RESUMEN
Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.
Asunto(s)
Aciclovir , Antivirales , Endodesoxirribonucleasas , Herpesvirus Humano 1 , Antivirales/farmacología , Células Vero , Chlorocebus aethiops , Animales , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Aciclovir/farmacología , Ganciclovir/farmacología , Foscarnet/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Cidofovir/farmacología , Humanos , Bromodesoxiuridina/análogos & derivadosRESUMEN
Human cytomegalovirus (HCMV) can cause serious diseases in immunocompromised patients. Use of current antivirals is limited by their adverse effects and emergence of drug resistance mutations. Thus, new drugs are an urgent need. The terminase complex (pUL56-pUL89-pUL51) represents a target of choice for new antivirals development. pUL51 was shown to be crucial for the cleavage of concatemeric HCMV DNA and viral replication. Its C-terminal part plays a critical role for the terminase complex assembly. However, no interaction domain is clearly identified. Sequence comparison of herpesvirus homologs and protein modelling were performed on pUL51. Importance of a putative interaction domain is validated by the generation of recombinant viruses with specific alanine substitutions of amino acids implicated in the domain. We identified a Leucine-Zipper (LZ) domain involving the leucine residues L126-X6-L133-X6-L140-X6-L147 in C-terminal part of pUL51. These leucines are crucial for viral replication, suggesting the significance for pUL51 structure and function. A mimetic-peptide approach has been used and tested in antiviral assays to validate the interaction domain as a new therapeutic target. Cytotoxicity was evaluated by LDH release measurement. The peptide TAT-HK29, homologous to the pUL51-LZ domain, inhibits HCMV replication by 27% ± 9% at 1.25 µM concentration without cytotoxicity. Our results highlight the importance of a leucine zipper domain in the C-terminal part of pUL51 involving leucines L126, L133, L140 and L147. We also confirm the potential of mimetic peptides to inhibit HCMV replication and the importance to target interaction domains to develop antiviral agents.
Asunto(s)
Antivirales , Materiales Biomiméticos , Citomegalovirus , Endodesoxirribonucleasas , Leucina Zippers , Proteínas Virales , Replicación Viral , Replicación Viral/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Antivirales/química , Antivirales/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Proteínas Virales/genética , Desarrollo de Medicamentos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/química , Humanos , Péptidos/química , Péptidos/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacologíaRESUMEN
Genotoxic insult causes nuclear and mitochondrial DNA damages with macroautophagy/autophagy induction. The role of mitochondrial DNA (mtDNA) damage in the requirement of autophagy for nuclear DNA (nDNA) stability is unclear. Using site-specific DNA damage approaches, we show that specific nDNA damage alone does not require autophagy for repair unless in the presence of mtDNA damage. We provide evidence that after IR exposure-induced mtDNA and nDNA damages, autophagy suppression causes non-apoptotic mitochondrial permeability, by which mitochondrial ENDOG (endonuclease G) is released and translocated to nuclei to sustain nDNA damage in a TET (tet methylcytosine dioxygenase)-dependent manner. Furthermore, blocking lysosome function is sufficient to increase the amount of mtDNA leakage to the cytosol, accompanied by ENDOG-free mitochondrial puncta formation with concurrent ENDOG nuclear accumulation. We proposed that autophagy eliminates the mitochondria specified by mtDNA damage-driven mitochondrial permeability to prevent ENDOG-mediated genome instability. Finally, we showed that HBx, a hepatitis B viral protein capable of suppressing autophagy, also causes mitochondrial permeability-dependent ENDOG mis-localization in nuclei and is linked to hepatitis B virus (HBV)-mediated hepatocellular carcinoma development.Abbreviations: 3-MA: 3-methyladenine; 5-hmC: 5-hydroxymethylcytosine; ACTB: actin beta; ATG5: autophagy related 5; ATM: ATM serine/threonine kinase; DFFB/CAD: DNA fragmentation factor subunit beta; cmtDNA: cytosolic mitochondrial DNA; ConA: concanamycin A; CQ: chloroquine; CsA: cyclosporin A; Dox: doxycycline; DSB: double-strand break; ENDOG: endonuclease G; GFP: green fluorescent protein; Gy: gray; H2AX: H2A.X variant histone; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HCC: hepatocellular carcinoma; I-PpoI: intron-encoded endonuclease; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOMP: mitochondrial outer membrane permeability; mPTP: mitochondrial permeability transition pore; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; 4-OHT: 4-hydroxytamoxifen; rDNA: ribosomal DNA; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TET: tet methylcytosine dioxygenase; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.
