ABSTRACT
Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.
Subject(s)
Aptamers, Nucleotide , Factor IXa , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Factor IXa/metabolism , Factor IXa/chemistry , Factor IXa/antagonists & inhibitors , Humans , Allosteric Regulation , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Protein Binding , Anticoagulants/chemistry , Anticoagulants/metabolism , Anticoagulants/pharmacologyABSTRACT
The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological rolesâsubstrate, receptor, and inhibitorâand were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.
Subject(s)
Peptides , Quantum Dots , Thrombin , Quantum Dots/chemistry , Peptides/chemistry , Peptides/metabolism , Thrombin/metabolism , Thrombin/analysis , Thrombin/chemistry , Factor Xa/metabolism , Factor Xa/chemistry , Proteolysis , Humans , Surface Properties , Peptide Hydrolases/metabolism , Peptide Hydrolases/chemistryABSTRACT
SARS-CoV-2 is the pathogen responsible for the most recent global pandemic, which has claimed hundreds of thousands of victims worldwide. Despite remarkable efforts to develop an effective vaccine, concerns have been raised about the actual protection against novel variants. Thus, researchers are eager to identify alternative strategies to fight against this pathogen. Like other opportunistic entities, a key step in the SARS-CoV-2 lifecycle is the maturation of the envelope glycoprotein at the RARR685↓ motif by the cellular enzyme Furin. Inhibition of this cleavage greatly affects viral propagation, thus representing an ideal drug target to contain infection. Importantly, no Furin-escape variants have ever been detected, suggesting that the pathogen cannot replace this protease by any means. Here, we designed a novel fluorogenic SARS-CoV-2-derived substrate to screen commercially available and custom-made libraries of small molecules for the identification of new Furin inhibitors. We found that a peptide substrate mimicking the cleavage site of the envelope glycoprotein of the Omicron variant (QTQTKSHRRAR-AMC) is a superior tool for screening Furin activity when compared to the commercially available Pyr-RTKR-AMC substrate. Using this setting, we identified promising novel compounds able to modulate Furin activity in vitro and suitable for interfering with SARS-CoV-2 maturation. In particular, we showed that 3-((5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic acid (P3, IC50 = 35 µM) may represent an attractive chemical scaffold for the development of more effective antiviral drugs via a mechanism of action that possibly implies the targeting of Furin secondary sites (exosites) rather than its canonical catalytic pocket. Overall, a SARS-CoV-2-derived peptide was investigated as a new substrate for in vitro high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound P3 as a promising hit with an innovative chemical scaffold. Given the key role of Furin in infection and the lack of any Food and Drug Administration (FDA)-approved Furin inhibitor, P3 represents an interesting antiviral candidate.
Subject(s)
Furin , SARS-CoV-2 , Small Molecule Libraries , Furin/antagonists & inhibitors , Furin/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Humans , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , COVID-19/virology , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Drug Evaluation, Preclinical/methodsABSTRACT
Background: Recombinant factor (F)VIIa (rFVIIa) has been approved by the US Food and Drug Administration for the treatment of hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the investigational uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence showing that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications. Objectives: To design a new rFVII with lower coagulant activity that could potentially be used as an alternative hemostatic agent aiming to minimize the risk of thrombogenicity. Methods: D60A was introduced into the F7 sequence by polymerase chain reaction-based mutagenesis. Wild type (WT) and D60A were generated in human embryonic kidney 293T cells by stable transfection. FVII coagulant activities were determined by amidolytic cleavage of the FVIIa-specific substrate, 2-step FXa generation, thrombin generation (TG), and clot-based assays. Results: WT and D60A demonstrated similar FVIIa amidolytic activity. However, D60A showed approximately 50% activity on FX activation and significantly longer lag time in the TG assay than that shown by WT. The clotting time produced by D60A spiked in FVII-deficient plasma was significantly prolonged than that of WT. Additionally, the ex vivo plasma half-lives of WT and D60A were comparable. Conclusion: D60A demonstrated lower coagulant activities, most likely due to the weakening of FX binding, leading to impaired FX activation and delayed TG and fibrin formation. Considering that a plasma FVII level of 15% to 25% is adequate for normal hemostasis, D60A is a molecule of interest for future development of an rFVII with a lesser extent of thrombogenicity.
