SUPPORTING INFORMATION

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and

Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms

Ana C. Puhl1*, Ethan J. Fritch2, Thomas R. Lane1, Longping V. Tse3, Boyd L. Yount3,

Carolina Q. Sacramento4,5, Natalia Fintelman-Rodrigues4,5, Tatyana Almeida Tavella6,

Fabio Trindade Maranhão Costa6, Stuart Weston7, James Logue7, Matthew Frieman7,

Lakshmanane Premkumar2, Kenneth H. Pearce8,9, Brett L. Hurst10,11, Carolina Horta

Andrade6,12, James A. Levi13, Nicole J. Johnson13, Samantha C. Kisthardt13, Frank

Scholle13, Thiago Moreno L. Souza4,5, Nathaniel John Moorman2,8,14, Ralph S. Baric2,3,14,

Peter B. Madrid15 and Sean Ekins1*

1Collaborations Pharmaceuticals, Inc., 840 Main Campus Drive, Lab 3510, Raleigh, NC

27606, USA.

2Department of Microbiology and Immunology, University of North Carolina School of

Medicine, Chapel Hill NC 27599, USA.

3Department of Epidemiology, University of North Carolina School of Medicine, Chapel

Hill NC 27599, USA.

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4Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo

Cruz (Fiocruz), Rio de Janeiro, RJ, Brazil

5Centro De Desenvolvimento Tecnológico Em Saúde (CDTS), Fiocruz, Rio de

Janeiro, Brasil

6Laboratory of Tropical Diseases – Prof. Dr. Luiz Jacinto da Silva, Department of

Genetics, Evolution, Microbiology and Immunology, University of Campinas-UNICAMP,

Campinas, SP, Brazil.

7Department of Microbiology and Immunology, University of Maryland School of

Medicine, Baltimore, Maryland, USA

8Center for Integrative Chemical Biology and Drug Discovery, Chemical Biology and

Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina,

Chapel Hill, North Carolina 27599, USA.

9UNC Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599,

USA.

10Institute for Antiviral Research, Utah State University, Logan, UT, USA.

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11Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan,

UT, USA.

12LabMol - Laboratory of Molecular Modeling and Drug Design, Faculdade de Farmácia,

Universidade Federal de Goiás, Goiânia, GO, 74605-170, Brazil.

13Department of Biological Sciences, North Carolina State University, Raleigh, NC,

USA.

14Rapidly Emerging Antiviral Drug Discovery Initiative, University of North Carolina at

Chapel Hill, Chapel Hill, NC, USA.

15SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA.

* To whom correspondence should be addressed: Ana C. Puhl, E-mail address:

ana@collaborationspharma.com; Sean Ekins, E-mail address:

sean@collaborationspharma.com, Phone: +1 215-687-1320

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 Table S1. Summary of all cell results infected with SARS-CoV-2, MHV and HCoV229E,

X = inactive, Y = active, blank = not tested. (*) indicates cells infected with SARS-CoV-

2.

Vero Vero Caco- Calu- A549- MHV/ Huh- Monocytes* HCoV229E Pseudovirus

E6 * 76* 2* 3* ACE2* DBT 7* /Hu-7

Pyronaridine X X Y X Y Y Y weak

Tilorone X Y Y Y Y Y X X X weak

Quinacrine X X Y X Y Y Y

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Table S2. Summary of mouse pharmacokinetics and in vitro average Ebola IC50 (3

strains1) *tilorone was dosed in vivo against Ebola at 25 mg/kg. Tilorone and

pyronaridine data is from male mice only. Pharmacokinetics data from recent

publications 2-4.

Drug Dose Cmax Cmax Ave IC50 CC50 Is the Cmax

(mg/kg) (ng/ml) (M) (M) (M) above IC50

Quinacrine 25 575* 1.44 1.31 8.62 Yes

Tilorone 10 135 0.33 1.28 18.04 No *

Pyronaridine 75 982 1.9 1.04 8.62 Yes

*previously unpublished data

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Table S3. Summary of human pharmacokinetics and in vitro average Ebola IC50 (3

strains 1). Pyronaridine pharmacokinetics from a single oral dose 5. Quinacrine

pharmacokinetics from an intrapleural dose 6.

Drug Dose Cmax Cmax Ave IC50 CC50 (M) Is the Cmax

(mg) (ng/ml) (M) (M) above IC50

Quinacrine 600 970 2.43 1.31 8.62 Yes

Tilorone 125 unknown Unknown 1.28 18.04 Unknown

Pyronaridine 400 495.8 1.01 1.04 8.62 Yes

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Table S4. In vitro and In vivo data for pyronaridine, quinacrine and tilorone 1-4.

