added followed by dropwise addition of Et₃N (15 mL, 0.102 mol). The system was refluxed for 5 h. After this time TLC analysis confirmed total consumption of starting material. The product was filtered. The crude product was recrystallized from EtOH, filtered and washed with cold EtOH. After drying in vacuo, the product was obtained as a white crystalline solid. Yield: 8.7 g (90 % yield), m.p. 152 -154 °C, mass spectra LC-MS (m/z): [M]⁺ = 348.15, [α]_D²⁵-60.7 (c 1.0, EtOH), ¹H NMR (80 MHz, DMSO-d₆) δ (ppm): 8.39 (s, 1H), 8.27 (d, J = 4.4 Hz, 2H), 7.54 (s, 1H), 6.50 – 6.30 (m, 1H), 6.23 (d, J = 3.2 Hz, 1H), 5.91 (d, J = 6.0 Hz, 1H), 5.46 (d, J = 6.1 Hz, 1H), 5.33 – 5.03 (m, 1H), 4.94 – 4.48 (m, 3H), 4.17 (s, 1H), 3.98 (dd, J = 3.4 Hz, 1H), 3.76 – 3.52 (m, 2H).¹³C NMR (20 MHz, DMSO-d₆) δ (ppm): 154.59, 152.98, 152.48, 148.97, 142.03, 140.28, 119.92, 110.65, 106.94, 88.28, 86.16, 73.81, 70.90, 61.89, 36.41, HPLC (X Terra RP18, 250x4.6x5mm, gradient 5-100% phase B [90% ACN/10% H2O (0,1% TFA)] : phase A [90% H2O (0,1% TFA)/10% ACN], 1.2 mL/min, 210 nm, 30 °C) t_r= 14.443 min, λmax 264 nm.

Phenyl (Isopropoxy-L-alaninyl) Kinetin Riboside Phosphoramidate, MB-711 (C): A flame dried round-bottom flask flushed with argon was charged with kinetin riboside 2’,3’ acetonide (prepared accordingly literature procedure - ACS Med. Chem. Lett. 2011, 2 (8), 577–582) (500 mg, 1.29 mmol). The flask was flushed again with argon, and anhydrous THF (10 mL) was added by syringe. The solution was cooled to 0-5 °C in ice bath, and t-BuMgCl (2 M in THF, 0.8 mL, 1.55 mmol) was slowly added by syringe. Next, the mixture was stirring at 0-5 °C for 40 minutes. After that time, the reagent for phosphoramidation was added neat, at once. The mixture was left to stir for 12 h at room temperature. AcOH 1M (5mL) was then added to quench the reaction before solvent was removed under reduced pressure to leave the crude product as a pale-yellow oil, that was used in the next step without further purification. Next, a solution of the crude product in DCM: ethylene glycol 1:1 (10 mL) was treated with 30 mol% of camphorsulfonic acid (89 mg, 0.38 mmol) at 5 ^oC, the mixture was left to stir for 16 h at this temperature. After that time, TLC analysis confirmed total consumption of starting material. The solvent was removed under reduce pressure and the crude product was purified by flash column chromatography and then recrystallized from ethanol. The resulting light yellowish crystalline solid was washed with cold ethanol, and dried under vacuum. Yield: 575 mg, 68% (after two steps), m.p. 138-140 °C, mass spectra LC-MS (m/z): [M]⁺ = 216.10 = 617.30, [α]_D²⁵-22.5 (c 0.2, CH₂Cl₂), ¹H NMR (500 MHz, CDCl₃) δ 8.31 (s, 1H), 7.99 (s, 1H), 7.33 (s, 1H), 7.17 (t, J = 7.8 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.65 (s, 1H), 6.28 (s, 2H), 5.96 (d, J = 3.2 Hz, 1H), 4.92 (hept, J = 6.2 Hz, 1H), 4.79 (sl, 2H), 4.42 (d, J = 3.0 Hz, 2H), 4.32 (dddd, J = 31.6, 14.0, 8.7, 3.1 Hz, 4H), 3.88 (ddt, J = 16.3, 9.2, 7.1 Hz, 1H), 3.70 (q, J = 7.0 Hz, 1H), 1.28 (d, J = 7.1 Hz, 3H), 1.22 (t, J = 7.0 Hz, 1H), 1.16 (t, J = 6.2 Hz, 6H) ppm. ¹³C NMR (DEPT-135) (126 MHz, CDCl₃) δ 173.2, 173.2, 154.4, 152.8, 151.6, 150.5, 149.7, 142.4, 138.6, 129.7, 125.1, 120.1, 120.0, 110.5, 107.7, 89.7, 83.7, 83.5, 75.2, 70.8, 69.5, 66.2, 58.4, 50.4, 37.6, 21.7, 21.7, 21,0, 20.9, 18.5 ppm, HPLC (X Terra RP18, 250x4.6x5mm, gradient 5-100% phase B [90% MeCN/10% H₂O (0,1% TFA)]: phase A [90% H₂O (0,1% TFA)/10% MeCN], 1.2 mL/min, 210 nm, 30 °C) t_r= 14.443 min, λmax 264 nm.

