【病毒外文文献】2004 Inhibition of Severe Acute Respiratory Syndrome-Associated Coronavirus (SARSCoV) by Calpain Inhibitors and _-D-N4-H

Introduction 15 2004 International Medical Press 0956 3202 02 17 00 Antiviral Chemistry Fouchier et al 2003 It is a life threatening and highly contagious febrile respiratory ill ness that was initially described in early 2003 in patients from Guangdong Province in southern China This was quickly followed by numerous cases in Vietnam Hong Kong Singapore Canada and the USA with some cases in other countries Hsueh et al 2003 Treatment for the disease is supportive as there are no approved or universally recommended therapies for SARS Initially systemic corticosteroids were used to suppress the production of the myriad inflammatory mediators that appear in response to the viral infection Ho et al 2003 Meng et al 2003 Combination therapy with ribavirin and corticosteroids has also been attempted Peiris et al 2003 Koren et al 2003 However the efficacy of these treat ments has not been demonstrated in controlled studies Wenzel et al 2003 Several in vitro studies have been undertaken in an attempt to find antiviral agents that suppress SARSCoV replication Included in the list of agents found inhibitory to the virus have been glycyrrhizin Cinatl et al 2003a interferons Cinatl et al 2003b and siRNA Zhang et al 2003 He et al 2003 It has also been suggested that certain products in the armamentarium of Chinese tradi tional medicine could be of value Jia Chou et al 2003 Theil et al 2003 and perhaps several homologues of cellular RNA processing enzymes Snijder et al 2003 In addition some have suggested that aminopeptidase N CD13 might be a putative receptor for the virus and that the interaction between virus glycoprotein and aminopep tidase N could be a target for a small molecule such as Inhibition of severe acute respiratory syndrome associated coronavirus SARSCoV by calpain inhibitors and D N 4 hydroxycytidine Dale L Barnard 1 Valerie D Hubbard 1 Jared Burton 1 Donald F Smee 1 John D Morrey 1 Michael J Otto 2 and Robert W Sidwell 1 1 Institute for Antiviral Research Utah State University Logan UT USA 2 Pharmasset Inc Tucker Ga USA Corresponding author E mail honery cc usu edu We evaluated two types of compounds for effica cy in inhibiting SARSCoV replication in vitro cal pain inhibitors a class of cellular cysteine pro teinases and a number of nucleoside analogues Cytopathic effect reduction assays visually deter mined with spectrophotometric verification by neutral red NR uptake assay were used to evalu ate cytotoxicity and antiviral potency of the com pounds Significantly inhibitory compounds were then evaluated in virus yield reduction assays Two calpain inhibitors Val Leu CHO calpain inhibitor VI and Z Val Phe Ala CHO calpain inhibitor III were the most potent inhibitors of SARSCoV By virus yield reduction assay calpain inhibitor VI had a 90 effective concentration EC 90 of 3 M and calpain inhibitor III had an EC 90 of 15 M D N 4 hydroxycytidine was the most selective nucleoside analogue inhibitor with an EC 90 of 6 M by virus yield reduction assay These compounds or analogues warrant further evalua tion as potential therapies for treating SARS or could be used as lead compounds for discovery of more potent SARSCoV inhibitors Keywords severe acute respiratory syndrome associated coronavirus SARSCoV calpain inhibitors D N 4 hydroxcytidine 16 2004 International Medical Press D Barnard et al ubenimix Kontoyiannis et al 2003 Most recently Li et al 2003 have shown that angiotensin convertase enzyme 2 is a functional receptor for SARSCoV Xiao et al 2003 have demonstrated that the angiotensin convertase enzyme 2 interacts with defined amino acid sequences of glycopro tein S of SARSCoV Thus this interaction between virus glycoprotein and cell receptor represents an additional and partially defined target for antiviral chemotherapy as well as for vaccine development Based on previous studies with other viruses and some of the potential targets described above we evaluated a num ber of compounds for inhibitory activity against SARSCoV in vitro Materials and methods Chemicals Calpain inhibitors Calpain inhibitors were obtained from CalBiochem