key: cord-0884592-lh1rw8w5 authors: Lo, Michael K.; Shrivastava-Ranjan, Punya; Chatterjee, Payel; Flint, Mike; Beadle, James R.; Valiaeva, Nadejda; Schooley, Robert T.; Hostetler, Karl Y.; Montgomery, Joel M.; Spiropoulou, Christina title: Broad-spectrum in vitro antiviral activity of ODBG-P-RVn: an orally-available, lipid-modified monophosphate prodrug of remdesivir parent nucleoside (GS-441524) date: 2021-08-10 journal: bioRxiv DOI: 10.1101/2021.08.06.455494 sha: 32b59a3e76de4bfd789e13a80f928a2a7731fe83 doc_id: 884592 cord_uid: lh1rw8w5 The intravenous administration of remdesivir for COVID-19 confines its utility to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524) against viruses that cause diseases of human public health concern, including SARS-CoV-2. ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had near-equivalent activity to remdesivir in primary-like human small airway epithelial cells. Our results warrant investigation of ODBG-P-RVn efficacy in vivo. . e r o -E 6 c e l l s ( 1 8 ) . 4 3 I n t h i s s t u d y , w e e x t e n d e d o u r i n v i t r o c o m p a r i s o n s t o i n c l u d e 1 4 v i r u s e s f r o m a c r o s s 7 v i r u s f a m i l i e s 4 4 r e s p o n s i b l e f o r c a u s i n g d i s e a s e s o f s i g n i f i c a n t h u m a n p u b l i c h e a l t h c o n c e r n . T h e s e w e r e F i l o v i r i d a e : e d 3 p r e v i o u s l y d e s c r i b e d a s s a y s t o c o m p a r e 5 0 t h e a n t i v i r a l a c t i v i t i e s o f R V n , R D V , a n d O D B G -P -R V n a g a i n s t t h i s p a n e l o f v i r u s e s : 1 ) d i r e c t l y m e a s u r i n g 5 1 f l u o r e s c e n c e o f a r e p o r t e r p r o t e i n e x p r e s s e d b y r e c o m b i n a n t v i r u s e s ( R E P ) ( 2 ) , ( F i g u r e 1 A ) ; 2 ) 5 2 q u a n t i t a t i n g f o c u s -f o r m i n g u n i t s ( F F U ) v i a f l u o r e s c e n t r e p o r t e r i m a g i n g ( 3 2 ) ( F i g u r e 1 B ) ; a n d 3 ) i n d i r e c t l y 5 3 m e a s u r i n g c y t o p a t h i c e f f e c t ( C P E ) b a s e d o n c e l l u l a r A T P l e v e l s ( C e l l T i t e r G l o 2 . 0 , P r o m e g a ) ( 2 ) ( F i g u r e 5 4 1 C ) , w h i c h w a s a l s o u s e d t o e v a l u a t e c o m p o u n d c y t o t o x i c i t y ( F i g u r e 1 D ) . A s s a y c o n d i t i o n s v a r i e d b a s e d 5 5 o n v i r u s r e p l i c a t i o n k i n e t i c s a n d o n t h e s p e c i f i c a s s a y u s e d ; m u l t i p l i c i t i e s o f i n f e c t i o n ( M O I . . I n s u m m a r y , o u r r e s u l t s d e m o n s t r a t e t h a t O D B G -P -R V n h a s g r e a t e r a n t i v i r a l a c t i v i t y t h a n R V n i n a l l c e l l 1 0 0 l i n e s t e s t e d a n d h a s c e l l -t y p e d e p e n d e n t a c t i v i t y l e v e l s t h a t r a n g e f r o m m o d e r a t e l y l e s s e r t h a n t o n e a r l y 1 0 a r r e n T K , J o r d a n R , L o M K , R a y A S , M a c k m a n R L , S o l o v e v a V , S i e g e l D , P e r r o n M , B a n n i s t e r R , 1 1 7 H u i H C , L a r s o n N , S t r i c k l e y R , W e l l s J , S t u t h m a n K S , V a n T o n g e r e n S A , G a r z a N L , D o n n e l l y G , 1 1 (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 10, 2021. ; 1 1 . T e m p e s t i l l i M , C a p u t i P , A v a t a n e o V , N o t a r i S , F o r i n i O , S c o r z o l i n i L , M a r c h i o n i L , A s c o l i B a r t o l i T , 1 6 2 C a s t i l l e t t i C , L a l l e E , C a p o b i a n c h i M R , N i c a s t r i E , D ' A v o l i o A , I p p o l i t o G , A g r a t i C , G r o u p C I S . 2 0 2 0 . 1 6 3 P h a r m a c o k i n e t i c s o f r e m d e s i v i r a n d G S -4 4 1 5 2 4 i n t w o c r i t i c a l l y i l l p a t i e n t s w h o r e c o v e r e d f r o m 1 6 4 C O V I D -1 9 . J A n t i m i c r o b C h e m o t h e r 7 5 : 2 9 7 7 -2 9 8 0 . 105 and is also made available for use under a CC0 license. (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 10, 2021. ; S 2 5 3 F i g u r e 1 . C o m p a r i s o n o f a n t i v i r a l a c t i v i t i e s o f R V n , R D V , a n d O D B G -P -R V n i n A f r i c a n g r e e n m o n k e y ( V e r o -2 5 4 E 6 ) , h u m a n h e p a t o m a ( H u h 7 ) , a n d h u m a n b r o n c h i o a l v e o l a r c a r c i n o m a ( N C I -H 3 5 8 ) c e l l l i n e s u s i n g 2 5 5 r e p o r t e r -b a s e d , i m a g e -b a s (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted August 10, 2021. ; c o l o r e d s h a p e / l i n e i n t h e l e g e n d r e p r e s e n t s a n i n d e p e n d e n t e x p e r i m e n t p e r f o r m e d i n b i o l o g i c a l 3 0 1 t r i p l i c a t e . 3 0 f 3 b i o l o g i c a l 3 2 3 r e p l i c a t e s ; e a c h c o l o r e d s h a p e / l i n e i n t h e l e g e n d r e p r e s e n t s a n i n d e p e n d e n t e x p e r i m e n t p e r f o r m e d i n 3 2 4 b i o l o g i c a l t r i p l i c a t e . 3 2 Malaysia-ZsG Filoviridae Paramxyovirdae NiV-M Rec. Malaysia-ZsG Filoviridae EBOV Rec. Makona-ZsG RVn SI, selective index = EC50/CC50; REP, reporter; CPE, cytopathic effect; FFU, focus-forming unit VTR, virus titer reduction; ND, not determined Mean values with ± standard deviation values were derived from a minimum of 3 independent experiments performed in biological triplicates. REP/FFU/CPE/VTR assays were conducted at 72 hpi. EC 50, EC90, and CC50 values were calculated using Graphpad Prism EC50, 50% effective inhibition concentration EC90, 90% effective inhibition concentration 50% cytotoxic concentration SI, selective index = EC50/CC50; REP, reporter; CPE, cytopathic effect FFU, focus-forming unit Mean values with ± standard deviation values were derived from 3 independent experiments performed in biological triplicates except for NiV-B (NCI-H358), HeV (NCI-H358), and YFV (Vero E6)which were performed twice in biological triplicates. Data in red text derived from Huh7 cells, data in blue derived from NCI-H358 cells. REP/FFU/CPE assays were conducted between 72-144 hpi