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1.
Sci Rep ; 11(1): 7429, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33795718

RESUMEN

The 2019 novel coronavirus pandemic caused by SARS-CoV-2 remains a serious health threat to humans and there is an urgent need to develop therapeutics against this deadly virus. Recent scientific evidences have suggested that the main protease (Mpro) enzyme in SARS-CoV-2 can be an ideal drug target due to its crucial role in the viral replication and transcription processes. Therefore, there are ongoing research efforts to identify drug candidates against SARS-CoV-2 Mpro that resulted in hundreds of X-ray crystal structures of ligand-bound Mpro complexes in the Protein Data Bank (PDB) describing the interactions of different fragment chemotypes within different sites of the Mpro. In this work, we performed rigorous molecular dynamics (MD) simulation of 62 reversible ligand-Mpro complexes in the PDB to gain mechanistic insights about their interactions at the atomic level. Using a total of over 3 µs long MD trajectories, we characterized different pockets in the apo Mpro structure, and analyzed the dynamic interactions and binding affinity of ligands within those pockets. Our results identified the key residues that stabilize the ligands in the catalytic sites and other pockets of Mpro. Our analyses unraveled the role of a lateral pocket in the catalytic site in Mpro that is critical for enhancing the ligand binding to the enzyme. We also highlighted the important contribution from HIS163 in the lateral pocket towards ligand binding and affinity against Mpro through computational mutation analyses. Further, we revealed the effects of explicit water molecules and Mpro dimerization in the ligand association with the target. Thus, comprehensive molecular-level insights gained from this work can be useful to identify or design potent small molecule inhibitors against SARS-CoV-2 Mpro.


Asunto(s)
Simulación de Dinámica Molecular , Inhibidores de Proteasas/química , Proteínas de la Matriz Viral/antagonistas & inhibidores , Sitios de Unión , /virología , Dominio Catalítico , Bases de Datos de Proteínas , Humanos , Ligandos , Mutagénesis Sitio-Dirigida , Análisis de Componente Principal , Inhibidores de Proteasas/metabolismo , Termodinámica , Proteínas de la Matriz Viral/metabolismo
2.
Nat Commun ; 12(1): 2079, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824320

RESUMEN

Human manganese superoxide dismutase is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers are used by the enzyme to rid the mitochondria of O2•-. The mechanisms of concerted transfer enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules at particular redox states. Here, neutron diffraction of two redox-controlled manganese superoxide dismutase crystals reveal the all-atom structures of Mn3+ and Mn2+ enzyme forms. The structures deliver direct data on protonation changes between oxidation states of the metal. Observations include glutamine deprotonation, the involvement of tyrosine and histidine with altered pKas, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond. We report a concerted proton and electron transfer mechanism for human manganese superoxide dismutase from the direct visualization of active site protons in Mn3+ and Mn2+ redox states.


Asunto(s)
Electrones , Protones , Superóxido Dismutasa/metabolismo , Aminoácidos/metabolismo , Aniones , Biocatálisis , Dominio Catalítico , Glutamina/metabolismo , Humanos , Ligandos , Neutrones , Multimerización de Proteína , Solventes , Superóxido Dismutasa/química
3.
Nat Commun ; 12(1): 2059, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824325

RESUMEN

Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase µ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutagénesis/genética , Nucleótidos/metabolismo , Adenina/metabolismo , Emparejamiento Base , Biocatálisis , Dominio Catalítico , Citosina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Insercional/genética , Oxidación-Reducción
4.
Molecules ; 26(5)2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807773

RESUMEN

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (Mpro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites-including xenobiotics-that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results-obtained in silico and in vitro-prove that silybin and silymarin, respectively, are able to inhibit Mpro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19.


Asunto(s)
Antivirales/farmacología , Metaboloma , Inhibidores de Proteasas/farmacología , Silimarina/farmacología , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Dominio Catalítico/efectos de los fármacos , Simulación por Computador , /química , Bases de Datos de Compuestos Químicos , Descubrimiento de Drogas , Pruebas de Enzimas , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Silimarina/química , Silimarina/metabolismo , Programas Informáticos
5.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33809335

RESUMEN

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in ß-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A ß-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on ß-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of ß-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.