Asunto(s)
Autofagia/genética , Daño del ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Endodesoxirribonucleasas/metabolismo , Inestabilidad Genómica , Transporte Activo de Núcleo Celular , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/antagonistas & inhibidores , Dioxigenasas/genética , Dioxigenasas/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Neoplasias Hepáticas/metabolismo , Mitocondrias/enzimología , Mitocondrias/genética , PermeabilidadRESUMEN
Human cytomegalovirus (HCMV) is an important ubiquitous opportunistic pathogen that belongs to the betaherpesviridae. Primary HCMV infection is generally asymptomatic in immunocompetent individuals. In contrast, HCMV infection causes serious disease in immunocompromised patients and is the leading cause of congenital viral infection. Although they are effective, the use of conventional molecules is limited by the emergence of resistance and by their toxicity. New antivirals targeting other replication steps and inducing fewer adverse effects are therefore needed. During HCMV replication, DNA packaging is performed by the terminase complex, which cleaves DNA to package the virus genome into the capsid. With no counterpart in mammalian cells, these terminase proteins are ideal targets for highly specific antivirals. A new terminase inhibitor, letermovir, recently proved effective against HCMV in phase III clinical trials. However, its mechanism of action is unclear and it has no significant activity against other herpesvirus or non-human CMV.
TITLE: Le complexe terminase, une cible de choix dans le traitement de l'infection à cytomégalovirus humain. ABSTRACT: Le cytomégalovirus humain (CMVH) est un pathogène opportuniste majeur en cas d'immunodépression et représente la principale cause d'infection congénitale d'origine virale. Bien qu'efficace, l'utilisation des molécules conventionnelles est limitée par leur toxicité et par l'émergence de résistance du virus, rendant nécessaire le développement de nouveaux traitements. Lors de la réplication du CMVH, l'encapsidation de l'ADN est réalisée par le complexe terminase qui clive l'ADN pour empaqueter le génome dans la capside. L'absence d'homologues dans les cellules des mammifères rend les protéines du complexe terminase des cibles idéales pour des antiviraux spécifiques. Une nouvelle molécule, le letermovir, cible une étape exclusivement virale en interagissant avec le complexe terminase. Son efficacité a été prouvée lors d'essais cliniques de phase III. Néanmoins, son mécanisme d'action n'est, à ce jour, pas élucidé et aucune activité n'est observée contre les autres herpèsvirus.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Endodesoxirribonucleasas/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Citomegalovirus/patogenicidad , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/inmunología , Endodesoxirribonucleasas/fisiología , Humanos , Huésped Inmunocomprometido , Terapia Molecular Dirigida/tendencias , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/fisiología , Ensamble de Virus/efectos de los fármacos , Ensamble de Virus/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.
Asunto(s)
Sistemas CRISPR-Cas/efectos de los fármacos , Edición Génica/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Virales/genética , Virus/genética , Archaea/genética , Archaea/inmunología , Archaea/virología , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Coevolución Biológica , Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , ADN/antagonistas & inhibidores , ADN/química , ADN/genética , ADN/metabolismo , División del ADN/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Familia de Multigenes , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/farmacología , Virus/metabolismo , Virus/patogenicidadRESUMEN
The effect of sulfated polysaccharides on the digestion of dietary DNA by pepsin was studied using in vitro simulated gastric juice. The results showed that fucoidan (FUC), dextran sulfate (DS) and chondroitin sulfate (CS) could inhibit the digestion of DNA in a dose-dependent manner. Polysaccharides with high sulfate group content have stronger inhibition ability. Fluorescence spectroscopy results showed that polysaccharides could bind to pepsin, and transmission electron microscopy (TEM) confirmed that polysaccharides can interact with DNA, which not only is the main reason that polysaccharides inhibit the digestion of DNA by pepsin but also causes the digestion of DNA by DNase II to be inhibited. The finding suggests that the digestion of DNA should be reevaluated when eating foods rich in sulfated polysaccharides. This study enriched the known pharmacological properties of sulfated polysaccharides as pepsin inhibitors and provided inspiration for the use of sulfated polysaccharides as oligonucleotide drug delivery carriers.
