Discovery Of Covalent Inhibitors Against Lp-PLA2 And SARS-CoV-2 PL Protease

Over the past 100 years,covalent inhibitors have proved to be successful therapies for various indications and had significant beneficial effects on human health.In recent years,the development of covalent drugs has attracted sustained attention of researchers,as more covalent drugs have been approved rapidly and the increasing importance of covalent drugs has been appreciated.These successful covalent drugs have shown several distinguished benefits including increased potency or binding efficiency,binding to otherwise“intractable”targets and prolonged duration of action.Covalent inhibitors targeting catalytic serine or cysteine of hydrolases are always the major field in the research and development history of covalent dugs.Herein,we reported the discovery of novel covalent inhibitors against two hydrolases including lipoprotein-associated phospholipase A2（Lp-PLA2）and SARS-Co V-2 papain-like protease(PL^pro)which are essential for the pathogenesis of related diseases.Our work not only provided the novel covalent inhibitors but also offered alternative strategies for the identification of covalent inhibitors.Lp-PLA2 plays an important role in the development of inflammation and is a promising therapeutic target for anti-inflammatory diseases,such as coronary heart diseases,Alzheimer’s disease and diabetic macular edema.As an important complement to the conventional strategy,we utilized a covalent fragment-based approach to design covalent inhibitors of Lp-PLA2.The enol-cyclocarbamate warhead（2-1）derived from natural products was selected as a starting point.The crystal structure of Lp-PLA2 in complex with fragment 2-1 revealed the covalent reaction mechanism,especially the reaction site on the warhead.We superimposed the crystal structures of Lp-PLA2 in complex with 2-1 as well as previously reported noncovalent inhibitors,indicating that the biphenyl ethanediamine moiety（2-2）of darapladib is suitable for fragment growing.Surprisingly,the resulting compound 2-3not only gained high inhibitory activity against Lp-PLA2 with an IC₅₀ value of 25 n M,more than four orders of magnitude of improvement compared to 2-1,but also exhibited more than 1000-fold selectivity toward Lp-PLA2 over PLA2VIIB.PLA2VIIB is the closest homologue of Lp-PLA2.It has to be noted that both the warhead 2-1 and the fragment 2-2 showed extremely weak potency and low selectivity,highlighting the power and advantage of the fragment-based strategy for drug design.The crystal structure of Lp-PLA2 in complex with 2-3 not only elucidated the binding mode of the inhibitor as we predicted but also revealed an unoccupied hydrophobic pocket.Accordingly,a linear trifluoroethoxyl group was introduced in compound 2-4 in order to occupy the hydrophobic pocket,which enhanced the inhibitory potency of 2-4 by～2-fold(IC₅₀=13 n M).In addition,2-4also showed excellent selectivity toward Lp-PLA2 over PLA2VIIB（3900-fold）,other serine hydrolases as well as the whole proteome in cells.It is the most selective inhibitor of Lp-PLA2 to the best of our knowledge.Furthermore,fluorescent probes with high selectivity and sensitivity were developed based on 2-4 so as to not only characterize Lp-PLA2 in vitro or even in living cells in a way more convenient than immunoblotting tests or immunofluorescence imaging,but also identify its enzymatic activity which cannot be achieved by the antibody-based approaches.These probes could be used as biochemical tools for elucidating the functional versatility of Lp-PLA2 under physiological or pathological conditions.PL^pro in coronavirus is one of key cysteine proteases responsible for the proteolytic processing of viral polyproteins,and plays an important role in dysregulation of host immune response by the cleavage of ubiquitin or ubiquitin-like protein from protein substrates.Thus,targeting PL^pro is an attractive strategy to both inhibit viral replication and prevent disruption of the host immune response against viral infection.A key issue from the standpoint of inhibitor design is the barrier derived from the restricted binding sites（S1/S2）for recognizing two consecutive glycine（P1/P2）in substrates of SARS-Co V-2 PL^pro.These restricted S1/S2 sites represented a significant challenge to the design of orthosteric inhibitors which occupy substrate binding pocket.Herein,we performed a high-throughput screening of compounds including FDA-approved drugs and candidates in clinical trials using an enzymatic assay.Though we embarked on our search for new inhibitors of the SARS-Co V-2 PL^pro,we unexpectedly found activators of the protease.Among them,dimesna and pyritinol both were potent activators which subsequently enabled us to identify C270 as a novel binding site on the SARS-Co V-2 PL^pro and elucidated an unrevealed allosteric and covalent binding mechanism to regulate activity of the protease.Moreover,the SARS-Co V-2 PL^pro was also activated by GSSG through this vital site in vitro as well as in cells,which suggested the activation of the protease by endogenous GSH/GSSG covalently binding to C270 under pathological conditions.To answer the question whether the modification of C270 can inhibit the activity of the protease and to aid the future efforts in developing novel inhibitors targeting this site,we explored the influence of different mutants of C270 on the K_m and V_max of the protease.The results suggested that the covalent modification by the positive-charged molecule may exhibit an inhibition effect on the protease.Inspired by this discovery,the first allosteric inhibitor with potency of 9.4μM was also identified to covalently modify the protease through this site.Our study thereby highlights a promising strategy for identification of the unrecognized allosteric modulation site based on the discovery of covalent probes and reveals an exquisite allosteric modulation mechanism for the protease,which offers a great opportunity for the development of novel SARS-Co V PL^pro inhibitors as well as anti-virus therapies.Herein,we not only identified two types of covalent inhibitors with a novel scaffold and binding mechanism,but also offered two alternative strategies according to the feature of targets interested so as to overcome the limitation of conventional approach.In the first project,given the limited space left for placement of an additional warhead due to the proximity between the catalytic serine and noncovalent inhibitors,we conducted a covalent fragment-based approach which resulted in highly potent and selective covalent inhibitors of Lp-PLA2.In the second project,the activators discovered accidently during inhibitor screening enabled us to explore C270 as an unrecognized regulation site,and further identify the first covalent and allosteric inhibitor targeting this site based on the influence of different mutants of C270 on the protease activity.Moreover,our work reinforces that covalent fluorescent probes or allosteric activators are invaluable biochemical tools for the discovery of novel inhibitors and the functional investigation of targets.