Asunto(s)
Proteínas Mutantes/genética , Conformación Proteica , Ingeniería de Proteínas , beta-Lactamasas/genética , Aminoácidos/genética , Bacterias/enzimología , Sitios de Unión/genética , Dominio Catalítico/genética , Biología Computacional , Estabilidad de Enzimas/genética , Escherichia coli/enzimología , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas Mutantes/ultraestructura , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , beta-Lactamasas/ultraestructura
6.
Nat Commun ; 12(1): 1347, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649331

RESUMEN

The human microbiome can produce metabolites that modulate insulin signaling. Type 2 diabetes patients have increased circulating concentrations of the microbially produced histidine metabolite, imidazole propionate (ImP) and administration of ImP in mice resulted in impaired glucose tolerance. Interestingly, the fecal microbiota of the patients had increased capacity to produce ImP, which is mediated by the bacterial enzyme urocanate reductase (UrdA). Here, we describe the X-ray structures of the ligand-binding domains of UrdA in four different states, representing the structural transitions along the catalytic reaction pathway of this unexplored enzyme linked to disease in humans. The structures in combination with functional data provide key insights into the mechanism of action of UrdA that open new possibilities for drug development strategies targeting type 2 diabetes.


Asunto(s)
Imidazoles/metabolismo , Oxidorreductasas/metabolismo , Shewanella/enzimología , Ácido Urocánico/metabolismo , Arginina/metabolismo , Dominio Catalítico , Flavina-Adenina Dinucleótido/metabolismo , Imidazoles/química , Cinética , Ligandos , Modelos Moleculares , Oxidorreductasas/química , Conformación Proteica , Dominios Proteicos , Especificidad por Sustrato , Termodinámica , Ácido Urocánico/química
7.
J Phys Chem B ; 125(10): 2533-2550, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33657325

RESUMEN

The novel RNA virus, severe acute respiratory syndrome coronavirus II (SARS-CoV-2), is currently the leading cause of mortality in 2020, having led to over 1.6 million deaths and infecting over 75 million people worldwide by December 2020. While vaccination has started and several clinical trials for a number of vaccines are currently underway, there is a pressing need for a cure for those already infected with the virus. Of particular interest in the design of anti-SARS-CoV-2 therapeutics is the human protein angiotensin converting enzyme II (ACE2) to which this virus adheres before entry into the host cell. The SARS-CoV-2 virion binds to cell-surface bound ACE2 via interactions of the spike protein (s-protein) on the viral surface with ACE2. In this paper, we use all-atom molecular dynamics simulations and binding enthalpy calculations to determine the effect that a bound ACE2 active site inhibitor (MLN-4760) would have on the binding affinity of SARS-CoV-2 s-protein with ACE2. Our analysis indicates that the binding enthalpy could be reduced for s-protein adherence to the active site inhibitor-bound ACE2 protein by as much as 1.48-fold as an upper limit. This weakening of binding strength was observed to be due to the destabilization of the interactions between ACE2 residues Glu-35, Glu-37, Tyr-83, Lys-353, and Arg-393 and the SARS-CoV-2 s-protein receptor binding domain (RBD). The conformational changes were shown to lead to weakening of ACE2 interactions with SARS-CoV-2 s-protein, therefore reducing s-protein binding strength. Further, we observed increased conformational lability of the N-terminal helix and a conformational shift of a significant portion of the ACE2 motifs involved in s-protein binding, which may affect the kinetics of the s-protein binding when the small molecule inhibitor is bound to the ACE2 active site. These observations suggest potential new ways for interfering with the SARS-CoV-2 adhesion by modulating ACE2 conformation through distal active site inhibitor binding.


