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: [email protected]
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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.
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