RESUMEN
Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.
Asunto(s)
Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Antivirales/farmacología , Antivirales/química , Química Farmacéutica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Cristalografía por Rayos X/métodos , Química Clic/métodos , Animales , Virus de la Influenza A/efectos de los fármacos , Células de Riñón Canino Madin Darby , Inhibidores de Proteínas Virales de Fusión/farmacología , Inhibidores de Proteínas Virales de Fusión/química , PerrosRESUMEN
Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a "link-and-release" two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3-phosphohistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.
Asunto(s)
Dominio Catalítico , Modelos Moleculares , Fosfolipasa D , Fosfolipasa D/metabolismo , Fosfolipasa D/química , Fosfolipasa D/genética , Humanos , Especificidad por Sustrato , Cristalografía por Rayos X , Mutación , Lisosomas/metabolismo , Lisosomas/enzimología , Fosforilación , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/química , Multimerización de Proteína , Unión Proteica , ExodesoxirribonucleasasRESUMEN
Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.