Asunto(s)
ADN , Modelos Biológicos , Pepsina A , Polisacáridos , Sulfatos , Animales , Sulfatos de Condroitina , ADN/química , ADN/metabolismo , Sulfato de Dextran , Digestión/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Jugo Gástrico/química , Jugo Gástrico/metabolismo , Pepsina A/antagonistas & inhibidores , Pepsina A/química , Pepsina A/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Polisacáridos/farmacología , Sulfatos/química , Sulfatos/metabolismo , Sulfatos/farmacologíaRESUMEN
Approximately 80% of adults are infected with a member of the herpesviridae family. Herpesviruses establish life-long latent infections within neurons, which may reactivate into lytic infections due to stress or immune suppression. There are nine human herpesviruses (HHV) posing health concerns from benign conditions to life threatening encephalitis, including cancers associated with viral infections. The current treatment options for most HHV conditions mainly include several nucleoside and nucleotide analogs targeting viral DNA polymerase. Although these drugs help manage infections, their common mechanism of action may lead to the development of drug resistance, which is particularly devastating in immunocompromised patients. Therefore, new classes of drugs directed against novel targets in HHVs are necessary to alleviate this issue. We analyzed the conservation rates of all proteins in herpes simplex virus 1 (HHV-1), a representative of the HHV family and one of the most common viruses infecting the human population. Furthermore, we generated a full-length structure model of the most conserved HHV-1 protein, the DNA packaging terminase pUL15. A series of computational analyses were performed on the model to identify ATP and DNA binding sites and characterize the dynamics of the protein. Our study indicates that proteins involved in HHV-1 DNA packaging and cleavage are amongst the most conserved gene products of HHVs. Since the packaging protein pUL15 is the most conserved among all HHV-1 gene products, the virus will have a lower chance of developing resistance to small molecules targeting pUL15. A subsequent analysis of the structure of pUL15 revealed distinct ATP and DNA binding domains and the elastic network model identifies a functionally important hinge region between the two domains of pUL15. The atomic information on the active and allosteric sites in the ATP- and DNA-bound model of pUL15 presented in this study can inform the structure-based drug discovery of a new class of drugs to treat a wide range of HHVs.
Asunto(s)
Antivirales/farmacología , Empaquetamiento del ADN/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Simplexvirus/efectos de los fármacos , Simplexvirus/enzimología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Sitio Alostérico/efectos de los fármacos , ADN Viral/metabolismo , Endodesoxirribonucleasas/metabolismo , Pruebas de Sensibilidad Microbiana , Simplexvirus/genética , Proteínas Virales/metabolismoRESUMEN
Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Bacterianas/química , Proteínas Asociadas a CRISPR/química , Microscopía por Crioelectrón , ADN/metabolismo , Endodesoxirribonucleasas/química , Inhibidores Enzimáticos/química , Edición Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , ARN Guía de Kinetoplastida/metabolismoRESUMEN
CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics.
Asunto(s)
Sistemas CRISPR-Cas , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/metabolismo , Inhibidores Enzimáticos/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Ribonucleasas/metabolismo , Microscopía por Crioelectrón , Endodesoxirribonucleasas/ultraestructura , Unión Proteica , Conformación Proteica , ARN Guía de Kinetoplastida/ultraestructura , Ribonucleasas/ultraestructuraRESUMEN
Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity, and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability.
