RESUMEN
Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688, show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline, however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307, an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71, a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.
Asunto(s)
Antivirales , Proteínas no Estructurales Virales , Antivirales/farmacología , Antivirales/química , Humanos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Animales , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , Ensayos Clínicos como Asunto , Serina Endopeptidasas/metabolismo , Replicación Viral/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Virus Zika/efectos de los fármacos , Virus del Nilo Occidental/efectos de los fármacosRESUMEN
COVID-19 has caused severe consequences in terms of public health and economy worldwide since its outbreak in December 2019. SARS-CoV-2 3C-like protease (3CLpro), crucial for the viral replications, is an attractive target for the development of antiviral drugs. In this study, several kinds of Michael acceptor warheads were utilized to hunt for potent covalent inhibitors against 3CLpro. Meanwhile, novel 3CLpro inhibitors with the P3-3,5-dichloro-4-(2-(dimethylamino)ethoxy)phenyl moiety were designed and synthesized which may form salt bridge with residue Glu166. Among them, two compounds 12b and 12c exhibited high inhibitory activities against SARS-CoV-2 3CLpro. Further investigations suggested that 12b with an acrylate warhead displayed potent activity against HCoV-OC43 (EC50 = 97 nM) and SARS-CoV-2 replicon (EC50 = 45 nM) and low cytotoxicity (CC50 > 10 µM) in Huh7 cells. Taken together, this study devised two series of 3CLpro inhibitors and provided the potent SARS-CoV-2 3CLpro inhibitor (12b) which may be used for treating coronavirus infections.
Asunto(s)
Acrilatos , Antivirales , Proteasas 3C de Coronavirus , SARS-CoV-2 , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , SARS-CoV-2/efectos de los fármacos , Humanos , Antivirales/farmacología , Antivirales/síntesis química , Antivirales/química , Acrilatos/farmacología , Acrilatos/química , Acrilatos/síntesis química , Relación Estructura-Actividad , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/síntesis química , Descubrimiento de Drogas , COVID-19/virología , Estructura MolecularRESUMEN
Chikungunya virus (CHIKV) is responsible for the most endemic alphavirus infections called Chikungunya. The endemicity of Chikungunya has increased over the past two decades, and it is a pathogen with pandemic potential. There is currently no approved direct-acting antiviral to treat the disease. As part of our antiviral drug discovery program focused on alphaviruses and the non-structural protein 2 protease, we discovered that J12 and J13 can inhibit CHIKV nsP2 protease and block the replication of CHIKV in cell cultures. Both compounds are metabolically stable to human liver microsomal and S9 enzymes. J13 has excellent oral bioavailability in pharmacokinetics studies in mice and ameliorated Chikungunya symptoms in preliminary efficacy studies in mice. J13 exhibited an excellent safety profile in in vitro safety pharmacology and off-target screening assays, making J13 and its analogs good candidates for drug development against Chikungunya.
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Antivirales , Fiebre Chikungunya , Virus Chikungunya , Modelos Animales de Enfermedad , Animales , Virus Chikungunya/efectos de los fármacos , Ratones , Fiebre Chikungunya/tratamiento farmacológico , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Humanos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/síntesis química , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Estructura Molecular , Cisteína Endopeptidasas/metabolismo , Microsomas Hepáticos/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (Mpro) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2WK521WT in VeroE6TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2WK521E166V), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of Mpro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to MproE166V, whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of Mpro, and compromised the enzymatic activity; the Ki values of recombinant MproE166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to MproE166V than nirmatrelvir. SARS-CoV-2WK521WT selected with 5h acquired A191T substitution in Mpro (SARS-CoV-2WK521A191T) and better replicated in the presence of 5h, than SARS-CoV-2WK521WT. However, no significant enzymatic or structural changes in MproA191T were observed. The replicability of SARS-CoV-2WK521E166V proved to be compromised compared to SARS-CoV-2WK521WT but predominated over SARS-CoV-2WK521WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2WK521A191T surpassed that of SARS-CoV-2WK521WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2's drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.
