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1.
Curr Top Microbiol Immunol ; 389: 203-41, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25778681

RESUMEN

Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy.


Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/fisiología , Humanos , Inhibidores de la Transcriptasa Inversa/farmacología , Transcripción Reversa/efectos de los fármacos , Internalización del Virus/efectos de los fármacos
2.
Am J Hum Genet ; 91(2): 343-8, 2012 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-22863190

RESUMEN

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.-14C>T in the 5'-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.


Asunto(s)
Proteínas de la Membrana/genética , Osteogénesis Imperfecta/genética , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Exoma/genética , Femenino , Ligamiento Genético , Humanos , Masculino , Datos de Secuencia Molecular , Osteogénesis Imperfecta/diagnóstico por imagen , Mutación Puntual/genética , Radiografía , Análisis de Secuencia de ADN
3.
J Eukaryot Microbiol ; 61(2): 182-203, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24372610

RESUMEN

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1-5.8S-ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3-10.9 µm long and 5.1-10.0 µm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4-0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1-5.8S-ITS2, and LSU rDNA region differed by 1.5-3.8%, 6.0-17.4%, and 9.1-17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.


Asunto(s)
Dinoflagelados/clasificación , Dinoflagelados/aislamiento & purificación , Agua de Mar/parasitología , Carotenoides/análisis , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Dinoflagelados/citología , Dinoflagelados/genética , Genes de ARNr , Microscopía , Datos de Secuencia Molecular , Orgánulos/ultraestructura , Fotosíntesis , Filogenia , ARN Protozoario/genética , ARN Ribosómico/genética , ARN Ribosómico 18S/genética , República de Corea , Análisis de Secuencia de ADN
4.
Open Forum Infect Dis ; 11(5): ofae212, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38756763

RESUMEN

Background: Persistence of HIV-1 in reservoirs necessitates life-long antiretroviral therapy (ART). There are conflicting data using genetic analysis on whether persistence includes an actively replicating reservoir with strong evidence arguing against replication. Methods: We investigated the possibility of ongoing viral evolution during suppressive therapy by comparing near full-length viral genomic sequences using phylogenetic analysis of viral RNA in plasma before therapy initiation early after infection and from virus induced to grow from the latent reservoir after a period of suppressive ART. We also focused our analysis on evidence of selective pressure by drugs in the treatment regimen and at sites of selective pressure by the adaptive immune response. Results: Viral genomes induced to grow from the latent reservoir from 10 participants with up to 9 years on suppressive ART were highly similar to the nearly homogeneous sequences in plasma taken early after infection at ART initiation. This finding was consistent across the entire genome and when the analysis focused on sites targeted by the drug regimen and by host selective pressure of antibody and cytotoxic T cells. The lack of viral evolution away from pretherapy sequences in spite of demonstrated selective pressure is most consistent with a lack of viral replication during reservoir persistence. Conclusions: These results do not support ongoing viral replication as a mechanism of HIV-1 persistence during suppressive ART.

5.
Biochemistry ; 52(29): 4929-40, 2013 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-23763575

RESUMEN

The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.


Asunto(s)
Cápside/metabolismo , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , Línea Celular , Fluoresceína/química , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Especificidad por Sustrato , Ensamble de Virus , Replicación Viral
6.
J Biol Chem ; 287(49): 40867-74, 2012 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-23043111

RESUMEN

HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt protein-protein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A).


Asunto(s)
VIH-1/metabolismo , Virión/genética , Dominio Catalítico , Resistencia a Medicamentos , Regulación Viral de la Expresión Génica , Productos del Gen gag/metabolismo , Genoma Viral , Humanos , Modelos Genéticos , Conformación Molecular , Mutación , Inhibidores de Proteasas/química , Mapeo de Interacción de Proteínas , Proteolisis , Receptores CCR5/metabolismo , Ensamble de Virus/genética , Replicación Viral/genética
7.
J Biol Chem ; 287(16): 13279-90, 2012 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-22334652

RESUMEN

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.


