RESUMEN
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
Asunto(s)
Sistemas CRISPR-Cas , Hexosiltransferasas , Lipopolisacáridos , Proteínas de la Membrana , FN-kappa B , Transducción de Señal , Receptor Toll-Like 4 , Hexosiltransferasas/metabolismo , Hexosiltransferasas/genética , FN-kappa B/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Receptor Toll-Like 4/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Células HEK293 , Inflamación/metabolismo , Inflamación/genética , Glicosilación , Microscopía por Crioelectrón , Dominio Catalítico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/química , Leptotrichia/genética , ARN Guía de Kinetoplastida/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Leptotrichia/metabolismo , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , División del ARN , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.
Asunto(s)
COVID-19/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Viral/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Proteínas no Estructurales Virales/metabolismo , Animales , COVID-19/genética , COVID-19/patología , Chlorocebus aethiops , Microscopía por Crioelectrón , Humanos , ARN Mensajero/genética , ARN Viral/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/virología , SARS-CoV-2/genética , SARS-CoV-2/ultraestructura , Células Vero , Proteínas no Estructurales Virales/genéticaRESUMEN
Self-replication of amyloid fibrils via secondary nucleation is an intriguing physicochemical phenomenon in which existing fibrils catalyze the formation of their own copies. The molecular events behind this fibril surface-mediated process remain largely inaccessible to current structural and imaging techniques. Using statistical mechanics, computer modeling, and chemical kinetics, we show that the catalytic structure of the fibril surface can be inferred from the aggregation behavior in the presence and absence of a fibril-binding inhibitor. We apply our approach to the case of Alzheimer's A[Formula: see text] amyloid fibrils formed in the presence of proSP-C Brichos inhibitors. We find that self-replication of A[Formula: see text] fibrils occurs on small catalytic sites on the fibril surface, which are far apart from each other, and each of which can be covered by a single Brichos inhibitor.
Asunto(s)
Péptidos beta-Amiloides , Amiloide , Péptidos beta-Amiloides/química , Amiloide/química , Simulación por Computador , Fragmentos de Péptidos/química , CinéticaRESUMEN
The N-terminal region of the human Lysine Specific Demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple post-translational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N-terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N-terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1's own electropositive NLS amino acids 107-120 inhibit demethylation activity on a model Histone 3 lysine 4 di-methyl (H3K4me2) peptide (Kiapp â¼ 3.3 µM) and H3K4me2 nucleosome substrates (IC50 â¼ 30.4 µM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, IDRs, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.
RESUMEN
ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L-cis hydroxyproline biphenyl ester (Lc-BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC , Antígenos de Histocompatibilidad Menor , Sistema de Transporte de Aminoácidos ASC/metabolismo , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/química , Cinética , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/química , Humanos , Sodio/metabolismo , Sodio/química , Animales , Unión CompetitivaRESUMEN
The cryo-EM resolution revolution has heralded a new era in our understanding of eukaryotic lipid flippases with a rapidly growing number of high-resolution structures. Flippases belong to the P4 family of ATPases (type IV P-type ATPases) that largely follow the reaction cycle proposed for the more extensively studied cation-transporting P-type ATPases. However, unlike the canonical P-type ATPases, no flippase cargos are transported in the phosphorylation half-reaction. Instead of being released into the intracellular or extracellular milieu, lipid cargos are transported to their destination at the inner leaflet of the membrane. Recent flippase structures have revealed multiple conformational states during the lipid transport cycle. Nonetheless, critical conformational states capturing the lipid cargo "in transit" are still missing. In this review, we highlight the amazing structural advances of these lipid transporters, discuss various perspectives on catalytic and regulatory mechanisms in the literature, and shed light on future directions in further deciphering the detailed molecular mechanisms of lipid flipping.
Asunto(s)
Adenosina Trifosfatasas , Humanos , Animales , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/química , Microscopía por Crioelectrón , Transporte Biológico , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Metabolismo de los Lípidos , Conformación ProteicaRESUMEN
The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.