NovaBiochem Corporation San Diego Calif USA and included N 4 fluorophenylsulfonyl L valyl L leucinal 4 fluorophenylsulfonyl Val Leu CHO calpain inhibitor VI carbobenzoxy valinyl phenylalaninal Z Val Phe CHO calpain inhibitor III N acetyl Leu Leu Nle CHO ALLN calpain inhibitor I Z Leu Leu Tyr CH 2 F Z LLY FMK calpain inhibitor IV benzyloxycar bonylleucyl norleucinal calpeptin Z Leu Nle CHO and the non selective calpain inhibitor 3 4 iodophenyl 2 mercapto Z 2 propenoic acid The calpain inhibitor 2 2S 3S trans epoxysuccinyl L leucylamido 3 methylbu tane ethyl ester E 64d was purchased from Sigma Aldrich St Louis Mo USA Nucleosides nucleotides and nucleoside analogues N 4 benzoyl 5 O dimethoxytrityl 5 methyl 2 O methylcytidine N 4 benzoyl 5 O dimethoxytrityl 3 deoxycytidine N 4 benzoyl 3 deoxycytidine N 4 acetyl 5 methyl 2 O methylcytidine N 4 benzoyl 2 O methylcy tidine 2 deoxycytidine hydrochloride 2 thiocytidine 5 bromo 2 deoxycytidine 7 deaza 2 deoxyguanosine and 7 deaza 2 deoxyadenosine were purchased from Berry 5 dideoxyguanosine chlorocytidine cyclic cytosine monophosphate cCMP and dideoxycytosine triphosphate ddCTP were from Axxora LLC San Diego Calif USA and 2 3 deoxyadenosine was from CalBiochem NovaBiochem Corp D N 4 hydroxycytidine was kindly provided by Pharmasset Inc Tucker Ga USA and 3 deazane planocin A was donated by Victor E M rquez National Cancer Institute Frederick Md USA Cidofovir was obtained from Gilead Sciences Foster City Calif USA Ribavirin and pyrazofurin were provided by ICN Pharmaceuticals Costa Mesa Calif USA and human leukocyte derived interferon alpha n3 Alferon N Injection by Hemispherx Biopharma Inc Philadelphia Pa USA CHO O N H H N OO S F Figure 1 Structures of the compounds most inhib itory of SARSCoV A Calpain inhibitor VI 4 fluorophenylsulfonyl Val Leu CHO B Calpain inhibitor III Z Val Phe Ala CHO C D N 4 hydroxycytidine O O O O H N H H N N O NHOH N OH O HO OH A B C 17 Inhibition of SARSCoV Antiviral Chemistry Manassas Va USA The cells were routinely grown in minimal essential medium MEM supplemented with 5 heat inactivated foetal bovine serum FBS Hyclone Laboratories Logan UT USA For antiviral assays the serum was reduced to 2 and gentamicin was added to the medium at a final concentration of 50 g ml SARSCoV strain Urbani 200300592 was obtained from the CDC and routinely passaged in Vero 76 cells Cytopathic effect CPE inhibition assay Barnard et al s 2001 protocol was used Compounds were tested at varying concentrations four 1 log 10 or seven 1 2 log 10 dilutions once or twice with this assay and the activ ity was then verified spectrophotometrically by neutral red NR uptake assay on the same plate see below Virus multiplicity of infection MOI 0 001 and compound were added in equal volumes to 80 90 confluent cell monolayers in 96 well tissue culture plates The MOI used was such that 100 of the cells in the virus controls showed cytopathic effects CPE within 3 5 days The plates were incubated at 37 C until the cells in the virus control wells showed complete viral CPE as observed by light microscopy Each concentration of drug was assayed for viral CPE inhibition in triplicate and for cytotoxicity in duplicate Six wells per microplate were set aside as unin fected untreated cell controls and six wells received virus in medium only per microplate and represented controls for virus replication Alferon was included as a positive control drug for each set of compounds tested For all CPE based assays the 50 effective concentrations EC 50 were calcu lated by linear regression analysis of the means of the CPE ratings expressed as percentages of untreated uninfected controls for each concentration Morphological changes resulting from a compound s cytotoxicity were graded on a scale of 0 5 with 5 being defined as complete cytotoxicity The 50 cytotoxic doses IC 50 were calculated by regression analysis and a selectiv ity index SI was calculated using the formula SI IC 50 EC 50 Neutral red NR uptake assay of CPE inhibition and compound cytotoxicity This assay was done on the same CPE inhibition test plates described above to verify the inhibitory activity and the cytotoxicity observed by visual observation The usual correlation between visual and NR assays in our hands has been greater than 95 Barnard et al 2001 The NR assay was performed using a modified method of Cavenaugh et al 1990 as described by Barnard et al 1999 Each well of the plate had medium removed and 0 034 NR added The plate was then incubated for 2 h at 37 C in the dark The NR solution was removed from the wells rinsed and the remaining dye extracted using ethanol buffered with S renson s citrate buffer Absorbances at 540 nm 450 nm were read with a microplate reader Bio Tek EL 1309 Bio Tek Instruments Inc Winooski Vt USA Absorbance values were expressed as percentages of untreated controls and EC 50 IC 50 and SI values were calculated as earlier described Virus yield reduction assay All compounds with an SI greater than 10 were evaluated in a virus yield reduction assay to confirm the results of the CPE inhibition NR uptake assays Infectious virus yields from each well from a second CPE inhibition assay were determined as previously described Barnard et al 2001 After CPE was scored each plate was frozen at 80 C and thawed Sample wells at each compound concentra tion tested were pooled and titred in Vero cells for infec tious virus by CPE assay as described previously by Barnard et al 2002 A 90 reduction in virus yield was then calculated by lin ear regression analysis This represented a 1 log 10 inhibition in titre when compared to untreated virus controls Evaluation of cytotoxicity in rapidly dividing cells Cytotoxicity in rapidly dividing cells was evaluated by deter mining the total number of cells as reflected by a NR uptake assay after a 3 day exposure to several concentrations of compound Barnard et al 2002 To quantitate cell growth after 72 h in the presence or absence of drug the plates were treated as described above for the NR assay Absorbance val ues were expressed as percentage of untreated controls and IC 50 values were calculated by regression analysis Results Using CPE reduction assays visually assessed and verified spectrophotometrically by NR uptake assay in the same plate we evaluated a number of metabolic inhibitors including calpain inhibitors and several nucleotides nucle osides and nucleoside analogues for anti SARSCoV activ ity in Vero 76 cells Compounds found active in those two assays were further evaluated for inhibition of infectious virus production The most inhibitory calpain inhibitor had the motif Val Leu CHO and an EC 50 of 1 M as determined by NR assay Table 1 Calpain inhibitor III Z Val Phe Ala CHO was also active against SARSCoV the EC 50 0 5 M by visual assay and 1 M by NR assay Of the cytosine type compounds D N 4 hydroxycyti dine was very active with an EC 50 of 5 M but with some 18 2004 International Medical Press D Barnard et al cytotoxicity detected the IC 50 was equal to 50 M by NR assay Table 2 N 4 Benzoyl 5 O dimethoxytrityl 5 methyl 2 O methylcytidine slightly but selectively inhib ited SARSCoV replication as assessed by NR assay EC 50 4 M However slight viral CPE was evident at every dilution of compound tested data not shown N 4 Benzoyl 5 O dimethoxytrityl 3 deoxycytidine N 4 ben zoyl 3 deoxycytidine N 4 acetyl 5 methyl 2 O methyl cytidine 2 deoxycytidine 3 deoxycytidine cytosine N 4 benzoyl 2 O methylcytidine 2 deoxycytidine hydro chloride 2 thiocytidine 5 bromo 2 deoxycytidine 5 chlorocytidine cidofovir cyclic cytosine monophosphate cCMP dideoxycytosine triphosphate and 2 3 dideoxy cytidine were inactive Of the guansosine like compounds evaluated ribavirin was found to be active against SARSCoV in the Vero cells at a very high concentration by NR assay with an EC 50 622 M Table 3 In this same group of compounds pyrazofu rin was also inhibitory but only near cytotoxic levels SI 3 by NR assay Dideoxyguanosine and 7 deaza 2 deoxyguanosine were inactive None of the adenosine derivatives 2 deoxyadenosine 2 3 deoxyadenosine 7 deaza 2 deoxyadenosine and 3 deazaneplanocin A was particularly selective in their in hibition of SARSCoV replication although 7 deaza 2 deoxyadenosine 2 deoxytubercidin was inhibitory at 100 50 1 100 100 Z Val Phe CHO III 0 5 20 40 1 10 10 N Acetyl Leu Leu Nle CHO I 2 20 10 4 11 3 Z Leu Leu Tyr CH 2 F IV 2 17 9 7 8 1 Z Leu Nle CHO I accepted 22 December 2003