key: cord-0937046-w8tirve0 authors: Guo, Wenrui; Porter, Linsey M; Crozier, Thomas WM; Coates, Matthew; Jha, Akhilesh; McKie, Mikel; Nathan, James A; Lehner, Paul J; Greenwood, Edward JD; McCaughan, Frank title: Topical TMPRSS2 inhibition prevents SARS-CoV-2 infection in differentiated primary human airway cells date: 2021-04-28 journal: bioRxiv DOI: 10.1101/2021.04.23.440619 sha: 8de7c53e9d0cd61a404d14d0da3ea4a0ee51fdd7 doc_id: 937046 cord_uid: w8tirve0 Background There are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection. Methods The likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection. Results We first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection. Conclusion These experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial. . I t f o l l o w s t h a t f o r p r e v e n t a t i v e t h e r a p i e s , t h e u s e o f d i f f e r e n t i a t e d a i r w a y c e l l s i s c r i t i c a l t o m o d e l l i n g t h e r a p e u t i c e f f i c a c y i n v i t r o . P r i o r w o r k s u g g e s t s t h a t s o l u b l e A C E 2 c o u l d a c t a s a d e c o y r e c e p t o r f o r s p i k e p r o t e i n a n d r e d u c e S A R S -C o V -2 v i r a l l o a d 8 , a n d c l i n i c a l t r i a l s t e s t i n g t h i s s t r a t e g y a r e u n d e r w a y 9 . A n a l t e r n a t i v e a p p r o a c h i s t o m o d u l a t e t h e c l e a v a g e o f s p i k e p r o t e i n b y i n h i b i t i n g T M P R S S 2 a c t i v i t y , b y ( 1 ) u s i n g a n d r o g e n r e c e p t o r ( A R ) a n t a g o n i s t s t o s u p p r e s s a n d r o g e n -s e n s i t i v e T M P R S S 2 e x p r e s s i o n 1 0 1 1 , o r ( 2 ) d i r e c t i n h i b i t i o n o f t h e s e r i n e p r o t e a s e a c t i v i t y o f T M P R S S 2 2 1 2 . T M P R S S 2 e x p r e s s i o n i s r e g u l a t e d b y a n d r o g e n s i g n a l l i n g v i a A R b i n d i n g a t 1 3 a n d 6 0 k b u p s t r e a m o f t h e T M P R S S 2 t r a n s c r i p t i o n a l s t a r t s i t e 1 3 . a n d r o g e n d e p r i v a t i o n i n e a r l y , n o n -h o s p i t a l i s e d m a l e C O V I D -1 9 p a t i e n t s h a v e s t a r t e d r e c r u i t m e n t ( C l i n i c a l T r i a l s . g o v I d e n t i f i e r : N C T 0 4 4 4 6 4 2 9 ; C l i n i c a l T r i a l s . g o v I d e n t i f i e r : N C T 0 4 4 7 5 6 0 1 ) . C a m o s t a t m e s y l a t e , a s e r i n e p r o t e a s e i n h i b i t o r , d i r e c t l y i n h i b i t s T M P R S S 2 1 8 . E x p e r i m e n t s m i m i c k i n g t h e s y s t e m i c d e l i v e r y o f c a m o s t a t h a v e s h o w n t h a t i t i s e f f e c t i v e i n r e d u c i n g v i r a l e n t r y . C a m o s t a t r e d u c e d p s e u d o t y p e d S A R S -C o V -2 v i r a l e n t r y i n d i f f e r e n t i a t e d a i r w a y e p i t h e l i a l c e l l s 2 a n d r e d u c e d t h e a l r e a d y m o d e s t l e v e l s o f w i l d t y p e v i r a l e n t r y i n d i s t a l a i r w a y o r g a n o i d s 1 0 1 9 . A p o t e n t i a l b e n e f i t o f c a m o s t a t i s t h a t i t i s o r a l l y d e l i v e r e d a s w e l l a s b e i n g w e l l -t o l e r a t e d , a n d i n e x p e n s i v e 1 . I t h a s b e e n l i c e n s e d i n J a p a n s i n c e 1 9 8 5 a n d i s i n r e g u l a r u s e