Search Results (99)

Metabolic dysfunction-associated steatotic liver disease (MASLD) has recently become the leading cause of chronic liver disease and can progress to hepatocellular carcinoma (HCC) through multiple pathogenic mechanisms. Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor activated by proteases such as trypsin, tryptase or coagulation factors VII and Xa. Recent studies have shown that PAR2 expression is increased in the liver of patients with MASLD or liver fibrosis. Its activation is linked to metabolic dysfunction through several pathways, including SREBP1c activation, AMPK inhibition and Akt-induced insulin resistance. Inhibition of PAR2 has been effective in reducing MASLD progression in different animal models. Notably, PAR2 blockade has also been effective in more advanced stages of the disease by dampening chronic inflammation and fibrogenesis through the inhibition of hepatic stellate cell activation and of TGF-β and SerpinB3 production. PAR2 also plays a role in cancer development, promoting tumour proliferation, angiogenesis and expression of immune checkpoint inhibitors (like PD-L1, CD47 and CD24). Due to its multifaceted involvement in liver disease, PAR2 is emerging as a key therapeutic target in this clinical context. This review aims to summarise current knowledge on PAR2′s role in MASLD and its potential as a therapeutic target. Full article

Ubiquitin-specific protease 7 (USP7), a deubiquitinase enzyme responsible for removing ubiquitin (Ub) from target proteins, plays a crucial role in oncogenic pathways and has been implicated in various human diseases. X-ray crystallography has revealed distinct conformations of USP7, including apo (ligand-free), allosteric inhibitor-, and Ub-bound states. However, the dynamic mechanisms underlying the allosteric inhibition of USP7 remain unclear. This study investigates the effect of allosteric inhibitor binding on the dynamics of USP7 through multiple replica molecular dynamics simulations. Our results demonstrate that Ub binding stabilizes the USP7 conformation, while allosteric inhibitor binding increases flexibility and variability in the fingers and palm domains of USP7. Furthermore, our analysis of USP7 local regions reveals that allosteric inhibitor binding not only restrains the dynamics of the C-terminal Ub binding site, thereby impeding the accessibility of Ub to USP7, but also disrupts the proper alignment of the catalytic triad (Cys223-His464-Asp481) in USP7. Additionally, community network analysis indicates that intra-domain communications within the fingers domain in USP7 are significantly enhanced upon allosteric inhibitor binding. This study reveals that the binding of an allosteric inhibitor induces a dynamic shift in enzyme’s conformational equilibrium, effectively disrupting its catalytic activity through allosteric modulation. Full article

ADAM (A Disintegrin and Metalloproteinase) family members are multifunctional transmembrane proteases that govern tumorigenesis and metastasis by cleaving membrane-bound substrates such as growth factors, cytokines, and cell adhesion molecules. Several ADAMs, including ADAM8, ADAM9, ADAM10, ADAM12, and ADAM17, are overexpressed in malignancies and are linked with a poor prognosis. These proteases contribute to tumour growth by regulating cell proliferation, cell fate, invasion, angiogenesis, and immune evasion. ADAM10 and ADAM17, especially, facilitate the shedding of critical developmental and growth factors and their receptors, as well as immuno-regulatory molecules, hence promoting tumour progression, immune escape, and resistance to therapy. Recent work has unveiled multiple regulatory pathways that modulate ADAM functions, which include trafficking, dimerization, and conformational modifications that affect substrate accessibility. These observations have rekindled efforts to produce selective ADAM inhibitors, avoiding the off-target consequences reported with early small molecule inhibitors targeting the enzyme active site, which is conserved also in matrix metalloproteinases (MMPs). Promising approaches tested in preclinical models and, in some cases, clinical settings include more selective small-molecule inhibitors, monoclonal antibodies, and antibody–drug conjugates designed to specifically target ADAMs. In this review, we will discuss the emerging roles of ADAMs in cancer biology, as well as the molecular processes that control their function. We further discuss the therapeutic potential of targeting ADAMs, with a focus on recent advances and future directions in the development of ADAM-specific cancer therapies. Full article

