Steroid Hormone Receptors · January 17, 2022


1997. severe disease are those under 6 weeks of age, those with bronchopulmonary dysplasia, congenital heart disease, ROR gamma modulator 1 or immunodeficiency, and those born prematurely. Hospital admission rates for these groups range between 5% and 30% (20, 25). The mortality rate among children admitted to hospital is approximately 3% for those with heart and lung problems and up to 1% for those without these risk factors (11, 25). In adults and the elderly, RSV pneumonia is increasingly recognized as a significant cause of morbidity and mortality, being associated with more than 17,000 deaths annually between 1991 and 1998 (9, 22). Among the hospitalized elderly, mortality can be as high as 10 to 20%, and among severely immunocompromised patients with RSV pneumonia, it can be on the order of 50 to 70% (10). There is therefore an urgent and unmet medical need for novel therapies to deal with infections caused by this virus. The development of new therapeutics for RSV was recently reviewed (17, 19). Although research into the prevention and treatment of RSV infection has been ongoing for almost 40 years, vaccine development is difficult (8, 13), and to date, there is no clinically approved vaccine. The development of RSV vaccines for use in young infants has been complicated by reduced immune NOS2A responses in this age group due to immunologic immaturity and the immunosuppressive effects of maternal antibodies. Passive immunization with the monoclonal antibody palivizumab (Synagis) has provided about 50% protection to high-risk children (21). These include infants born prematurely and those with congenital conditions. Because the antibody has to be given prophylactically and treatment is very expensive, its use is limited mainly to developed countries. The effectiveness of ribavirin (16), the only licensed antiviral small molecule against RSV, is questionable (24). Ribavirin has to be given by prolonged aerosol and has been shown to be potentially mutagenic and teratogenic. The need for an effective ROR gamma modulator 1 and safe treatment for RSV infection is paramount. RSV, a pneumovirus of the paramyxovirus family, is an enveloped nonsegmented negative-strand RNA virus (6). The 15.2-kb genome has been sequenced fully and contains 10 mRNA species encoding 11 distinct proteins. The genome is encapsidated by the nucleocapsid (N) protein, which forms a helical nucleocapsid and protects the RNA from ribonucleases. The N protein is also associated with the viral polymerase, phosphoprotein, and M2-1 protein, which together form the transcriptase complex. The ribonucleoprotein (RNP) is essential for transcription, and naked RNA does not provide a template for the viral polymerase. As with all single-stranded RNA viruses, the virus does not have a proofreading mechanism during replication, resulting in a relatively high error rate and frequent mutations. Promising inhibitors targeting the fusion event were unsuccessful, partly because of the rapid emergence of resistant mutants mapping to the F gene (5). Therefore, an effective antiviral compound should ideally target essential genes of the replication complex, as these are often more highly conserved due to their functional role (19). In this study, we describe the identification of RSV604, a potent RSV inhibitor, and outline the experiments performed to determine its mechanism of action (MOA). The results suggest that RSV604 targets the nucleocapsid protein and is active post-RSV infection. RSV604 is currently in phase II trials. MATERIALS AND METHODS Cells and viruses. HEp-2 cells and RSV strains Long and B were obtained from the American Type Culture Collection (Manassas, VA). Vero cells were obtained from the European Collection of Cell Cultures (Porton Down, United Kingdom). The RSV RSS and A2 strains and pneumonia virus of mice strain 15 were gifts from A. Easton (University of Warwick, United Kingdom). Bovine RSV (Snook strain) was kindly supplied by ROR gamma modulator 1 G. Taylor (Institute of Animal Health, Compton, United Kingdom). Cells and virus stocks were grown as reported previously (4). Clinical isolates of RSV were obtained from G. Toms (University of Newcastle, United Kingdom), P. Rice (St. Georges Hospital, London, United Kingdom), J. De Vincenzo (University of Tennessee, Memphis, TN), and the Health Protection Agency (Colindale, London, United Kingdom). Synthesis of compounds. RSV604 and related compounds were synthesized as previously described (4). BMS-433771 (5) was synthesized in-house. Ribavirin was ROR gamma modulator 1 purchased from Sigma. Antiviral.