COVID19 (Coronavirus), is not a predicament for mankind, it is just an RNA molecule which causes this illness. At Atakam Medical our scientists took upon the challenge to destroy this molecule and had succeeded!

At ATAKAM Medical, our scientists have been battling SARS and MERS viruses for years, finding innovative ways to target the essence of this molecule. We have explored enzyme-based and host-based strategies to destroy this nemesis, we have developed a whole new subgroup of compounds to screen against the SARS COV2. Finally, on July 1st, we received our final data from the Southern Research Institute in Alabama (our screening facility), and it was beyond impressive:

Evaluation of Antivirals against SARS-CoV-2 by Cytopathic Effect (CPE) Assay (Vero-E6 cells) report-public.pdf   |   2.81 MB

Evaluation of Antivirals against SARS-CoV-2 by Cytopathic Effect (CPE) Assay (Vero-E6 cells)

Introduction

The purpose of the study was to evaluate the inhibitory effect of 320 compounds from Atakam Inc, against SARS-CoV2 coronavirus by CPE assay in Vero E6 Cells.

Materials and Methods

320 compounds were solubilized in DMSO at 10mM and stored at 4°C until the day of the assays. A set of positive control antiviral compounds were included in each of the assays performed.

Virus Strains and Cell Lines

The viruses and cell lines utilized for these evaluations were obtained from WRCEVA and American Type Culture Collection (ATCC) (followed by sorting and sub-cloning cells for high expression of ACE2). The evaluation was performed using a CPE reduction assay to measure antiviral effect and a cell viability assay to measure cytotoxic effect of compounds

Compound preparation

Compound solutions (80 µL of either 10 mM stock solutions in 100% DMSO) were transferred into wells in columns 3-22 of an empty ECHO plate (stock plate). Compounds were diluted 3-fold by transferring 17 µL of each sample from the stock plate into another ECHO plate with 34 µL DMSO in each well and mixing. This process was repeated to create 8 more plates of serially diluted sample, each plate containing a 3-fold diluted sample of the previous plate. A 30 nL aliquot for each diluted sample was dispensed into corresponding wells of assay plates using an ECHO550 acoustic liquid handling system. An equal amount of DMSO was added to control wells in columns 1, 2, 23, and 24. The final assay concentration range was 30 - 0.002 µM prepared from the 10mM stocks in the initial screen and 1000 – 0.05 nM prepared from the 333 µM stocks in the second screen.

Method for measuring antiviral effect of compounds:

Vero E6 cells were grown in MEM supplemented with 10% HI FBS and harvested in MEM/1% PS supplemented 2% HI FBS on the day of assay. Assay ready plates pre-drugged with test compounds were prepared in the BSL-2 lab by adding 5μL assay media to each well. The plates and cells were then passed into the BSL-3 facility. Cells were batch inoculated with SARS CoV-2 (M.O.I. ~ 0.002) which results in 5% cell viability 72 hours post infection. A 25μL aliquot of virus inoculated cells (4,000 Vero E6 cells/well) was added to each well in columns 3-24 of the assay plates. The wells in columns 23-24 contained only virus infected cells for the 0% CPE reduction controls. Prior to virus inoculation, a 25μL aliquot of cells was added to columns 1-2 of each plate for the cell only 100% CPE reduction controls. After incubating plates at 37°C/5%CO2 and 90% humidity for 72 hours, 30μL of Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a BMG CLARIOstar plate reader following incubation at room temperature for 10 minutes to measure cell viability. Plates were sealed with a clear cover and surface decontaminated prior to luminescence reading.

Method for measuring cytotoxic effect of compounds:

Compound cytotoxicity was assessed in a BSL-2 counter screen as follows: VeroE6 cells in media were added in 25μl aliquots (4,000 cells/well) to each well of assay ready plates prepared with test compounds as above. Cells only (100% viability) and cells treated with hyamine at 100µM final concentration (0% viability) serve as the high and low signal controls, respectively, for cytotoxic effect in the assay. DMSO was maintained at a constant concentration for all wells as dictated by the dilution factor of stock test compound concentrations. After incubating plates at 37°C/5%CO2 and 90% humidity for 72 hours, 30μl Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a BMG PHERAstar plate reader following incubation at room temperature for 10 minutes to measure cell viability.

Data analysis

For all assays the raw data from plate readers were imported into ActivityBase where values were associated with compound IDs and test concentrations.

For the antiviral CPE reduction assay, raw signal values are converted to % CPE reduction by the following formula: % CPE reduction = 100 x (test cmpd value – mean value infected cell controls)/(mean value uninfected cell controls – mean value infected cell controls).