The emergence of HIV strains resistant to the current anti-retroviral drugs has necessitated the search for new anti-retroviral medications. Methanolic and aqueous T. ferdinandiana fruit extracts have potent inhibitory activity against several phases of the HIV-1 replicative cycle. Cell infectivity studies using a non-resistant HIV-1 pseudovirus demonstrated that the methanolic (IC₅₀ 16 µg/mL) and aqueous extracts (IC₅₀ 19 µg/mL) were potent inhibitors of viral infection in a non-replicating HIV-1 assay. Both extracts also inhibited HIV-1 reverse transcriptase (IC₅₀ values of 35 and 33 µg/mL for methanolic and aqueous extracts, respectively) and HIV-1 protease (IC₅₀ values of 19 and 27 µg/mL, respectively) in recombinant enzyme assays. Given their inhibitory activities against multiple phases of HIV-1 replication, T. ferdinandiana fruit extracts may be particularly useful as HIV-1 therapeutics. Furthermore, both extracts displayed good safety profiles and therapeutic indices, indicating their suitability for therapeutic usage. LC-MS metabolomic profiling analysis of the methanolic extract identified several interesting constituents, including a relative abundance of tannins, as well as several flavonoids and stilbenes. All of these compounds have previously been reported to have bioactivities consistent with the anti-HIV-1 activities reported herein. Based on these studies, methanolic and aqueous T. ferdinandiana fruit extracts are promising potential therapies for the prevention, treatment and management of HIV-1. Full article

The ongoing emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to resistance against multiple coronavirus disease 2019 (COVID-19) vaccines and therapeutic medications, making the development of effective therapeutics against SARS-CoV-2 a high priority. Studies have shown that bioactive polyphenols, particularly those with triphenol groups, can effectively inhibit the activity of SARS-CoV-2 3-chymotrypsin-like protease (3CL^pro). However, the structural instability of polyphenols necessitates further research. To address this, we conducted a literature review to identify triphenol compounds that are either approved or currently undergoing clinical trials, assessing their potential to inhibit SARS-CoV-2 3CL^pro. Exifone and benserazide hydrochloride were identified as the inhibitors of SARS-CoV-2 3CL^pro among these compounds, using a fluorescence resonance energy transfer (FRET)-based assay. Benserazide hydrochloride was confirmed as a covalent binder to SARS-CoV-2 3CL^pro through time-dependent inhibition and kinetic analysis, with its binding mode elucidated by molecular docking. Notably, exifone not only inhibited the protease activity but also blocked the interaction between the host cell receptor angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor binding domain (S-RBD), as identified by surface plasmon resonance (SPR) and flow cytometry. Additionally, exifone demonstrated antiviral activity against various SARS-CoV-2-S pseudovirus variants. In conclusion, the discovery of exifone and benserazide hydrochloride underscores the potential of polyphenols in developing conserved 3CL^pro inhibitors for coronaviruses, offering new strategies for the rapid development of effective drugs against both current and future coronavirus pandemics. Full article

Canine distemper is a severe and lethal viral disease of dogs and wild carnivores with an urgent need for the identification of effective antiviral agents against canine distemper virus (CDV). We assessed multiple agents for their ability to block the replication of three different lineages of CDV isolated from wild carnivores in the United States. Six antiviral compounds were selected after preliminary experiments that excluded ribavirin, hesperidin and rutin: a protease inhibitor (nirmatrelvir), a polymerase inhibitor (favipiravir) and four nucleoside analogs (remdesivir, GS-441524, EIDD2801 and EIDD1931). Antiviral efficacy was determined by the attenuation of the cytopathic effect in a CDV-susceptible cell line and the inhibition of viral RNA replication. The nucleoside analog GS-441524 effectively blocked the replication of CDV at pharmacologically relevant concentrations. Four other antiviral agents inhibited CDV replication to a lesser degree (remdesivir, nirmatrelvir, EIDD2801 and EIDD1931). The replication of different viral lineages was differentially inhibited by the antivirals. Several of the nucleoside analogs have been safely used previously in carnivore species for the treatment of other viral diseases, suggesting that they may be promising candidates for the treatment of canine distemper in dogs. Our results emphasize the need to consider different viral lineages in the screening of antiviral compounds. Full article

Treatment options for viral infections are limited and viruses have proven adept at evolving resistance to many existing therapies, highlighting a significant vulnerability in our defenses. In response to this challenge, we explored the modulation of cellular RNA metabolic processes as an alternative paradigm to antiviral development. Previously, the small molecule 5342191 was identified as a potent inhibitor of HIV-1 replication by altering viral RNA accumulation at doses that minimally affect host gene expression. In this report, we document 5342191 as a potent inhibitor of adenovirus, coronavirus, and influenza replication. In each case, 5342191-mediated reduction in virus replication was associated with altered viral RNA accumulation and loss of viral structural protein expression. Interestingly, while resistant viruses were rapidly isolated for compounds targeting either virus-encoded proteases or polymerases, we have not yet isolated 5342191-resistant variants of coronavirus or influenza. As with HIV-1, 5342191’s inhibition of coronaviruses and influenza is mediated through the activation of specific cell signaling networks, including GPCR and/or MAPK signaling pathways that ultimately affect SR kinase expression. Together, these studies highlight the therapeutic potential of compounds that target cellular processes essential for the replication of multiple viruses. Not only do these compounds hold promise as broad-spectrum antivirals, but they also offer the potential of greater resilience in combating viral infections. Full article