ABSTRACT
Rheumatoid arthritis is a systemic autoimmune inflammatory disease that affects millions of people worldwide. There are multiple disease-modifying anti-rheumatic drugs available; however, many patients do not respond to any treatment. A disintegrin and metalloproteinase 10 has been suggested as a potential new target for RA due to its role in the release of multiple pro- and anti-inflammatory factors from cell surfaces. In the present study, we determined the pharmacokinetic parameters and in vivo efficacy of a compound CID3117694 from a novel class of non-zinc-binding inhibitors. Oral bioavailability was demonstrated in the blood and synovial fluid after a 10 mg/kg dose. To test efficacy, we established the collagen-induced arthritis model in mice. CID3117694 was administered orally at 10, 30, and 50 mg/kg/day for 28 days. CID3117694 was able to dose-dependently improve the disease score, decrease RA markers in the blood, and decrease signs of inflammation, hyperplasia, pannus formation, and cartilage erosion in the affected joints compared to the untreated control. Additionally, mice treated with CID 3117694 did not exhibit any clinical signs of distress, suggesting low toxicity. The results of this study suggest that the inhibition of ADAM10 exosite can be a viable therapeutic approach to RA.
ABSTRACT
Activation-by-inhibition is a biochemical paradox seldom observed in exosite enzymes, wherein active site-bound inhibitors unexpectedly lead to enzyme activation. This intriguing phenomenon occurs at low, undersaturating substrate concentrations, posing a significant challenge in drug discovery, especially when targeting enzymes such as protein kinases, proteases, and other posttranslational modification enzymes. These enzymes often rely on accessory recognition sites known as exosites, which contribute to complex substrate binding mechanisms and unique kinetic behaviors. This study aims to provide a theoretical kinetic explanation for nonallosteric mechanism-based activation-by-inhibition, shedding light on the complexities of inhibiting exosite enzymes solely through active site targeting. Notably, the dual activator-inhibitor behavior of active site-bound inhibitors manifests in a nonmonotonic biphasic dose-response, emphasizing the importance of understanding the role of the inhibitor concentration at low substrate levels. Our findings underscore the potential widespread occurrence of activation by inhibition, a phenomenon that may have been overlooked in the past, and thus advocate for novel strategies in drug design that consider the impact of exosites on enzyme behavior to effectively target exosite enzymes.
ABSTRACT
This comprehensive review addresses the intricate and multifaceted regulation of peptidase activity in human health and disease, providing a comprehensive investigation that extends well beyond the boundaries of the active site. Our review focuses on multiple mechanisms and highlights the important role of exosites, allosteric sites, and processes involved in zymogen activation. These mechanisms play a central role in shaping the complex world of peptidase function and are promising potential targets for the development of innovative drugs and therapeutic interventions. The review also briefly discusses the influence of glycosaminoglycans and non-inhibitory binding proteins on enzyme activities. Understanding their role may be a crucial factor in the development of therapeutic strategies. By elucidating the intricate web of regulatory mechanisms that control peptidase activity, this review deepens our understanding in this field and provides a roadmap for various strategies to influence and modulate peptidase activity.
Subject(s)
Glycosaminoglycans , Peptide Hydrolases , Humans , Catalytic Domain , Allosteric Site , Proteolysis , Binding Sites , Allosteric RegulationABSTRACT
The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of ~109 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning, we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a noncompetitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest a mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect that these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity.
Subject(s)
Bacteriophages , Peptides , Humans , Amino Acid Sequence , N-Acetylglucosaminyltransferases/metabolism , Mutation , Bacteriophages/metabolismABSTRACT
Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform ß (AtLEGß) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGß and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGß. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGß suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGß regulation and its broader physiological significance.
Subject(s)
Arabidopsis , Papain , Papain/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Cysteine Endopeptidases/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Plants/metabolismABSTRACT
A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6 × 103 M-1 s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in ß3-ß4 (R756Q/R759Q/R762Q), ß9-ß10 (residues 828-835), and ß6-ß7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.
Subject(s)
ADAMTS1 Protein , Animals , Mice , ADAMTS1 Protein/chemistry , ADAMTS1 Protein/metabolism , ADAMTS4 Protein/metabolism , ADAMTS5 Protein/metabolism , Aggrecans/metabolism , Versicans/metabolismABSTRACT
Triabin, a lipocalin-like thrombin inhibitor from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, exhibits effective inhibition comparable to hirudin despite binding exclusively at exosite I. Interestingly, it was reported that higher triabin doses would not inhibit thrombin completely, which makes it a promising antithrombotic candidate agent with a larger therapeutic window. However, few structural and functional studies about triabin have been reported in the past three decades, mostly due to the lack of a reliable and practicable recombinant expression technology for this seemingly small protein. In this work, we have adopted the SUMO fusion technology for the expression of triabin in E. coli cells-with facile refolding and purification procedures-and the bioactive triabin was produced in â¼12 mg/L culture medium. Subsequently, the structure-function studies through extensive site-directed mutagenesis reveal that triabin's Phe-106 involved in the hydrophobic contacts plays a surprisingly important role in the thrombin inhibition, in contrast to the negatively charged residues Asp-135 or Glu-128 involved in the salt-bridge interaction. As such, this study complements our understanding of the interaction mechanism of natural thrombin inhibitors, which should facilitate the development of anticoagulant drugs with a novel mode of action against thrombin.
