Enterovirus A71 antivirals: Past, present, and future

Abstract

Enterovirus A71 (EV-A71) is a significant human pathogen, especially in children. EV-A71 infection is one of the leading causes of hand, foot, and mouth diseases (HFMD), and can lead to neurological complications such as acute flaccid myelitis (AFM) in severe cases. Although three EV-A71 vaccines are available in China, they are not broadly protective and have reduced efficacy against emerging strains. There is currently no approved antiviral for EV-A71. Significant progress has been made in developing antivirals against EV-A71 by targeting both viral proteins and host factors. However, viral capsid inhibitors and protease inhibitors failed in clinical trials of human rhinovirus infection due to limited efficacy or side effects. This review discusses major discoveries in EV-A71 antiviral development, analyzes the advantages and limitations of each drug target, and highlights the knowledge gaps that need to be addressed to advance the field forward.

Keywords: 2C protein; Acute flaccid myelitis; Antivirals; EV-A71; Enterovirus A71; Foot and mouth disease (HFMD); Hand; Picornavirus.

Graphical abstract

Figure 1

Schematic overview of the life cycle of EV-A71. EV-A71 viral particles attach to the host cell surface by binding to its specific receptor and entering the host cell through endocytosis. Upon uncoating, the viral genome RNA is released and serves as a template for translation into viral polyprotein or the synthesis of negative-strand RNA, which is further used as a template for viral genome RNA replication. Viral polyprotein is cleaved into structural and non-structural proteins by 2Apro and 3Cpro. Viral capsid proteins VP0, VP1 and VP3 first self-organize into a protomer, five of which assemble into a pentamer. Twelve pentamer and viral genome RNA assemble into a provirion, which mature into progeny virion upon the cleavage of VP0 into VP2 and VP4, a process induced by viral genome RNA. Mature virions release and exit from host cells.

Figure 2

Three CNS penetration pathways exploited by enteroviruses. (A) Enterovirus can cross the BBB and reach the CNS by directly infecting BMECs. (B) Enterovirus can infect leukocytes, which act as carriers to transport virus into the CNS. (C) Enteroviruses hijack the retrograde axonal transport to enter the CNS. Viruses first infect muscles, then motor neurons, and finally reach the spinal cord.

Figure 3

EV-A71 capsid inhibitors targeting the VP1 hydrophobic pocket. (A) Cryo-EM structure of EV-A71 capsid proteins in complex with sphingosine (PDB: 6UH6) and (B) NLD-22 (PDB: 6LQD). VP1, VP2, VP3, and NLD-22 were colored in gray, tint, yellow, and magenta, respectively. (C) Chemical structures of EV-A71 capsid inhibitors targeting the VP1 hydrophobic pocket.

Figure 4

EV-A71 antivirals targeting the five-fold axis of the capsid proteins. (A) Chemical structure of EV-A71 capsid inhibitors targeting the five-fold axis of the capsid proteins. (B) Chemical structure of CB-30. (C) Binding pose of EV-A71 capsid inhibitor CB-30 (compound 30 in the original publication). Reprinted with permission from Ref. 68. Copyright © 2020, American Chemical Society.

Figure 5

EV-A71 2A protease inhibitors. (A) Chemical structure of the EV-A71 2Apro inhibitors. (B) Modeling of the substrate peptide on the X-ray crystal structure of EV-A71 2Apro C110A mutant (PDB: 4FVB). Reprinted with permission from Ref. 115. Copyright © 2013, American Society for Microbiology.

Figure 6

EV-A71 2B inhibitor.

Figure 7

EV-A71 2C inhibitors. (A) X-ray crystal structure of EV-A71 2C protein (PDB: 5GRB). One monomer is colored in cyan, and another monomer is colored in gray and shown in surface. ATP is shown in sticks. (B) X-ray crystal structure of CV-B3 2C (117-329) in complex with (S)-fluoxetine (PDB: 6T3W). (C) Cryo-electron micrographs of CV-B3 2C with (S)-fluoxetine (SFX) and ATP. Reprinted from Ref. 132. (D) Chemical structure of EV-A71 2C inhibitors.

Figure 8

EV-A71 3A structure and inhibitors. (A) Model of the EV-A71 3A protein and the associated proteins in the replication organelle. (B) X-ray crystal structure of the C-terminal Golgi-dynamics domain (GOLD) of ACBD3 in complex with the cytoplasmic domain of the EV-A71 3A protein (PDB: 6HLW). The ACBD3 and 3A proteins are colored in grey and rainbow, respectively. (C) Chemical structure of EV-A71 3A inhibitors.

Figure 9

EV-A71 3C inhibitors. (A) Chemical structures of EV-A71 3Cpro inhibitors. (B) X-ray crystal structure of EV-A71 3Cpro in complex with rupintrivir (AG7088) (PDB: 3SJO). (C) X-ray crystal structure of EV-A71 3Cpro in complex with 3C-18p (PDB: 7DNC). Reprinted with permission from Ref. 157. Copyright © 2021, American Chemical Society. (D) X-ray crystal structure of EV-A71 3Cpro in complex with macrocyclic inhibitor 3C-4 (PDB: 6LKA).

Figure 10

EV-A71 3D polymerase inhibitors. (A) Chemical structures of EV-A71 3D polymerase inhibitors. (B) X-ray crystal structure of CV-B3 3Dpol in complex with BPR-3P0128 (PDB: 4Y2A).

Figure 11

EV-A71 IRES inhibitors. (A) Chemical structures of EV-A71 IRES inhibitors. (B) Solution NMR structure of EV-A71 IRES in complex with DMA-135 (PDB: 6XB7).

Figure 12

Host-targeting EV-A71 inhibitors.

Figure 13

EV-A71 inhibitors with unknown mechanism of action.