Asunto(s)
Proteasa del VIH/metabolismo , VIH-1/enzimología , VIH-1/crecimiento & desarrollo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Duplicado del Terminal Largo de VIH/fisiología , Proteasa del VIH/genética , VIH-1/genética , Especificidad por Sustrato/fisiología , Virión/enzimología , Ensamble de Virus/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genética
8.
Acta Derm Venereol ; 93(5): 557-61, 2013 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-23388687

RESUMEN

The effectiveness of intermittent topical tacrolimus to prevent relapse in patients with stabilized facial seborrhoeic dermatitis has not been evaluated. The aim of this study was to determine whether proactive use of 0.1% tacrolimus ointment can keep adult facial seborrhoeic dermatitis in remission. A total of 75 patients who had stabilized facial seborrhoeic dermatitis after 2 weeks' (open-label induction) treatment with 0.1% tacrolimus were randomized in a double-blind fashion to treatment with 0.1% tacrolimus once a week, twice a week, or vehicle twice a week, for 10 weeks (maintenance). Significant improvement in erythema, scaling and pruritus compared with baseline was maintained during the maintenance phase in both tacrolimus groups, but not in the vehicle group. The mean recurrence rate according to global assessment was significantly higher in the tacrolimus once-weekly group than in the twice-weekly group. In conclusion, twice-weekly treatment with 0.1% tacrolimus ointment had superior effects in keeping facial seborrhoeic dermatitis in remission.


Asunto(s)
Dermatitis Seborreica/tratamiento farmacológico , Fármacos Dermatológicos/administración & dosificación , Dermatosis Facial/tratamiento farmacológico , Tacrolimus/administración & dosificación , Administración Cutánea , Adulto , Anciano , Distribución de Chi-Cuadrado , Dermatitis Seborreica/diagnóstico , Fármacos Dermatológicos/efectos adversos , Método Doble Ciego , Esquema de Medicación , Dermatosis Facial/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pomadas , Inducción de Remisión , República de Corea , Prevención Secundaria , Tacrolimus/efectos adversos , Factores de Tiempo , Resultado del Tratamiento
9.
J Korean Med Sci ; 28(1): 145-51, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23341725

RESUMEN

Tinea incognito (TI) is a dermatophytic infection which has lost its typical clinical appearance because of improper use of steroids or calcineurin inhibitors. The incidence of TI is increasing nowadays. We conducted retrospective review on 283 patients with TI from 25 dermatology training hospitals in Korea from 2002-2010 to investigate the demographical, clinical, and mycological characteristics of TI, and to determine the associated risk factors. More than half (59.3%) patients were previously treated by non-dermatologists or self-treated. The mean duration of TI was 15.0 ± 25.3 months. The most common clinical manifestations were eczema-like lesion, psoriasis-like, and lupus erythematosus-like lesion. The trunk and face were frequently involved, and 91 patients (32.2%) also had coexisting fungal infections. Among 67 isolated strains, Trichophyton rubrum was the most frequently detected (73.1%). This is the largest study of TI reported to date and the first investigational report concerning TI in Korea. We suggest that doctors should consider TI when a patient has intractable eczema-like lesions accompanied by tinea pedis/unguium. Furthermore, there should be a policy change, which would make over-the-counter high-potency topical steroids less accessible in some countries, including Korea.


Asunto(s)
Tiña/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Demografía , Eccema/patología , Cara/patología , Femenino , Humanos , Lupus Eritematoso Cutáneo/patología , Masculino , Persona de Mediana Edad , Psoriasis/patología , República de Corea , Estudios Retrospectivos , Factores de Riesgo , Tiña/microbiología , Trichophyton/aislamiento & purificación , Adulto Joven
10.
Eur J Med Chem ; 257: 115501, 2023 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-37244161

RESUMEN

Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants. The phosphonate moiety substantially improved potency against highly mutated and resistant HIV-1 protease variants, but only when combined with more hydrophobic moieties at the P1' and P2' positions. Phosphonate analogs with a larger hydrophobic P1' moiety maintained excellent antiviral potency against a panel of highly resistant HIV-1 variants, with significantly improved resistance profiles. The cocrystal structures indicate that the phosphonate moiety makes extensive hydrophobic interactions with the protease, especially with the flap residues. Many residues involved in these protease-inhibitor interactions are conserved, enabling the inhibitors to maintain potency against highly resistant variants. These results highlight the need to balance inhibitor physicochemical properties by simultaneous modification of chemical groups to further improve resistance profiles.


Asunto(s)
Inhibidores de la Proteasa del VIH , VIH-1 , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/química , Darunavir/farmacología , Péptido Hidrolasas , Proteasa del VIH/genética , Cristalografía por Rayos X
11.
Elife ; 122023 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-36920025

RESUMEN

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.


