Articleshttps://doi.org/10.1038/s41564-020-0752-7

Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymeraseRobert M. Cox   1, Julien Sourimant   1, Mart Toots1, Jeong-Joong Yoon   1, Satoshi Ikegame2, Mugunthan Govindarajan3, Ruth E. Watkinson2, Patricia Thibault2, Negar Makhsous4, Michelle J. Lin4, Jose R. Marengo3, Zachary Sticher   3, Alexander A. Kolykhalov3, Michael G. Natchus3, Alexander L. Greninger4, Benhur Lee2 and Richard K. Plemper   1 ✉

1Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA. 2Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 3Emory Institute for Drug Development, Emory University, Atlanta, GA, USA. 4Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA. ✉e-mail: rplemper@gsu.edu

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Supplementary Figure 1. Paramyxovirus L sequence alignments. Covered are confirmed GHP-88309

resistance sites and positions of GHP-88309 resistance mutations are highlighted (red boxes). A single residue

critical for susceptibility to GHP-88309 is not conserved in NiV L at position 1156. Genera and subfamilies are

color-coded (respirovirus, green; morbillivirus, blue; henipavirus, orange; Ferlavirus, yellow; avulavirinae, pink;

aquaparamyxovirus, light green; rubulavirinae, light blue).

Supplementary Figure 2. Preparation and optimization of the MeV in vitro RdRP assay. a-b, SDS-PAGEs of

MeV P-L (a) and MeV P-LN774A (b) protein preparations, purified on Ni-NTA resin. Aliquots of eluted proteins

used in biochemical RdRP assays were fractioned on 7.5% gels, followed by visualization through Coomassie

blue staining. c-d, Optimization of the MeV in vitro RdRP assay. Autoradiograms of in vitro synthesis products

after polyacrylamide gel fractionation; labeling of products through 32P-GTP tracer. The sequence of the

synthetic RNA template is shown. A range of MgCl2 and MgCl2 concentrations were explored to identify nature

and concentration of the bivalent cation cofactor associated with maximal in vitro polymerase activity. Large-

scale protein production batches and condition-finding RdRP assays were conducted once to meet the needs

of the study. Originals are shown in Source Data file (Plemper_SourceData_SI_Fig2_Originals.pdf).

HPIV3-JS-

Nano [% inhibit.]

RSV-firefly [% inhibit.]

IAV-Nano [% inhibit.]

HPIV3-JS-Nano

EC50 [µM] Cytotoxicity

CC50 [µM] Swiss ADME Badapple

HPIV3-JS-Nano

EC50 [µM] Cytotoxicity

CC50 [µM]

assay type direct

[10 µm] (n = 2)

direct [10 µm] (n = 1)

reporter interference

(n = 1)

direct dose response

(n = 2)

direct dose response

(n = 2)

PAINS filter

aggregator predictor

sourced dose response

(n ≥3)

sourced dose response

(n ≥3) cutoff >85 <25 <25 <1 >10 pass pass <1 >20

GHP-88309 98 2 2 0.6 >10 pass pass 0.69 >20 GHP-64627 88 70 -7 0.44 >10 pass pass 6.8 0.84 Supplementary Table 1. HPIV3 HTS Counterscreens. Overview of direct and orthogonal counterscreens

applied to hit identification after primary HTS. Requested cut-offs for further advance of a hit candidate and

performance of GHP-88309 and GHP-64627 are shown.

Virus Assay EC50 [µM] EC90 [µM] SI HPIV3-JS TCID50 0.4 0.7 >2,500 HPIV-1-5F6 TCID50 0.8 2.9 >1,250 SeV-F1R-eGFP TCID50 2.0 7.0 >1,250 SeV-F1R-gausiLuc luciferase 2.0 18.7 >500 MeV/Alaska.USA/16.00 TCID50 0.6 1.1 >1,666 CDV-5804p TCID50 0.4 0.9 >2,127 HPIV-2 piva/ok/283/09 TCID50 >100 >100 NA RSV A2-L19F-firefly luciferase >100 >100 NA VSV-nanoLuc luciferase >100 >100 NA IAV-WSN-nanoLuc luciferase >100 >100 NA

Supplementary Table 2. GHP-88309 indication spectrum. Summary of assay types used, calculated EC50 and

EC90 concentrations (four parameter variable slope regression modeling) and corresponding SI values for the

different target viruses examined in Fig. 1d (NA; not applicable).

Compound ID Structure HPIV3

EC50 [µM]A MeV

EC50 [µM]A Toxicity

CC50 [µM]

GHP-88309-001

R = X = F

21.45 (16.11-28.2)

7.6 (4.1-14.0) >300B

GHP-88309-002

R = X= F

1.94 (1.3-2.7)

8.03 (5.4-11.8)

>300B

GHP-88309-003

R = X= H

>100 n.d. >100C

GHP-88309-004

R = X= H

>100 n.d. >100C

GHP-88309-

005 R1 = -(CO)NH2, R2 = R3 = R4 = R5 = H 0.62

(0.52-0.72) 11.8 >300B

GHP-88309-006

R1 = -(CO)NH2, R2 = NH2, R3 = R4 = R5 = H 1.8 (1.6-2.1) 10.2 >100C

GHP-88309-007

R1 = -(CO)NH2, R2 = F, R3 = OCH3, R4 = R5 = H 15.9 >100 >100C

GHP-88309-008

R1 = -(CO)NH2, R3 = Cl, R2 = R4 = R5 = H 0.63 (0.42-0.85) 10.3 >300A

GHP-88309-009

R1 = -(CO)NH2, R3 = -O(CH2)2N3, R2 = R4 = R5 = H 1.3 (0.82-1.8)

