Non-Peptidyl Non-Covalent Cathepsin C Inhibitor Bearing Novel Thiophene Fragment:Design,Synthesis And Anti-Inflammatory Activity Evaluation

Cathepsin C（Cat C）is one the most important cysteine proteases that activate neutrophil serine proteases（NSPs）by cleaving the dipeptide part from the N-terminal of NSP zymogens.NSPs are secreted by polymorphonuclear neutrophils（PMNs）as part of innate immune mechanisms.However,excessive secretion and activation of active NSPs can lead to tissue damage and inflammation.Because organ damage in NSPs-related inflammatory diseases is mediated by multiple NSP proteases,early inhibition of a single NSP is not an effective method,and later development of inhibitors targeting multiple NSPs is difficult due to the overlapping effects of multiple factors.Therefore,compared with highly selective small molecule inhibitors targeting a single NSP,blocking the upstream maturation enzymes of NSPs by targeting Cat C in the promyelocytic stage of neutrophil maturation in the bone marrow can better inhibit NSPs activity level.However,existing Cat C inhibitors are based on peptidyl-covalent inhibitors,but the highly reactive nature of the electrophilic"warhead"structure leads to poor selectivity and subsequent off-target effects,and the peptidyl backbone brings metabolic defects.Therefore,we hope to develop a new generation of"non-peptidyl non-covalent Cat C inhibitors".Based on the non-peptidyl non-covalent Cat C inhibitor previously reported by us,in this study,starting from compound 77(IC₅₀=57.2 n M,HLM CL_int=9.4),through structure-based drug design and structure-activity relationship（SAR）study,compound SF38,a novel Cat C inhibitor bearing a unique thiophene structure was identified,which exhibited a strong inhibitory activity against Cat C(IC₅₀=59.9 n M),with improved metabolic stability in vitro(HLM CL_int=6.3).Docking analysis of compound SF38 and Cat C using the CDOCKER module in Discovery Studio 2018 R2 showed that compound SF38 had a similar binding mode to compound 77 and formed multiple interactions with amino acid residues in the S1 and S2 regions of Cat C.Cellular thermal shift assays showed that compound SF38increased the temperature tolerance of the target Cat C protein in a concentration-dependent manner.In an acute toxicity study,no significant weight loss or toxicity was observed in mice within 7 days of a single dose of SF38 at 1500 mg/kg.In addition,no pathological changes in different tissues（heart,liver,spleen,lung and kidney）were observed by H＆E staining.Further in vivo experiments were carried out.In vivo pharmacokinetic（PK）studies showed a bioavailability（F）of 42.07%.In vivo evaluation showed that SF38 inhibited the activity of Cat C in bone marrow and blood,thereby reducing the activation of NSPs,and exhibited anti-inflammatory activity.In the highest dose group of SF38（50 mg/kg）,the activities of Cat C and NSPs（NE,Cat G,PR3）in bone marrow and blood were suppressed to about 50%of the normal group.Compound SF38 was able to inhibit the activity levels of Cat C and downstream NSPs in bone marrow and blood in a dose-and time-dependent manner.And it exhibits potent anti-inflammatory activity and potential protection in animal models of acute lung injury（ALI）.Significant dose-dependent reductions in Cat C activity and downstream NSPs activation were observed in the bone marrow and blood of mice in the experimental group,the levels of pro-inflammatory cytokines were significantly reduced,and anti-inflammatory cytokines were significantly increased in a dose-dependent manner,and the survival rate of mice was improved.The compound SF38 was a potent Cat C inhibitor,which can exert anti-inflammatory activity and potential protection in vivo by inhibiting the activation of NSPs for the treatment of NSPs-related inflammatory diseases.These results not only enriched the SAR of Cat C inhibitor with thiophene structure characteristic,but also proved the broad prospect of non-peptidyl non-covalent Cat C inhibitor.