ADME property Pyronaridine Quinacrine Tilorone

Solubility 168 µM at pH 7.4 80.5 µM at pH 7.4 465 µM at pH 7.4
CYP inhibition 1A2, 2C9, 2C19 (>50 1A2 (6.66 µM), 2C9 CYPs 1A2, 2C9,
µM), 3A4 (42.9 µM), (>50 µM), 2C19 (8.66 2C19, 3A4, 2D6
2D6 (2.23 µM) µM), 3A4 (3.96 µM), (>50 µM)
2D6 (0.013 µM)
Mouse liver t1/2 = >186 min, t1/2 = 12.6 min, t1/2 = 102.7 min
microsomes CLint = <7.4 Clint= 110.0µL/min/mg CLint = 13.5
µL/min/mg protein protein µL/min/mg
protein
Guinea Pig liver t1/2 = 66.1 min, t1/2 = 17.1min, t1/2 = 12.2 min,
microsomes CLint = 21.0 µL CLint = 81.3µL CLint = 113.7 µL
/min/mg protein /min/mg protein /min/mg protein
Non-Human primate t1/2 = 89.7 min, t1/2 = 10.1 min, t1/2 = 94.3 min,
liver microsomes CLint = 15.5 µL CLint = 137.4 µL CLint = 14.7 µL
/min/mg protein /min/mg protein /min/mg protein
Human liver T1/2 = 122.2 min, T1/2 = 27.5 min, t1/2 127.1 min,
microsomes CLint = 11.4 µL CLint = 50.5 µL CLint 10.9
/min/mg protein /min/mg protein µL/min/mg
protein

Mouse plasma 96.5% 91.8% 61.4 %

protein binding

Human plasma 95.1% 89.9% 52 %

protein binding

Caco-2 Papp A-B = 6.46; B-A Papp A-B = 30.6xE-6 Papp A-B =20.4;
= 4.8 (x10-6 cm/s) cm/s B-A = 8.87(x10-
Efflux ratio = 0.74 B-A = 19.0xE-6 cm/s 6cm/s) Efflux
Efflux ratio = 0.92 ratio= 0.435

CYP3A4 Induction 1.5x at 10 µM Not tested Not tested

Maximum Tolerated 100 mg/kg 50mg/kg* 100 mg/kg
dose in mice (Balb/c)

*previously unpublished data

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Figure S1. Dose response data from Vero E6 cells. % inhibition is shown in blue and

% cytotoxicity is shown in red. A) Pyronaridine, B) Tilorone and C) Quinacrine.

A Pyronaridine
(MOI = 0.01)
B Tilorone
(MOI = 0.01)
C Quinacrine
(MOI = 0.01)

150 150 150

125 125 125
Percent (%)

Percent (%)
Percent (%)
100 100 100
75 75 75
50 50 50
25 25 25
0 0 0
-25 -25 -25
0.1 1 10 100 0.1 1 10 100 0.1 1 10 100
Drug Concentration (µM) Drug Concentration (µM) Drug Concentration (µM)
IC50 = ND
inhibition % IC50 = ND IC50 = ND inhibition %
inhibition % CC50 = 2.11 uM
cytotoxicity %
CC50 = 5.62 uM CC50 = 6.34 uM cytotoxicity % SI = ND
SI = ND cytotoxicity % SI = ND

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Figure S2: In vitro antiviral SARS-CoV-2 testing in Calu-3 cells. Calu-3 (ATCC, HTB-

55) cells were pretreated with test compounds for 2 hours prior to continuous infection

with SARS-CoV-2 (isolate USA WA1/2020) at a MOI=0.5. Forty-eight hours post-

infection, cells were fixed, immunostained, and imaged by automated microscopy for

infection (dsRNA+ cells/total cell number) and cell number. Sample well data was

normalized to aggregated DMSO control wells and plotted versus drug concentration to

determine the IC50 (infection: blue) and CC50 (toxicity: green). Percentage of Control

(POC)=(sample well measurement /aggregated DMSO avg)*100 for n=3 replicates. A.

Pyronaridine, B. Quinacrine, C. Tilorone, D. Remdesivir.

A B
IC50 > 50 IC50 > 50
CC50 6.49 CC50 7.63
SI 1 SI 1

C D
IC50 10.77 IC50 0.016
CC50 19.87 CC50 > 10
SI 2 SI > 622

Tilorone

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Figure S3. Yield-reduction assays in Calu-3 cells. A and B. Lung epithelial cell line

(CALU-3) at MOI of 0.1 for 1 h at 37 °C and treated with different concentrations of

tilorone. Lysis of cell monolayer was performed 48 h post infection and virus was titrated

by plaque-forming units (PFU) assays and reported as % inhibition (A) and PFU/mL (B).