Asunto(s)
Infecciones por VIH , Inhibidores de la Proteasa del VIH , VIH-1 , Humanos , Inhibidores de la Proteasa del VIH/química , VIH-1/genética , Darunavir/farmacología , Darunavir/uso terapéutico , Mutación , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico
12.
Hum Mutat ; 33(1): 91-4, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21990045

RESUMEN

Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects without any other nonoral symptoms. Recently, a disease-causing nonsense mutation (c.406C>T) in a novel gene, FAM20A, was identified in a large consanguineous family affected by AI with gingival hyperplasia. We performed mutational analyses on nine AI families with similar phenotypes and identified three homozygous mutations (c.34_35delCT, c.813-2A>G, c.1175_1179delGGCTC) in three families and a compound heterozygous mutation (c.[590-2A>G] + [c.826C>T]) in one family. An in vitro splicing assay with a minigene confirmed the mutations located in the splicing acceptor site caused the deletion of exons 3 and 6, respectively. Taking into consideration the locations of the nonsense and frameshift mutations, the mutant transcripts are most likely degraded by nonsense-mediated mRNA degradation and it results in a loss of the FAM20A protein. This study confirms the importance of the FAM20A protein in enamel biomineralization as well as tooth eruption.


Asunto(s)
Amelogénesis Imperfecta/genética , Proteínas del Esmalte Dental/genética , Mutación del Sistema de Lectura , Eliminación de Secuencia , Secuencia de Bases , Codón sin Sentido , Consanguinidad , Análisis Mutacional de ADN , Exones , Heterocigoto , Homocigoto , Humanos , Datos de Secuencia Molecular , Linaje , Fenotipo , República de Corea
13.
Antimicrob Agents Chemother ; 56(2): 623-33, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22083488

RESUMEN

Resistance-associated mutations in the HIV-1 protease modify viral fitness through changes in the catalytic activity and altered binding affinity for substrates and inhibitors. In this report, we examine the effects of 31 mutations at 26 amino acid positions in protease to determine their impact on infectivity and protease inhibitor sensitivity. We found that primary resistance mutations individually decrease fitness and generally increase sensitivity to protease inhibitors, indicating that reduced virion-associated protease activity reduces virion infectivity and the reduced level of per virion protease activity is then more easily titrated by a protease inhibitor. Conversely, mutations at more variable positions (compensatory mutations) confer low-level decreases in sensitivity to all protease inhibitors with little effect on infectivity. We found significant differences in the observed effect on infectivity with a pseudotype virus assay that requires the protease to cleave the cytoplasmic tail of the amphotropic murine leukemia virus (MuLV) Env protein. Additionally, we were able to mimic the fitness loss associated with resistance mutations by directly reducing the level of virion-associated protease activity. Virions containing 50% of a D25A mutant protease were 3- to 5-fold more sensitive to protease inhibitors. This level of reduction in protease activity also resulted in a 2-fold increase in sensitivity to nonnucleoside inhibitors of reverse transcriptase and a similar increase in sensitivity to zidovudine (AZT), indicating a pleiotropic effect associated with reduced protease activity. These results highlight the interplay between enzyme activity, viral fitness, and inhibitor mechanism and sensitivity in the closed system of the viral replication complex.


Asunto(s)
Farmacorresistencia Viral/genética , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Mutación , Animales , Línea Celular , Farmacorresistencia Viral/efectos de los fármacos , Productos del Gen env/genética , Productos del Gen env/metabolismo , Proteasa del VIH/efectos de los fármacos , Proteasa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Virión/efectos de los fármacos , Virión/enzimología , Virión/genética , Virión/patogenicidad , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología
14.
Am J Hum Genet ; 82(2): 489-94, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18252228

RESUMEN

Amelogenesis imperfecta (AI) is a collection of diverse inherited disorders featuring dental-enamel defects in the absence of significant nondental symptoms. AI phenotypes vary and are categorized as hypoplastic, hypocalcified, and hypomaturation types. Phenotypic specificity to enamel has focused research on genes encoding enamel-matrix proteins. We studied two families with autosomal-dominant hypocalcified AI and have identified nonsense mutations (R325X and Q398X) in the FAM83H gene on chromosome 8q24.3. The mutations perfectly cosegregate with the disease phenotype and demonstrate that FAM83H is required for proper dental-enamel calcification.