Asunto(s)
Vía Alternativa del Complemento , Humanos , Vía Alternativa del Complemento/efectos de los fármacos , Complemento C3/metabolismo , Complemento C3/antagonistas & inhibidores , Inactivadores del Complemento/farmacología , Complemento C3b/metabolismoRESUMEN
Kinesin-1 is a microtubule motor that transports cellular cargo along microtubules. KIF5A is one of three kinesin-1 isoforms in humans, all of which are autoinhibited by an interaction between the motor and an IAK motif in the proximal region of the C-terminal tail. The C-terminal tail of KIF5A is â¼80 residues longer than the other two kinesin-1 isoforms (KIF5B and KIF5C) and it is unclear if it contributes to autoinhibition. Mutations in KIF5A cause neuronal diseases and could affect autoinhibition, as reported for a mutation that skips exon 27, altering its C-terminal sequence. Here, we combined negative-stain electron microscopy, crosslinking mass spectrometry (XL-MS) and AlphaFold2 structure prediction to determine the molecular architecture of the full-length autoinhibited KIF5A homodimer, in the absence of light chains. We show that KIF5A forms a compact, bent conformation, through a bend between coiled-coils 2 and 3, around P687. XL-MS of WT KIF5A revealed extensive interactions between residues in the motor, between coiled-coil 1 and the motor, between coiled-coils 1 and 2, with coiled-coils 3 and 4, and the proximal region of the C-terminal tail and the motor in the autoinhibited state, but not between the distal C-terminal region and the rest of the molecule. While negative-stain electron microscopy of exon-27 KIF5A splice mutant showed the presence of autoinhibited molecules, XL-MS analysis suggested that its autoinhibited state is more labile. Our model offers a conceptual framework for understanding how mutations within the motor and stalk domain may affect motor activity.
Asunto(s)
Cinesinas , Humanos , Exones , Cinesinas/química , Cinesinas/genética , Mutación , Isoformas de Proteínas/química , Isoformas de Proteínas/genéticaRESUMEN
Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.
Asunto(s)
Hidrolasas Diéster Fosfóricas , Humanos , Biología Computacional/métodos , Cristalografía por Rayos X , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Motivos de Unión al ARN/genéticaRESUMEN
CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY15, that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens.
Asunto(s)
Antígenos CD40 , Péptidos , Transducción de Señal , Animales , Ratones , Aminoácidos , Antígenos CD40/metabolismo , Ligando de CD40 , Antígeno-1 Asociado a Función de Linfocito , Transducción de Señal/efectos de los fármacos , Integrinas/metabolismoRESUMEN
Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels.
Asunto(s)
Canales de Potasio Éter-A-Go-Go , Imipramina , Fenómenos Electrofisiológicos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/química , Canales de Potasio Éter-A-Go-Go/genética , Imipramina/química , Imipramina/farmacología , Unión Proteica , Animales , Dominios Proteicos , Ratones , XenopusRESUMEN
We report a novel small-molecule screening approach that combines data augmentation and machine learning to identify Food and Drug Administration (FDA)-approved drugs interacting with the calcium pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) from skeletal (SERCA1a) and cardiac (SERCA2a) muscle. This approach uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, thus allowing to screen with high precision large databases of small molecules, including approved and investigational drugs. We chose SERCA because it plays a major role in the excitation-contraction-relaxation cycle in muscle and it represents a major target in both skeletal and cardiac muscle. The machine learning model predicted that SERCA1a and SERCA2a are pharmacological targets for seven statins, a group of FDA-approved 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in the clinic as lipid-lowering medications. We validated the machine learning predictions by using in vitro ATPase assays to show that several FDA-approved statins are partial inhibitors of SERCA1a and SERCA2a. Complementary atomistic simulations predict that these drugs bind to two different allosteric sites of the pump. Our findings suggest that SERCA-mediated Ca2+ transport may be targeted by some statins (e.g., atorvastatin), thus providing a molecular pathway to explain statin-associated toxicity reported in the literature. These studies show the applicability of data augmentation and machine learning-based screening as a general platform for the identification of off-target interactions and the applicability of this approach extends to drug discovery.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Miocardio/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Aprendizaje AutomáticoRESUMEN
Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.
Asunto(s)
Histona Desacetilasas , Oxadiazoles , Animales , Ratones , Histona Desacetilasa 6/química , Histona Desacetilasas/genética , Oxadiazoles/farmacología , Ratones Noqueados , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasa 1RESUMEN
Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.