Cells and Virus. African green monkey kidney (Vero, subtype E6), human hepatoma (Huh-7) and human lung epithelial cell line (Calu-3) were cultured in this study. Vero and Calu-3 were kept in high glucose DMEM, whereasHuh-7 in low glucose DMEM medium. Either DMEM were complemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin and 100 μg/mL streptomycin (Pen/Strep; ThermoFisher) at 37 °C in a humidified atmosphere with 5% CO₂.

Human primary monocytes were obtained after 3 h of plastic adherence of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy donors by density gradient centrifugation (Ficoll-Paque, GE Healthcare). PBMCs (2.0 x 10⁶cells) were plated onto 48-well plates (NalgeNunc) in RPMI-1640 without serum for 2 to 4 h. Non-adherent cells were removed and the remaining monocytes were maintained in DMEM with 5% human serum (HS; Millipore) and penicillin/streptomycin. The purity of human monocytes was above 95%, as determined by flow cytometric analysis (FACScan; Becton Dickinson) using anti-CD3 (BD Biosciences) and anti-CD16 (Southern Biotech) monoclonal antibodies.

The SARS-CoV-2 B.1 lineage (GenBank #MT710714) and gamma variant (also known as P1 or B.1.1.28 lineage; #EPI_ISL_1060902) were isolated on Vero E6 cells from nasopharyngeal swabs of confirmed cases. All procedures related to virus culture were handled at biosafety level 3 (BSL3) multiuser facility according to WHO guidelines. Virus titers were determined as plaque forming units (PFU)/mL. Virus stocks were kept in - 80 °C ultralow freezers.

Cytotoxicity assay. Monolayers of 1.5 x 10⁴ cells in 96-well plates were treated for 3 days with various concentrations (semi-log dilutions from 1000 to 10 µM) of the antiviral drugs. Then, 5 mg/ml 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) in DMEM was added to the cells in the presence of 0.01% of N-methyl dibenzopyrazine methyl sulfate (PMS). After incubating for 4 h at 37 °C, the plates were measured in a spectrophotometer at 492 nm and 620 nm. The 50% cytotoxic concentration (CC₅₀) was calculated by a non-linear regression analysis of the dose–response curves.

Yield-reduction assay. Vero E6 cells were infected with a multiplicity of infection (MOI) of 0.01. HuH-7, Calu-3 and monocytes were infected at MOI of 0.1. Cells were infected at densities of 5 x 10⁵ cells/well in 48-well plates for 1h at 37 °C. The cells were washed, and various concentrations of compounds were added to DMEM with 2% FBS. After 24 (Vero E6), or 48-72h (HuH-7 and Calu-3) supernatants were collected and harvested virus was quantified by PFU/mL or real time RT-PCR. A variable slope non-linear regression analysis of the dose-response curves was performed to calculate the concentration at which each drug inhibited the virus production by 50% or 90% (EC₅₀ and EC₉₀, respectively).

Virus titration. Monolayers of Vero E6 cells (2 x 10⁴ cell/well) in 96-well plates were infected with serial dilutions of supernatants containing SARS-CoV-2 for 1h at 37°C. Fresh semi-solid medium containing 2.4 % of carboxymethylcellulose (CMC) was added and culture was maintained for 72 h at 37 ºC. Cells were fixed with 10 % formaline for 2 h at room temperature and then, stained with crystal violet (0.4 %). The virus titers were determined by plaque-forming units (PFU) per milliliter.

