Site Of Protease Research Articles

Furin-Mediated Modification Is Required for Epithelial Sodium Channel-Activating Activity of Soluble (Pro)Renin Receptor in Cultured Collecting Duct Cells.

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M-1 cells. M-1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin-site Mut) or without (WT) mutagenesis of the furin cleavage site. As compared with empty vector control (EM), Furin-site Mut showed the attenuation effect on WT-induced α-ENaC expression and amiloride-sensitive short circuit current. In a separate experiment, M-1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin), indicating overexpression of the two types of sPRR induced a significant and comparable increase in the release of sPRR, but only sPRR-furin showed an increase of ENaC activity. Single channel analysis of ENaC activity in Xenopus A6-2F3 cells confirms sPRR-furin activation of ENaC open probability. Lastly, HEK-293 cells were pretreated with furin inhibitor α1-antitrypsin Portland (α1-PDX) followed by transfection with EM, WT PRR. sPRR in the conditioned medium was enriched by using protein centrifugal filter devices and applied to M-1 cells followed by measurement of ENaC activity, demonstrating that pretreatment with α1-PDX attenuated ENaC-acting activity induced by overexpression of WT PRR. In summary, we conclude that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na+ transport in the CD cells.

Effects of walnut kernel pellicle on the composition and properties of enzymatic hydrolysates of walnut meal by peptidomics and bioinformatics.

The purpose of this article is to investigate the effects of walnut (Juglans regia L.) kernel pellicle on the composition and properties of enzymatic hydrolysis products of walnut meal using peptidomics and bioinformatics. In this study, a total of 3423 peptide sequences were identified in peeled walnut protein hydrolysates (PWPH) and unpeeled walnut protein hydrolysates (UWPH). Due to the presence of the walnut kernel pellicle, the enzyme cleavage sites of alkaline proteases on walnut precursor proteins were altered, resulting in differences in the number and length of the peptides obtained. Principal component analysis indicates significant differences between PWPH and UWPH. Combined with bioinformatics analysis, it was shown that walnut kernel peeling improved the release of peptides, formed more bioactive peptides, reduced allergenicity, and improved water solubility. Seven peptides with acetylcholinesterase (AChE) inhibitory activity were identified, and the peptide Val-Gly-Ala-Pro-Phe-Asp-Gly-Ala (VGAPFDGA) has the strongest inhibitory activity with an IC50 of 0.38±0.01mg/mL. These results confirmed that walnut kernel peeling could greatly change the composition of the walnut protein hydrolysates, and seven novel peptides were reported that showed significant AChE inhibitory activity.

Identification of a potential anti-viral drug targeting allosteric site of papain-like protease against rubella using a molecular modeling approach.

Rubella virus (RUBV) is responsible for causing rashes, lymphadenopathy, and fever which are the hallmarks of an acute viral illness called Rubella. For RUBV replication, the non-structural polyprotein p200 must be cleaved by the rubella papain-like protease (RubPro) into the multifunctional proteins p150 and p90. Hence, RubPro is an attractive target for anti-viral drug discovery. Moreover, the binding of host Calmodulin 1 (CaM) to RubPro modulates the protease activity and infectivity of RUBV. However, the binding mode of CaM and RubPro remain uncertain. Therefore, our investigation not only delves into understanding the interaction between CaM and the RubPro but also aims to recognize the allosteric site for the development of antiviral protease inhibitors. In this study, we interestingly identified the allosteric site in close vicinity with the CaM binding domain of RubPro. Considering the allosteric site of RubPro, we employed a computational modelling approach to identify the potential antiviral compounds. Leveraging ChemDiv protease inhibitors database, we employed structure-based virtual screening, ADME, pass prediction, and docking studies, unveiling three potent compounds: C073-2897, C073-3328, and C073-3368. Moreover, molecular dynamics simulation analysis revealed that these compounds affect the RubPro structure and dynamics and may also influence the binding of CaM with RubPro. Notably, binding energy calculation showed that the compound C073-3328 exhibits higher binding affinity, while C073-3368 displays a lower binding affinity with RubPro. These compounds signify potential for managing RUBV infections and pioneering effective antiviral treatments. This computational study could pave the way for improved methods of managing or controlling rubella infections.

Molecular Insights into the Regulation of GNPTAB by TMEM251.

