The use of Fpocket and virtual screening techniques enabled us to identify potential allosteric druggable pockets within the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Of the compounds screened, compound 1 was identified as a promising inhibitor, lowering a SARS-CoV-2 RdRp activity to 57 % in an enzymatic assay at 10 μM concentration. The structure of compound 1 was subsequently optimized in order to preserve or enhance inhibitory activity. This involved the substitution of problematic ester and aromatic nitro groups with more inert functionalities. The N,N'-diphenylurea scaffold with two NH groups was identified as essential for the compound's activity but also exhibited high toxicity in Calu-3 cells. To address this issue, a scaffold hopping approach was employed to replace the urea core with potentially less toxic urea isosteres. This approach yielded several structural analogues with notable activity, specifically 2,2'-bisimidazol (in compound 55 with residual activity RA=42 %) and (1H-imidazol-2-yl)urea (in compounds 59 and 60, with RA=50 and 28 %, respectively). Despite these advances, toxicity remained a major concern. These compounds represent a promising starting point for further structure-activity relationship studies of allosteric inhibitors of SARS-CoV-2 RdRp, with the goal of reducing their cytotoxicity and improving aqueous solubility.

• Klíčová slova
• RdRp; remdesivir, SAR study, SARS-CoV-2, allosteric inhibitor, scaffold hopping,
• MeSH
• alosterická regulace účinky léků   MeSH
• antivirové látky * farmakologie   chemie   chemická syntéza   MeSH
• inhibitory enzymů farmakologie   chemie   chemická syntéza   MeSH
• koronavirová RNA-replikasa antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• molekulární struktura MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   metabolismus   MeSH
• SARS-CoV-2 * účinky léků   enzymologie   MeSH
• simulace molekulového dockingu MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• inhibitory enzymů MeSH
• koronavirová RNA-replikasa MeSH
• RNA-dependentní RNA-polymerasa MeSH

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

• Klíčová slova
• RNA‐dependent RNA polymerase (RdRp), SARS‐CoV‐2, mutations, phenotypic effect,
• MeSH
• COVID-19 virologie   MeSH
• koronavirová RNA-replikasa * genetika   metabolismus   chemie   MeSH
• lidé MeSH
• mutace * MeSH
• RNA-dependentní RNA-polymerasa genetika   chemie   metabolismus   MeSH
• SARS-CoV-2 * genetika   enzymologie   MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny * genetika   chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• koronavirová RNA-replikasa * MeSH
• NS8 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP12 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP7 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA-dependentní RNA-polymerasa MeSH
• virové nestrukturální proteiny * MeSH

An enigmatic localized pneumonia escalated into a worldwide COVID-19 pandemic from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This review aims to consolidate the extensive biological minutiae of SARS-CoV-2 which requires decipherment. Having one of the largest RNA viral genomes, the single strand contains the genes ORF1ab, S, E, M, N and ten open reading frames. Highlighting unique features such as stem-loop formation, slippery frameshifting sequences and ribosomal mimicry, SARS-CoV-2 represents a formidable cellular invader. Hijacking the hosts translational engine, it produces two polyprotein repositories (pp1a and pp1ab), armed with self-cleavage capacity for production of sixteen non-structural proteins. Novel glycosylation sites on the spike trimer reveal unique SARS-CoV-2 features for shielding and cellular internalization. Affording complexity for superior fitness and camouflage, SARS-CoV-2 challenges diagnosis and vaccine vigilance. This review serves the scientific community seeking in-depth molecular details when designing drugs to curb transmission of this biological armament.

• Klíčová slova
• 2019-nCoV, COVID-19, RNA, bats, coronavirus, pandemic, virus,
• MeSH
• COVID-19 genetika   metabolismus   virologie   MeSH
• fylogeneze MeSH
• lidé MeSH
• otevřené čtecí rámce MeSH
• pandemie MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 genetika   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• RNA virová MeSH
• virové proteiny MeSH