The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.

Efforts to mitigate the COVID-19 pandemic include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer cells (unmodified Calu-1 and A549). The adapted virus exhibited mutations in the spike protein, including a 9 amino acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6 cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to SR-B1 receptor blocking and adapted-SARS2 exhibited significantly less dependency of ACE2. Both variants were similarly neutralized by COVID-19 convalescent plasma but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of nucs. Among the tested nucs, only remdesivir, molnupiravir and to a limited extent galidesivir, showed antiviral effect across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations in vivo, the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.

The oral protease inhibitor nirmatrelvir is expected to play a pivotal role for prevention of severe cases of coronavirus disease 2019 (COVID-19). To facilitate monitoring of potentially emerging resistance, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir. Resistant variants selected in cell culture harbored different combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetic studies in a homologous infectious cell culture system revealed up to 80-fold resistance conferred by the combination of substitutions L50F and E166V. Resistant variants had high fitness increasing the likelihood of occurrence and spread of resistance. Molecular dynamics simulations revealed that E166V and L50F+E166V weakened nirmatrelvir-Mpro binding. The SARS-CoV-2 polymerase inhibitor remdesivir retained activity against nirmatrelvir resistant variants and combination of remdesivir and nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatment programs with high efficacy against SARS-CoV-2 and potentially emerging coronaviruses.

The glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1, liraglutide has an added fatty acid (FA) moiety that causes oligomerization of liraglutide as suggested by small-angle x-ray scattering (SAXS) and multiangle static light scattering (MALS) results. SAXS data suggested a global shape of a hollow elliptical cylinder of size hexa-, hepta-, or octamer, whereas MALS data indicate a hexamer. To elaborate further on the stability of these oligomers and the role of the FA chains, a series of molecular-dynamics simulations were carried out on 11 different hexa-, hepta-, and octameric systems. Our results indicate that interactions of the fatty acid chains contribute noticeably to the stabilization. The simulation results indicate that the heptamer with paired FA chains is the most stable oligomer when compared to the 10 other investigated structures. Theoretical SAXS curves extracted from the simulations qualitatively agree with the experimentally determined SAXS curves supporting the view that liraglutide forms heptamers in solution. In agreement with the SAXS data, the heptamer forms a water-filled oligomer of elliptical cylindrical shape.

The lipidation of peptide drugs is one strategy to obtain extended half-lives, enabling once-daily or even less frequent injections for patients. The half-life extension results from a combination of self-association and association with human serum albumin (albumin). The self-association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration-dependent self-association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin-detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin-degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size-exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin-detemir and albumin-degludec mixtures are proposed.

Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log10 increase in infectivity titers (TCID50) in Huh7.5 cells, with titers of ~8 Log10TCID50/mL, and >2-Log10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log10TCID50/mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log10TCID50/mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity.ImportanceThe cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.

Insulin detemir is a lipidated insulin analogue that obtains a half-life extension by oligomerization and reversible binding to human serum albumin. In the present study, the complex between a detemir hexamer and albumin is investigated by an integrative approach combining molecular dynamics (MD) simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations and dynamic light scattering (DLS) experiments. Recent reported small angle X-ray scattering data could not unambiguously resolve the exact binding site of detemir on albumin. We therefore applied MD simulations to deduce the binding site and key protein-protein interactions. MD simulations were started from initial complex structures based on the SAXS models and free energies of binding were estimated from the simulations by using the MM-PBSA approach for the different binding positions. The results suggest that the overlapping FA3-FA4 binding site (named FA4) is the most favorable site with a calculated free energy of binding of -28±6 kcal/mol and a good fit to the reported SAXS data throughout the simulations. Multiple salt bridges, hydrogen bonds and favorable van der Waals interactions are observed in the binding interface that promote complexation. The binding to FA4 is further supported by DLS competition experiments with the prototypical FA4 ligand, ibuprofen, showing displacement of detemir by ibuprofen. This study provides information on albumin-detemir binding on a molecular level, which could be utilized in a rational design of future lipidated albumin-binding peptides.

The sirtuins are NAD+-dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biology, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ε-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms; in part, because of a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors and de novo cyclic peptide inhibitors selected from mRNA-display library screening; compounds that represent starting points for development of the needed SIRT7-specific chemical tools.