?.Design,Synthesis And Characterization Of Inhibitors Of P53-MDM2/MDMX Interactions?.Synthesis And Characterization Of Glycopeptides With Novel Glycans As Immunogens For Antibody Production

Malignant neoplasms have become one of the most common and serious diseases,threatening human life extremely.The interactions between tumor suppressor protein p53 and its negative regulators MDM2/MDMX are closely related to the occurrence and development of many tumors.Therefore,inhibition of p53-MDM2/MDMX interactions has become an important target in anticancer drug design.At present,MDM2/MDMX antagonists are mainly classified as peptides and small molecules.However,these two types of antagonists exhibit their own obvious shortcomings.Small molecular antagonists fail to showcase potent affinities to the taegrt proteins,and they also seldomly can bind with MDM2 and MDMX simultaneously,whereas two notable weaknesses of peptide antagonists are susceptibility to proteolytic degradation and poor membrane permeability.In the first section,we developed two five-point pharmacophore models for filtering the 2012 National Cancer Institute database,from which molecular docking was conducted to identify dual inhibitors.We found 38 virtual hits and subjected them to a fluorescence polarization-based competitive binding assay,resulting in 10 active compounds of different scaffolds.To further expand the chemical diversity of the initial hits,we performed a hit-based substructure search and identified NSC148171 from pharmacophore 1 as the most potent dual-specificity inhibitor with Ki values for MDM2 and MDMX at 0.62 and 4.6 m M.All hits were subjected to inhibition assay of cancer cellular vitality and showed anti-proliferative activity roughly correlated with their Ki values.This work not only yields several novel scaffolds for further structural and functional optimization of dual-specificity inhibitors of the p53-MDM2/MDMX interactions,but also showcases the power of our computational methods for small molecule anticancer drug discovery.In the second section,we obtained PMI-M3(LTFLEYWAQLMQ)as the arguably most potent peptide activator of p53 reported to date,with single-digit picomolar binding affinities for both MDM2 and MDMX.PMI-M3,substantially differing in amino acid sequence from its phage-selected parent peptide PMI,was three orders of magnitude more potent in binding with both MDM2 and MDMX.Structural studies suggested that systematic mutational analysis prevailed over structure-based rational design in this particular case as the latter would not have necessarily informed the sequence of PMI-M3 based on known structures of peptide-MDM2/MDMX complexes.Aided by the systematic mutations coupled with addictivity analysis,we reported an ultra-potent MDM2/MDMX antagonist,which provides a strong sequence and structural basis for the follow-up study.In the third section,we designed a series of novel stapled peptides based on DTC(dithiocarbamate)reaction,of which the binding affinities of PS3-1-3 and PS5-1-3 for both MDM2 and MDMX gained enhancements by one order of magnitude.It is worth pointing out that these novel stapled peptides displayed obvious advantages in the properties of proteolytic degradation and membrane permeability when compared to the parent PMI and solubility compared to regular stapled peptide.Structural studies revealed the detailed structure of these stapled peptides and their corresponding binding mode with MDM2 and MDMX.The analog derived from PS3-1-3 and PS5-1-3,termed as PS6-3,showed the significantly better inhibitory activities against tumor cell line HCT116 p53+/+ than HCT116 p53-/-,demonstrating its role as p53 activaotr for anticancer therapy.We firstly applied DTC reaction in the stapling stragety for the improvement of peptide drug availability and the corresponding novel staple peptides with DTC side chains exhibited moderate advantages when compared with the linear peptides.Such novel staple strategy can be extended in a variety of peptides.Finally,we designed a series of novel PMI hybrid analogs using D-amino acid mutation strategy,which can defense proteolytic degradation moderately and matained original binding affinities and helix.Of note,the inhibitory activitiy of PMI-1 aganist U87 tumor cells was significantly better than that of the positive control Nutlin-3 when encapsulated in liposomes.Above results shed light for exploring the structural basis of p53-MDM2/MDMX interfaces and developing new classes of MDM2/MDMX antagonists as p53 activators for anticancer therapy.Glycosylation is one of the most important protein post-translational modifications in the biological system.Abnormal glycosylation is often associated with many biological processes including viral and bacterial infections,cancer metastasis,inflammatory response,innate and adaptive immunity,and many other signaling pathways.It is difficult to eradicate bacterial infections owing to the abuse of antibiotics in clinical practice and such bacteria seriously threaten the health of human beings.Recently,two novel glycosylations were discovered and closely related to bacterial infection.One of them was arginine rhamnosylation of the transcriptional elongation factor(EF-P)in many bacteria including P.aeruginosa,S.oneidensis and N.meningitidis.EF-P could be activated once its Arg-32 was glycosylated,thereby mitigating the stasis of ribosome attachment in the polyproline sequence.The other novel glycosylation was serine heptosylation catalyzed by the heptosyltransferase family from Gram-negative bacterial pathogens(BAHT),which could induce the biosynthesis of bacterial autotransporter and mediate the adhesion of bacteria to host cells,thereby playing an important role in intestinal infection.Arginine rhamnosylation may be a potential target for the design of new antibiotics,and BAHT-mediated heptosylation also could provide important theoretical guidance for anti-adherence therapy as a new antimicrobial strategy.It is urgent to develop related specific antibody to further understand these particular glycosylations.As an important molecular tool,antibody plays an irreplaceable role in the detection of proteins.In the first section,we used NMR spectroscopy to show for the first time that the a anomer of rhamnose is attached to Arg32 of EF-P,demonstrating that the corresponding glycosyltransferase Ear P inverts the sugar of its cognate substrate d TDP-b-L-rhamnose.Based on this finding we describe the synthesis of an a-rhamnosylated arginine containing peptide antigen in order to raise the first anti-rhamnosyl arginine specific antibody(anti-Arg Rha).Using ELISA and Western Blot analyses we demonstrated both its high affinity and specificity without any cross-reactivity to other N-glycosylated proteins.Having the anti-Arg Rha at hand we were able to visualize endogenously produced rhamnosylated EF-P.Thus,we expect the antibody to be not only important to monitor EF-P rhamnosylation in diverse bacteria but also to identify further rhamnosyl arginine containing proteins.As EF-P rhamnosylation is essential for pathogenicity,our antibody might also be a powerful tool in drug discovery.In the second section,we constructed a seven-carbon sugar framework using Wittig reaction,and then asymmetric Sharpless oxidation affords the heptose with two absolute configurations at C-6.Mediated by glucoside and Trichloroimidate glycoreaction,we successfully obtained the serine heptose that is consistent with the nature glycosylated modification.Using the glycopeptide obtained by the solid phase peptide synthesis as a hapten,polyclonal antibodies were generated by animal immunization,which could specifically recognize serine heptosylation.Sebsequent western blot assay showcased the extremly specifiticy of antibodies produced by 6-D and 6-L heptosylated antigen.We firstly reported the synthesis of specific serine heptosylated peptides and the production of the corresponding antibodies,which provided a powerful molecular tool for the subsequent study of this special glycosylation,and the antibody itself have the potential to treat bacterial infection as a novel anti-adhesion stragtegy.The polyclonal antibodies obtained in this work not only enhance our understanding of arginine rhamnosylation and serine heptacosylation,but also help us to further indentify glycosylated modified proteins.It serves a powerful tool for the development of new highly effective antibody-based antibiotics and screening of small molecule antibiotics.