Asunto(s)
/metabolismo , Inhibidores de Proteasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , /antagonistas & inhibidores , Sitios de Unión , /virología , Dominio Catalítico , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Inhibidores de Proteasas/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Termodinámica
8.
Methods Mol Biol ; 2266: 155-170, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33759126

RESUMEN

Medicinal chemistry society has enough arguments to justify the usage of fragment-based drug design (FBDD) methodologies for the identification of lead compounds. Since the FDA approval of three kinase inhibitors - vemurafenib, venetoclax, and erdafitinib, FBDD has become a challenging alternative to high-throughput screening methods in drug discovery. The following protocol presents in silico drug design of selective histone deacetylase 6 (HDAC6) inhibitors through a fragment-based approach. To date, structural motifs that are important for HDAC inhibitory activity and selectivity are described as: surface recognition group (CAP group), aliphatic or aromatic linker, and zinc-binding group (ZBG). The main idea of this FBDD method is to identify novel and target-selective CAP groups by virtual scanning of publicly available fragment databases. Template structure used to search for novel heterocyclic and carbocyclic fragments is 1,8-naphthalimide (CAP group of scriptaid, a potent HDAC inhibitor). Herein, the design of HDAC6 inhibitors is based on linking the identified fragments with the aliphatic or aromatic linker and hydroxamic acid (ZBG) moiety. Final selection of potential selective HDAC6 inhibitors is based on combined structure-based (molecular docking) and ligand-based (three-dimensional quantitative structure-activity relationships, 3D-QSAR) techniques. Designed compounds are docked in the active site pockets of human HDAC1 and HDAC6 isoforms, and their docking conformations used to predict their HDAC inhibitory and selectivity profiles through two developed 3D-QSAR models (describing HDAC1 and HDAC6 inhibitory activities).


Asunto(s)
Descubrimiento de Drogas/métodos , Histona Desacetilasa 6/química , Inhibidores de Histona Desacetilasas/química , Simulación del Acoplamiento Molecular/métodos , Naftalimidas/química , Secuencias de Aminoácidos , Dominio Catalítico , Bases de Datos de Compuestos Químicos , Diseño de Fármacos , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/química , Histona Desacetilasa 6/antagonistas & inhibidores , Técnicas In Vitro , Ligandos , Conformación Molecular , Simulación de Dinámica Molecular , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad
9.
Science ; 372(6537): 52-56, 2021 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-33707221

RESUMEN

Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.


Asunto(s)
Complejo Mediador/química , ARN Polimerasa II/química , Factores Generales de Transcripción/química , Iniciación de la Transcripción Genética , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Complejo Mediador/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismo , Factores Generales de Transcripción/metabolismo
10.
Phys Chem Chem Phys ; 23(10): 5852-5863, 2021 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-33688867

RESUMEN

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Cianuros/química , Nucleótidos/metabolismo , /fisiología , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Alanina/química , Alanina/metabolismo , Alanina/farmacología , Alanina/uso terapéutico , /patología , Dominio Catalítico , Humanos , Simulación de Dinámica Molecular , Ribosa/química , /metabolismo , Electricidad Estática , Replicación Viral/efectos de los fármacos
11.
Nat Struct Mol Biol ; 28(3): 319-325, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33674802

RESUMEN

The COVID-19 pandemic caused by nonstop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report that suramin, a 100-year-old drug, is a potent inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and acts by blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for treatment of COVID-19. The 2.6 Å cryo-electron microscopy structure of the viral RdRp bound to suramin reveals two binding sites. One site directly blocks the binding of the RNA template strand and the other site clashes with the RNA primer strand near the RdRp catalytic site, thus inhibiting RdRp activity. Suramin blocks viral replication in Vero E6 cells, although the reasons underlying this effect are likely various. Our results provide a structural mechanism for a nonnucleotide inhibitor of the SARS-CoV-2 RdRp.