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Proteasas 3C de Coronavirus , Farmacorresistencia Viral , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Humanos , Chlorocebus aethiops , Animales , Farmacorresistencia Viral/genética , Células Vero , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , COVID-19/virología , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Cristalografía por Rayos X , Lactamas , Leucina , Nitrilos , ProlinaRESUMEN
The skin of amphibians is a rich source of peptides with a wide range of biological activities. They are stored in secretory granules in an inactive form. Upon stimulation, they are secreted together with proteases into the skin. Once activated, they rapidly exert their biological effects, including fighting microorganisms and predators, while their excess is immediately destroyed by the released proteases. To keep bioactive peptides in their initial form, it is necessary to inhibit these enzymes. Several inhibitors for this purpose have previously been mentioned; however, there has not been any reliable comparison of their efficiency so far. Here, we studied the efficiency of methanol and hydrochloric and formic acids, as well as phenylmethylsulfonyl fluoride, in the inhibition of nine frog peptides with the known sequence, belonging to five families in the secretion of Pelophylax esculentus. The results demonstrated that methanol had the highest inhibitory efficiency, while phenylmethylsulfonyl fluoride was the least efficient, probably due to its instability in aqueous media. Possible cleavages between certain amino acid residues in the sequence were established for each of the inhibitors. These results may be helpful for future studies on the nature of proteases and on prediction of the possible cleavage sites in novel peptides.
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Péptido Hidrolasas , Péptidos , Piel , Animales , Piel/metabolismo , Piel/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Proteínas Anfibias/metabolismo , Secuencia de Aminoácidos , Anfibios/metabolismo , Metanol/química , Fluoruro de Fenilmetilsulfonilo/farmacologíaRESUMEN
Respiratory viral infections (VRTIs) rank among the leading causes of global morbidity and mortality, affecting millions of individuals each year across all age groups. These infections are caused by various pathogens, including rhinoviruses (RVs), adenoviruses (AdVs), and coronaviruses (CoVs), which are particularly prevalent during colder seasons. Although many VRTIs are self-limiting, their frequent recurrence and potential for severe health complications highlight the critical need for effective therapeutic strategies. Viral proteases are crucial for the maturation and replication of viruses, making them promising therapeutic targets. This review explores the pivotal role of viral proteases in the lifecycle of respiratory viruses and the development of protease inhibitors as a strategic response to these infections. Recent advances in antiviral therapy have highlighted the effectiveness of protease inhibitors in curtailing the spread and severity of viral diseases, especially during the ongoing COVID-19 pandemic. It also assesses the current efforts aimed at identifying and developing inhibitors targeting key proteases from major respiratory viruses, including human RVs, AdVs, and (severe acute respiratory syndrome coronavirus-2) SARS-CoV-2. Despite the recent identification of SARS-CoV-2, within the last five years, the scientific community has devoted considerable time and resources to investigate existing drugs and develop new inhibitors targeting the virus's main protease. However, research efforts in identifying inhibitors of the proteases of RVs and AdVs are limited. Therefore, herein, it is proposed to utilize this knowledge to develop new inhibitors for the proteases of other viruses affecting the respiratory tract or to develop dual inhibitors. Finally, by detailing the mechanisms of action and therapeutic potentials of these inhibitors, this review aims to demonstrate their significant role in transforming the management of respiratory viral diseases and to offer insights into future research directions.