i n J a p a n a n d S o u t h K o r e a f o r p a t i e n t s w i t h c h r o n i c p a n c r e a t i t i s . S e v e r a l c l i n i c a l t r i a l s i n p a t i e n t s w i t h C O V I D -1 9 a r e u n d e r w a y ( C l i n i c a l T r i a l s . g o v I d e n t i f i e r : N C T 0 4 4 5 s e s u s e d i n t h i s s t u d y a r e t h e c l i n i c a l i s o l a t e s n a m e d " S A R S - . T m a g n i f i c a t i o n x 4 0 o i l , o r e n t i r e t r a n s w e l l i n s e r t s w e r e i m a g e d u s i n g a n A r r a y s c a n X T I ( T h e r m o - a t i s t i c a l a n a l y s e s o f m R N A e x p r e s s i o n a s s a y s a n d i n f e c t i o n q u a n t i f i c a t i o n d a t a w e r e p e r f o r m e d u s i n g P r i s m 8 s o f t w a r e ( G r a T M P R S S 2 e x p r e s s i o n i n d i f f e r e n t i a t e d h u m a n a i r w a y c e l l s i n r e s p o n s e t o D H T a n d e n z a l u t a a n d r o g e n r e c e p t o r t h a n t e s t o s t e r o n e . E n z a l u t a m i d e i s a n a n d r o g e n s i g n a l l i n g a n t a g o n i s t . O n t r e a t m e n t w i t h D H T t h e r e w a s a m o d e s t , b u t n o t s i g n i f i c a n t , i n d u c t i o p o t e n t i a l i m p a c t o f s y s t e m i c t h e r a p y , a l t h o u g h i n v i v o t h e c o m p o u n d i s m e t a b o l i s e d w i t h i n a m i n u t e o f r e a c h i n g t h e c i r c u l a t i o n . W h e n d e l i v e r e d i n t h i s w a y , c a m o s t a o f s o d i u m c h a n n e l f l u x p e r s i s t e d a t l e a s t 6 h o u r s a f t e r t r e a t m e n t , d e s p i t e m u l t i p l e w a s h e s w i t h w a r m e d P B S , d e m o n s t r a t i n g t h a t t o p i c a l c a m o s t a t c a n h a v e a s u s t a i n e a d m i n i s t e r e d 2 0 0 ยต g o r l e s s . C a m o s t a t w a s n o t d e t e c t e d i n t h e s y s t e m i c c i r c u l a t i o n a f t e r n a s a l d o s i n g . T h e p r i m a r y m e t a b o l i t e w a s d e t e c t e d a t 3 h o u r s , o n l y i n t h o s C a m o s t a t m e s y l a t e i s g e n e r a l l y a d m i n i s t e r e d a s a n o r a l p r e p a r a t i o n , b u t i s a l m o s t i n s t a n t a n e o u s l y m e t a b o l i s e d i n t h e c i r c u l a t i o n t o a n i n t e r m e d i a t e m e t a b o l i t e w i t h s i g n i f i c a n t l y l e s s a c t i v i t y 1 3 6 . A l t h o u g h w e l l t o l e r a t e d a t h i g h o r a l d o s e s , t h e m a x i m u m c o n c e n t r a t i o n o f t h e m e t a b o l i t e a t t a i n e d i n t h e c i r c u l a t i o n a t s t a n d a r d d o s i n g i T h e r e r e m a i n s a n u r g e n t u n m e t n e e d f o r a s a f e , e a s i l y a d m i n i s t e r e d , e f f e c t i v e t h e r a p e u t i c w i t h p o t e n t i a l e f f i c a c y i n p r e -o r p o s t -e x p o s u r e p r o p h y l a x K r u s k a l -W a l i l i s t e s t w i t h D u n n ' s c o r r e c t i o n f o r m u l t i p l e c o m p a r i s o n s ( b e t w e e n a l l c o n d i t i o n s ) . L D H r e l e a s e a s s a y a s a m e a s u r e o f c y t o t o x i c i t y f o l l o w i n g 4 8 h b a s a l c a m o s t a t o f h A E C -A L I c u l t u r e s d e r i v e d f r o m b r o n c h i a l o r n a s a l c e l l s . 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