18.5 (12.4-27.7) >100C

GHP-88309-010

R1 = -(CO)NH2, R3 = -CH2OCH2C≡CH, R2 = R4 = R5 = H

1.2 (1.16-1.33)

29.8 (25.5-34.6) >100C

GHP-88309-011

R1 = -(CO)NH2, R4 = F, R2 = R3 = R5 = H 0.94 (0.83-1.1) 9.4 >300B

GHP-88309-012

R1 = -(CO)NH2, R2 = F, R5 = CH3, R3 = R4 = H >100 >100 >100C

GHP-88309-013

R1 = -(CO)OCH3, R2 = F, R3 = R4 = R5 = H 88.6 (75.7-103) 60.6 >200D

GHP-88309-014

R1 = -CH3, R2 = F, R3 = R4 = R5 = H 43.3 (38.4-47.6)

32.3 (25.4-39.9) >200D

GHP-88309-015

R1 = -(CO)NHOH, R3 = OCH3, R2 = R4 = R5 = H 14.45 46.6 (38.7-56.9) >100C

ABased on 4-parameter variable slope regression modeling; 95% CIs were calculated when possible

BHighest concentration assessed 300 µM

CHighest concentration assessed 100 µM

DHighest concentration assessed 200 µM

n.d. not determined

Supplementary Table 3. GHP-88309 SAR development. Chemical elaboration and bioactivity testing of the

GHP-88309 scaffold against HPIV3 and MeV reporter viruses. EC50 and EC90 concentrations were calculated

through 4-parameter variable slope regression modeling of dose-response data. Cytotoxicity was assessed

through PrestoBlue assay (n = 3).

ID Peptide Localization Coverage PSM Score (%) 1 55-73 58-61 1 27.743 2 404-422 413 1 28.064 3 966-1011 975-995 1 21.017

Supplementary Table 4. Photoaffinity labeling results. Three distinct peptides were detected by LC-MS/MS

that contained additional mass corresponding to that of GHP-88309-016. Predicted localization of crosslinks,

coverage, and the peptide spectrum match (PSM) score calculated in pFind are shown.

Mutation KD [µM] R2 wild type 6.2 ± 0.2 0.93 E858D 8.6 ± 0.3 0.93 S869P >300 0.96 I1009F 68.3 ± 4 0.90 Y1106S >300 0.85

E858D/I1009F >300 0.95 S869P/Y942H 298 ± 3 0.96

Supplementary Table 5. GHP-88309 BLI binding parameters to standard and resistant L variants. KD values

of all L constructs tested in Fig. 2f are shown with SD where applicable.

Dose [mg/kg]

Tmax [hours]

Cmax [ng/mL]

AUC¥ [h´ng/mL]

t1/2 [hours]

F (%)

50 0.5 12873 20422 0.8 88.8 150 0.5 36450 260058 1.66 -

Supplementary Table 6. Selected PK parameters and oral bioavailability of GHP-88309. Calculations are

based on single-dose pK studies of GHP-88309 in mice shown in Fig. 4b.

inoculum virus

Pre-existing mutation

GHP-88309 dose

[mg/kg]

passage I passage II

N Lung titer [TCID50/g tissue]

Sanger validation of res. site

N Lung titer [TCID50/g tissue]

Resistance (EC50 fold-change)

Sanger validation of L 844-

1156

recSeV n/a 50 b.i.d. 5

2.12 ´ 106

6.80 ´ 105

1.41 ´ 105

4.55 ´ 105

1.78 ´ 106

n/a 5

8.60 ´ 104

6.16 ´ 104

1.92 ´ 105

£ LoD £ LoD

<1 <1 <1 n.d. n.d.

unchanged unchanged unchanged

n/a n/a

resistant recSeV LE863V 50 b.i.d. 2 £ LoD n.d. discontinued

resistant recSeV LY942H 50 b.i.d. 2 3.05 ´ 106

1.78 ´ 106 LH942Y

revertants discontinued

resistant recSeV LI1009L 50 b.i.d. 2 £ LoD n.d. discontinued

resistant recSeV LY1106S 50 b.i.d. 2 6.33 ´ 104

7.01 ´ 104 LS1106Y

revertants discontinued

n/a, not applicable n.d., not determined LoD, level of detection

Supplementary Table 7. In vivo adaptation of recSeV to GHP-88309. Serial passaging of recSeV in mice in

the presence of 50 mg/kg body weight GHP-88309.

AvgMax SD Max AvgMed SDMed AvgMin SDMin S/B Z’ Plate 1 1001965 268256 826334 207037 2098 946 464 0.53 Plate 2 862469 244811 667041 223756 1591 761 542 0.51 Plate 3 953548 270753 763529 211977 1774 747 537 0.51

Supplementary Table 8. Validation of the HPIV3-JS-NanoLuc 384-well HTS screening protocol. Control

plates were arranged as described in the legend to Extended Data 1b. Average (Avg) signals, signal-to-

background (S/B) and Z’ scores for each reference plate are shown.