Cells were washed, fresh medium added with 2% FBS and 3 to 5 days post infection the cytopathic effect was scored in at least 3 replicates per dilution by independent readers. The reader was blind with respect to source of the supernatant.

Molecular detection of virus RNA levels. The total viral RNA from a culture supernatant and/or monolayers was extracted using QIAamp Viral RNA (Qiagen®), according to manufacturer’s instructions. Quantitative RT-PCR was performed using GoTaq® Probe qPCR and RT-qPCR Systems (Promega) in an StepOne™ Real-Time PCR System (Thermo Fisher Scientific) ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Amplifications were carried out in 25 µL reaction mixtures containing 2× reaction mix buffer, 50 µM of each primer, 10 µM of probe, and 5 µL of RNA template. Primers, probes, and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect the SARS-CoV-2⁴⁰. The standard curve method was employed for virus quantification. For reference to the cell amounts used, the housekeeping gene RNAse P was amplified. The Ct values for this target were compared to those obtained to different cell amounts, 10⁷ to 10², for calibration. Alternatively, genomic (ORF1) and subgenomic (ORFE) were detected, as described elsewhere⁴¹.

Generation of mutant virus. Calu-3 cells were infected with SARS-CoV-2 at a MOI 0.1 for 1h at 37 ºC and then treated with MB905 at increasing concentrations, from 0.5 to 9 µM, after each passage. That is, after infection and initial treatment, cells were accompanied daily up to the observation of cytophatic effects (CPE) and virus was recovered from the culture supernatant, titered and used in a next round of infection in the presence of higher drug concentration of the drug. As a control, SARS-CoV-2 was also passaged in the absence of treatments to monitor genetic drifts associated with culture passage. Virus RNA virus was extracted by Qiamp viral RNA (Qiagen) and quantified using Qbit 3 Fluorometer (Thermo Fisher Scientific) according to manufacters recommendations.

RNA virus was submitted to unbiased sequence using a MGI-2000 and a metatranscriptomics approach. To do so, at least 4.2 ng of purified total RNA of each sample was used for libraries construction using the MGIEasy RNA Library Prep Set (MGI, Shenzhen, China). All libraries were constructed through RNA‐fragmentation (250 bp), followed by reverse‐transcription and second‐strand synthesis. After purification with MGIEasy DNA Clean Beads (MGI, Shenzhen, China), were submitted to end‐repair, adaptor‐ligation, and PCR amplification steps. After purification as previously described, samples were quantified with Qubit 1X dsDNA HS Assay Kit using an Invitrogen Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Foster City, CA) and homogeneously pooled (1 pmol/ pool of PCR products) and submitted to denaturation and circularization steps to be transformed into a single‐stranded circular DNA library. Purified libraries were quantified with Qubit ssDNA Assay Kit using Invitrogen Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Foster City, CA) and DNA nanoballs were generated by rolling circle amplification of a pool (40 fmol/ reaction), then quantified as described for the libraries and loaded onto the flow cell and sequenced with PE100 (100-bp paired-end reads).

Sequencing data were initially analyses in the usegalaxy.org platform. Next, aligned through clustalW, using the Mega 7.0 software.

In sislico studies. The 3D structure corresponding to the genetic sequence of SARS-CoV-2 at position 1681 (GATCGCCATT, upon RNA-dependent RNA polymerase reaction)⁴⁷ was built at pH 7.4 and the structure was energy minimized in terms of energy by Density Functional Theory (DFT) under dielectric constant of water by Spartan'18 software (Wavefunction, Inc., Irvine, CA, USA; https://www.wavefun.com/). For the structure with the lowest energy was replaced the nucleobase adenine by kinetin moiety and a novel energy minimization under the same conditions described above was conducted. The same procedure for the lowest energy structure of the template GATCGCCATT was also done upon replacement of guanine by kinetin. The superposition between the genetic sequence of SARS-CoV-2 and each replacement condition corresponding to the three (for adenine to kinetin) and two (for guanine to kinetin) lowest energy positions were generated with the PyMOL Delano Scientific LLC software (DeLano Scientific LLC; https://pymol.org/2/) with the statistical approximations through the commands “align”, “cealign” and “super” and was considered the approximation with the lowest root mean square deviation (RMSD) value. This same software was also used to generate the figures and detect the main interactions among kinetin and nucleobases through a cutoff for the interaction of 3.30 Å and analysis of van der Waals radius superposition.

