Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.

Papain a protease enzyme naturally present in the Carica papaya has gained significant interest across several industries due to its unique properties and versatility. The unique structure of papain imparts the functionality that assists in elucidating how papain enzyme works and making it beneficial for a variety of purposes. This review highlights recent advancements in papain extraction techniques to enhance production efficiency to meet market demand. The extraction of papain from the Carica papaya plant offers various advantages such as cost-effectiveness, biodegradability, safety, and the ability to withstand a wide range of pH and temperature conditions. Key findings reveal that non-conventional papain extraction techniques offer significant advantages in terms of efficiency, product quality, and environmental sustainability. Furthermore, papain treatment enhances the value of final products due to its anti-bacterial, anti-oxidant, and anti-obesity properties. The ability of papain to hydrolyze a wide range of proteins across various conditions makes it a suitable protease enzyme. While the study emphasizes the advantages of papain, the study also acknowledges limitations such as the continuous research and development to optimize extraction processes which will help unlock papain\'s potential and meet the growing demand. © 2024 Society of Chemical Industry.

Optimized syntheses of (E)-5-(2-ethoxyphenyl)-N-(3-(methylsulfonyl)allyl)-1H-pyrazole-3-carboxamide (RA-0002034, 1), a promising antiviral covalent cysteine protease inhibitor lead, were developed. The syntheses avoid the contamination of 1 with the inactive cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-one 2, which is formed by the intramolecular aza-Michael reaction of the vinyl sulfone warhead under basic conditions and slowly at pH 7.4 in phosphate buffer. The pure cysteine protease inhibitor 1 could be synthesized using either modified amide coupling conditions or through the introduction of a MOM-protecting group and was stable as a TFA or HCl salt. Although acyclic 1 demonstrated poor pharmacokinetics with high in vivo clearance in mice, inactive cyclic 2 showed improved plasma exposure. The potential use of cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-ones as prodrugs for the acyclic β-amidomethyl vinyl sulfone warhead was demonstrated by GSH capture experiments with an analog of 2.

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pKi = 7.3 - 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK.

Proteases are implicated in every hallmark of cancer and have complicated functions. For cancer cells to survive and thrive, the process of controlling intracellular proteins to keep the balance of the cell proteome is essential. Numerous natural compounds have been used as ligands/ small molecules to target various proteases that are found in the lysosomes, mitochondria, cytoplasm, and extracellular matrix, as possible anticancer therapeutics. Promising protease modulators have been developed for new drug discovery technology through recent breakthroughs in structural and chemical biology. The protein structure, function of significant tumor-related proteases, and their natural compound inhibitors have been briefly included in this study. This review highlights the most current frontiers and future perspectives for novel therapeutic approaches associated with the list of anticancer natural compounds targeting protease and the mode and mechanism of proteinase-mediated molecular pathways in cancer.

OBJECTIVE: Cysteine protease caspase-1 (Casp1) plays a crucial role in the conversion of pro-cytokines to active cytokines (CYTs). The purpose of this work was to determine Casp1 blood levels in a cohort of 114 cholecystectomy patients and assess their association with other CYTs and numeric rating scale (NRS) pain scores, postoperatively.
METHODS: Blood levels of Casp1 and seven CYTs (IL-18, IL-18BP, IL-1ra, IL-6, IL-10, IL-1β, and IL-8) were measured at three time points; before operation, immediately after operation, and six hours after operation in 114 patients with cholelithiasis (Chole).
RESULTS: Casp1 blood levels correlated with NRS pain scores at 24 h following surgery (p=0.016). In addition, Casp1 blood levels correlated significantly to IL-18 blood levels (p<0.001).
CONCLUSIONS: This is the first report to evaluate Casp1 blood levels in Chole patients in correlation with other CYTs. The findings confirm a significant correlation between Casp1 blood levels and NRS pain scores. Moreover, this study provides initial evidence suggesting that inhibition of the activity of Casp1 may reduce postsurgical acute phase immune response possibly through the Casp1/pro-Il-18 pathway.

The main cysteine protease (Mpro) of coronavirus SARS-CoV-2 has become a promising target for computational development in anti-COVID-19 treatments. Here, we benchmarked the performance of six biomolecular molecular dynamics (MD) force fields (OPLS-AA, CHARMM27, CHARMM36, AMBER03, AMBER14SB and GROMOS G54A7) and three water models (TIP3P, TIP4P and SPC) for reproducing the native fold and the enzymatic activity of Mpro as monomeric and dimeric units. The MD sampling up to 1 μs suggested that the proper choice of the force fields and water models plays an essential role in reproducing the tertiary structure and the inter-residue distance between the catalytic dyad His41-Cys145. We found that while most benchmarked all-atom force fields reproduce well the native fold of Mpro, the CHARMM27/TIP3P and OPLS-AA/TIP4P setups revealed a good performance in reproducing the structure of the catalytic domain. In addition, these FF setups were also well-adopted for MD sampling of Mpro at the physiologic conditions by mimicking the presence of 100 mM NaCl and the elevated temperature of 310 K. Finally, both FFs were also performed well in reproducing the native fold of Mpro in a dimeric form. Therefore, comparing the preservation of the native fold of Mpro and the stability of its catalytic site architecture, our MD benchmarking suggests that the OPLS-AA/TIP4P and CHARMM27/TIP3P MD setups at the physiologic conditions may be well-suited for rapid in silico screening and developing broad-spectrum anti-coronaviral therapeutic agents.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1\' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.
Protein degradation in skeletal muscle is a key component of protein turnover and maintenance of muscle function. Protein degradation in postmortem muscle is commonly observed and is associated with the accumulation of degradation products and improved meat tenderness. Because there is significant evidence that calpain-1 is involved with proteolysis of muscle proteins in both situations, defining the factors that regulate calpain activity will position scientists to improve calpain-1 activity in both contexts. Calpain-1 is a neutral calcium-dependent proteinase that is inhibited by calpastatin, oxidation, and slightly acidic pH environments. Because oxidation of the calpain/calpastatin complex with hydrogen peroxide appeared to activate calpain-1, we hypothesize that calpastatin binding to calpain may protect the active site cysteine. In the current study, we tested this hypothesis and investigated how n-ethyl maleimide (NEM), an alkylating agent, affects the regulation of calpain in the presence and absence of calpastatin molecules. The results suggest that calpastatin can protect calpain-1 from reacting with maleimide-polyethylene glycol but that exposure of calpain-1/calpastatin complex to NEM or hydrogen peroxide resulted in autolysis and activation of calpain. Under some circumstances, calpastatin appears to protect calpain-1 from inhibition by modification of active site cysteine. These novel observations show a different role for calpastatin and give reason to interpret calpastatin abundance and activity data in a different light.

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.