A Calu-3 Cells- inhibition B Calu-3 Cells (PFU/mL)

150
Tilorone 1×107
Tilorone
RDV
8×106
Remdesivir
% inhibition

100
6×106

PFU/mL
4×106
50
2×106

0
0 -6.0 -5.5 -5.0 -4.5
-6.5 -6.0 -5.5 -5.0 -4.5 -2×106
log compound [M]
log compound [M]

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Figure S4. Yield-reduction assays in Human hepatoma lineage (HUH-7) cells and

Monocytes. HUH-7 cells were infected at MOI of 0.1 for 1 h at 37 °C and treated with

different concentrations of tilorone. Lysis of cell monolayer was performed 48 h post

infection and cell-associated viral RNA was quantified by real time RT-PCR.

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Figure S5. Tilorone impairs SARS-CoV-2 replication in human primary monocytes.

A) Human primary monocytes were infected at the indicated MOI of 0.01 and treated with

10 µM of tilorone. After 24h, cell-associated viral RNA levels were measured by real time

RT-PCR. B) Cytotoxicity assays for Tilorone in monocytes after 24 h. The effect of

inhibition of SARS-Cov-2 RNA levels is likely due to toxicity as SI=0.

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Figure S6. Cytotoxicity assays in human monocytes and Vero CCL81 cells. Human

monocytes and/ or Vero CCL81 cells were incubated with different concentrations of the

compounds A. quinacrine and B. pyronaridine, for 24 h and 72 h respectively, for further

cellular metabolic evaluation by measuring the activity of a mitochondrial enzyme

succinate dehydrogenase using MTT test. CC50 values obtained for monocytes or Vero

CCL-81 cells are described in blue and red respectively. One independent experiment

was performed in duplicate.

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Figure S7. Additional coronavirus testing. A. HCoV229E antiviral assay and B.

cytotoxicity in Huh-7 cell line.

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Figure S8. Pseudovirus testing. Neutralization activity for A. tilorone and B.

pyronaridine. None of the concentrations tested reached 50% neutralization.

A Tilorone B Pyronaridine
Antiviral effect of CPI1072 against rVSV-dG 2019-CoV-2-18AA S in Antiviral effect of CPI1058 against rVSV-dG 2019-CoV-2-18AA S in
Vero cells on day 1 Vero cells on day 1
IC50 = 79.22 IC50 = 6.50
110 110

90 90

70 70

% Inhibition
% Inhibition

50 50

30 30

10 10

-10 -10
0.1 1 10 0.1 1
Concentration (µM) Concentration (µM)

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Supplemental References

1. Lane, T. R.; Massey, C.; Comer, J. E.; Freiberg, A. N.; Zhou, H.; Dyall, J.;
Holbrook, M. R.; Anantpadma, M.; Davey, R. A.; Madrid, P. B.; Ekins, S., Efficacy of
Oral Pyronaridine Tetraphosphate and Favipiravir Against Ebola Virus Infection in
Guinea Pig Antiviral Research 2020, 181, 104863.

2. Ekins, S.; Lingerfelt, M. A.; Comer, J. E.; Freiberg, A. N.; Mirsalis, J. C.;
O'Loughlin, K.; Harutyunyan, A.; McFarlane, C.; Green, C. E.; Madrid, P. B., Efficacy of
Tilorone Dihydrochloride against Ebola Virus Infection. Antimicrob Agents Chemother
2018, 62 (2). DOI: 10.1128/AAC.01711-17.

3. Lane, T. R.; Comer, J. E.; Freiberg, A. N.; Madrid, P. B.; Ekins, S., Repurposing
Quinacrine Against Ebola Virus Infection In vivo. Antimicrob Agents Chemother 2019,
63, e01142-19. DOI: 10.1128/AAC.01142-19.

4. Lane, T. R.; Massey, C.; Comer, J. E.; Anantpadma, M.; Freundlich, J. S.; Davey,
R. A.; Madrid, P. B.; Ekins, S., Repurposing the antimalarial pyronaridine
tetraphosphate to protect against Ebola virus infection. PLoS Negl Trop Dis 2019, 13
(11), e0007890. DOI: 10.1371/journal.pntd.0007890.

5. Blessborn, D.; Kaewkhao, K.; Song, L.; White, N. J.; Day, N. P. J.; Tarning, J.,
Quantification of the antimalarial drug pyronaridine in whole blood using LC-MS/MS -
Increased sensitivity resulting from reduced non-specific binding. J Pharm Biomed Anal
2017, 146, 214-219. DOI: 10.1016/j.jpba.2017.08.023.

6. Bjorkman, S.; Elisson, L. O.; Gabrielsson, J., Pharmacokinetics of quinacrine

after intrapleural instillation in rabbits and man. J Pharm Pharmacol 1989, 41 (3), 160-3.
DOI: 10.1111/j.2042-7158.1989.tb06421.x.

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