Discovery And Development Of Subtype-selective Covalent Pyruvate Dehydrogenase Kinase Inhibitors And Candidate Drug For Anti-drug-resistance Staphylococcus Aureus

There are two parts in my dissertation for Ph.D.degreee.Part 1 presents the discovery of subtype-selective covalent pyruvate dehydrogenase kinase 1(PDK1)inhibitors.Unlike their normal counterparts,most cancer cells feature a unique metabolic profile of high aerobic glycolysis,which also known as "Warburg effect".It describes the phenomenon of the enhanced conversion of glucose into lactate even in the presence of oxygen,which contributes to tumor initiation and progression.Aerobic glycolysis confers a significant growth advantage of cancer cells not only by supplying essential ATP production and generating biosynthesis precursors for cell growth,but also by providing reducing equivalents for antioxidant defense and generating lactate for micro environmental maintenance.Mitochondrial pyruvate dehydrogenase kinase(PDK)functions as a molecular switch in glucose metabolism that activates aerobic glycolysis and diminishes mitochondrial respiration via phosphorylating and inactivating pyruvate dehydrogenase(PDH),a gate-keeping mitochondrial enzyme.To date,four pyruvate dehydrogenase kinase isoenzymes(PDK1-4)have been identified in mammals with tissue-specific expression.PDK1 is closely associated with cancer malignancy,including controlling metabolic and malignant phenotypes,promotion of the Warburg effect and tumor growth,lowering the metastatic potential.These evidences suggest that the inhibition of PDK 1 may offer a new therapeutic option targeting cancer glucose metabolism for the treatment of cancer.Before our recent works,three major classes of PDK inhibitors have been reported and distinguished by different binding sites.The well-known PDK inhibitor dichloroacetate(DCA),an analog of pyruvate,belongs to the first class.However,the high effective dosage(100 mg/kg)limits its further clinical application.Nov3r,AZD7545 and CPI613 represent a class of PDK inhibitors that target the lipoyl-binding pocket of the enzymes among which only CPI613 demonstrates certain anticancer effects,but the homologous structure to lipoate may cause some expectable side-effect.Radicicol and M77976 belong to a class of ATP-competitive inhibitors.However,the high similarity of the ATP-binding pocket of kinases may lead to off-target effects,resulting in side effects and toxicity issues.In our previous works,we carried out a PDK1 enzymatic screen using an in-house library of-600 old drugs in our team,and identified JX06 as the first covalent inhibitor of PDK1.Using JX06 as a chemical probe,we found that the covalent inhibitory action mode of JX06 was through targeting a highly conserved cysteine residue(Cys240,a new interaction site)and illustrated the mechanism of covalent inhibition.In this thesis,based on the therapeutic potential of JX06,to obtain novel analogs and explore the SAR of the JX06 analogs,we designed two types of novel disulfide-based analogs(la-m,2a-s,3a-j),derived from JX06.Firstly we tested the IC50 values of the forty-two analogues against PDK1,and obtained preliminary SARs on IC50 data and identified twenty-two analogs(lc-d,1f-m,2a-d,2f,2i,2j-k,2m-n,2p,and 3a)with potent PDK1 inhibitory activity(IC50<0.3 ?M).Considering the specialty of covalent inhibitors,IC50 value alone may be not sufficient for SAR discussion.In order to validate the SAR concluded above,we introduced the second-order rate constant of target inactivation(kinact/Ki)as the quantitative assessment of potency,which were well consistent with IC50.Encouraged by the favorable PDK1 inhibitory activity in the biochemical kinase activity assay,we assessed the cellular activity of twenty-two best analogs using immunoblotting analysis.Compounds 2k and 3a showed best activities on both site of S232 and S293,and they represent the best combination of SAR factors.The following pharmacology experiments of 2k and 3a showed same covalent inhibition to PDK1 and similar changes to the metabolism to that of JX06.Due to the most significant role of PDK1 among the PDK subtypes in cancer therapy,we measured the kinact/Ki value of JX06,2k and 3a against all PDK isoforms using a biochemical enzymatic assay.Compound 3a was the most potent inhibitor of PDK1(IC50=26 nM,kinact/Ki=5.14×103 M-1s-1),and exhibited-40-fold selectivity towards PDK1 over PDK2 and 3,better than 2k and even 10-fold improvement than that of 16.To better understand the binding mode of JX06 analogues with PDKs,we carried out molecular docking of JX06,2k and 3a with PDK 1-4,respectively.Detailed analysis of molecular docking of JX06,2k and 3a with PDK 1-4 gained a better understanding of the subtype-selectivity for PDK 1-4 of these compounds.Moreover,the in vitro antiproliferative assays showed that 3a