Asunto(s)
Antivirales/farmacología , /química , Inhibidores Enzimáticos/farmacología , Suramina/farmacología , Animales , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Dominio Catalítico , Chlorocebus aethiops , Microscopía por Crioelectrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , Suramina/química , Suramina/metabolismo , Células Vero , Replicación Viral/efectos de los fármacos
12.
Nat Commun ; 12(1): 1621, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712579

RESUMEN

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and ß-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.


Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Simulación de Dinámica Molecular , Mutación , Catálisis , Dominio Catalítico/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hidroxilación , Cinética , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por Sustrato
13.
Phys Chem Chem Phys ; 23(11): 6746-6757, 2021 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-33711090

RESUMEN

COVID-19, the disease caused by the newly discovered coronavirus-SARS-CoV-2, has created a global health, social, and economic crisis. As of mid-January 2021, there are over 90 million confirmed cases and more than 2 million reported deaths due to COVID-19. Currently, there are very limited therapeutics for the treatment or prevention of COVID-19. For this reason, it is important to find drug targets that will lead to the development of safe and effective therapeutics against the disease. The main protease (Mpro) of the virus is an attractive target for the development of effective antiviral therapeutics because it is required for proteolytic cleavage of viral polyproteins. Furthermore, the Mpro has no human homologues, so drugs designed to bind to this target directly have less risk for off-target effects. Recently, several high-resolution crystallographic structures of the Mpro in complex with inhibitors have been determined-to guide drug development and to spur efforts in structure-based drug design. One of the primary objectives of modern structure-based drug design is the accurate prediction of receptor-ligand binding affinities for rational drug design and discovery. Here, we perform rigorous alchemical absolute binding free energy calculations and QM/MM calculations to give insight into the total binding energy of two recently crystallized inhibitors of SARS-CoV-2 Mpro, namely, N3 and α-ketoamide 13b. The total binding energy consists of both covalent and non-covalent binding components since both compounds are covalent inhibitors of the Mpro. Our results indicate that the covalent and non-covalent binding free energy contributions of both inhibitors to the Mpro target differ significantly. The N3 inhibitor has more favourable non-covalent interactions, particularly hydrogen bonding, in the binding site of the Mpro than the α-ketoamide inhibitor. Also, the Gibbs energy of reaction for the Mpro-N3 covalent adduct is greater than the Gibbs reaction energy for the Mpro-α-ketoamide covalent adduct. These differences in the covalent and non-covalent binding free energy contributions for both inhibitors could be a plausible explanation for their in vitro differences in antiviral activity. Our findings are consistent with the reversible and irreversible character of both inhibitors as reported by experiment and highlight the importance of both covalent and non-covalent binding free energy contributions to the absolute binding affinity of a covalent inhibitor towards its target. This information could prove useful in the rational design, discovery, and evaluation of potent SARS-CoV-2 Mpro inhibitors for targeted antiviral therapy.


Asunto(s)
Peptidomiméticos/química , Inhibidores de Proteasas/química , Proteínas de la Matriz Viral/antagonistas & inhibidores , Amidas/química , Amidas/metabolismo , Sitios de Unión , /virología , Dominio Catalítico , Diseño de Fármacos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Simulación de Dinámica Molecular , Peptidomiméticos/metabolismo , Inhibidores de Proteasas/metabolismo , Teoría Cuántica , Termodinámica , Proteínas de la Matriz Viral/metabolismo
14.
Nat Commun ; 12(1): 1852, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767175

RESUMEN

TEM-1 ß-lactamase degrades ß-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of ß-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian ß-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 ß-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.


Asunto(s)
beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , Biología Computacional , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Evolución Molecular , Simulación de Dinámica Molecular , Penicilinas/metabolismo , Conformación Proteica , Especificidad por Sustrato
15.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-33670016

RESUMEN

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins' function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.


