Identification, Characterization and Synthesis of Natural Parasitic Cysteine Protease Inhibitors — More Potent Falcitidin Analogs

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain 2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogs of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogs by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogs followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and additionally low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

Screening of Zinc Database against Streptococcal Cysteine Protease Enzyme for Identification of Novel Group A Streptococcus Inhibitors

Inhibition of streptococcal cysteine protease has recently emerged as quite a promising target to treat severe cases of Group A Streptococcus infections. For the identification of streptococcal cysteine protease inhibitors, structure-based virtual screening (SBVS) of the ZINC Database was performed. The docking protocol was performed with the help of AutoDock Tools and AutoDock Vina software. Based on binding affinity and similarity of interactions with our target receptor streptococcal cysteine protease, 4 hit compounds were identified, which were further subjected to ADMET (Adsorption, Distribution, Metabolism, Excretion, Toxicity) and Drug-likeness to identify the best hit compound. The most potent compound showed binding of -7.7 KJ/mol with receptor streptococcal cysteine protease. It also showed 6 similar amino acid interactions with the receptor’s native ligand along with good ADME and Drug-likeness properties. Furthermore, the molecular dynamics simulation analysis revealed that the complex formed between the protein streptococcal cysteine protease and the hit compound ZINC000205429716 had good structural stability. The current study reveals the successful use of in silico SBVS methods for the identification of novel and possible streptococcal cysteine protease inhibitors, with compound ZINC000205429716 serving as a potential lead for the creation of Group A Streptococcus inhibitors.

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide translates ORF1a and ORF1ab in human host cells to form 15 functionally important, mature nonstructural proteins. One of the two enzymes, papain-like protease or PLpro, also possesses deubiquitination and deISGylation activities that suppresses host innate immune responses toward SARS-CoV-2 infection. Therefore, PLpro is a potential COVID-19 drug target. To repurpose drugs for PLpro, we experimentally screened 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLpro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit potent inhibition of PLpro at 200 uM. More comprehensive characterizations revealed 7 inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 60 uM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 uM. Among four inhibitors with an IC50 value below 10 uM, SJB2-043 is the most unique in that it doesn't fully inhibit PLpro but has an outstanding IC50 value of 0.56 uM. SJB2-043 likely binds to an allosteric site of PLpro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLpro holds promises but in-depth analysis of repurposed drugs is necessary to avoid omitting allosteric inhibitors.

Cystatin-like protein of sweet orange (CsinCPI-2) modulates pre-osteoblast differentiation via β-Catenin involvement

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.

A QSAR study of peptidyl vinyl sulfone cysteine protease inhibitors using Topomer CoMFA and Molecular Docking

Background: Malaria is one of the most important infectious diseases in the world. The most severe form of malaria in humans is caused by Plasmodium falciparum. Malaria is a worldwide health problem, with 214 million new cases in 2015 and 438,000 deaths, most of which in Africa. Therefore, there is an urgent need for novel, low-toxic, more specific inhibitors to find new antimalarial agents. A promising target for antimalarial drug design is falcipain-2, a cysteine protease from P. falciparum, that has received considerable attention due to its key role in the life cycle of the parasite. Methods: Three-dimensional quantitative structure-activity relationship (3D-QSAR) models of 39 peptidyl vinyl sulfone cysteine protease inhibitors was constructed using Topomer CoMFA. Topomer Search was employed to virtually screen lead-like compounds in the ZINC database. Molecular docking was employed to further explore the binding requirements between the ligands and the receptor protein which included several hydrogen bonds between peptidyl vinyl sulfone cysteine protease inhibitors and active site residues. Results: The non-cross correlation coefficient (r 2 ), the interaction validation coefficient (q2 ) and the external validation (r 2 pred) were 0.902, 0.685 and 0.763, respectively. The results showed that the model not only had good estimation stability but also good prediction capability. 22 new molecules were obtained, whose predicted activity are higher than template molecules. The results showed that the Topomer Search technology can be effectively applied to screen and design new peptidyl vinyl sulfone cysteine protease inhibitors. Molecular docking showed extensive interactions between peptidyl vinyl sulfone cysteine protease inhibitors and residues of LYS24, ASP21, LYS59 and ASP17 in the active site. Conclusion: 39 peptidyl vinyl sulfone cysteine protease inhibitors were used in the 3D-QSAR study. Topomer CoMFA 3DQSAR method was used to build the model, and the model was well predicted and statistically validated. The design of potent new inhibitors of cysteine protease can get useful insights from these results.

Legumes Protease Inhibitors as Biopesticides and Their Defense Mechanisms against Biotic Factors

Legumes are affected by biotic factors such as insects, molds, bacteria, and viruses. These plants can produce many different molecules in response to the attack of phytopathogens. Protease inhibitors (PIs) are proteins produced by legumes that inhibit the protease activity of phytopathogens. PIs are known to reduce nutrient availability, which diminishes pathogen growth and can lead to the death of the pathogen. PIs are classified according to the specificity of the mechanistic activity of the proteolytic enzymes, with serine and cysteine protease inhibitors being studied the most. Previous investigations have reported the efficacy of these highly stable proteins against diverse biotic factors and the concomitant protective effects in crops, representing a possible replacement of toxic agrochemicals that harm the environment.

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.