Application Of Drug Repurposing Strategy In The Research And Development Of New Drugs Against Malaria And Rare Disease Conjunctival Melanoma

This doctoral dissertation focus on the repurposing of launched drug or clinical drug candidate.In this dissertation,based on the anti-tumor clinical drug candidate Quisinostat as lead compound,novel anti-malarial HDAC inhibitors have been elaborately designed,synthesized and their antimalarial effects,safety,druggability and mechanism of action have also been systematically evaluated.Meanwhile,the anti-arrhythmia drug Propafenone was identified as a novel anti-conjunctival melanoma agent and primary structure modifications of Propafenone have also been performed.Overall,this doctoral dissertation consists of 2 Sections.Section 1.Design,synthesis and biological investigations of novel anti-malarial HDAC inhibitors based on Quisinostat.Malaria is a global virulent infectious disease induced by Plasmodium spp.,among which Plasmodium falciparum(Pf)have caused the most severe endemic and devastation.Since the growing threat of drug-resistant malaria,anti-malarial drugs endowed with novel structures and mechanisms of action and multi-stage efficacy are urgently needed in clinic.Among the published results of several research groups,Plasmodium falciparum histone deacetylase(PfHDAC)have demonstrated the potential to become novel anti-malarial drug target.Furthermore,Professor Jiang Lubin’s research group at the Institut Pasteur of Shanghai have also discovered Quisinostat,a clinical phase-II anti-tumor HDAC inhibitor,as a potent multi-stage anti-malarial agent.Although the severe cytotoxicity and narrow anti-malarial therapeutic window of this anti-tumor agent have prevented it from direct anti-malarial clinical application,Quisinostat could still be envisaged as an excellent lead compound of structure modification.Therefore,in this section we performed anti-malarial structure modification based on Quisinostat as lead compound,aiming at enhancing the safety of derivatives while reserving the original potency of Quisinostat.According to the structural feature of HDAC inhibitors and the molecular docking results of Quisinostat with PfHDAC1,the structure of Quisinostat can be divided into three part:the Zn2+-binding group(ZBG),linker and CAP group.In the preliminary stage,based on the scaffold hopping strategy we synthesized six novel diamine linker derivatives A1～A6 while the pyrimidyl hydroxamic acid(ZBG)and N-methylindolyl(CAP)groups of Quisinostat were reserved.Based on two derivative A2 and A5 which displayed enhanced selectivity toward human cells,we further synthesized 69 novel derivative B1-B39 and C1-C30 via elaborate modification of CAP groups.Among these derivatives,compounds B35,B39 and C9 displayed anti-malarial potency with inhibitory IC50 values of 11.3 nM,11.5 nM and 3.19 nM against erythrocytic wild-type P.falciparum 3D7 respectively,which were similar to that of Quisinostat(IC50(3D7)=5.2 nM).Meanwhile the selectivity of B35,B39 and C9 toward two human cells(HepG2 and 293T)were also enhanced by 60～80,100～140,8～10 folds compared to that of Quisinostat.These results demonstrated that the safety of novel derivatives could be significantly improved by structure modifications.In the further researches,B35,B39 and C9 suppressed the proliferation of several multi-drug resistant clinical P.falciparum isolates without demonstration of cross-resistance with common anti-malaria drugs.Especially ring-stage survival assays further proved the potent inhibition of C9 against two Artemisinin-resistant P.falciparum isolates 6218 and 6320.These results demonstrated the potential of our novel derivatives in overcoming the clinical drug-resistant malaria.B35,B39 and C9 displayed enhanced metabolic stability and several pharmacokinetic properties compared to that of Quisinostat in the liver microsome evaluations and mouse pharmacokinetic assays,and they also demonstrated improved in vivo safety among mouse acute toxicity assay and other experiments.In several in vivo therapeutic efficiency assays,B35 and B39 displayed partial erythrocytic therapeutic effect at the dosages of 75～150 mg/kg,and C9 could not only cure mouse