RESUMEN
A triazolylsialoside-human serum albumin conjugate was prepared as a multivalent hemagglutinin and neuraminidase inhibitor using a di-(N-succinimidyl) adipate strategy. Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) indicated that five tetravalent sialyl galactosides were grafted onto the protein backbone resulting in an eicosavalent triazolylsialoside-protein complex. Compared with monomeric sialic acid, molecular interaction studies showed that the synthetic pseudo-glycoprotein bound tightly not only to hemagglutinin (HA)/neuraminidase (NA) but also to mutated drug-resistant NA on the surface of the influenza virus with a dissociation constant (KD) in the 1 µM range, attributed to the cluster effect. Moreover, this glycoconjugate exhibited potent antiviral activity against a broad spectrum of virus strains and showed no cytotoxicity towards Human Umbilical Vein Endothelial Cells (HUVECs) and Madin-Darby canine kidney (MDCK) cells at high concentrations. Further mechanistic studies demonstrated this multivalent sialyl conjugate showed strong capture and trapping of influenza virions, thus disrupting the ability of the influenza virus to infect host cells. This research lays the experimental foundation for the development of new antiviral agents based on multivalent sialic acid-protein conjugates.
Asunto(s)
Gripe Humana , Animales , Perros , Humanos , Antivirales/química , Células Endoteliales/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Hemaglutininas/metabolismo , Células de Riñón Canino Madin Darby , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Albúmina Sérica Humana , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Proteínas Virales/metabolismo , Virión/metabolismoRESUMEN
A guanidinothiosialoside-human serum albumin conjugate as mucin mimic was prepared via a copper-free click reaction. Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) indicated that three sialoside groups were grafted onto the protein backbone. The synthetic glycoconjugate exhibited strong influenza virion capture and trapping capability. Further mechanistic studies showed that this neomucin bound tightly to neuraminidase on the surface of influenza virus with a dissociation constant (KD) in the nanomolar range and had potent antiviral activity against a broad spectrum of virus strains. Most notably, the glycoconjugate acted as a biobarrier was able to protect Madin-Darby canine kidney (MDCK) cells from influenza viral infection with 50% effective concentrations (EC50) in the nanomolar range and showed no cytotoxicity towards Human Umbilical Vein Endothelial Cells (HUVEC) at high concentrations. This research establishes an attractive strategy for the development of new multivalent antiviral agents based on mucin structure. Moreover, the method for the functionalization of the natural biological macromolecular scaffold with bioactive small molecules also lays the experimental foundation for potential biomedical and biomaterial applications.
Asunto(s)
Antivirales/química , Bencenosulfonatos/química , Bencenosulfonatos/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Albúmina Sérica Humana/uso terapéutico , Animales , Antivirales/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Química Clic , Perros , Hexanos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Gripe Humana/virología , Ligandos , Células de Riñón Canino Madin Darby , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Mucinas , Neuraminidasa/metabolismo , Albúmina Sérica Humana/química , Virión/efectos de los fármacosRESUMEN
We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses. Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS), virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced agglutination of viral particles, the high capture efficiency and affinity constant indicate that this neoprotein is comparable to natural glycosylated mucin, suggesting that this material could potentially be used as anti-infective barriers to prevent virus from invading host cells. The study also rationalizes the feasibility of antiviral drug development based on OA or other antiviral small molecules conjugated protein strategies.