Asunto(s)
Antivirales , Inhibidores de Proteasas , Infecciones del Sistema Respiratorio , SARS-CoV-2 , Humanos , SARS-CoV-2/efectos de los fármacos , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/virología , Antivirales/uso terapéutico , Antivirales/farmacología , Inhibidores de Proteasas/uso terapéutico , Inhibidores de Proteasas/farmacología , Tratamiento Farmacológico de COVID-19 , Proteasas Virales/metabolismo , COVID-19/virología , Rhinovirus/efectos de los fármacos , Rhinovirus/enzimologíaRESUMEN
Linseed, also known as flax is an important oilseed crop with many potential uses in paint, textile, food and pharmaceutical industries. Susceptibility to bud fly (Dasyneura lini Barnes) infestation is a serious biotic concern leading to severe yield penalty in linseed. Protease inhibitors (PIs) are potential candidates that activate during the insect-pest attack and modulate the resistance. In the present study, we explored the PI candidates in the linseed genome and a total of 100 LuPI genes were identified and grouped into five distinct subgroups. The analysis of cis-acting elements revealed that almost all LuPI promoters contain several regulatory elementary related to growth and development, hormonal regulation and stress responses. Across the subfamilies of PIs, the specific domains are consistently found conserved in all protein sequences. The tissue-specific in-silico expression pattern via RNA-seq revealed that all the genes were regulated during different stress. The expression through qRT-PCR of 15 genes revealed the significant up-regulation of LuPI-24, LuPI-40, LuPI-49, LuPI-53, and LuPI-63 upon bud fly infestation in resistant genotype EC0099001 and resistant check variety Neela. This study establishes a foundation resource for comprehending the structural, functional, and evolutionary dimensions of protease inhibitors in linseed.
Asunto(s)
Dípteros , Lino , Regulación de la Expresión Génica de las Plantas , Inhibidores de Proteasas , Lino/genética , Lino/metabolismo , Animales , Dípteros/genética , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/metabolismo , Mapas de Interacción de Proteínas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Familia de Multigenes , FilogeniaRESUMEN
Vaccines and first-generation antiviral therapeutics have provided important protection against COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of the two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease have demonstrated clinical efficacy, known PLpro inhibitors have, to date, lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Here, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , Proteasas Similares a la Papaína de Coronavirus , Modelos Animales de Enfermedad , SARS-CoV-2 , Animales , Ratones , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Antivirales/farmacocinética , Antivirales/uso terapéutico , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Humanos , COVID-19/virología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Aprendizaje Automático , Femenino , Replicación Viral/efectos de los fármacosRESUMEN
Emergence of newer variants of SARS-CoV-2 underscores the need for effective antivirals to complement the vaccination program in managing COVID-19. The multi-functional papain-like protease (PLpro) of SARS-CoV-2 is an essential viral protein that not only regulates the viral replication but also modulates the host immune system, making it a promising therapeutic target. To this end, we developed an in vitro interferon stimulating gene 15 (ISG15)-based Förster resonance energy transfer (FRET) assay and screened the National Cancer Institute (NCI) Diversity Set VI compound library, which comprises 1584 small molecules. Subsequently, we assessed the PLpro enzymatic activity in the presence of screened molecules. We identified three potential PLpro inhibitors, namely, NSC338106, 651084, and 679525, with IC50 values in the range from 3.3 to 6.0 µM. These molecules demonstrated in vitro inhibition of the enzyme activity and exhibited antiviral activity against SARS-CoV-2, with EC50 values ranging from 0.4 to 4.6 µM. The molecular docking of all three small molecules to PLpro suggested their specificity towards the enzyme's active site. Overall, our study contributes promising prospects for further developing potential antivirals to combat SARS-CoV-2 infection.
Asunto(s)
Antivirales , Proteasas Similares a la Papaína de Coronavirus , Citocinas , Ensayos Analíticos de Alto Rendimiento , SARS-CoV-2 , Ubiquitinas , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Antivirales/farmacología , Antivirales/química , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Citocinas/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/química , Ubiquitinas/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Tratamiento Farmacológico de COVID-19 , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Transferencia Resonante de Energía de Fluorescencia , COVID-19/virologíaRESUMEN
Natural antioxidants have become the subject of many investigations due to the role that they play in the reduction of oxidative stress. Their main scavenging mechanisms concern the direct inactivation of free radicals and the coordination of metal ions involved in Fenton-like reactions. Recently, increasing attention has been paid to non-covalent inhibition of enzymes involved in different diseases by the antioxidants. Here, a computational investigation on the primary antioxidant power of (+)-catechin against the â¢OOH radical has been performed in both lipid-like and aqueous environments, taking into account the relevant species present in the simulated acid-base equilibria at the physiological pH. Hydrogen Atom Transfer (HAT), Single Electron Transfer (SET), and Radical Adduct Formation (RAF) mechanisms were studied, and relative rate constants were estimated. The potential inhibitory activity of the (+)-catechin towards the most important proteases from SARS-CoV-2, 3C-like (Mpro) and papain-like (PLpro) proteases was also investigated by MD simulations to provide deeper atomistic insights on the binding sites. Based on the antioxidant and antiviral properties also unravelled by comparison with other molecules having similar chemical scaffold, our results propose that (+)-CTc satisfies can explicate a dual action as antioxidant and antiviral in particular versus Mpro from SARS-CoV-2.