Glutamic proteases (GPs) represent one of the seven peptidase families described in the MEROPS database of peptidases (also known as proteases, proteinases, and proteolytic enzymes). Currently, the GP family is divided into six sub-families (G1–G6) distributed across three clans (GA, GB, and GC). A glutamic acid and another variable amino acid are the catalytic residues in this family. Members of the GP family are involved in a wide variety of biological functions. For example, they act as bacterial and plant pathogens, and are involved in cancer and celiac disease. These enzymes are considered potential drug targets given their crucial roles in numerous biological processes. Characterizing GPs provides insights into their structure–function relationships, enabling the design of specific inhibitors or modulators. Such advancements directly contribute to drug discovery by identifying novel therapeutic targets and guiding the development of potent and selective drugs for various diseases, including cancers and autoimmune disorders. To address the challenges associated with labor-intensive experimental methods, we developed GPpred, an innovative support vector machine (SVM)-based predictor to identify GPs from their primary sequences. The workflow involves systematically extracting six distinct feature sets from primary sequences, and optimization using a recursive feature elimination (RFE) algorithm to identify the most informative hybrid encodings. These optimized encodings were then used to evaluate multiple machine learning classifiers, including K-Nearest Neighbors (KNNs), Random Forest (RF), Naïve Bayes (NB), and SVM. Among these, the SVM demonstrated a consistent performance, with an accuracy of 97% during the cross-validation and independent validation. Computational methods like GPpred accelerate this process by analyzing large datasets, predicting potential enzyme targets, and prioritizing candidates for experimental validation, thereby significantly reducing time and costs. GPpred will be a valuable tool for discovering GPs from large datasets, and facilitating drug discovery efforts by narrowing down viable therapeutic candidates. Full article

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (M^pro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a K_i value of 48 ± 5 μM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds. Full article

Outcomes for glioblastoma (GBM) remain poor despite standard-of-care treatments including surgical resection, radiation, and chemotherapy. Intratumoral heterogeneity contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. Drug repositioning studies on antiretroviral therapy (ART) have shown promising potent antineoplastic effects in multiple cancers; however, its efficacy in GBM remains unclear. To better understand the pleiotropic anticancer effects of ART on GBM, we conducted a comprehensive drug repurposing analysis of ART in GBM to highlight its utility in translational neuro-oncology. To uncover the anticancer role of ART in GBM, we conducted a comprehensive bioinformatic and in vitro screen of antiretrovirals against glioblastoma. Using the DepMap repository and reversal of gene expression score, we conducted an unbiased screen of 16 antiretrovirals in 40 glioma cell lines to identify promising candidates for GBM drug repositioning. We utilized patient-derived neurospheres and glioma cell lines to assess neurosphere viability, proliferation, and stemness. Our in silico screen revealed that several ART drugs including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) demonstrated marked anti-glioma activity with the capability of reversing the GBM disease signature. RTIs effectively decreased cell viability, GBM stem cell markers, and proliferation. Our study provides mechanistic and functional insight into the utility of ART repurposing for malignant gliomas, which supports the current literature. Given their safety profile, preclinical efficacy, and neuropenetrance, ARTs may be a promising adjuvant treatment for GBM. Full article

Viruses are minuscule infectious agents that reproduce exclusively within the living cells of an organism and are present in almost every ecosystem. Their continuous interaction with humans poses a significant threat to the survival and well-being of everyone. Apart from the common cold or seasonal influenza, viruses are also responsible for several important diseases such as polio, rabies, smallpox, and most recently COVID-19. Besides the loss of life and long-term health-related issues, clinical viral infections have significant economic and social impacts. Viral enzymes, especially proteases which are essential for viral multiplication, represent attractive drug targets. As a result, screening of viral protease inhibitors has gained a lot of interest in the development of anti-viral drugs. Despite the availability of anti-viral therapeutics, there is a clear need to develop novel curative agents that can be used against a given virus or group of related viruses. This review highlights the importance of yeasts as an in vivo model for screening viral enzyme inhibitors. We also discuss the advantages of yeast-based screening platforms over traditional assays. Therefore, in the present article, we discuss why yeast is emerging as a model of choice for in vivo screening of anti-viral molecules and why yeast-based screening will become more relevant in the future for screening anti-viral and other molecules of clinical importance. Full article