For the cell viability assay measuring compound cytotoxicity, % cell viability is calculated as follows: % viability = 100*(test cmpd value - mean low signal control)/(mean high signal control – mean low signal control).

EC₅₀ and CC₅₀ values were calculated from four parameters logistic fit of data using the Xlfit module of ActivityBase.

Raw data for both antiviral activity and toxicity with a graphical representation of the data are provided in separate excel files submitted to the client.

Table 1. Antiviral activity of Reference Compounds in the SARS-CoV-2 CPE assay (First round of Testing)

Compound ID Batch Supplier Supplier ID Antiviral IC₅₀ (µM) Cytotoxicity CC₅₀ (µM) Selectivity Index (CC₅₀/IC₅₀)
AB01968659 01 Sigma Calpain Inhibitor IV 0.626 >7.17 >11.45
AB00053436 10 SRI Repository Chloroquine 4.181 >30.00 >7.175
AB01952209 01 Selleck Remdesivir 7.275 >30.00 >4.123

Many of the compounds tested in the first round of the assays showed IC₅₀ less than the lowest concentration of the compounds tested. To identify the precise IC₅₀ of these most potent compounds, testing was performed the second time with lower concentrations.

Table 2. Antiviral activity of Reference Compounds in the SARS-CoV-2 CPE assay (Second round of Testing)

Compound ID Batch Supplier Supplier ID Antiviral IC₅₀ (µM) Cytotoxicity CC₅₀ (µM) Selectivity Index (CC₅₀/IC₅₀)
AB01968659 01 Sigma Calpain Inhibitor IV 0.24 >7.17 >29.9
AB00053257 08 SRI Repository Hydroxychloroquine 2.1 >30.00 >14.3
AB00053436 10 SRI Repository Chloroquine 4.03 >30.00 >7.4
AB01952209 04 Selleck Remdesivir 10.8 >30.00 >2.8

Table 3. Antiviral activity some of sponsor’s Compounds in the SARS-CoV-2 CPE assay

Compound ID Supplier ID Antiviral IC₅₀ (µM) Cytotoxicity CC₅₀ (µM) Selectivity Index (CC₅₀/IC₅₀)
AB01952209 Remdesivir 7.275 >30.00 >4.123
AB01969905 ATK-xxxxx <0.0015 >30.00 >20000
AB01969907 ATK-xxxxx 0.002 >30.00 >15000
AB01969916 ATK-xxxxx <0.0015 >30.00 >20000
AB01970030 ATK-xxxxx 0.002 29.678 14839
AB01969936 ATK-xxxxx 0.002 >30.00 >15000
AB01969957 ATK-xxxxx 0.003 >30.00 >10000
AB01969964 ATK-xxxxx <0.0015 >30.00 >20000
AB01969965 ATK-xxxxx <0.0015 >30.00 >20000
AB01969966 ATK-xxxxx <0.0015 >30.00 >20000
AB01969980 ATK-xxxxx <0.0015 >30.00 >20000
AB01969981 ATK-xxxxx <0.0015 >30.00 >20000
AB01970003 ATK-xxxxx 0.004 >30.00 >7500
AB01970004 ATK-xxxxx 0.002 >30.00 >15000
AB01970005 ATK-xxxxx <0.0015 >30.00 >20000
AB01970007 ATK-xxxxx <0.0015 >30.00 >20000
AB01970008 ATK-xxxxx <0.0015 >30.00 >20000
AB01970011 ATK-xxxxx <0.0015 >30.00 >20000
AB01970014 ATK-xxxxx <0.0015 >30.00 >20000
AB01970033 ATK-xxxxx <0.0015 >30.00 >20000
AB01970036 ATK-xxxxx <0.0015 >30.00 >20000
AB01970048 ATK-xxxxx <0.0015 >30.00 >20000
AB01970054 ATK-xxxxx <0.0015 >30.00 >20000
AB01970058 ATK-xxxxx <0.0015 >30.00 >20000
AB01970079 ATK-xxxxx <0.0015 >30.00 >20000
AB01970061 ATK-xxxxx <0.0015 >30.00 >20000
AB01970073 ATK-xxxxx <0.0015 >30.00 >20000
AB01970079 ATK-xxxxx <0.0015 >30.00 >20000
AB01970080 ATK-xxxxx <0.0015 >30.00 >20000
AB01970116 ATK-xxxxx <0.0015 >30.00 >20000
AB01970128 ATK-xxxxx <0.0015 >30.00 >20000
AB01970132 ATK-xxxxx <0.0015 >30.00 >20000
AB01970136 ATK-xxxxx <0.0015 >30.00 >20000

