Screening And Bioactivity Of Small Molecule Inhibitors For Prenyltransferase

The antibiotic resistance becomes more and more serious owing to the abuse and overuse of antibiotics.Aspergillus is reported to be a conditional pathogenic fungus among the pathogens.Invasive aspergillosis（IA）is a life-threatening disease impacting immunocompromised individuals.Standard treatments of IA,including polyenes and azoles,suffer from high toxicity and emerging resistance,leading to the need to develop new antifungal agents with novel mechanisms of action.Similarly,Mycobacterium tuberculosis and Staphylococcus aureus.etc have evolved the resistance to some standard antibiotics（eg.rifampicin and methicillin）,and even there is some“superbugs”emerging in clinic without effective treatment.So,it is urgent to develop noval antibiotic with new mechanism of action.Prenyltransferase（PT）is an important target for antibiotics.For example,ergosterol biosynthesis is a classic target for antifungals,and squalene syn thase（SQS）catalyzes the first committed step in ergosterol biosynthesis in Aspergillus spp.SQS is a potential target to develop antifungals.Cis-long chain prenyltransferases（cis-LCPTs）are essential enzymes used in cell wall biosynthesis in all bacteria including pathogens such as M.tuberculosis and S.aureus.Because they are essential for bacterial survival and have highly conserved structure across many different species,cis-LCPTs are thought to be targets for antibiotic development.In this project,two important proteins of A.flavus and M.tuberculosis were taken as targets respectively to screen the potential inhibitors.The main contents are as follows:（1）Here,we cloned,expressed,purified and characterized SQS from the pathogen Aspergillus flavus（AfSQS）,confirming that it produced squalene.To identify potential leads targeting AfSQS,we tested known squalene synthase inhibitors,zaragozic acid and the phosphonosulfonate BPH-652,finding that they were potent inhibitors.We then screened a library of 744 compounds from the National Cancer Institute（NCI）Diversity Set V for inhibition activity.20 hits were identified and IC₅₀0 values were determined using dose-response curves.14 compounds that interfered with the assay were excluded and the remaining 6 compounds were analyzed for drug-likeness,resulting in one compound,celastrol,which had an AfSQS IC₅₀0 value of 830 nM.Enzyme inhibition kinetics revealed that celastrol binds to AfSQS in a noncompetitive manner,but did not bind covalently.Since celastrol is also known to inhibit growth of the highly virulent Aspergillus fumigatus by inhibiting flavin-dependent monooxygenase siderophore A（SidA,under iron starvation conditions）,it may be a promising multi-target lead for antifungal development.（2）To identify potential leads targeting cis-LCPTs we screened a library of 750compounds from the NCI Diversity Set V for inhibition activity against M.tuberculosis decaprenyl diphosphate synthase（cis-DPPS,Rv2361c,named as MtDPPS）.17 hits were identified and IC₅₀s were then determined using dose-response curves.Compounds were then tested for inhibition of cell growth against a panel of bacteria as well as for human cell toxicity and in vivo activity in a S.aureus/Caenorhabdytis elegans model.The most active compound found was benzobromanone which had IC₅₀¹7μM against MtDPPS and was active against Clostridiodes difficile（MIC²μg/mL）and S.aureus（MIC⁵μg/mL）and had low toxicity against HEK293T cells（¹00μM）and no effect on C.elegans survival while reducing S.aureus burden in C.elegans more effectively than did ampicillin.We also carried out a series of quantum chemical and molecular docking investigations aimed at probing the mechanism of cis-LCPTs inhibition.Overall,our results indicate that benzobromanone and redoxal act,at least in part,by targeting cis-LCPTs.