RNA immunoprecipitation. Calu-3 cells (2x10⁶) were infected with SARS-CoV-2 at MOI 0.1 and treated with 10 µM of the compounds. After 24 hours the supernatant was removed, and monolayer was washed with PBS. Monolayer RNA was extracted by commercial kit (Qiagen), following manufacture recommendation. To the extracted RNA, we added 25 µL anti-kinetin/protein A/magnetic beads (1:1:1). Anti-kinetin polyclonal antibody was purchased from Agrisera LTD, Sweden (www.grisera.com; #AS09444), as isotype control unspecific rabbit sera as used. After, overnight shaking at 4°C, magnetic racks allowed us to wash the materials with 175 µL of TBS-T (Tris Buffer Solution 1M, MgCl₂1M and NaCl 5M) buffer. After that, RNA was re-extracted using viral RNA mini kit (Qiagen) and samples submitted to RT-PCR to detect ORFN mRNA and housekeeping gene (GAPDH; Origene Tech inc). One step real time RT-PCR conditions are described above.

SARS-CoV-2 RNA polymerase inhibition assay. The SARS-CoV-2 RNA polymerase, nsp12, 7 and 8 (BPSBiosciences # 100839) was purchased and incubated at assay conditions described elsewhere¹³, with modifications in the assay readout. In brief, 500 nM of the 33-1-mer template and 10-mer primer described by Wang et al. 2020¹³ and synthesized by IDT (www.idtdna.com) was incubated with 125 nM of the viral RNA polymerase in the volume of 20 μL containing 250 μM of each nucleotide triphosphate (NTP), 50 mM HEPES (pH 7.0), 50 mM NaCl, 5 mM MgCl₂, 4 mM DTT and the antiviral nucleotide analogues for 3h at 25°C. Kinetin ribose 5’-triphosphate (MB-801TP) and GS-443902 (equivalent to remdesivir triphosphate) were purchased (https://www.biolog.de/6-fu-atp; https://www.medchemexpress.com/gs-443902.html). The reaction mixture was quenched with an equal volume of 20 mM EDTA (pH 8.0). Next, serial dilutions of the stopped reaction were incubated with a pyrophosphate luminescent detection kit (PPIlight # LT07-610; https://bioscience.lonza.com/lonza_bs/BR/en/). Reaction luminescence was quantified in a Glomax Discover plate reader (www.promega.com).

5’-Nucleotidase assay – Theis enzymatic activity was measured by a commercial assay #ab235945 from www.abcam.com. The either the commercial substrate, AMP, or the MB-711 were incubated with mice liver extracts potterized in 10 % sucrose from euthanized mock-infected mice. Liver extracts were a source of 5’-nucletidase and, in the case of MB-711, the enzymes cathepsin A or carboxylesterase 1 and histidine triad nucleotide-binding protein 1 (HINT1) to release its nucleotide monophosphate.

Pre-clinical development of MB-905: Animals. All in vivo procedures were performed in accordance with the Ethical Principles in Animal Experimentation adopted by the National Council for the Control of Animal Experimentation (CONCEA). The protocol used in all in vivo studies reported here were reviewed and approved by the Ethics Committee on Animal Use (CEUA # 210, 214, 215, 217, 241 and 306) of the Center of Innovation and Preclinical Studies (CIEnP). Mice and rats used in the following assays were obtained from the CIEnP bioterium, whose breeding colonies were purchased from Charles River Laboratories (USA), and were maintained under SPF (Specific Pathogen Free) conditions.

