The potential for zoonotic transmission and global spread demonstrated by the pandemic SARS-CoV-2, and its burden on public health, have emphasized the critical need to develop highly efficacious... Show moreThe potential for zoonotic transmission and global spread demonstrated by the pandemic SARS-CoV-2, and its burden on public health, have emphasized the critical need to develop highly efficacious strategies for prophylaxis and therapy of infections with coronavirus at large. This thesis was largely dedicated to the search of coronavirus inhibitors by phenotypic cell-based screenings using different classes of compounds including immunosuppressive and non-immunosuppressive derivatives of cyclosporin A, hits from FDA-approved drug libraries and molecules synthesized by collaborators. Although, no effective therapies were found, this work warranted the further clinical investigation of a non-immunosuppressive compound in SARS-CoV-2-infected kidney transplant recipients, which is currently in progress. The development of antiviral therapies requires a detailed understanding of CoV replication and its interplay with host cells. Here, an in-depth characterization of the viral replicase subunit non-structural protein 14 provides evidence for its importance for virus viability and fitness, while establishing that nsp14 might be a good target for drug design with a potential pan-coronaviral activity spectrum. In the discussion, the history of unsuccessful CoV-targeting antivirals is briefly summarized, together with possible new approaches in antiviral research. Lastly, some prospects for future research are outlined. Show less
Kovacikova, K.; Gorostiola González, M.; Jones, R.; Requera, J.; Gigante, A.; Pérez-Pérez, M.J.; ... ; Hemert, M.J. van 2021
Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising... Show moreChikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-β-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target. Show less