Asunto(s)
Endodesoxirribonucleasas/química , Neoplasias Pancreáticas/tratamiento farmacológico , Péptidos/química , Termodinámica , Aminoácidos/química , Supervivencia Celular/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Humanos , Simulación de Dinámica Molecular , Mutación/genética , Neoplasias Pancreáticas/genética , Péptidos/genética , Péptidos/farmacología , Unión ProteicaRESUMEN
A key step in the replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. Enzymes required for this process are so-called terminases, first described for double-stranded DNA bacteriophages. The HCMV terminase consists of the two subunits, the ATPase pUL56 and the nuclease pUL89, and a potential third component pUL51. The terminase subunits are essential for virus replication and are highly conserved throughout the Herpesviridae family. Together with the portal protein pUL104 they form a powerful biological nanomotor. It has been shown for tailed dsDNA bacteriophages that DNA translocation into preformed capsid needs an extraordinary amount of energy. The HCMV terminase subunit pUL56 provides the required ATP hydrolyzing activity. The necessary nuclease activity to cleave the concatemers into unit-length genomes is mediated by the terminase subunit pUL89. Whether this cleavage is mediated by site-specific duplex nicking has not been demonstrated, however, it is required for packaging. Binding to the portal is a prerequisite for DNA translocation. To date, it is a common view that during translocation the terminase moves along some domains of the DNA by a binding and release mechanism. These critical structures have proven to be outstanding targets for drugs to treat HCMV infections because corresponding structures do not exist in mammalian cells. Herein we examine the HCMV terminase as a target for drugs and review several inhibitors discovered by both lead-directed medicinal chemistry and by target-specific design. In addition to producing clinically active compounds the research also has furthered the understanding of the role and function of the terminase itself.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/prevención & control , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Endodesoxirribonucleasas/antagonistas & inhibidores , Acetatos/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Citomegalovirus/genética , Infecciones por Citomegalovirus/tratamiento farmacológico , Humanos , Quinazolinas/uso terapéutico , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Human cytomegalovirus (HCMV) can cause severe disease in patients with compromised or immature immune systems. Currently approved pharmacotherapies for the treatment of systemic HCMV infections [ganciclovir (GCV), cidofovir (CDV), foscarnet] are limited by a high incidence of adverse effects and/or the development of drug resistance. Given that many of these drugs have the same viral target (HCMV-encoded DNA polymerase), cross-resistance is relatively common. The primary means to combat drug resistance is combination pharmacotherapy using therapeutics with different molecular mechanisms of action with the expectation that those combinations result in an additive or synergistic enhancement of effect; combinations that result in antagonism can, in many cases, be detrimental to the outcome of the patient. We therefore tested select combinations of approved (GCV, CDV, letermovir (LMV)) and experimental (brincidofovir (BCV), cyclopropavir (CPV), maribavir (MBV), BDCRB) drugs with the hypothesis that combinations of drugs with different and distinct molecular mechanisms of action will produce an additive and/or synergistic enhancement of antiviral effect against HCMV in vitro. Using MacSynergy II (a statistical package that measures enhancement or lessening of effect relative to zero/additive), select drug combination studies demonstrated combination indices ranging from 160 to 372 with 95% confidence intervals greater than zero indicating that these combinations elicit a synergistic enhancement of effect against HCMV in vitro. These data suggest that administration of a viral DNA polymerase inhibitor, MBV, and/or a viral terminase inhibitor in combination has the potential to address the resistance/cross-resistance problems associated with currently available therapeutics.
Asunto(s)
Antivirales/administración & dosificación , Antivirales/farmacología , Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Citomegalovirus/efectos de los fármacos , Bencimidazoles/farmacología , Línea Celular , Cidofovir/farmacología , Ciclopropanos/farmacología , Citosina/análogos & derivados , Citosina/farmacología , ADN Polimerasa Dirigida por ADN/efectos de los fármacos , Antagonismo de Drogas , Combinación de Medicamentos , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Endodesoxirribonucleasas/antagonistas & inhibidores , Fibroblastos , Foscarnet/farmacología , Ganciclovir/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Organofosfonatos/farmacología , Ribonucleósidos/farmacología , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Human cytomegalovirus (HCMV) infection poses a major health threat to immunocompromised individuals. Until recently, treatment of HCMV infection has relied solely on polymerase inhibitors that have safety and resistance issues. pUL89 provides the enzymatic functions for the HCMV terminase complex in viral DNA packaging and is an attractive target for developing a new class of HCMV drugs. However, inhibitors of the endonuclease activity of the Câ terminus of pUL89 (pUL89-C) were unknown before our recently characterized hydroxypyridonecarboxylic acid (HPCA) hit 7 r (1-(3-chloro-4-fluorobenzyl)-5-hydroxy-4-oxo-1,4-dihydropyridine-3-carboxylic acid; numbered as 10 k in our previous publication: Y. Wang, L. Mao, J. Kankanala, Z. Wang, R.â J. Geraghty, J. Virol. 2017, 91, e02152-16). Herein, we explored the structure-activity relationship (SAR) of the HPCA chemotype mainly with regard to the N1 site through the synthesis of 35 analogues. The SAR studies, along with molecular modeling, identified a possible pharmacophore model minimally consisting of a chelating triad and a hydrophobic phenyl or biphenyl methyl substituent at N1. Lastly, our best compounds consistently inhibited pUL89-C in the low micromolar range in biochemical assays and exhibited strong antiviral activity without cytotoxicity, laying a solid medicinal chemistry foundation for further HCMV drug discovery efforts targeting pUL89-C.