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Antioxidantes , Catequina , Simulación de Dinámica Molecular , Inhibidores de Proteasas , SARS-CoV-2 , Catequina/química , Catequina/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Antioxidantes/metabolismo , SARS-CoV-2/efectos de los fármacos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Antivirales/química , Antivirales/farmacología , Humanos , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/químicaRESUMEN
The SARS-CoV-2 papain-like protease (PLpro), essential for viral processing and immune response disruption, is a promising target for treating acute infection of SARS-CoV-2. To date, there have been no reports of PLpro inhibitors with both submicromolar potency and animal model efficacy. To address the challenge of PLpro's featureless active site, a noncovalent inhibitor library with over 50 new analogs was developed, targeting the PLpro active site by modulating the BL2-loop and engaging the BL2-groove. Notably, compounds 42 and 10 exhibited strong antiviral effects and were further analyzed pharmacokinetically. 10, in particular, showed a significant lung accumulation, up to 12.9-fold greater than plasma exposure, and was effective in a mouse model of SARS-CoV-2 infection, as well as against several SARS-CoV-2 variants. These findings highlight the potential of 10 as an in vivo chemical probe for studying PLpro inhibition in SARS-CoV-2 infection.
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Antivirales , Tratamiento Farmacológico de COVID-19 , Proteasas Similares a la Papaína de Coronavirus , SARS-CoV-2 , Animales , Humanos , Ratones , Antivirales/farmacología , Antivirales/química , Antivirales/farmacocinética , Antivirales/síntesis química , Dominio Catalítico , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/metabolismo , COVID-19/virología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/síntesis química , SARS-CoV-2/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
DNA damage triggers cell signaling cascades that mediate repair. This signaling is frequently dysregulated in cancers. The proteins that mediate this signaling are potential targets for therapeutic intervention. Ubiquitin-specific protease 1 (USP1) is one such target, with small-molecule inhibitors already in clinical trials. Here, we use biochemical assays and cryo-electron microscopy (cryo-EM) to study the clinical USP1 inhibitor, KSQ-4279 (RO7623066), and compare this to the well-established tool compound, ML323. We find that KSQ-4279 binds to the same cryptic site of USP1 as ML323 but disrupts the protein structure in subtly different ways. Inhibitor binding drives a substantial increase in thermal stability of USP1, which may be mediated through the inhibitors filling a hydrophobic tunnel-like pocket in USP1. Our results contribute to the understanding of the mechanism of action of USP1 inhibitors at the molecular level.
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Microscopía por Crioelectrón , Humanos , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Dengue fever, prevalent in Southeast Asian countries, currently lacks effective pharmaceutical interventions for virus replication control. This study employs a strategy that combines machine learning (ML)-based quantitative-structure-activity relationship (QSAR), molecular docking, and molecular dynamics simulations to discover potential inhibitors of the NS3 protease of the dengue virus. We used nine molecular fingerprints from PaDEL to extract features from the NS3 protease dataset of dengue virus type 2 in the ChEMBL database. Feature selection was achieved through the low variance threshold, F-Score, and recursive feature elimination (RFE) methods. Our investigation employed three ML models - support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost) - for classifier development. Our SVM model, combined with SVM-RFE, had the best accuracy (0.866) and ROC_AUC (0.964) in the testing set. We identified potent inhibitors on the basis of the optimal classifier probabilities and docking binding affinities. SHAP and LIME analyses highlighted the significant molecular fingerprints (e.g. ExtFP69, ExtFP362, ExtFP576) involved in NS3 protease inhibitory activity. Molecular dynamics simulations indicated that amphotericin B exhibited the highest binding energy of -212 kJ/mol and formed a hydrogen bond with the critical residue Ser196. This approach enhances NS3 protease inhibitor identification and expedites the discovery of dengue therapeutics.