erythrocytic P.yoelii infection at the dosage of 60 mg/kg,but also partially cure mouse liver-stage P.berghei infection at the dosage of 30 mg/kg,while the mouse survival was not affected at the effective dosages.These results preliminarily displayed the potential multi-stage(erythrocytic and liver-stage)therapeutic effects of C9.In the erythrocytic stage-specific parasite-killing assay C9 showed similar properties to Quisinostat in eliminating both ring,trophozoite and schizont erythrocytic parasites with most potent activity against schizont.To investigate the anti-malarial mechanism of action of new derivatives,we performed the Western blot experiments to detect the change of acetylation level in P.falciparum histone H3 after compound treatment,via which B35,B39 and C9 proved to be PfHDAC inhibitors.We also constructed two PfHDAC1/2 gene knock-down parasites using glmS ribozyme,and further demonstrated PfHDAC1 as the anti-malarial target of C9 via inhibitory sensitivity assays against these two gene knock-down parasite.Inhibiton of recombinant PfHDAC1 in vitro also displayed that the IC50 value of C9 was 0.34 nM,which was stronger than other reported PfHDAC1 inhibitors and only weaker than Quisinostat(IC50=3 pM).Finally,human HDAC inhibition assay showed that C9 significantly suppressed four class-I hHDACs with similar potency to Quisinostat,and the inhibitory effects of B35 and B39 toward class-I hHDACs declined by 10～20 folds.Overall,in this section 75 pyrimidyl hydroxamic acid derivatives were synthesized and their therapeutic effects and druggability in vitro and in vivo were also systematically evaluated.Among these derivatives,we finally identified compound C9 as a novel PfHDAC1 inhibitor with multi-stage(erythrocytic and liver-stage)therapeutic effects and potency against multi-drug resistant P.falciparum.Our results further demonstrated the potential of PfHDAC1 as a novel anti-malarial drug target.Currently structure modification and mechanism study of C9 are in progress.Meanwhile,we also discovered the pan-PfHDAC inhibitors B35 and B39 with significantly improved safety and partial anti-malarial activities,which were also endowed with potential for further researchSection 2.Design,synthesis and biological investigations of novel anti-conjunctival melanoma derivatives based on PropafenoneConjunctival melanoma(CM),a life-threatening malignant ocular tumor,is a rare disease only gradually gaining attention in recent studies.Currently neither widely accepted clinical option nor effective chemotherapy for CM treatment is available,and systematic novel drug development and clinical research report for CM is also absent.Therefore,discovering safe and effective novel anti-CM drugs is an imminent task.In previous research,we have systematically evaluated the anti-proliferation potency of our drug library against CM cells with the collaboration of Professor Jia Renbing’s research group at the Shanghai Ninth People’s Hospital.From our drug library we first identified the novel anti-CM application of type 1C anti-arrhythmic drug Propafenone hydrochloride.However,the insufficient anti-CM activities of Propafenone could not fulfill the clinical demand.To promote the development of novel anti-CM drug,in this section we further performed anti-CM repurposing of Propafenone,aiming at enhancing the safety and anti-CM potency of new derivatives via structure modification.We divided the structure of Propafenone into three domains,and synthesized 46 derivatives D1～D46 through three-stage structural modifications.In vitro studies revealed that the antiproliferation activities of compounds D33 and D34 against CRMM1 cell(IC50 values were 0.57 and 0.13 μM)were enhanced by 43 and 190 folds compared to Propafenone(IC50(CRMM1)=24.70(μM),meanwhile the selectivity of D33 and D34 toward human PIG1 cell(SI values were 14.5 and 19.1)also improved by 6.3 and 8.3 folds compared to Propafenone(SI=2.3).Overall,in this section we preliminarily improving the in vitro anti-CM potency and safety of new derivatives through structural modification of Propafenone as lead compound.Further structure modification and mechanism study based on new derivative are currently in progress.