Asunto(s)
Amelogénesis Imperfecta/genética , Cromosomas Humanos Par 8/genética , Fenotipo , Proteínas/genética , Amelogénesis Imperfecta/patología , Secuencia de Bases , Codón sin Sentido/genética , Cartilla de ADN/genética , Humanos , Hibridación in Situ , Escala de Lod , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN
15.
Eur J Oral Sci ; 119 Suppl 1: 324-8, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22243263

RESUMEN

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders with regard to genetic aetiology and clinical phenotype and affects tooth enamel with no other non-oral syndromic conditions. X-linked AI is caused by mutations in the amelogenin (AMELX) gene, the only AI candidate gene located on the X chromosome. To date, 15 mutations in the AMELX gene have been found to cause AI. We identified a proband with generalized hypoplastic enamel and unusual multiple crown resorption in premolars and molars. Pedigree analysis suggested an X-linked hereditary pattern. We performed mutational analysis for the AMELX gene based on the candidate gene approach. Sequencing analysis revealed a novel mutation in exon 6 (g.4090delC, c.517delC, p.Pro173LeufsX16). This frameshift mutation produces a premature stop codon within exon 6 and is predicted to replace 33 amino acids at the C-terminus with 15 novel amino acids if the mutant mRNA escapes the nonsense-mediated decay system. Although crown resorptions occur frequently in patients with the hypoplastic type of A1, an association with the AMELX mutation has not been previously reported. We believe that these findings will broaden our understanding of the clinical phenotype and pathogenesis of X-linked AI.


Asunto(s)
Amelogénesis Imperfecta/genética , Amelogenina/genética , Resorción Dentaria/genética , Amelogénesis Imperfecta/complicaciones , Niño , Codón sin Sentido , Análisis Mutacional de ADN , Mutación del Sistema de Lectura , Humanos , Masculino , Mordida Abierta/complicaciones , Corona del Diente/patología
16.
J Virol ; 83(17): 8536-43, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19515760

RESUMEN

The human immunodeficiency virus type 1 (HIV-1) protease (PR) makes five obligatory cleavages in the viral Gag polyprotein precursor. The cleavage events release the virion structural proteins from the precursor and allow the virion to undergo maturation to become infectious. The protease cleavage between the matrix protein (MA) domain and the adjacent capsid protein (CA) domain releases CA from the membrane-anchored MA and allows the N terminus of CA to refold into a structure that facilitates the formation of hexamer arrays that represent the structural unit of the capsid shell. In this study, we analyzed the extent to which each of the HIV-1 Gag processing sites must be cleaved by substituting the P1-position amino acid at each processing site with Ile. A mutation that blocks cleavage at the MA/CA processing site (Y132I) displayed a strong transdominant effect when tested in a phenotypic mixing strategy, inhibiting virion infectivity with a 50% inhibitory concentration of only 4% of the mutant relative to the wild type. This mutation is 10- to 20-fold more potent in phenotypic mixing than an inactivating mutation in the viral protease, the target of many successful inhibitors, and more potent than an inactivating mutation at any of the other Gag cleavage sites. The transdominant effect is manifested as the assembly of an aberrant virion core. Virus containing 20% of the Y132I mutant and 80% of the wild type (to assess the transdominant effect on infectivity) was blocked either before reverse transcription (RT) or at an early RT step. The ability of a small amount of the MA/CA fusion protein to poison the oligomeric assembly of infectious virus identifies an essential step in the complex process of virion formation and maturation. The effect of a small-molecule inhibitor that is able to block MA/CA cleavage even partially would be amplified by this transdominant negative effect on the highly orchestrated process of virion assembly.


Asunto(s)
Proteasa del VIH/metabolismo , VIH-1/fisiología , Mutación Missense , Ensamble de Virus , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Sustitución de Aminoácidos/genética , Animales , Antígenos VIH/metabolismo , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/genética , Humanos , Mutagénesis Sitio-Dirigida , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética
17.
J Med Chem ; 63(15): 8296-8313, 2020 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-32672965

RESUMEN

The design, synthesis, and X-ray structural analysis of hybrid HIV-1 protease inhibitors (PIs) containing bis-tetrahydrofuran (bis-THF) in a pseudo-C2-symmetric dipeptide isostere are described. A series of PIs were synthesized by incorporating bis-THF of darunavir on either side of the Phe-Phe isostere of lopinavir in combination with hydrophobic amino acids on the opposite P2/P2' position. Structure-activity relationship studies indicated that the bis-THF moiety can be attached at either the P2 or P2' position without significantly affecting potency. However, the group on the opposite P2/P2' position had a dramatic effect on potency depending on the size and shape of the side chain. Cocrystal structures of inhibitors with wild-type HIV-1 protease revealed that the bis-THF moiety retained similar interactions as observed in the darunavir-protease complex regardless of the position on the Phe-Phe isostere. Analyses of cocrystal structures and molecular dynamics simulations provide insights into optimizing HIV-1 PIs containing bis-THF in non-sulfonamide dipeptide isosteres.


Asunto(s)
Furanos/química , Furanos/farmacología , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/metabolismo , VIH-1/enzimología , Cristalografía por Rayos X , Darunavir/análogos & derivados , Darunavir/farmacología , Dipéptidos/química , Dipéptidos/farmacología , Diseño de Fármacos , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Proteasa del VIH/química , VIH-1/efectos de los fármacos , Humanos , Modelos Moleculares , Relación Estructura-Actividad
18.
Handb Exp Pharmacol ; (189): 85-110, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19048198

RESUMEN

This review provides an overview of the development of viral protease inhibitors as antiviral drugs. We concentrate on HIV-1 protease inhibitors, as these have made the most significant advances in the recent past. Thus, we discuss the biochemistry of HIV-1 protease, inhibitor development, clinical use of inhibitors, and evolution of resistance. Since many different viruses encode essential proteases, it is possible to envision the development of a potent protease inhibitor for other viruses if the processing site sequence and the catalytic mechanism are known. At this time, interest in developing inhibitors is limited to viruses that cause chronic disease, viruses that have the potential to cause large-scale epidemics, or viruses that are sufficiently ubiquitous that treating an acute infection would be beneficial even if the infection was ultimately self-limiting. Protease inhibitor development is most advanced for hepatitis C virus (HCV), and we also provide a review of HCV NS3/4A serine protease inhibitor development, including combination therapy and resistance. Finally, we discuss other viral proteases as potential drug targets, including those from Dengue virus, cytomegalovirus, rhinovirus, and coronavirus.


Asunto(s)
Antivirales/farmacología , Péptido Hidrolasas/química , Inhibidores de Proteasas/farmacología , Virus/efectos de los fármacos , Virus/enzimología , Proteasas Virales 3C , Animales , Cisteína Endopeptidasas , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Farmacorresistencia Viral , Quimioterapia Combinada , Inhibidores de la Proteasa del VIH/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Humanos , Modelos Moleculares , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores
19.
Gut Liver ; 13(3): 342-348, 2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30600675

RESUMEN

Background/Aims: Sorafenib remains the only approved molecular targeted agent for hepatocellular carcinoma (HCC); however, reliable biomarkers that predict its efficacy are still lacking. The aim of this study was to explore whether cancer stem cell (CSC) markers have a predictive role with regard to the sorafenib response in HCC patients. Methods: We enrolled 47 patients with HCC for whom tumor samples obtained before starting sorafenib treatment were available. RNA was extracted from formalin-fixed, paraffin-embedded samples, and real-time polymerase chain reaction was used to quantify mRNA expression of the CSC genes EpCAM, CD13, CK8, CD24, CD44, CD90, CD133, SALL4, ALDH1A1, ALB, and AFP . Results: Of 47 patients, 14.9% and 74.5% had vascular invasion and extrahepatic spread, respectively. Patients with low CD133 expression tended to have longer progression-free survival (PFS) than those with high CD133 expression (5.5 months vs 4.0 months), although without statistical significance. The expression levels of other markers were not associated with PFS. When examining markers in combination, patients with high CD133 and CD90 expression had shorter PFS rates than those with low expression (2.7 months vs 5.5 months; p=0.04). Patients with low CD133 and EpCAM expression demonstrated better PFS than those with high expression (7.0 months vs 4.2 months; p=0.04). Multivariable analysis indicated that an Eastern Cooperative Oncology Group performance status score of 1 and high CD133/CD90 expression were significantly associated with shorter PFS. Conclusions: Overexpression of the CSC markers CD133 and CD90 in HCC was associated with poorer response to sorafenib. These two genes may serve as predictive biomarkers for sorafenib therapy.


Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Sorafenib/uso terapéutico , Adulto , Anciano , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Femenino , Humanos , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Pruebas de Farmacogenómica , ARN Mensajero/metabolismo , Resultado del Tratamiento
20.
J Med Chem ; 62(17): 8062-8079, 2019 09 12.
Artículo en Inglés | MEDLINE | ID: mdl-31386368

RESUMEN

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.


Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Línea Celular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Células HEK293 , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , VIH-1/enzimología , Humanos , Enlace de Hidrógeno , Ligandos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por Sustrato
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