Design,Synthesis And Evaluation Of Anti-inflammatory Activity In Vitro And In Vivo Of Novel Non-peptidyl Non-covalent Cathepsin C Inhibitors

Polymorphonuclear neutrophils(PMNs)are the first immune cells recruited to the site of inflammation and constitute the earliest defense line of immune system against the invasion of pathogenic microorganisms.One of the major mechanisms for PMNs to exert immune protection is the secretion of neutrophil serine proteases(NSPs),including neutrophil elastase(NE),cathepsin G(Cat G),and protease 3(PR3).NSPs play regulatory roles in inflammatory diseases.Excessive secretion of active NSPs caused by the accumulation and activation of PMNs leads to tissue damage and inflammation.NSPs are involved in the progression of a variety of inflammatory diseases,such as sepsis,acute pancreatitis,rheumatoid arthritis,and chronic respiratory disorders,including chronic obstructive pulmonary disease(COPD),non-cystic fibrosis bronchiectasis(NCFBE)and Corona Virus Disease 2019(COVID-19).Therefore,NSPs are considered as biological targets for the treatment of PMN-related inflammatory diseases.Drugs that directly target NSPs are the most direct way to regulate the NSPs activity,but there are many difficulties in the actual drug development.This prompted researchers to find an indirect mean and a therapeutic target to inhibit the activation of NSPs.Cathepsin C(CatC),an important lysosomal cysteine protease,mediates the maturation process of NSPs and participates in the inflammation and immune regulation process associated with PMN.Therefore,CatC is considered to be an attractive target for the treatment of PMN-related inflammatory diseases and autoimmune diseases,and drug development with CatC as a therapeutic target has a broad prospect.CatC inhibitors have been developed for more than thirty years.Almost all inhibitors are peptide derivatives containing an electrophilic“warhead”moiety that can form a covalent bond with Cys234.However,no CatC inhibitor has been successfully marketed as a drug.The main reason is:the metabolic instability and potential off-target toxicity caused by the highly reactive electrophilic“warhead”structure and the peptide-based structure.During the development of novel CatC inhibitors,in order to avoid the defects of peptidyl-covalent derivatives and explore the possibility of developing CatC inhibitor with new mechanism of action,we proposed the concept of“non-peptidyl non-covalent CatC inhibitor”.We decided to develop this new type of CatC inhibitor,hoping that while avoiding the poor metabolic stability and potential safety hazards caused by electrophilic“warhead”moiety,non-peptidyl non-covalent CatC inhibitor should inhibit the CatC activity and the activation of downstream NSPs and also can exhibit effective in vivo anti-inflammatory activity.In this study,the complete discovery process of the first potent non-peptidyl non-covalent CatC inhibitor was described involving the hit discovery,medicinal chemistry optimization,structure-based design,acute toxicity study,pharmacokinetic(PK)study,in vivo CatC and NSP inhibitory activity,and anti-inflammatory activity.Using enzymatic activity assay,we screened the in-house compound library and found hit 14 was identified as a preliminary hit with moderate inhibitory activity against CatC,with an IC₅₀of 985 n M.Through medicinal chemistry optimization,structure-based drug design and structure-activity relationship(SAR)study,from this,hit 14 led to the discovery of the lead compound 54,which showed a strong inhibitory activity against CatC,with an IC₅₀of 57 n M.The docking study pointed out that the binding mode of 54 with CatC was different from that of any covalent CatC inhibitors previously reported,which may be attributed to the absence of electrophilic“warhead”moiety.The compound was embedded in the S1 site and S2 pocket and maintained the binding with active site through multiple types of interactions.In order to verify the cysteine cathepsin family selectivity of non-peptidyl non-covalent CatC inhibitors,we tested the inhibitory effect of compound 54 on multiple cysteine cathepsins,which met the expected target.Compound 54 showed extremely high selectivity within the cysteine cathepsin family.Cellular thermal shift assay study indicated that compound 54 increased the temperature tolerance of CatC in a concentration-dependent manner,indicating that it can bind to the target well.The prolonged pharmacological inhibition of CatC leads to the elimination of NSPs.In order to evaluate the effect of compound on the active NSPs,we detected the inhibitory effects of compound 54 on CatC and active NSPs.In HL-60cells,compound 54 inhibited CatC and NSPs activities in a dose dependent manner.Next,we evaluated the stability of NSPs in C57BL/6 bone marrow cells treated with compound 54 by western blotting.The results showed that after 48 h,NE,Cat G,and PR3 were degraded to 47.8,27.8,and 19.9%of the original levels,respectively.These results indicated that compound 54 can inhibit the CatC activity and the activation of NSPs in cells.Further physicochemical property research and PK research investigated the drug property of the compound 54.The compound in water,phosphate buffered saline(PBS),artificial gastric juice,and artificial intestinal juice was determined as 0.035,0.034,6.500,and 0.016 mg/m L,respectively.The experimental Log P was found to be 1.87,which follows the Lipinski rule of five.In Caco-2 permeability experiment,this compound exhibited moderate permeability and no obvious efflux phenomena with the apparent permeability coefficient(P_app)value of 1.6×10^-6cm/s.54 showed acceptable stability against the liver microsome in both human and mice.The effects of 54 against Cytochrome P450 proteins(CYPs)showed that it exhibited non-significant effects on2C9,2D6,and 3A4 and weak inhibition against 1A2 and 2C19.In vivo PK study showed that by oral administration,the bioavailability of 54 was approximately 16.7%.Finally,the pharmacodynamic(PD)properties of 54 in normal mice and COPD rat models were investigated.The effects of 54 on CatC inhibition and downstream NSP inhibition were assessed in vivo by oral administration twice a day to mice(5,15,45mg/kg)for 6 days.An obvious dose-dependent decreases in CatC activity and downstream NSP activation were observed in both the bone marrow and blood.In the highest dose group(45 mg/kg),the activities of CatC and NSPs(NE,Cat G,PR3)in the bone marrow were inhibited to 16.2,28.3,17.2,and 30.8%of the normal group levels.In a rat model of COPD induced by cigarette smoke and lipopolysaccharide(LPS)stimulation,54 showed a good therapeutic effect on inflammation.Significant dose-dependent decreases in CatC activity and NSPs activation were also observed in the bone marrow,blood,lung and bronchoalveolar lavage fluid(BALF)of rats.Significant pro-inflammatory alterations including alveolar hemorrhage and dilatation,partial alveolar fusion,inflammatory cell infiltration,and destruction of the alveolar structure could be observed in the model group.54 could significantly improve these histopathological changes.In addition,the levels of pro-inflammatory cytokines(IFN-?,IL-1?,IL-6,IL-17,TNF-?,GM-CSF)in the tissues all showed varying degrees of reduction.This study has initially completed the complete and systematic discovery and evaluation of non-peptidyl non-covalent CatC inhibitors.The experimental results verified to a certain extent the initially envisage.These results encouraged us to continue to discover more effective non-peptidyl non-covalent CatC inhibitors,carry out more in-depth mechanism research,and expand the indications of non-peptidyl non-covalent CatC inhibitors.