Pharmacokinetics in rodent plasma and lung tissue. Pharmacokinetics in rodent plasma was performed in CD1 mice (20-30 g) and Sprague-Dawley rats (250- 300g) of both sexes. The following pre-formulations were used to dissolve MB-905: dose of 3 mg/kg (i.v.): 1% DMSO + 4% PEG400 + 0.5% Tween80 + 94.5% Saline; dose of 30 mg/kg (p.o.): 10% DMSO + 40% PEG400 + 5% Tween80 + 45% Saline; dose of 550 mg/kg (p.o.): 5% Tween 80 + 95% PEG400. This trial consisted of administering MB-905 at doses of 10, 30 or 550 mg/kg, orally or 3 mg/kg intravenously. After oral or intravenous administration, blood samples were collected at times of 0.25, 0.5, 1, 2, 4, 8 and 24 and 0.083, 0.25, 0.5, 1, 2 and 4 hours, respectively. Parameters such as area under the curve (AUC), time taken to reach the maximum concentration (T_max), time taken for C_max to drop in half (T _1/2), volume of distribution, clearance, elimination constant and bioavailability (F) were evaluated. Bioavailability was calculated using the following equation: F (%)= [(Intravenous dose x oral AUC) / (Oral dose x Intravenous AUC)] x 100. To assess the putative metabolites of MB-905 in the lungs, Sprague- Dawley rats (8- 12 weeks - 250 – 300g) of both sexes were used. Animals were treated orally with MB-905 as previously described. Animals were sacrificed at different time points after treatments and lungs were immediately removed, placed in liquid nitrogen at – 80 ^oC and processed for further analysis. For the analysis of plasma and lung supernatant, UPLC-MS/MS equipment was used.

Plasma protein binding. The assay consists in the incubation of 200 µL blood plasma fractions from male and female mice containing MB-905 (0.26, 2.6 or 26 µM) and PBS using the equilibrium dialysis (RED-Thermo Fisher®) with an 8 kDa molecular weight cutoff membranes. After 4 hours of incubation at 37 °C and 30 rpm (orbital shaker), samples were matrix-matched and quenched by protein precipitation, following by LC-MS/MS analysis.

Metabolic stability of compounds in HLM and rCYPs inhibition. Substrate stocks solutions were prepared in DMSO and diluted in methanol and PBS, for a final organic composition of 0.2% DMSO and 0.5% methanol. MB-905 (0.1, 1 or 10 µM) was incubated in HLM (0.2 mg/mL) diluted in PBS (100 mM, pH 7.4) in the absence or in presence of NADPH (0.6 mM) in a final volume of 100 µL. Incubations were conducted at 37 °C, in triplicates. At various time points (0, 5, 15, 30 and 60 minutes), a 15 µL aliquot was removed and quenched with 100 µL of methanol containing the internal standard minoxidil (50 ng/mL). rCYPs inhibition assay was performed using standard substrates midazolam and dextromethorphan for rCYPA4 and rCYP2D6, respectively. Samples were vortexed, centrifuged (14,000 rpm, 5 minutes, 4 °C) and the supernatant used for measured the metabolites 4-OH-midazolam and O-demetyl-dextromethorphan by UPLC-MS/MS. Data expressed represents the comparison of metabolites production in the presence of MB-905 (100 µM) versus the presence of standard enzyme inhibitors, ketoconazole (0.5 µM, for CYP3A4) and quinidine (0.2 µM, for CYP2D6).

Maximum tolerated dose toxicity study (MTD) and dose selection in mice. For the MTD assay (OECD 425), mice (3 animals of each sex/group) were divided into five experimental groups and the up and down procedure was applied to which 175 mg/kg was used as the first dose. Then, since the MTD assay pointed out 550 mg/kg as the recommended dose for repeated exposure, tolerability of MB-905 was assessed by submitting two experimental groups (5 of each sex/group) to an oral treatment with the vehicle (group 1) or with MB-905 (550 mg/kg) for 7 consecutive days. Mortality, morbidity, body weight, food consumption, general and detailed clinical signs of toxicity were evaluated. At necropsy, vital (brain, heart, liver, spleen, kidneys and adrenal glands) and reproductive organs (ovaries, testicles and epididymis) were carefully removed, weighed and stored for further analysis (if necessary).

28-day repeated dose oral toxicity in mice. Male and female CD1 mice (6-8 weeks, 10 mice/group/sex) were treated orally with vehicle (45% polyethylene glycol 400 - PEG 400, 30% propylene glycol, 20% filtered water and 5% ethanol) or with different doses of MB-905 (10 mg/kg, 80 mg/kg or 250 mg/kg), once daily for 28 days (main animals). Additionally, recovery groups (5 males and 5 females) were established to which the same treatment regimen was applied but animals (Vehicle or MB-905 250 mg/kg) remained untreated for another 14 days in order to observe persistence, reversibility or delayed occurrence of toxic effects related to the administration of the Test Item. These experiments were conducted in compliance with the GLP principles. Morbidity, mortality, body weight, food consumption as well as general and detailed clinical signs were evaluated. In the last week, urine samples from all animals were collected for analysis. At necropsy, blood samples were collected for hematological, biochemical and coagulation analyses, followed by tissue and organ collection for macroscopic and histopathological analyses.

Prolonged toxicokinetics in mice. Mice (5 of each sex/group) were treated orally with MB-905 at doses of 10, 80 or 250 mg/kg, for 28 consecutive days. Blood samples on the 0^th and on the 28^th day after treatments were collected at 0.25, 1, 3, 8 and 24 hours and analyzed by UPLC-MSMS. Plasma AUC_all and C_max for both time points periods were calculated.

Cardiovascular safety pharmacology in rats. Conscious and freely-moving male Sprague-Dawley rats (9-12 weeks), previously submitted to surgery for placement of the DSI™ PhysioTel hardware system implant in the abdominal aorta, were treated orally with Vehicle (5 ml/kg) or MB-905 (50 or 250 mg/kg) once a day for 7 consecutive days. Cardiovascular parameters such as systolic blood pressure, diastolic blood pressure, heart rate and electrocardiogram were evaluated before treatments (baseline) and at 0.5, 1, 2, 3, 4, 5, 6, 7, 12 and 24 hours after the treatments on days 1 and 7.

Inhibition of voltage-dependent potassium channels of the hERG type (human ether-a-go-go related).

HEK293 cell line (4x10e5 cell) (BPS Bioscience, San Diego, CA, USA) expressing recombinant human ERG potassium channel (ether-a-gogo-related gene, Kv11.1) was used. The channel activity was determined using FLIPR Potassium assay kit (Molecular Devices - San Jose, CA, USA). Cells were cultivated in microplate and incubated with a loading buffer for one hour at room temperature in the dark. Then, MB-905 (0.01 - 300 µM) or Dofetilide (0.0001 - 1 µM, used as positive control drug) were added to the wells and incubated for thirty minutes at room temperature in the dark. After that, the microplate was transferred to FlexStation 3 (Molecular Devices - San Jose, CA, USA) with the addition of 1 mM thallium + 10 mM potassium using automated pipetting. Data analysis was performed using SoftMax Pro Software (Molecular Devices - San Jose, CA, USA) and GraphPad Prism. The results were expressed as percentage of inhibition of the hERG channel and the mean inhibitory concentration (IC₅₀) was determined.

Ames test. This test was performed using the bacterial reverse mutation assay in accordance to the OECD guideline 471 and conducted in compliance with the GLP principles. The preliminary assay with the Salmonella typhimurium TA 100 strain, with or without metabolic activation (S9), was carried out with MB-905 at concentrations of 8, 40, 200, 1,000, and 5,000 µg/plate. As cytotoxicity and mutagenicity were not observed in the absence and presence of S9, the definitive test with the strains TA 97a, TA 98, TA 100, TA 102 and TA 1535, with or without S9, was performed with the aforementioned concentrations.

Mouse bone marrow micronucleus test. This assay was performed in mouse bone marrow in accordance to the OECD guideline 474 and conducted in compliance with the GLP principles. Male and female Swiss mice (5-10 weeks) were divided into 5 experimental groups and were treated orally with vehicle (5% Tween + 95% PEG E 400), three different doses of MB-905 (32, 125 or 500 mg/kg) for three consecutive days or with cyclophosphamide (25 mg/kg, i.p.) for 2 consecutive days. Bone marrow cells were collected and processed according to a methodology described by Schmid (1975). The ratio of polychromatic to normochromatic erythrocytes and the count of micronuclei were determined. This assay was conducted in compliance with the GLP principles.

In vivo assays – Mice infections and treatment. Experiments with transgenic mice expressing human ACE-2 receptor (K18-hACE2- mice) were performed in Animal Biosafety Level 3 (ABSL-3) multiuser facility, according to the animal welfare guidelines of the Ethics Committee of Animal Experimentation (CEUA-INCa, License 005/2021) and WHO guidelines. The animals were obtained from the Oswaldo Cruz Foundation breeding colony and maintained with free access to food and water at 29–30°C under a controlled 12 h light/dark cycle. Experiments were performed during the light phase of the cycle.

For infection procedures, mice were anaesthetized with 60 mg/kg of ketamine and 4 mg/kg of xylazine and inoculated intranasally with DMEM high glucose (Mock-infected) or 10⁵ PFU of SARS-CoV-2 gamma strain in 10 µl of DMEM high glucose. A total number of 18 mice per experimental group was used along three independent experiments

The animals were monitored daily during seven days for survival and body-weight analysis. In the case of weight loss higher than 25%, euthanasia was performed to alleviate animal suffering. In the last day, the bronchoalveolar lavage (BAL) from both lungs was harvested by washing the lungs once with 1 mL of cold PBS. After centrifugation of BAL (1500 rpm for 5 minutes), the pellet was used for total and differential leukocytes counts (diluted in Turk’s 2% acetic acid fluid) using a Neubauer chamber. The lactate dehydrogenase (LDH) quantification was performed with centrifuged BAL supernatant to evaluated cell death (CytoTox96, Promega, USA). Differential cell counts were performed by cytospin (Cytospin3; centrifugation of 350xg for 5 minutes at room temperature) and stained by the May-Grünwald-Giemsa method.

After BAL harvesting, lungs were perfused with 20 mL of saline solution to remove the circulating blood. Lungs were then collected, pottered, and homogenized in 500 µL of a phosphatase and protease inhibitor cocktail Complete, mini EDTA-free Roche Applied Science (Mannheim, Germany) for 30 sec, using an Ultra-Turrax Disperser T-10 basic IKA (Guangzhou, China), for virus titration, RNA and cytokine quantification. Alternatively, lungs were preserved for histology.

Histological procedure. Histological features related to the injury caused by SARS-CoV-2 infection were analyzed in the lungs of K18-hACE2 mice. Inflammatory and vascular infiltrates and evidence of cell degeneration was evaluated to characterize the level of the tissue damage. The collected material was fixed with formaldehyde (4%), dehydrated and embedded in paraffin to the obtention of tissue slices through the use of a microtome. The slices were fixed and stained with hematoxylin and eosin for microphotographs analysis.

Statistical analysis. The assays were performed blinded by one professional, codified and then read by another professional. All experiments were carried out at least three independent times, including a minimum of two technical replicates in each assay. The dose-response curves used to calculate EC₅₀, EC₉₀and CC₅₀ values were generated by variable slope plot from Prism GraphPad software 8.0. The equations to fit the best curve were generated based on R² values ≥ 0.9. Student’s T-test was used to access statistically significant P values <0.05. The statistical analyses specific to each software program used in the bioinformatics analysis are described above.

Funding:

This Project is being financed by the Brazilian National Research Council (CNPq, finance code 403543/2020-7) and the Brazilian Company of Research and Industrial Innovation (EMBRAPII, finance code 015/2020). Additional support is being received from Coordination for the Improvement of Higher Education Personnel (CAPES) and from the Foundation for Research Support in the State of Rio de Janeiro (FAPERJ, finance code E-26/210.775/2021).

Acknowledgements:

We thank the Oswaldo Cruz Foundation (Fiocruz) and the National Cancer Institute (INCa) for allowing the use of their cellular and animal biosafety level 3 Platforms, respectively. The authors are grateful to the Hemotherapy Service of the Clementino Fraga Filho Hospital (Federal University of Rio de Janeiro, UFRJ).

We also wish to acknowledge Dr. Patricia Machado Rodrigues e Silva Martins and Dr. Tatiana Paula Teixeira Ferreira from the Fiocruz Foundation for the microphotographs analysis. National Institutes of Science and Technology Program (INCT) on Diseases of Neglected Populations (INCT-IDPN, # 465313/2014-0), on Innovation in Medicines and Identification of New Therapeutics Targets (INCT-INOVAMED) and on Neuroimmunomodulation (INCT/NIM).

Our special thanks to Ms. Andrea Yamagata (Quality Assurance Director at Microbiológica) for her essential leadership and guidance in the developing of the active substances and regulatory issues and to Dr. Marcelo Morales (Ministry of Science Technology and Innovation, MCTI) for his interest, enthusiasm and continued support.

Conflict of interest: The corresponding authors are among the inventors of the patent request PCT/BR2021/050136 and PCT/BR2022/050120, on the topic of this study.

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Yes there is potential Competing Interest. The corresponding authors are among the inventors of the patent request PCT/BR2021/050136 and PCT/BR2022/050120, on the topic of this study.

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Identification and preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral and anti-inflammatory therapy

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