| Influenza virus is a respiratory pathogen which can give rise to substantial socio-economic loss and burden on medical treatment due to its rapid global spread and high mortality.Influenza spreads around the world in yearly outbreaks,resulting in about 3 to 5 million cases of severe illness and approximately 250,000 to 500,000 deaths.Therefore,the prevention and treatment of influenza infection has become an urgent problem to public health all over the world.Neuraminidase(NA)is functionally indispensable in the lifecycle of influenza virus and becoming a great target for anti-influenza drugs design.By now,four NA inhibitors have been licensed.However,the widespread drug-resistant issue and inconvenient administration of these drugs are still a concern for years.Consequently,it is imperative to develop novel NA inhibitors to overcome drug resistance and to treat influenza infection effectively.According to new research,the amino acids Arg430-Thr439 which are adjacent to the active site of NA are highly flexible and form a large pocket named as 430-cavity.This cavity is directly connected with active site and has large molecular volumes,making it a promising binding site for NA inhibitors design.The crystal structure of oseltamivir carboxylate bounding with NA revealed that the C-1 carboxyl group was well exposed toward the newly discovered 430-cavity and could serve as the potential modification site for designing specific inhibitors to improve the antiviral efficacy,anti-resistance profiles and to react with various of NA subtypes.Based on the above analysis and "Multiple-Sites Binding" concept,in this research,our proposed strategy is to extend the structure of C-1 side chain of oseltamivir by attaching additional groups of suitable shape,size,and hydrophobicity to fill the 430-cavity,making the compounds can occupy the active site and 430-cavity at the same time to achieve"Multiple-Sites Binding" effect and to strengthen the affinity with NA.Meanwhile,this modification can make the side chain interact with the residues around the 430-cavity and form additional action to compensate the affinity loss caused by amino acids mutation to enhance anti-resistance profiles.In the research we have designed and synthesized 38 C-1 modified oseltamivir analogs and evaluated their biological activity.This dissertation is divided into five chapters,mainly containing three parts,which are as follow:Part 1:It has been reported that 430-cavity is composed of many hydrophobic amino acids,thus we introduce various hydrophobic substituents into the scaffold of oseltamivir to fill the 430-cavity,hoping the side chain could form hydrophobic interactions with these amino acids.We obtained 21 target compounds and tested their inhibitory activities against H5N1 and H5N6 NAs.The result showed that most derivatives in this series can inhibit the enzyme,and compound A-17 which had a biphenyl group was the greatest inhibitor(H5N1:IC50=0.96?M;H5N6:IC50=0.99 ?M),showing equal inhibition to both NA subtypes.,The docking analysis indicated the biphenyl group increased the length of the side chain and could occupy the 430-cavity well,and this is the reason for NA inhibitory activity of A-17 being superior to that of other compounds.Part 2:On the basis of the work in the first part,we continued improving the interaction between the compounds and 430-cavity by increasing the length of C-1 side chain and enhancing the interaction at linker region.We decided to introduce the 1,2,3-triazole group which has a length of 5 A into the side chain through Click-reaction.This fragment can form hydrogen-bonding interactions with Arg118,Arg371 and Arg152 in the channel of 430-cavity,because it is both H-bond donor and H-bond acceptor.Eventually,we have designed and synthesized 11 target compounds.Anti-NA activities of these compounds showed that most compounds were potent against H5N1 and H5N6 NAs,especially B-5(H5N1:IC50=0.24 ?M;H5N6:IC50=0.18 ?M)was more powerful than series A.But its potency was lower than positive drug OSC,including the oseltamivir-resistant H274Y-H5N1 NA.Part 3:The loss of hydrogen-bonding and salt bridge between target compounds and Arg118,Arg371 and Arg152 is the main reason for the decline of inhibitory activity in part 1 and 2.To overcome this issue,in this part,we introduced kinds of amino acids into the C-1 side chain,because amino acids can remain the hydrogen-bonding interaction with the tree arginine and extend into the 430-cavity at the same time.Subsequently,we have designed and synthesized 6 target compounds.Among them,compound C-2 exerted the greatest inhibition,with IC50 values(H5N1:IC50=0.088 ?M;H5N6:IC50=0.096 pM)and EC50 values(H5N1:EC50=4.26 ?M;H5N6:EC50=1.31?M)are similar to OSC.And its potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC.Notably,although compound C-2 was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model,it displayed greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L.Overall,according to the novel discovered 430-cavity,we used "Multiple-Sites Binding" strategy to design and synthesize three classes of 38 compounds.This exploratory process targeting 430-cavity is logical and step-by-step,and eventually we found compound C-2 which has similar inhibitory activity against influenza virus to lead compound.We believe this work will provide valuable information for the development of anti-influenza drugs research. |
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