Asunto(s)
Atovacuona/farmacología , Citocromos b/genética , Resistencia a Medicamentos/genética , Proteínas con Hierro-Azufre/genética , Membrana Dobles de Lípidos/metabolismo , Mutación/genética , Fosfolípidos/metabolismo , Plasmodium falciparum/genética , Animales , Atovacuona/química , Dominio Catalítico , Bovinos , Resistencia a Medicamentos/efectos de los fármacos , Entropía , Proteínas con Hierro-Azufre/metabolismo , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plasmodium falciparum/efectos de los fármacos , Conformación Proteica , Mapas de Interacción de Proteínas , Estabilidad Proteica
16.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671286

RESUMEN

CpdB is a 3'-nucleotidase/2'3'-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3',5'-cyclic diadenosine monophosphate) and c-di-GMP (3',5'-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-ß response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell-wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3'-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2',3'-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.


Asunto(s)
2',3'-Nucleótido Cíclico Fosfodiesterasas/química , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Adenosina/metabolismo , Biocatálisis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfatos/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , Sitios de Unión , Dominio Catalítico , Proteínas de Escherichia coli/genética , Histidina/metabolismo , Hidrólisis , Cinética , Modelos Moleculares , Mutación Puntual/genética , Dominios Proteicos , Relación Estructura-Actividad , Especificidad por Sustrato
17.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652925

RESUMEN

Two new minor Amaryllidaceae alkaloids were isolated from Hippeastrum × hybridum cv. Ferrari and Narcissus pseudonarcissus cv. Carlton. The chemical structures were identified by various spectroscopic (one- and two-dimensional (1D and 2D) NMR, circular dichroism (CD), high-resolution mass spectrometry (HRMS) and by comparison with literature data of similar compounds. Both isolated alkaloids were screened for their human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) inhibition activity. One of the new compounds, a heterodimer alkaloid of narcikachnine-type, named narciabduliine (2), showed balanced inhibition potency for both studied enzymes, with IC50 values of 3.29 ± 0.73 µM for hAChE and 3.44 ± 0.02 µM for hBuChE. The accommodation of 2 into the active sites of respective enzymes was predicted using molecular modeling simulation.


Asunto(s)
Alcaloides/química , Alcaloides de Amaryllidaceae/química , Inhibidores de la Colinesterasa/química , Colinesterasas/ultraestructura , Alcaloides/farmacología , Enfermedad de Alzheimer , Alcaloides de Amaryllidaceae/farmacología , Butirilcolinesterasa/química , Butirilcolinesterasa/ultraestructura , Dominio Catalítico/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Colinesterasas/química , Dicroismo Circular , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad
18.
Nat Commun ; 12(1): 1728, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741927

RESUMEN

Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.


Asunto(s)
Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Humanos , Leucotrieno C4/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Estrés Oxidativo , Conformación Proteica
19.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-33670179

RESUMEN

Fumarylacetoacetate hydrolase (FAH) is the fifth enzyme in the tyrosine catabolism pathway. A deficiency in human FAH leads to hereditary tyrosinemia type I (HT1), an autosomal recessive disorder that results in the accumulation of toxic metabolites such as succinylacetone, maleylacetoacetate, and fumarylacetoacetate in the liver and kidney, among other tissues. The disease is severe and, when untreated, it can lead to death. A low tyrosine diet combined with the herbicidal nitisinone constitutes the only available therapy, but this treatment is not devoid of secondary effects and long-term complications. In this study, we targeted FAH for the first-time to discover new chemical modulators that act as pharmacological chaperones, directly associating with this enzyme. After screening several thousand compounds and subsequent chemical redesign, we found a set of reversible inhibitors that associate with FAH close to the active site and stabilize the (active) dimeric species, as demonstrated by NMR spectroscopy. Importantly, the inhibitors are also able to partially restore the normal phenotype in a newly developed cellular model of HT1.


Asunto(s)
Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/farmacología , Hidrolasas/antagonistas & inhibidores , Hidrolasas/metabolismo , Tirosinemias/tratamiento farmacológico , Tirosinemias/enzimología , Animales , Dominio Catalítico , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Hidrolasas/genética , Ratones , Tirosinemias/genética
20.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-33670267

RESUMEN

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Eliminación de Secuencia
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