Subject(s)
Escherichia coli , Thrombin , Thrombin/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Salivary Proteins and Peptides/pharmacology , Insect Proteins/metabolism , Binding SitesABSTRACT
The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Engineering of ScpA to enhance its potential as a therapeutic will require detailed examination of the basis for its highly selective activity. The emerging view of ScpA and related subtilases is that selection of their substrates is a dynamic two-step process involving flexibility in the domains around the active site and in the C-ter of the substrate. Surface plasmon resonance (SPR) analyses of the ScpA-hC5a interaction have shown that high affinity binding of the substrate is driven by electrostatic interactions between an exosite on the Fn2 domain of the enzyme and the bulky N-ter cleavage product (PN, 'core' residues 1-67) of C5a [1]. Introduction of a D783A mutation in the Fn2 exosite, located approximately 50 Å from the catalytic serine, was shown to significantly reduce substrate binding affinity and k cat of the enzyme. X-ray crystallographic studies on the D783A mutant (ScpAD783A) were undertaken to better interpret the impact of this mutation on the specificity and activity of ScpA. Here we present the 1.9 Å X-ray diffraction data for ScpAD783A and the molecular replacement solution for the structure. Both raw diffraction images and coordinates have been made available on public databases. Additional details on the related SPR and enzyme kinetics analyses on ScpAD783A reported in Jain et al. [2].
ABSTRACT
Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family-termed ElpB and ElpQ-that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. In vitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using binding assays and complement component C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks complement component C4 cleavage by competing with C1s-C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp-C1s binding, we performed site-directed mutagenesis, which showed that ElpB and ElpQ binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.
Subject(s)
Borrelia burgdorferi , Lyme Disease , Humans , Complement C1s/chemistry , Complement C1s/metabolism , Borrelia burgdorferi/genetics , Complement C4/chemistry , Complement System Proteins/metabolism , Serine Proteases , Lipoproteins/geneticsABSTRACT
The C5a peptidase from Streptococcus pyogenes (ScpA) is a highly specific enzyme with potential therapeutic value. ScpA is a good model for studying determinants of specificity in the multidomain immunomodulatory enzymes (IMEs), which comprise a large family of bacterial surface proteases. The surface exposed region of ScpA has 5 main domains which includes 3 C-terminal Fn3-like domains (Fn1, Fn2 and Fn3) (Kagawa et al. 2009). Progressive deletion of the Fn3-like domains from the C-ter resulted in loss of enzyme activity and showed an important role for the Fn2 domain in enzyme function. Functional investigation of specific acidic residues on the Fn2 domain identified 3 residues 30-50 Å from the catalytic site (D783, E864 and D889) which impacted to differing degrees on binding and on catalysis, supporting the presence of an exosite on the Fn2. In particular, residue D783 was observed to impact on both substrate binding affinity and the activity of ScpA. A double mutant cycle analysis showed energetic coupling between the targeted ScpA residues and residues in the core portion (residues 1-67) of the C5a substrate. The data supports the presence of a communication network between the active site and the exosite on Fn2. These findings provide a basis for rational engineering of this important enzyme family to enhance stability, activity and/or specificity.
ABSTRACT
Thrombin has long been considered a desirable antithrombotic target, but anti-thrombin therapy without anti-platelet therapy has never achieved the ideal effect. HY023016, derived from dabigatran etexilate, exhibited a potent antithrombotic efficacy. In the present study, mechanisms underlying this effect were explored. HY023016 strongly decreased the binding of thrombin to recombinant GPIbα N-terminal sequence, which was confirmed by surface plasmon resonance. Flow cytometry revealed that HY023016 selectively decreased the binding of antibody to GPIbα and inhibited the washed human platelet aggregation induced by thrombin. Fluorescence experiment showed that HY023016 remarkably inhibited exosite II by a loss of affinity for the γ'-peptide of fibrinogen. Using intravital microscopy, we observed and recorded the dynamic process of thrombus formation and found that HY023016 effectively prevented thrombus formation in rat arteriovenous shunt thrombosis model. On the basis of these findings, we propose that HY023016 provides a novel insight into the antithrombotic mechanism, which exerts synergistic anticoagulant and antiplatelet effects through thrombin and GPIbα.