Asunto(s)
Proteínas Bacterianas , Metaloproteasas , Termolisina/metabolismo , Proteínas Bacterianas/metabolismo , Metaloproteasas/genética , Espectroscopía de Resonancia Magnética , Péptido HidrolasasRESUMEN
The SARS-CoV-2 coronavirus is characterized by high mutation rates and significant infectivity, posing ongoing challenges for therapeutic intervention. To address potential challenges in the future, the continued development of effective drugs targeting SARS-CoV-2 remains an important task for the scientific as well as the pharmaceutical community. The main protease (Mpro) of SARS-CoV-2 is an ideal therapeutic target for COVID-19 drug development, leading to the introduction of various inhibitors, both covalent and non-covalent, each characterized by unique mechanisms of action and possessing inherent strengths and limitations. Natural products, being compounds naturally present in the environment, offer advantages such as low toxicity and diverse activities, presenting a viable source for antiviral drug development. Here, we identified a natural compound, rosmarinic acid, which exhibits significant inhibitory effects on the Mpro of the SARS-CoV-2. Through detailed structural biology analysis, we elucidated the precise crystal structure of the complex formed between rosmarinic acid and SARS-CoV-2 Mpro, revealing the molecular basis of its inhibitory mechanism. These findings not only enhance our understanding of the antiviral action of rosmarinic acid, but also provide valuable structural information and mechanistic insights for the further development of therapeutic strategies against SARS-CoV-2.
Asunto(s)
Antivirales , Cinamatos , Proteasas 3C de Coronavirus , Depsidos , Ácido Rosmarínico , SARS-CoV-2 , Depsidos/química , Depsidos/farmacología , Cinamatos/química , Cinamatos/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Humanos , Antivirales/farmacología , Antivirales/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Modelos Moleculares , Cristalografía por Rayos X , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Sitios de Unión , Unión ProteicaRESUMEN
Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.
Asunto(s)
Aciltransferasas , Glycine max , Aciltransferasas/química , Aciltransferasas/metabolismo , Aciltransferasas/genética , Glycine max/enzimología , Especificidad por Sustrato , Coenzima A/metabolismo , Coenzima A/química , Modelos Moleculares , Conformación Proteica , Chalconas/química , Chalconas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMEN
PURPOSE: The purpose of this study was to develop an assay for simultaneous determination of lapatinib and its metabolites (N-dealkylated lapatinib and O-dealkylated lapatinib) by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and to determine the interaction between shikonin and lapatinib in vitro, in vivo, in silico and its mechanism of action. METHODS: A new UPLC-MS/MS method for the determination of the concentrations of lapatinib and its metabolites was developed. In vivo, Sprague-Dawley (SD) rats were given lapatinib with or without shikonin. In vitro, to study the interaction mechanism, rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4.1 were used for determining enzyme kinetics. Lastly, we used in silico molecular docking to investigate the molecular mechanism of inhibition. RESULTS: The selectivity, precision, accuracy, stability, matrix effect and recovery of UPLC-MS/MS all met the requirements of quantitative analysis of biological samples. Administration of lapatinib combined with shikonin resulted in significantly increased pharmacokinetic parameters (AUC(0-t) and Cmax) of lapatinib, indicating that shikonin increased the exposure of lapatinib in rats. Moreover, in vitro kinetic measurements indicated that shikonin was a time-independent inhibitor, which inhibited the metabolism of lapatinib through a competitive mechanism in RLMs, while noncompetitive inhibition type in both HLMs and CYP3A4.1. Molecular docking analysis further verified the non-competitive inhibition of shikonin on lapatinib in CYP3A4.1. CONCLUSION: We developed an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites. It could be successfully applied to the study of pharmacokinetic interaction of shikonin on the inhibition of lapatinib metabolism in vivo and in vitro. In the end, further studies are needed to determine if such interactions are indeed valid in humans and if the interaction is clinically relevant.
Asunto(s)
Citocromo P-450 CYP3A , Naftoquinonas , Espectrometría de Masas en Tándem , Ratas , Humanos , Animales , Lapatinib/metabolismo , Ratas Sprague-Dawley , Cromatografía Liquida , Espectrometría de Masas en Tándem/métodos , Citocromo P-450 CYP3A/metabolismo , Simulación del Acoplamiento Molecular , Cromatografía Líquida de Alta Presión/métodos , Microsomas Hepáticos/metabolismoRESUMEN
Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.
Asunto(s)
Quitinasas , Quitinasas/química , Quitinasas/metabolismo , Quitina/química , Quitina/metabolismo , Dominio Catalítico , Unión Proteica , Serratia marcescens/metabolismoRESUMEN
Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.