Discussion

In the first round of testing, compounds were tested at 30µM high test concentration. Sponsor’s compounds showed spectrum of activity ranging from <0.0015µM to >30µM. 163 compounds showed dose dependent antiviral activity. 57 of the compounds showed antiviral activity with efficacy as low as <0.0015µM; 73 compounds showed IC₅₀ of <0.1µM. 12 compounds showed IC₅₀ of 0.1-1µM, 12 compounds showed IC₅₀ of 1-10µM, 9 compounds showed IC₅₀ of 10-30µM, 157 compounds were inactive. Since 57 compounds showed antiviral potency (IC₅₀) below the lowest concentration tested (0.02 µM) the calculated IC₅₀ values are not accurate for those compounds. Therefore, all of the compounds were tested again (second round of testing) over a concentration range of 1000 – 0.05 nM. In the second round of testing the IC₅₀ values for the high potency compounds are more accurate. In this round of screening 22 compounds showed IC₅₀ of <0.1µM, 90 compounds showed IC₅₀ of 0.1-0.5µM and 17 compounds showed IC₅₀ of 0.51-0.99µM. Thus, 129 compounds showed IC₅₀ values < 1 µM in the second round and 21 compounds exhibited IC₅₀ values between 1 µM and 30 µM in the first round.

Overall these results show impressive antiviral potency of the sponsor’s compounds in the in vitro antiviral assays. Some of the sponsor’s compounds showed significantly better antiviral efficacy compared to Remdesivir (SARS-CoV-2 polymerase inhibitor), tested in the assay as a positive control (IC₅₀ of <0.1µM vs 7-10µM respectively). It might be useful to focus on the data from 21 compounds exhibited IC₅₀ values between 1 µM and 30 µM in the first round and 129 compounds which showed IC₅₀ values < 1 µM in the second round for further analysis.

22 compounds which showed the most potent activity at lower concentrations (<0.1µM) could be subjected to further preclinical testing to determine molecular target, mechanism of action, antiviral spectrum, PK parameters, and in vivo efficacy.

Our Letter to the World

Antiviral activity of several of our compounds was more than 5000 times higher compared to Remdesivir (or Hydroxychloroquine) and acute toxicity in mice was shown extremely low. For example, 57 compounds showed antiviral activity with efficacy as low as EC₅₀ <0.0015µM and have shown selectivity index more than Si> 20,000. (Si for Remdesivir >4.12)

This was our first, ‘in vivo’ (cellular) study on the cells of the monkeys.

Comparing to the vaccine

  1. It is a known fact that a vaccine temporarily stops the spread of infection caused by influenza or related SARS COV2 virus.
  2. If you are already sick - the vaccine cannot help.
  3. If you have a chronic condition(s), a vaccine can weaken that.
  4. Your individual algorithm of illness is unpredictable which is determined by prior conditions, demographics, region, etc. – vaccine cannot guarantee you an endogenic defense based on the multitude variations of these factors. Symptoms, as we see, are individual for everyone, and the virus mutates rapidly with each wave and vaccinations must be readjusted and be performed seasonally
  5. Antibodies in those who had illness dissipate within 2-3 months after being infected.
  6. Individuals who are 65+ are NOT recommended vaccination, the same reason they are more prone to oncologic conditions.

We need the CURE, and now we know the right way for the solution!

The next steps for us are to conduct preclinical testing to determine the molecular target, antiviral spectrum, in vivo efficacy, animal toxicity, clinical studies.

Dear Colleagues!

We, together with scientists from Ukraine, found several compounds that are highly active in relation to the SARS-CoV-2 virus. The results of biological tests carried out in a laboratory of level BSL-3 (Birmingham, Alabama) showed extremely high activity for many compounds in nanomolar concentrations of EC₅₀ >0.0015 uM with their low cytotoxicity. As a result, their selectivity index was Si>20,000. For comparison, we present data for the preparations (obtained in the same laboratory in Birmingham, Alabama) of Remdesevir Si>4.12 and hydroxychloroquine Si>1.

Acute toxicity in mice was determined for one of the compounds of the series. It showed an LD₅₀ of about 2000 mg/kg.

Considering your rich experience and the availability of a powerful base for creating new drugs, we would like to discuss the possibility of joint creation of medicine based on our compounds for the treatment of COVID-19 disease.

Thank you very much and looking forward to hearing from you.

Best regards

Anatolii Demchenko Chief Scientific Officer
Eugene Stavtsev Chief Executive Officer
Andrew Osichnuk President
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