Asunto(s)
Antivirales/farmacología , Citomegalovirus/enzimología , Endodesoxirribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piridonas/farmacología , Proteínas Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/química , Dominio Catalítico , Línea Celular , Endodesoxirribonucleasas/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Piridonas/síntesis química , Piridonas/química , Relación Estructura-Actividad , Proteínas Virales/química , Replicación Viral/efectos de los fármacosRESUMEN
Human cytomegalovirus (HCMV) is responsible for life-threatening infections in immunocompromised individuals and can cause serious congenital malformations. Available antivirals target the viral polymerase but are subject to cross-resistance and toxicity. New antivirals targeting other replication steps and inducing fewer adverse effects are therefore needed. During HCMV replication, DNA maturation and packaging are performed by the terminase complex, which cleaves DNA to package the genome into the capsid. Identified in herpesviruses and bacteriophages, and with no counterpart in mammalian cells, these terminase proteins are ideal targets for highly specific antivirals. A new terminase inhibitor, letermovir, recently proved effective against HCMV in phase III clinical trials, but the mechanism of action is unclear. Letermovir has no significant activity against other herpesvirus or non-human CMV. This review focuses on the highly conserved mechanism of HCMV DNA-packaging and the potential of the terminase complex to serve as an antiviral target. We describe the intrinsic mechanism of DNA-packaging, highlighting the structure-function relationship of HCMV terminase complex components.
Asunto(s)
Citomegalovirus/enzimología , Sistemas de Liberación de Medicamentos , Endodesoxirribonucleasas/antagonistas & inhibidores , Antivirales/administración & dosificación , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Infecciones por Citomegalovirus/tratamiento farmacológico , Endodesoxirribonucleasas/metabolismoRESUMEN
Letermovir is a human cytomegalovirus (CMV) terminase inhibitor that was clinically effective in a Phase III prevention trial. In vitro studies have shown that viral mutations conferring letermovir resistance map primarily to the UL56 component of the terminase complex and uncommonly to UL89. After serial culture of a baseline CMV laboratory strain under letermovir, mutation was observed in a third terminase component in 2 experiments, both resulting in amino acid substitution P91S in gene UL51 and adding to a pre-existing UL56 mutation. Recombinant phenotyping indicated that P91S alone conferred 2.1-fold increased letermovir resistance (EC50) over baseline, and when combined with UL56 mutation S229F or R369M, multiplied the level of resistance conferred by those mutations by 3.5-7.7-fold. Similarly a combination of UL56 mutations S229F, L254F and L257I selected in the same experiment conferred 54-fold increased letermovir EC50 over baseline, but 290-fold when combined with UL51 P91S. The P91S mutant was not perceptibly growth impaired. Although pUL51 is essential for normal function of the terminase complex, its biological significance is not well understood. Letermovir resistance mutations mapping to 3 separate genes, and their multiplier effect on the level of resistance, suggest that the terminase components interactively contribute to the structure of a letermovir antiviral target. The diagnostic importance of the UL51 P91S mutation arises from its potential to augment the letermovir resistance of some UL56 mutations at low fitness cost.