In vertebrates, newly synthesized lysosomal enzymes traffick to lysosomes through the mannose-6-phosphate (M6P) pathway. The Golgi membrane protein TMEM251 was recently discovered to regulate lysosome biogenesis by controlling the level of GlcNAc-1-phosphotransferase (GNPT). However, its precise function remained unclear. In this study, we demonstrate that TMEM251 is a two-transmembrane protein indispensable for GNPT stability, cleavage by Site-1-Protease (S1P), and enzymatic activity. We reconcile conflicting models by showing that TMEM251 enhances GNPT cleavage and prevents its mislocalization to lysosomes for degradation. We further establish that TMEM251 achieves this by interacting with GOLPH3 and retromer complexes to anchor the TMEM251-GNPT complex at the Golgi. Alanine mutagenesis identified F4XXR7 motif in TMEM251's N-tail for GOLPH3 binding. Together, our findings uncover TMEM251's multi-faceted role in stabilizing GNPT, retaining it at the Golgi, and ensuring the fidelity of the M6P pathway, thereby providing insights into its molecular function.

Postproline Cleaving Enzymes also Show Specificity to Reduced Cysteine.

In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

Design of non-covalent dual-acting inhibitors for proteases MPRO and PLPRO of coronavirus SARS-CoV-2 through evolutionary library generation, pharmacophore profile matching, and molecular docking calculations

The proteases of the SARS-CoV-2 coronavirus are crucial for the virus's life cycle, making them a prime target for developing antiviral drugs to combat COVID-19. Currently, there is a priority to develop new antiviral drugs that can target multiple viral proteins at once. In this study, we analyze the molecular mechanisms of how non-covalent inhibitors interact with the main protease (Mpro) and papain-like (PLpro) protease of SARS-CoV-2 to create a computer modelling algorithm for discovering ligands that can inhibit both Mpro and PLpro simultaneously. Aim of the study. We aim to analyze the molecular structures involved in the interactions between current non-covalent inhibitors and the Mpro and PLpro proteases of SARS-CoV-2. The goal is to identify a common molecular structure that could be used to discover new inhibitors with a dual-acting mode using computer simulations. Materials and Methods. LigandScout 4.5 software was used for 3D-pharmacophore analysis, virtual screening and molecular docking. AutoDock Vina 1.1.2 tools was utilized for molecular docking. Web-servers PLIP (Protein-Ligand Interaction Profiler) and Pharmit were used for studying molecular binding mechanisms. Generation of evolutionary libraries was performed by DataWarrior 6.0. Analysis and visualization were performed by Discovery Studio 2024 Suite. Results. Our study analyzed various models of SARS-CoV-2 protease binding sites available in the Protein Data Bank (PDB) and their corresponding non-covalent inhibitor ligands. This analysis helped identify important features of the Mpro and PLpro ligands. By comparing the pharmacophore models of Mpro ligands with the structural features of PLpro inhibitors, we identified ligands that could potentially match the binding sites of both proteases. Using the structures of these ligands, an evolutionary library was created in the DataWarrior program. Virtual screening of this library using both Mpro and PLpro pharmacophores revealed several new hit molecules. Molecular docking of these molecules into the active sites of the Mpro and PLpro proteases and calculating their binding energetics led to the identification of several molecules and their corresponding scaffolds with dual inhibition potential. These findings can be further studied in vitro with the aim of discovering drugs for COVID-19. Conclusions. We used computer-based screening to search for ligands that could bind to both Mpro and PLpro proteases. After identifying these potential compounds, we developed synthesis methods to obtain them for further in vitro biological activity studies

Alpha-1-antitrypsin as novel substrate for S. aureus' Spl proteases - implications for virulence.