Neural regeneration and neuroprotection represent strategies for future management of neurodegenerative disorders such as Alzheimer’s disease (AD) or glaucoma. However, the complex molecular mechanisms that are involved in neuroprotection are not clearly understood. A promising candidate that maintains neuroprotective signaling networks is neuroserpin (Serpini1), a serine protease inhibitor expressed in neurons which selectively inhibits extracellular tissue-type plasminogen activator (tPA)/plasmin and plays a neuroprotective role during ischemic brain injury. Abnormal function of this protein has been implicated in several conditions including stroke, glaucoma, AD, and familial encephalopathy with neuroserpin inclusion bodies (FENIB). Here, we explore the potential biochemical roles of Serpini1 by comparing proteome changes between neuroserpin-deficient (NS^−/−) and control mice, in the retina (RE), optic nerve (ON), frontal cortex (FC), visual cortex (VC), and cerebellum (CB). To achieve this, a multiple-plex quantitative proteomics approach using isobaric tandem mass tag (TMT) technology was employed followed by functional enrichment and protein–protein interaction analysis. We detected around 5000 proteins in each tissue and a pool of 6432 quantified proteins across all regions, resulting in a pool of 1235 differentially expressed proteins (DEPs). Principal component analysis and hierarchical clustering highlighted similarities and differences in the retina compared to various brain regions, as well as differentiating NS^−/− proteome signatures from control samples. The visual cortex revealed the highest number of DEPs, followed by cerebellar regions. Pathway analysis unveiled region-specific changes, including visual perception, focal adhesion, apoptosis, glutamate receptor activation, and supramolecular fiber organization in RE, ON, FC, VC, and CB, respectively. These novel findings provide comprehensive insights into the region-specific networking of Serpini1 in the central nervous system, further characterizing its potential role as a neuroprotective agent. Data are available via ProteomeXchange with identifier PXD046873. Full article

The human genome is estimated to encode more than 500 proteases performing a wide range of important physiological functions. They digest proteins in our food, determine the activity of hormones, induce cell death and regulate blood clotting, for example. During viral infection, however, some proteases can switch sides and activate viral glycoproteins, allowing the entry of virions into new target cells and the spread of infection. To reduce unwanted effects, multiple protease inhibitors regulate the proteolytic processing of self and non-self proteins. This review summarizes our current knowledge of endogenous protease inhibitors, which are known to limit viral replication by interfering with the proteolytic activation of viral glycoproteins. We describe the underlying molecular mechanisms and highlight the diverse strategies by which protease inhibitors reduce virion infectivity. We also provide examples of how viruses evade the restriction imposed by protease inhibitors. Finally, we briefly outline how cellular protease inhibitors can be modified and exploited for therapeutic purposes. In summary, this review aims to summarize our current understanding of cellular protease inhibitors as components of our immune response to a variety of viral pathogens. Full article

SARS-CoV-2 Main Protease (Mpro) is an enzyme that cleaves viral polyproteins translated from the viral genome, which is critical for viral replication. Mpro is a target for anti-SARS-CoV-2 drug development. Herein, we performed a large-scale virtual screening by comparing multiple structural descriptors of reference molecules with reported anti-coronavirus activity against a library with >17 million compounds. Further filtering, performed by applying two machine learning algorithms, identified eighteen computational hits as anti-SARS-CoV-2 compounds with high structural diversity and drug-like properties. The activities of twelve compounds on Mpro’s enzymatic activity were evaluated by fluorescence resonance energy transfer (FRET) assays. Compound 13 (ZINC13878776) significantly inhibited SARS-CoV-2 Mpro activity and was employed as a reference for an experimentally hit expansion. The structural analogues 13a (ZINC4248385), 13b (ZNC13523222), and 13c (ZINC4248365) were tested as Mpro inhibitors, reducing the enzymatic activity of recombinant Mpro with potency as follows: 13c > 13 > 13b > 13a. Then, their anti-SARS-CoV-2 activities were evaluated in plaque reduction assays using Vero CCL81 cells. Subtoxic concentrations of compounds 13a, 13c, and 13b displayed in vitro antiviral activity with IC₅₀ in the mid micromolar range. Compounds 13a–c could become lead compounds for the development of new Mpro inhibitors with improved activity against anti-SARS-CoV-2. Full article

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease–serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease–serpin in the framework of heparin that we review includes the following: thrombin–antithrombin III, plasmin–anti-plasmin, C1 esterase/kallikrein–C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)–alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer. Full article