had more potent inhibitory effects than that of JX06 on the proliferation of two human cancer cell lines(A549:IC50=0.225?M and Kelly:IC50 0.057 ?M),without inhibition activity against normal cell lines GM00637 and L02(IC50>10 ?M).To further prove the therapeutic potential of compound 3a,we compared antitumor efficacy of JX06,2k and 3a in A549 subcutaneous xenograft mice models.A 21-day continuous treatment of JX06 and 3a considerably reduced tumor volume compared with the vehicle control(JX06,TGI = 50.4%@ 80 mg/kg;3a,TGI ?47.8%@ 80 mg/kg).Likewise,endpoint tumor weights in 3a treated group were significantly less than those treated with vehicle control.Altogether,these data demonstrate that 3a may be a promising starting point for the discovery of potential therapeutic drugs to specifically inhibit PDK1.Considering the small number of PDK1 inhibitors reported thus far,3a could be regarded as the first generation of subtype-selective and covalent PDK1 inhibitors for chemical biology study.Part 2 presents the development of candidate drug for anti-drug-resistance Staphylococcus aureus.In the previously works of our team,we discovered naftifine hydrochloride,an antifungal drug,could inhibit the enzymatic activity of CrtN,and exhibited the antibacterial efficacy(pre-infection administration)in vivo.In this thesis,Base on the structure of C13.,a naftifine analogue as CrtN inhibitor reported by our group,we attempted to perform chemical modification to reduce the inhibition of hERG potassium channel,and to explore antibacterial efficacy(post-infection administration)in vivo.We hoped to identify a good candidate drug for anti-drug-resistance Staphylococcus aureus.Firstly,by scaffold hopping,we replaced the benzofuran scaffold in C13 by indole scaffold,chemical modifications were performed in four chemical modification regions of the lead compound C13.Based on the in vitro pigment inhibitory activities,a preliminary analysis revealed some noteworthy observations of the SAR.Compound 44 was identified with the most potent pigment inhibitory activities(IC50=3.3 nM).As expected,the pigment inhibitory activities of compound 44 against three MRSA strains(USA300.USA400 and Mu50)are comparable with that against S.aureus Newman.Additionally,neither the growth characteristics nor the survival of the Newman or MRSA strains was suppressed when incubated with 44(up to 0.2 mM).Thus,we could conclude that 44 inhibited the production of pigment rather than inhibited the survival of the bacteria.Similar to naftifine,a HPLC experiment was conducted and verified that CrtN is the target of the indole analogue 44.To further explore potential therapeutic target,44 was assayed for CrtN enzymatic inhibitory activity using our previous protocol,and showed submicromolar inhibition against CrtN.We next determined whether the inhibition of pigment of all strains translated to susceptibility of the four colonies to innate immune clearance using two assay systems:hydrogen peroxide killing and human whole blood killing.The results suggested that 44 indeed make S.aureus more susceptible to immune clearance in hydrogen peroxide and human blood.Next,we extended our assessment on the contribution of 44 to abscess formation in a systematic S.aureus Newman infection model.The 44-treated group effectively reduced bacterial survival in the livers,kidneys and hearts(inhibitory rate>95%)not only in pre-infection administration but aslo post-infection administration group.These results clearly demonstrated that in vivo 44 treatments attenuated the pathogenicity of S.aureus.The efficiency of 44 in lethal S.aureus infection model was further evaluated;the untreated mice died out within 4 days postchallenge,while 44 exhibited the better protective effect than naftifine,resulting in 60%animal survival.Different from naftifine,44 abated the anti-fungal activities and performed good selective antibacterial efficacies.Finally,we tested the hERG inhibition activity of 44,which showed 44 possessed no hERG inhibition activity(IC50>40 ?M),10-fold less than that of C13(IC50 = 3.7 ?M).At present,we are exploring if 44 is also effective against MRS A in vivo.The above encouraging results showed that our structural modification was successful.Compound 44 not only maintained good in vitro and in vivo antibacterial activities similar to C13,but also significantly improved the safety profile(hERG).In the newly designed animal experiments,44 can still exhibit a stable antibacterial efficacy in post-infection administration mode,which provides a more convincing data for subsequent preclinical research.Thus the anti-virulence active compound 44 with novel scaffold structure is of good development advantages,and could be carried out in the progress of further structural optimization and druggability evaluation as an anti-MRS A drug candidate.