Asunto(s)
Antivirales , Virus del Dengue , Reposicionamiento de Medicamentos , Serina Endopeptidasas , Proteínas no Estructurales Virales , Antivirales/química , Antivirales/farmacología , Virus del Dengue/enzimología , Virus del Dengue/efectos de los fármacos , Aprendizaje Automático , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad Cuantitativa , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteasas ViralesRESUMEN
Pseudomonas aeruginosa has become a top-priority pathogen in the health sector because it is ubiquitous, has high metabolic/genetic versatility, and is identified as an opportunistic pathogen. The production of numerous virulence factors by P. aeruginosa was reported to act individually or cooperatively to make them robots invasion, adherences, persistence, proliferation, and protection against host immune systems. P. aeruginosa produces various kinds of extracellular proteases such as alkaline protease, protease IV, elastase A, elastase B, large protease A, Pseudomonas small protease, P. aeruginosa aminopeptidase, and MucD. These proteases effectively allow the cells to invade and destroy host cells. Thus, inhibiting these protease activities has been recognized as a promising approach to controlling the infection caused by P. aeruginosa. The present review discussed in detail the characteristics of these proteases and their role in infection to the host system. The second part of the review discussed the recent updates on the multiple strategies for attenuating or inhibiting protease activity. These strategies include the application of natural and synthetic molecules, as well as metallic/polymeric nanomaterials. It has also been reported that a propeptide present in the middle domain of protease IV also attenuates the virulence properties and infection ability of P. aeruginosa.
Asunto(s)
Péptido Hidrolasas , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/química , Humanos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Factores de Virulencia/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteínas Bacterianas/metabolismo , Animales , Productos Biológicos/farmacología , Productos Biológicos/química , Antibacterianos/farmacología , Antibacterianos/químicaRESUMEN
BACKGROUND AND AIMS: Inflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. METHODS: We designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. RESULTS: HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe-/- mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr-/- mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. CONCLUSIONS: These data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.
Asunto(s)
Aterosclerosis , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Lipoproteínas HDL , Animales , Aterosclerosis/patología , Aterosclerosis/prevención & control , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Aterosclerosis/tratamiento farmacológico , Lipoproteínas HDL/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones , Ratones Noqueados para ApoE , Placa Aterosclerótica , Masculino , Elastasa Pancreática/metabolismo , Aorta/patología , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/metabolismo , Inhibidores de Proteasas/farmacología , alfa 1-Antitripsina/farmacología , alfa 1-Antitripsina/metabolismoRESUMEN
SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (Mpro). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including Mpro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC50 values in the low nanomolar range, kinact/Ki values of >100,000 M-1 s-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho-fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p-ethoxy-o-fluorobenzoyl chloropyridyl ester (PSB-21110, 29b, MW 296 g/mol; EC50 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.
Asunto(s)
Antivirales , Proteasas 3C de Coronavirus , SARS-CoV-2 , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Humanos , Relación Estructura-Actividad , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/síntesis química , Cristalografía por Rayos X , Chlorocebus aethiops , Animales , Células Vero , Diseño de FármacosRESUMEN
The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (â¼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.
Asunto(s)
Antivirales , Proteasas 3C de Coronavirus , Pruebas de Enzimas , Ensayos Analíticos de Alto Rendimiento , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Antivirales/farmacología , Antivirales/química , Pruebas de Enzimas/métodos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , COVID-19/virología , Tratamiento Farmacológico de COVID-19RESUMEN
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.
Asunto(s)
Antivirales , Proteasas 3C de Coronavirus , Ensayos Analíticos de Alto Rendimiento , SARS-CoV-2 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , SARS-CoV-2/efectos de los fármacos , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/genética , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Inhibidores de Proteasas/farmacología , Descubrimiento de Drogas/métodos , COVID-19/virología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The single-stranded RNA genome of SARS-CoV-2 encodes several structural and non-structural proteins, among which the papain-like protease (PLpro) is crucial for viral replication and immune evasion and has emerged as a promising therapeutic target. The current study aims to discover new inhibitors of PLpro that can simultaneously disrupt its protease and deubiquitinase activities. Using multiple computational approaches, six compounds (CP1-CP6) were selected from our in-house compounds database, with higher docking scores (-7.97 kcal/mol to -8.14 kcal/mol) and fitted well in the active pocket of PLpro. Furthermore, utilizing microscale molecular dynamics simulations (MD), the dynamic behavior of selected compounds was studied. Those molecules strongly binds at the PLpro active site and forms stable complexes. The dynamic motions suggest that the binding of CP1-CP6 brought the protein to a closed conformational state, thereby altering its normal function. In an in vitro evaluation, CP2 showed the most significant inhibitory potential for PLpro (protease activity = 2.71 ± 0.33 µM and deubiquitinase activity = 3.11 ± 0.75 µM), followed by CP1, CP5, CP4 and CP6. Additionally, CP1-CP6 showed no cytotoxicity at a concentration of 30 µM in the human BJ cell line.
Asunto(s)
Proteasas Similares a la Papaína de Coronavirus , Enzimas Desubicuitinizantes , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , SARS-CoV-2 , SARS-CoV-2/enzimología , SARS-CoV-2/efectos de los fármacos , Humanos , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/química , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Dominio Catalítico , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Antivirales/farmacología , Antivirales/química , Productos Biológicos/farmacología , Productos Biológicos/química , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Unión ProteicaRESUMEN
Since 2020, many compounds have been investigated for their potential use in the treatment of SARS-CoV-2 infection. Among these agents, a huge number of natural products and FDA-approved drugs have been evaluated as potential therapeutics for SARS-CoV-2 using virtual screening and docking studies. However, the identification of the molecular targets involved in viral replication led to the development of rationally designed anti-SARS-CoV-2 agents. Among these targets, the main protease (Mpro) is one of the key enzymes needed in the replication of the virus. The data gleaned from the crystal structures of SARS-CoV-2 Mpro complexes with small-molecule covalent inhibitors has been used in the design and discovery of many highly potent and broad-spectrum Mpro inhibitors. The current review focuses mainly on the covalent type of SARS-CoV-2 Mpro inhibitors. The design, chemistry, and classification of these inhibitors were also in focus. The biological activity of these inhibitors, including their inhibitory activities against Mpro, their antiviral activities, and the SAR studies, were discussed. The review also describes the potential mechanism of the interaction between these inhibitors and the catalytic Cys145 residue in Mpro. Moreover, the binding modes and key binding interactions of these covalent inhibitors were also illustrated. The covalent inhibitors discussed in this review were of diverse chemical nature and origin. Their antiviral activity was mediated mainly by the inhibition of SARS-CoV-2 Mpro, with IC50 values in the micromolar to the nanomolar range. Many of these inhibitors exhibited broad-spectrum inhibitory activity against the Mpro enzymes of other coronaviruses (SARS-CoV-1 and MERS-CoV). The dual inhibition of the Mpro and PLpro enzymes of SARS-CoV-2 could also provide higher therapeutic benefits than Mpro inhibition. Despite the approval of nirmatrelvir by the FDA, many mutations in the Mpro enzyme of SARS-CoV-2 have been reported. Although some of these mutations did not affect the potency of nirmatrelvir, there is an urgent need to develop a second generation of Mpro inhibitors. We hope that the data summarized in this review could help researchers in the design of a new potent generation of SARS-CoV-2 Mpro inhibitors.