The serine protease like (Spl) proteases of Staphylococcus aureus are a family of six proteases whose function and impact on virulence are poorly understood. Here we propose alpha-1-antitrypsin (AAT), an important immunomodulatory serine protease inhibitor as target of SplD, E and F. AAT is an acute phase protein, interacting with many proteases and crucial for prevention of excess tissue damage by neutrophil elastase during the innate immune response to infections. We used MALDI-TOF-MS to identify the cleavage site of Spl proteases within AAT's reactive center loop (RCL) and LC-MS/MS to quantify the resulting peptide cleavage product in in vitro digestions of AAT and heterologous expressed proteases or culture supernatants from different S. aureus strains. We further confirmed proteolytic cleavage and formation of a covalent complex with Western Blots, investigated AAT's inhibitory potential against Spls and examined the NETosis inhibitory activity of AAT-Spl-digestions. SplD, E and F, but not A or B, cleave AAT in its RCL, resulting in the release of a peptide consisting of AAT's C-terminal 36 amino acids (C36). Synthetic C36, as well as AAT-SplD/E/F-digestions exhibit NETosis inhibition. Only SplE, but not D or F, was partly inhibited by AAT, forming a covalent complex. We unraveled a new virulence trait of S. aureus, where SplD/E/F cleave and inactivate AAT while the cleavage product C36 inhibits NETosis.

Structural insights into regulated intramembrane proteolysis by the positive alginate regulator MucP from Pseudomonas aeruginosa

Regulated intramembrane proteolysis (RIP) is a fundamentally conserved mechanism involving sequential cleavage by a membrane-bound Site-1 protease (S1P) and a transmembrane Site-2 protease (S2P). In the opportunistic pathogen Pseudomonas aeruginosa, the alternate sigma factor σ22 activates alginate production and in turn is regulated by the MucABCD system. The anti-sigma factor MucA, which inhibits σ22, is sequentially cleaved via RIP by AlgW (S1P) and MucP (S2P) respectively. In this study, we report high-resolution crystal structures of the MucP PDZ1 and PDZ2 domains. Structural and binding analysis confirms that MucP PDZ2 recognizes the carboxy-terminal Ala136 residue of MucA following Site-1 cleavage by AlgW, while the peptide binding groove of PDZ1 is obstructed by a short α-helix. A structure of MucP PDZ2 with bound MucA peptide shows how PDZ2 binds the newly exposed carboxyl terminus of MucA following AlgW cleavage. The ability of a ΔmucP strain of P. aeruginosa to form biofilms was reduced to a similar extent as a ΔalgW strain. This work paves the way for further studies of MucP and other PDZ-containing S2Ps in regulated intramembrane proteolysis.

S1P regulates intervertebral disc aging by mediating endoplasmic reticulum-mitochondrial calcium ion homeostasis.

As the aging process progresses, age-related intervertebral disc degeneration (IVDD) is becoming an emerging public health issue. Site-1 protease (S1P) has recently been found to be associated with abnormal spinal development in patients with mutations and has multiple biological functions. Here, we discovered a reduction of S1P in degenerated and aging intervertebral discs, primarily regulated by DNA methylation. Furthermore, through drug treatment and siRNA-mediated S1P knockdown, nucleus pulposus cells were more prone to exhibit degenerative and aging phenotypes. Conditional KO of S1P in mice resulted in spinal developmental abnormalities and premature aging. Mechanistically, S1P deficiency impeded COP II-mediated transport vesicle formation, which leads to protein retention in the endoplasmic reticulum (ER) and subsequently ER distension. ER distension increased the contact between the ER and mitochondria, disrupting ER-to-mitochondria calcium flow and resulting in mitochondrial dysfunction and energy metabolism disturbance. Finally, using 2-APB to inhibit calcium ion channels and the senolytic drug dasatinib and quercetin (D + Q) partially rescued the aging and degenerative phenotypes caused by S1P deficiency. In conclusion, our findings suggest that S1P is a critical factor in causing IVDD in the process of aging and highlight the potential of targeting S1P as a therapeutic approach for age-related IVDD.

Effect of protease hydrolysis on the structure of acidic heating-induced soy protein amyloid fibrils

The effectiveness of using amyloid fibrillation to improve the functional qualities of soy protein had drawn growing attention. However, the relationship between protein subunits and the structural polymorphism of soy protein-derived amyloid fibrils (SAFs) was not yet completely understood. In this study, soy protein subunits were hydrolyzed to different degrees according to the different action sites of different proteases (Pepsin, Papain and Alcalase). The impact of subunits on the amyloid fibrillation of soy protein was investigated through various techniques including atomic force microscopy, thioflavin T fluorescence, 1-anililo-naphthalene-8-sulfonate, and Fourier transform infrared spectrometer. The findings showed that the α and α’ subunits were associated with the formation of fibril branch chains. The degree of hydrolysis of β subunits was found to be proportional to the number of fibrils. The presence of the 11S component was identified as a necessary condition for the formation of long-rigid fibrils. Furthermore, enzymatic hydrolysis unfolded the protein structure, exposing hydrophobic groups, loosening the protein structure, and altering the proportion of parallel and antiparallel β-sheet structures. This promoted the formation of amyloid fibrils and accelerated the development of stable SAFs gel. This study advances the knowledge of the function of subunits in amyloid fibrillation.

