ProLectin M production
Galectin antagonist was produced in a GMP facility located in Pune, Maharashtra. ProLectin M (PL-M) formulation was prepared and sent to the in vitro lab facility in Hyderabad, India. Details of the facility and process are shared in the Supplementary information file. Briefly, PL-M is an oral chewable tablet containing guar gum with added flavouring agents and other excipients.
Cell lines, virus and reagents
A virus stock of SARS-CoV-2 a3i clade sourced from original isolation from an infected patient in India was prepared according to company standard protocols for intact virus enrichment https://www.thermofisher.com. The viral stock was prepared by infecting Vero 76 cells (ATCC, CRL-1587) until a cytopathic effect (CPE) was visible two days post-inoculation. Vero 76 cells were cultured in Dulbecco Minimal Essential Medium (DMEM) (Thermo Scientific, USA), supplemented with 2% (v/v) Fetal Bovine Serum (FBS) (Sigma-Aldrich, St. Louis, USA), and 50 µg/mL gentamicin (Gemini Bio-products, USA).
Cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich] assay. Vero cells were plated in triplicate in 96 well culture plates and incubated at 37°C under 5% CO2. After reaching 90–95% cell confluency, different concentrations of PL-M were added to the cells for 24 hours to assess the cytotoxic effect on these cells. After 24 hours, 100 µL of MTT substrate (final concentration 50 µg/mL) was added to the cells, and the plate was incubated for 3 hours at 37ºC under 5% CO2. Later, the formed formazan crystals were dissolved in 100 µL DMSO, and a multimode microplate reader, Synergy HI (Agilent Technologies Inc., USA), was used to measure absorbance at 570 nm from which the percentage of viable cells was calculated.
Vero cells were cultured in 96-well plates at 37°C under 5% CO2 in DMEM supplemented media with 10% (v/v) FBS and 3.7 g/L sodium bicarbonate. At 90–95% confluency, cells were primed with a complete medium containing different concentrations of PL-M for 2 hours. The wells containing the test compound were replaced with the virus (~ MOI 0.1) in DMEM culture media (without FBS) for 3 hours. Later, the virus-containing medium was aspirated and replaced with fresh DMEM containing 10% FBS and test compound. Culture supernatant was collected for real time-PCR analysis of viral RNA copy.
Viral RNA extraction
Viral RNA was extracted from 200 µL aliquots of culture supernatants using the MagMAXTM viral/pathogen extraction kit (Applied Biosystems, Thermo Scientific). Viral supernatants from the test groups were mixed with a lysis buffer containing 260 µL of MagMAXTM viral/pathogen binding solution, 10 µL of MVP II binding beads, and 5 µL of MagMAXTM viral /pathogen proteinase-K for a total of 200 µL of sample in a deep well plate (KingFisherTM, Thermo Scientific). RNA extraction was performed using a KingFisher Flex system (version 1.01, Thermo Scientific) according to manufacture’s instructions. The eluted RNA was stored at -80oC until used.
RT-qPCR for detection of SARS-CoV-2
Quantitative PCR was performed using a Meril Covid-19 one step Real-Time PCR kit to detect the ORF1ab (FAM labeled) and nucleoprotein N (HEX labeled) genes of SARS-CoV-2 in the isolated RNA samples. Reaction conditions were set up according to manufacturer's protocol: 15 min at 50ºC (reverse transcription), 3 min at 95ºC (cDNA initial denaturation) followed by 15 sec at 95ºC (45 cycles of denaturation), 40 sec at 55ºC (annealing, extension and fluorescence measurement), and 10 sec at -25ºC (cooling). The program was set up using a QuantStudio-5 instrument (Thermo fisher). The threshold cycle (Ct) values of N gene (gene specific to SARS-CoV-2) were considered when plotting the graphs.
To determine anti-viral effects from PL-M against SARS-CoV-2, Vero cells were first treated with various concentrations of the test compound. After 2 hours, the compound in the culture medium was removed, and viral stock (~ MOI 0.1 in DMEM culture media without FBS) was used to infect Vero cells at 37°C for 3 hours. The unabsorbed virus in the culture medium was then removed, and cells were washed and overlaid with 1 mL of fresh DMEM containing 10% FBS and test compound. After 48 hours, the viral supernatant was collected, and qRT-PCR was used to determine the reduction in viral RNA copy number as previously described. Uninfected Vero cells and those infected with viral stock were used as cell and infection controls, respectively.
For these experiments, two protocols were used. In Protocol 1, Vero cells on the plate were initially treated with PL-M prior to being infected with SARS-Cov-2 (DMSO was used as the control). In Protocol 2, Vero cells were initially cultured with the SARS-CoV-2 virus prior to being treated with drug-spiked media. FBS was used as the control.
Cells released from and bound to SARS-CoV-2 virus were analyzed to calculate the half-maximal effective concentration (EC50) and half-inhibitory concentration (IC50) values of our test compound by plotting % viral reduction or viral presence vs. log concentration of the test compound.
All experiments were conducted in a Biosafety Level − 3 accredited laboratory as per standard operating principles of Good Laboratory Practices and in a facility approved by the Government of India and Department of Science and Technology affiliated facility. No laboratory personnel were exposed more than expected risk in carrying out our experiments.
Uniformly 15N-labeled galectin-3 (Gal-3) was dissolved at a concentration of 20 µM in 20 mM potassium phosphate buffer at pH 6.9, made up using a 95% H2O/ 5% D2O mixture. 1H-15N HSQC NMR experiments were performed to investigate binding of PL-M and its individual polysaccharide components (galactomannans AG and R) to Gal-3. 1H and 15N resonance assignments for recombinant human Gal-3 were previously reported.21
NMR experiments were carried out at 30oC on a Bruker 850 MHz spectrometer equipped with a H/C/N triple-resonance probe and an x/y/z triple-axis pulse field gradient unit. A gradient sensitivity-enhanced version of two-dimensional 1H-15N HSQC was applied with 256 (t1) x 2048 (t2) complex data points in nitrogen and proton dimensions, respectively. Raw data were converted and processed by using NMRPipe22 and were analyzed by using NMRview23.
PFU determined stock titers (ranging from Log107) were used to develop a regression equation and to calculate the percentage of viral reduction. After 72 hours of infection, the viral inhibition effect of the compound was assessed. The endpoint was calculated as the percentage of viral reduction using the following formula:
% Viral reduction = number of viral particles in infection control − number of viral particles exposed number of viral particles in infection control
The experiment was repeated twice, and results were averaged to calculate % viral reduction. The regression equation for viral particles vs. Ct value of the N- gene specific to SARS-CoV-2 virus (y = 3.5422x + 40.786; R2 = 0.99, x = viral particle number, and y = Ct value).
The number of viral particles calculated as
X = (40.786 – Ct Rd/Rp-genes at different time points)/3.5422.
The log reduction in % viral particles was interpreted as the efficacy of test compound in blocking viral infectivity in Vero cells. The student t-test was used to compare data to the control, and all data are presented as the mean ± SD.