Asunto(s)
Acetatos/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Farmacorresistencia Viral/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Quinazolinas/farmacología , Antivirales/farmacología , Células Cultivadas , Infecciones por Citomegalovirus/virología , Endodesoxirribonucleasas/antagonistas & inhibidores , Fibroblastos , Genes Virales/genética , Humanos , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Mutación , Proteínas Virales/genéticaRESUMEN
Protein kinase B2 (AKT2) is implicated in diverse process of cardiomyocyte signaling including survival and metabolism. However, the role of AKT2 in myocardium development and the signaling pathway is rarely understood. Therefore, we sought to determine the effect of AKT2 deletion on heart development and its downstream targets. By using experimental animal models and neonatal rat cardiomyocytes (NRCMs), we observed that AKT2 deficiency induces retardation of heart development and increased systemic blood pressure (BP) without affecting cardiac function. Further investigation suggested that deficiency of AKT2 in myocardium results in diminished MEF2A abundance, which induced decreased size of cardiomyocytes. We additionally confirmed that EndoG, which is also regulated by AKT2, is a suppressor of MEF2A in myocardium. Finally, our results proved that AKT2 deficiency impairs the response to ß-adrenergic stimuli that normally causes hypertrophy in cardiomyocytes by downregulating MEF2A expression. Our data are the first to show the important role of AKT2 in determining the size of myocardium, its deficiency causes retardation of cardiomyocyte development. We also proved a novel pathway of heart development involving EndoG and MEF2A regulated by AKT2.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-akt/deficiencia , Animales , Diferenciación Celular , Tamaño de la Célula , Células Cultivadas , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Técnicas de Silenciamiento del Gen , Corazón/crecimiento & desarrollo , Factores de Transcripción MEF2/antagonistas & inhibidores , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Ratones Noqueados , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/genética , Ratas , Transducción de SeñalRESUMEN
Developmental genetic studies of Antirrhinum majus demonstrated that two transcription factors from the MYB gene family, RADIALIS (RAD) and DIVIRICATA (DIV), interact through antagonism to regulate floral dorsoventral asymmetry. Interestingly, similar antagonistic interaction found among proteins of FSM1 (RAD-like) and MYBI (DIV-like) in Solanum lycopersicum is involved in fruit development. Here, we report the reconstruction of the phylogeny of I-box-like and R-R-type clades, where RAD- and DIV-like genes belong, respectively. We also examined the homology of these antagonistic MYB proteins using these phylogenies. The results show that there are likely three paralogs of RAD-/I-box-like genes, RAD1, RAD2, and RAD3, which originated in the common ancestor of the core eudicots. In contrast, R-R-type sequences fall into two major clades, RR1 and RR2, the result of gene duplication in the common ancestor of both monocots and dicots. RR1 was divided into clades RR1A, RR1B, and RR1C, while RR2 was divided into clades RR2A/DIV1, RR2B/DIV2, and RR2C/DIV3. We demonstrate that among similar antagonistic interactions in An. Majus and So. lycopersicum, RAD-like genes originate from the RAD2 clade, while DIV-like genes originate from distantly related paralogs of the R-R-type lineage. The phylogenetic analyses of these two MYB clades lay the foundation for future comparative studies including testing the evolution of the antagonistic relationship of proteins.
Asunto(s)
Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Variación Genética , Filogenia , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Convolvulaceae/genética , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Duplicación de Gen , Magnoliopsida/genética , Oryza/genética , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Solanaceae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Daño del ADN , Desoxirribonucleasa I/metabolismo , Endodesoxirribonucleasas/metabolismo , Células Epiteliales/efectos de los fármacos , Grafito/toxicidad , Túbulos Renales/efectos de los fármacos , Nanopartículas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Desoxirribonucleasa I/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células Epiteliales/enzimología , Células Epiteliales/patología , Proteínas HSP90 de Choque Térmico/metabolismo , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Túbulos Renales/enzimología , Túbulos Renales/patología , Estrés Oxidativo/efectos de los fármacos , Ratas , Medición de Riesgo , Factores de TiempoRESUMEN
Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity at the low pH environment of lysosomes where it is typically found in higher eukaryotes. Interestingly, DNase II has also been identified in a few genera of bacteria and is believed to have arisen via horizontal transfer. Here, we demonstrate that recombinant Burkholderia thailandensis DNase II is highly active at low pH in the absence of divalent metal ions, similar to eukaryotic DNase II. The crystal structure of B. thailandensis DNase II shows a dimeric quaternary structure which appears capable of binding double-stranded DNA. Each monomer of B. thailandensis DNase II exhibits a similar overall fold as phospholipase D (PLD), phosphatidylserine synthase (PSS) and tyrosyl-DNA phosphodiesterase (TDP), and conserved catalytic residues imply a similar mechanism. The structural and biochemical data presented here provide insights into the atomic structure and catalytic mechanism of DNase II.
Asunto(s)
Proteínas Bacterianas/química , Burkholderia/enzimología , Endodesoxirribonucleasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cobre/farmacología , Cristalografía por Rayos X , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/metabolismo , Células Eucariotas/enzimología , Evolución Molecular , Transferencia de Gen Horizontal , Concentración de Iones de Hidrógeno , Modelos Moleculares , Simulación del Acoplamiento Molecular , Filogenia , Células Procariotas/enzimología , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. IMPORTANCE: Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.