Deciphering the cleavage sites of 3C-like protease in Gammacoronaviruses and Deltacoronaviruses

Coronaviruses replicate by using the 3C-like protease (3CLpro) to cleave polyprotein precursors and host proteins. However, current tools for identifying 3CLpro cleavage sites are limited, particularly in Gammacoronaviruses (GammaCoV) and Deltacoronaviruses (DeltaCoV). This study aims to fill this gap by identifying 3CLpro cleavage sites in these viruses to provide deeper insights into their pathogenic mechanisms. By integrating sequence alignments and structural model comparisons, we developed a position-specific scoring matrix (PSSM) based on self-cleavage motifs, revealing specific preferences for each residue. Utilizing AlphaFold2's predicted alignment error (PAE) and predicted local distance difference test (pLDDT), we found that most cleavage sequences are located in regions with high PAE and low pLDDT values. KEGG pathway analysis showed that potential host protein cleavage targets are mainly concentrated in pathways related to nucleo-cytoplasmic transport and endocytosis. Through in vitro cleavage experiments and mutational analysis, we identified and validated three high-scoring proteins—nucleoporin 58 (NUP58), cell division cycle 73 (CDC73), and signal transducing adaptor molecule 2 (STAM2). These findings suggest that 3CLpro not only plays a vital role in viral replication but may also influence host cell functions by cleaving host proteins. This study provides an effective tool for identifying 3CLpro cleavage sites, revealing the pathogenic mechanisms of coronaviruses, and offering new insights for developing potential therapeutic targets.

A Structural Investigation of the Interaction between a GC-376-Based Peptidomimetic PROTAC and Its Precursor with the Viral Main Protease of Coxsackievirus B3

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CLPro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3CPro protease of enteroviruses, specifically characterizing its interaction with CVB3 3CPro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3CPro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3CPro and SARS-CoV-2 3CLPro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3CPro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3CPro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3CPro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent.

SARS-CoV-2 S protein harbors furin cleavage site located in a short loop between antiparallel β-strand

Furin cleavage site (FCS) of the SARS-CoV-2 S protein, which connects the S1/S2 junction, is essential for facilitating fusion with the host cells. Wild-type (Wt) SARS-CoV-2 S protein, PDB ID: 6yvb, lacks a sequence of amino acid residues, including the FCS that links the S1/S2 junction. For the first time, we demonstrated that a stretch of 14 amino acid residues (677QTNSPRRARSVASQ689) forms an antiparallel β-sheet comprising of PRRAR sequence in the FCS within a short loop. Upon comparing the loop content of the S1/S2 junction with that of Wt SARS-CoV-2 containing PRRAR in the FCS, we observed a decrease in antiparallel β-sheet content and an increase in loop content in the B.1.1.7 variant with HRRAR in the FCS. This short loop within antiparallel β-sheet can serve as a docking site for various proteases, including TMPRSS2 and α1AT. We performed a 300-ns simulation of the SARS-CoV-2 receptor binding domain (RBD) using several antibacterial and antiviral ligands commonly used to treat various infections. Our findings indicate that the receptor binding domain (RBD) comprising the receptor binding motif (RBM) utilizes β6 and a significant portion of the loop to bind with ligands, suggesting its potential for treating SARS-CoV-2 infections.

Collecting Duct Pro(Renin) Receptor Contributes to Unilateral Ureteral Obstruction-Induced Kidney Injury via Activation of the Intrarenal RAS.

