Enteroviruses infect multiple human tissues and cause diseases including meningitis, the common cold, myocarditis, pancreatitis, hepatitis, poliomyelitis, sepsis, type 1 diabetes, hand, foot, and mouth disease. Despite this burden, no antiviral therapy has been approved to date. Progress has been limited by the structural and topical diversity of enteroviruses because many variants are intrinsically insensitive to candidate agents and sensitive strains develop resistance rapidly. Here, we report that the approved anticancer drug vemurafenib inhibited replication of some tested enteroviruses in cell cultures. Passage of echovirus EV1 and coxsackievirus CVB5 for six cycles in cell culture yielded vemurafenib-resistant virus variants harboring mainly missense mutations in the viral 3A and VP1 proteins, underscoring the need for combination therapy. We therefore evaluated cocktails, combining vemurafenib with the VP1 inhibitor pleconaril and the 3C protease inhibitor AG7404. In cell culture, the cocktails suppressed replication of all seven tested enteroviruses. The combination was also effective in human pancreatic, retinal, and brain organoids. In infected mice, the triple regimen reduced viral titers in the pancreas. These findings support multi-stage targeting of the enterovirus life cycle as a promising path toward broadly active therapeutic cocktails.

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Centre for Embryology and Healthy Development University of Oslo Oslo 0373 Norway

Department of Clinical and Molecular Medicine Norwegian University of Science and Technology 7028 Trondheim Norway

Department of Experimental Medical Science Lund University 22100 Lund Sweden

Department of Microbiology Oslo University Hospital and University of Oslo 0372 Oslo Norway

Department of Surgery Faculty of Medicine University of Helsinki and Helsinki University Hospital 00014 Helsinki Finland

Department of Virology University of Helsinki 00014 Helsinki Finland

HUS Diagnostic Center Clinical Microbiology Helsinki University Hospital University of Helsinki 00029 Helsinki Finland

iCAN Digital Precision Cancer Medicine Flagship 00014 Helsinki Finland

Institute for Molecular Medicine FIMM Helsinki Institute for Life Science University of Helsinki 00014 Helsinki Finland

Laboratoire de virologie ULR3610 Univ Lille et CHU Lille 59000 Lille France

The Stephan Angeloff Institute of Microbiology Bulgarian Academy of Sciences 1113 Sofia Bulgaria

Translational Cancer Medicine Research Program Faculty of Medicine University of Helsinki 00014 Helsinki Finland

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