Subject(s)
Dabigatran , Fibrinolytic Agents , Animals , Anticoagulants , Dabigatran/pharmacology , Dabigatran/therapeutic use , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/therapeutic use , Rats , Thrombin/metabolismABSTRACT
Understanding the mechanisms of modulators' action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17's broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New 'exosite' (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity.
Subject(s)
ADAM17 Protein/antagonists & inhibitors , ADAM17 Protein/drug effects , ADAM17 Protein/metabolism , ADAM Proteins/metabolism , Binding Sites/drug effects , Catalytic Domain/drug effects , Computer Simulation , Drug Evaluation, Preclinical/methods , HEK293 Cells , High-Throughput Screening Assays/methods , Humans , Kinetics , Protease Inhibitors/pharmacology , Substrate Specificity/drug effectsABSTRACT
Members of the gasdermin family contain positively charged N-terminal domains (NTDs) capable of binding phospholipids and assembling membrane pores, and C-terminal domains (CTDs) that bind the NTDs to prevent pore formation in the resting states. The flexible NTD-CTD linker regions of gasdermins are highly variable in length and sequences, which may be attributable to gasdermin recognition by diverse proteases. In addition, protease cleavage within the NTDs is known to inactivate several gasdermin family members. Recognition and cleavage of the gasdermin family members by different proteases share common and distinct features at the protease active sites, as well as exosites recently identified for the inflammatory caspases. Utilization of exosites may strengthen enzyme-substrate interaction, improve efficiency of proteolysis, and enhance substrate selectivity. It remains to be determined if the dual site recognition of gasdermin D (GSDMD) by the inflammatory caspases is employed by other GSDMD-targeting proteases, or is involved in proteolytic processing of other gasdermins. Biochemical and structural approaches will be instrumental in revealing how potential exosites in diverse proteases engage different gasdermin substrates. Different features of gasdermin sequence, structure, expression characteristics, and post-translational modifications may dictate distinct mechanisms of protease-dependent activation or inactivation. Such diverse mechanisms may underlie the divergent physiological and pathological functions of gasdermins, and furnish opportunities for therapeutic targeting of gasdermins in infectious diseases and inflammatory disorders.
Subject(s)
Peptide Hydrolases , Phosphate-Binding Proteins , Pore Forming Cytotoxic Proteins , Caspases/metabolism , Humans , Peptide Hydrolases/metabolism , Phosphate-Binding Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Protein Processing, Post-Translational , ProteolysisABSTRACT
We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100â¯nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation.
Subject(s)
Dronabinol/analogs & derivatives , Dronabinol/pharmacology , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Binding, Competitive , HEK293 Cells , Humans , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB2/geneticsABSTRACT
Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.
Subject(s)
Aptamers, Nucleotide/metabolism , Hemostatics/metabolism , Thrombin/metabolism , Animals , Aptamers, Nucleotide/chemistry , Binding Sites , Hemostatics/chemistry , Humans , Ligands , Models, Molecular , Protein Binding , Substrate Specificity , Thrombin/chemistryABSTRACT
Thrombin is a crucial regulatory serine protease in hemostasis and thrombosis and has been a therapeutic target of thrombotic events. A novel oyster-derived thrombin inhibitory dodecapeptide (IEELEELEAER, P-2-CG) was identified and characterized. P-2-CG prolonged thrombin time from 9.6 s to 23.3 s at 5 mg/mL in vitro. P-2-CG bound to thrombin Exosite-I domain spontaneously. The occupied Exosite-I blocked fibrinogen binding, which prolonged fibrinogen clotting time to 28 s from 18.5 s. Molecule dynamics demonstrated the interaction of P-2-CG and thrombin Exosite-I involved in eight hydrogen bonds and lots of electrostatic forces. The residue Tyr76 at thrombin Exosite-I is one critical amino acid for fibrinogen binding. The Glu11 in P-2-CG was bound with Tyr76 through strong hydrogen bonds and hydrophobic action. P-2-CG also significantly reduced the mortality of mice that suffered an acute pulmonary embolism induced by thrombin and inhibited mice tail thrombosis induced by κ-carrageenan. The thrombin inhibitory efficiency in vitro and antithrombosis in vivo of P-2-CG provided insight for further applications to serve as an antithrombotic agent.