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) tr= 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) tr= 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) tr= 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) tr= 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 106 cells) 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, MgCl2 1M 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 (Tmax), time taken for Cmax 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 0th and on the 28th day after treatments were collected at 0.25, 1, 3, 8 and 24 hours and analyzed by UPLC-MSMS. Plasma AUCall and Cmax 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, EC90 and 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.