Although the concept of the intrarenal renin-angiotensin system (RAS) in renal disease is well-described in the literature, the precise pathogenic role and mechanism of this local system have not been directly assessed in the absence of confounding influence from the systemic RAS. The present study used novel mouse models of collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) or renin together with pharmacological inhibition of soluble PRR production to unravel the precise contribution of the intrarenal RAS to renal injury induced by unilateral ureteral obstruction. We examined the impact of CD-specific deletion of PRR, CD-specific deletion of renin, and S1P (site-1 protease) inhibitor PF429242 treatment on renal fibrosis and inflammation and the indices of the intrarenal RAS in a mouse model of unilateral ureteral obstruction. After 3 days of unilateral ureteral obstruction, the indices of the intrarenal RAS including the renal medullary renin content, activity and mRNA expression, and Ang (angiotensin) II content in obstructed kidneys of floxed mice were all increased. That effect was reversed with CD-specific deletion of PRR, CD-specific deletion of renin, and PF429242 treatment, accompanied by consistent improvement in renal fibrosis and inflammation. On the other hand, renal cortical renin levels were unaffected by unilateral ureteral obstruction, irrespective of the genotype. Similar results were obtained via pharmacological inhibition of S1P, the key protease for the generation of soluble PRR. Our results reveal that PRR-dependent/soluble PRR-dependent activation of CD renin represents a key determinant of the intrarenal RAS and, thus, obstruction-induced renal inflammation and fibrosis.

A method for producing protease pS273R of the African swine fever virus

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates.

Elucidating the Substrate Envelope of Enterovirus 68-3C Protease: Structural Basis of Specificity and Potential Resistance.

Enterovirus-D68 (EV68) has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Unfortunately, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. For other viral proteases, we have demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. However, the structural characterization of EV68-3C protease bound to its substrates has been lacking. Here, we have determined the substrate specificity of EV68-3C protease through molecular modeling, molecular dynamics (MD) simulations, and co-crystal structures. Molecular models enabled us to successfully characterize the conserved hydrogen-bond networks between EV68-3C protease and the peptides corresponding to the viral cleavage sites. In addition, co-crystal structures we determined have revealed substrate-induced conformational changes of the protease which involved new interactions, primarily surrounding the S1 pocket. We calculated the substrate envelope, the three-dimensional consensus volume occupied by the substrates within the active site. With the elucidation of the EV68-3C protease substrate envelope, we evaluated how 3C protease inhibitors, AG7088 and SG-85, fit within the active site to predict potential resistance mutations.

Design of novel and highly selective SARS-CoV-2 main protease inhibitors.

We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro, including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy.

From docking to dynamics: Unveiling the potential non-peptide and non-covalent inhibitors of Mpro from natural products

MotivationThis study aims to investigate non-covalent and non-peptide inhibitors of Mpro, a crucial protein target, by employing a comprehensive approach that integrates molecular docking, molecular dynamics simulations, and top-hits activity predictions. The focus is on elucidating the non-covalent and non-peptide binding modes of potential inhibitors with Mpro. MethodsWe employed a semi-flexible molecular docking methodology, binding score and ADME screening, which are based on structure, to screen compounds from CMNPD and HERB in silico. These methodologies allowed us to find potential candidates depending on their binding values and interactions with the binding site of main protease. To further evaluate the stability of these interactions, we conducted molecular dynamics simulations and calculated binding energies. Ultimately, a top-hits activity prediction method was employed to prioritize compounds based on their predicted inhibitory potential. ResultsThrough a combination of binding energy calculations and activity predictions, we identified six potential inhibitor molecules exhibiting promising activity against Mpro. These compounds demonstrated favorable binding interactions and stability profiles, making them attractive candidates for further experimental validation and drug development efforts targeting Mpro.

Development of a new experimental NMR strategy for covalent cysteine protease inhibitors screening: toward enhanced drug discovery.

In the development of antiviral drugs, proteases and polymerases are among the most important targets. Cysteine proteases, also known as thiol proteases, catalyze the degradation of proteins by cleaving peptide bonds using the nucleophilic thiol group of cysteine. As part of our research, we are examining how cysteine, an essential amino acid found in the active site of the main protease (Mpro) enzyme in SARS-CoV-2, interacts with electrophilic groups present in ethacrynic acid (EA) and compounds 4, 6, and 8 to form sulfur-carbon bonds. Nuclear magnetic resonance (NMR) spectroscopy was used to monitor the reaction rate between cysteine and Michael acceptors. We found that the inhibitory activity of these compounds towards Mpro is correlated to their chemical reactivity toward cysteine. This approach may serve as a valuable tool in drug development for detecting potential covalent inhibitors of Mpro and other cysteine proteases.

Substitutions in the nonactive site of the passenger domain on the activity of Haemophilus influenzae immunoglobulin A1 protease.

Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the β-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease.