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1.
Int J Mol Sci ; 25(10)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38791130

RESUMO

The increase in multi-drug resistant Candida strains has caused a sharp rise in life-threatening fungal infections in immunosuppressed patients, including those with SARS-CoV-2. Novel antifungal drugs are needed to combat multi-drug-resistant yeasts. This study aimed to synthesize a new series of 2-oxazolines and evaluate the ligands in vitro for the inhibition of six Candida species and in silico for affinity to the CYP51 enzymes (obtained with molecular modeling and protein homology) of the same species. The 5-(1,3-diphenyl-1H-pyrazol-4-yl)-4-tosyl-4,5-dihydrooxazoles 6a-j were synthesized using the Van Leusen reaction between 1,3-diphenyl-4-formylpyrazoles 4a-j and TosMIC 5 in the presence of K2CO3 or KOH without heating, resulting in short reaction times, high compound purity, and high yields. The docking studies revealed good affinity for the active site of the CYP51 enzymes of the Candida species in the following order: 6a-j > 4a-j > fluconazole (the reference drug). The in vitro testing of the compounds against the Candida species showed lower MIC values for 6a-j than 4a-j, and for 4a-j than fluconazole, thus correlating well with the in silico findings. According to growth rescue assays, 6a-j and 4a-j (like fluconazole) inhibit ergosterol synthesis. The in silico toxicity assessment evidenced the safety of compounds 6a-j, which merit further research as possible antifungal drugs.


Assuntos
Antifúngicos , Candida , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Antifúngicos/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Candida/efeitos dos fármacos , Humanos , Oxazóis/química , Oxazóis/farmacologia , Oxazóis/síntese química , Pirazóis/farmacologia , Pirazóis/química , Pirazóis/síntese química , Simulação por Computador , SARS-CoV-2/efeitos dos fármacos
2.
Menopause ; 31(7): 641-646, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38688462

RESUMO

OBJECTIVE: Musculoskeletal disorders frequently affect postmenopausal women. This study aims to compare muscle disorders between women according to the type of experienced menopause: premature (PM) or normal age of menopause (NAM). METHODS: This was a cross-sectional study conducted in nine Latin American countries in which late postmenopausal women (55 to 70 years) were surveyed with a general questionnaire, the Menopause Rating Scale (MRS: item #4 exploring musculoskeletal discomfort), and strength, assistance with walking, rising from a chair, climbing stairs, and falling questionnaire (risk of sarcopenia). RESULTS: A total of 644 women were included: 468 who had NAM, and 176 who had PM (116 spontaneous and 60 surgical). The overall mean age of the participants was 60.9 ± 4.2 years. Women who had PM experienced more musculoskeletal discomfort (33.5% vs 20.9%, P < 0.001) and a higher likelihood of sarcopenia (35.2% vs 19.9%, P < 0.001) than women who had a NAM. Women who had surgical PM exhibited a higher prevalence of severe musculoskeletal discomfort (46.7% vs 29.3%, P < 0.02) and a higher likelihood of sarcopenia (45.0% vs 27.6%, P < 0.02) than women who had a NAM. After adjusting for covariates (age, body mass index, menopausal hormone therapy use, physical activity, education, cigarette consumption, use of antidepressants, sexual activity, comorbidities, and having a partner), our logistic regression model determined that spontaneous PM was not associated with higher odds of musculoskeletal discomfort and higher odds of sarcopenia. On the other hand, women who had surgical PM were more likely to experience musculoskeletal discomforts (odds ratio: 2.26; 95% confidence interval: 1.22-4.17) and higher odds for sarcopenia (odds ratio: 2.05; 95% confidence interval: 1.16-3.65) as compared to women who experienced a NAM. CONCLUSIONS: Women experiencing surgical PM have a higher likelihood of developing muscle disorders. This underscores the potential significance of hormonal levels in influencing musculoskeletal health during postmenopause.


Assuntos
Menopausa , Pós-Menopausa , Sarcopenia , Humanos , Feminino , Pessoa de Meia-Idade , Estudos Transversais , Pós-Menopausa/fisiologia , Idoso , Menopausa/fisiologia , Sarcopenia/epidemiologia , Inquéritos e Questionários , Menopausa Precoce , América Latina/epidemiologia , Prevalência , Força Muscular
3.
bioRxiv ; 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38617369

RESUMO

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions required for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin and catalyzes the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. Here, we report the Cryo-EM structures of Rep68 bound to double-stranded DNA (dsDNA) containing the sequence of the AAVS1 integration site in different nucleotide-bound states. In the apo state, Rep68 forms a heptameric complex around DNA, with three Origin Binding Domains (OBDs) bound to the Rep Binding Site (RBS) sequence and three other OBDs forming transient dimers with them. The AAA+ domains form an open ring with no interactions between subunits and with DNA. We hypothesize the heptameric quaternary structure is necessary to load onto dsDNA. In the ATPγS-bound state, a subset of three subunits binds the nucleotide, undergoing a large conformational change, inducing the formation of intersubunit interactions interaction and interaction with three consecutive DNA phosphate groups. Moreover, the induced conformational change positions three phenylalanine residues to come in close contact with the DNA backbone, producing a distortion in the DNA. We propose that the phenylalanine residues can potentially act as a hydrophobic wedge in the DNA melting process.

4.
Molecules ; 28(20)2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37894702

RESUMO

As a new approach, pyrrolo[1,2-a]pyrazines were synthesized through the cyclization of 2-formylpyrrole-based enaminones in the presence of ammonium acetate. The enaminones were prepared with a straightforward method, reacting the corresponding alkyl 2-(2-formyl-1H-pyrrol-1-yl)acetates, 2-(2-formyl-1H-pyrrol-1-yl)acetonitrile, and 2-(2-formyl-1H-pyrrol-1-yl)acetophenones with DMFDMA. Analogous enaminones elaborated from alkyl (E)-3-(1H-pyrrol-2-yl)acrylates were treated with a Lewis acid to afford indolizines. The antifungal activity of the series of substituted pyrroles, pyrrole-based enaminones, pyrrolo[1,2-a]pyrazines, and indolizines was evaluated on six Candida spp., including two multidrug-resistant ones. Compared to the reference drugs, most test compounds produced a more robust antifungal effect. Docking analysis suggests that the inhibition of yeast growth was probably mediated by the interaction of the compounds with the catalytic site of HMGR of the Candida species.


Assuntos
Antifúngicos , Indolizinas , Antifúngicos/farmacologia , Pirróis/farmacologia , Indolizinas/farmacologia , Pirazinas/farmacologia , Testes de Sensibilidade Microbiana , Candida
5.
Molecules ; 28(10)2023 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-37241920

RESUMO

Current antidiabetic drugs have severe side effects, which may be minimized by new selective molecules that strongly inhibit α-glucosidase and weakly inhibit α-amylase. We have synthesized novel alkoxy-substituted xanthones and imidazole-substituted xanthones and have evaluated them for their in silico and in vitro α-glucosidase and α-amylase inhibition activity. Compounds 6c, 6e, and 9b promoted higher α-glucosidase inhibition (IC50 = 16.0, 12.8, and 4.0 µM, respectively) and lower α-amylase inhibition (IC50 = 76.7, 68.1, and >200 µM, respectively) compared to acarbose (IC50 = 306.7 µM for α-glucosidase and 20.0 µM for α-amylase). Contrarily, derivatives 10c and 10f showed higher α-amylase inhibition (IC50 = 5.4 and 8.7 µM, respectively) and lower α-glucosidase inhibition (IC50 = 232.7 and 145.2 µM, respectively). According to the structure-activity relationship, attaching 4-bromobutoxy or 4'-chlorophenylacetophenone moieties to the 2-hydroxy group of xanthone provides higher α-glucosidase inhibition and lower α-amylase inhibition. In silico studies suggest that these scaffolds are key in the activity and interaction of xanthone derivatives. Enzymatic kinetics studies showed that 6c, 9b, and 10c are mainly mixed inhibitors on α-glucosidase and α-amylase. In addition, drug prediction and ADMET studies support that compounds 6c, 9b, and 10c are candidates with antidiabetic potential.


Assuntos
Inibidores de Glicosídeo Hidrolases , Xantonas , Inibidores de Glicosídeo Hidrolases/farmacologia , Simulação de Acoplamento Molecular , alfa-Glucosidases/metabolismo , alfa-Amilases , Hipoglicemiantes/farmacologia , Relação Estrutura-Atividade , Imidazóis/farmacologia , Xantonas/farmacologia , Estrutura Molecular
6.
Cell Mol Biol (Noisy-le-grand) ; 68(6): 1-8, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-36227685

RESUMO

Colorectal cancer (CRC) is the third most common cancer in the world. Overall survival is related to clinical stage: more advanced stages show lower survival rates; therefore, they need to be monitored regularly with new, less invasive and more specific biomarkers. The concentration and integrity index of circulating cell-free DNA (ccfDNA) have been proposed as potential diagnostic and prognostic biomarkers for CRC, however, inconsistent results are still observed in different reports. Here we analyze these potential CRC biomarkers in a Mexican population. In this study, 124 patients with sporadic CRC and 37 healthy individuals were examined as a reference group. The ccfDNA was isolated from plasma samples of all included subjects. The ccfDNA concentration was determined by fluorometry and the integrity index (ALU247/ALU115 ratio) by quantitative PCR amplification (qPCR) of ALU sequences. The results show that ccfDNA concentration was higher in CRC patients than in the reference group (P=0.001). The integrity index showed no significant differences between these groups (P=0.258), except for histological type (P=0.012). A higher ccfDNA concentration was also associated with patients younger than 50 years (P=0.030). The ccfDNA concentration showed significant discriminatory power (AUC: 0.854, C.I.: 0.78-0.92, P=0.001) between patients and the reference group and between tumor-node-metastasis (TNM) stages. In conclusion, ccfDNA concentration proves to be a good diagnostic biomarker for CRC patients, whereas the integrity index did not show diagnostic utility.


Assuntos
Ácidos Nucleicos Livres , Neoplasias Colorretais , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA , Humanos , Reação em Cadeia da Polimerase em Tempo Real
7.
J Org Chem ; 87(19): 13034-13052, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36153994

RESUMO

Palladium-catalyzed functionalization was presently performed on two building blocks: 4-oxazolin-2-ones and 4-methylene-2-oxazolidinones. Direct Heck arylation of 4-oxazolin-2-ones led to a series of 5-aryl-4-oxazolin-2-ones, including analogues with N-chiral auxiliary, in an almost quantitative yield. The Pd(II)-catalyzed homocoupling reaction of 4-oxazolin-2-ones provided novel heterocyclic across-ring dienes. Meanwhile, the intramolecular cross-coupling of N-aryl-4-methylene-2-oxazolidinones furnished a series of oxazolo[3,4-a]indol-3-ones. Further functionalization of 4-methylene-2-oxazolidinones afforded substituted indoles and heterocyclic-fused indoles with aryl, bromo, carbinol, formyl, and vinyl groups. A computational study was carried out to account for the behavior of the formylated derivatives. The currently developed methodology was applied to a new formal total synthesis of ellipticine.


Assuntos
Elipticinas , Oxazolidinonas , Catálise , Indóis , Metano/análogos & derivados , Metanol , Paládio
8.
STAR Protoc ; 3(3): 101615, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36035794

RESUMO

eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).


Assuntos
Fator de Iniciação 5 em Eucariotos , Doenças Neurodegenerativas , Calorimetria , Fator de Iniciação 2 em Eucariotos , Fator de Iniciação 3 em Eucariotos , Humanos , Interferometria
9.
Microbiol Spectr ; 10(2): e0164221, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35377226

RESUMO

Due to the emergence of multidrug-resistant strains of yeasts belonging to the Candida genus, there is an urgent need to discover antifungal agents directed at alternative molecular targets. The aim of the current study was to evaluate the capacity of three different series of synthetic compounds to inhibit the Candida glabrata enzyme denominated 3-hydroxy-methyl-glutaryl-CoA reductase and thus affect ergosterol synthesis and yeast viability. Compounds 1c (α-asarone-related) and 5b (with a pyrrolic core) were selected as the best antifungal candidates among over 20 synthetic compounds studied. Both inhibited the growth of fluconazole-resistant and fluconazole-susceptible C. glabrata strains. A yeast growth rescue experiment based on the addition of exogenous ergosterol showed that the compounds act by inhibiting the mevalonate synthesis pathway. A greater recovery of yeast growth occurred for the C. glabrata 43 fluconazole-resistant (versus fluconazole-susceptible) strain and after treatment with 1c (versus 5b). Given that the compounds decreased the concentration of ergosterol in the yeast strains, they probably target ergosterol synthesis. According to the docking analysis, the inhibitory effect of 1c and 5b could possibly be mediated by their interaction with the amino acid residues of the catalytic site of the enzyme. Since 1c displayed higher binding energy than α-asarone and 5b, it is the best candidate for further research, which should include structural modifications to increase its specificity and potency. The derivatives could then be examined with in vivo animal models using a therapeutic dose. IMPORTANCE Within the context of the COVID-19 pandemic, there is currently an epidemiological alert in health care services due to outbreaks of Candida auris, Candida glabrata, and other fungal species multiresistant to conventional antifungals. Therefore, it is important to propose alternative molecular targets, as well as new antifungals. The three series of synthetic compounds herein designed and synthesized are inhibitors of ergosterol synthesis in yeasts. Of the more than 20 compounds studied, two were selected as the best antifungal candidates. These compounds were able to inhibit the growth and synthesis of ergosterol in C. glabrata strains, whether susceptible or resistant to fluconazole. The rational design of antifungal compounds derived from clinical drugs (statins, fibrates, etc.) has many advantages. Future studies are needed to modify the structure of the two present test compounds to obtain safer and less toxic antifungals. Moreover, it is important to carry out a more in-depth mechanistic approach.


Assuntos
COVID-19 , Candida glabrata , Acil Coenzima A , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida glabrata/metabolismo , Farmacorresistência Fúngica , Ergosterol/metabolismo , Ácidos Fíbricos/metabolismo , Fluconazol/metabolismo , Fluconazol/farmacologia , Humanos , Hidroximetilglutaril-CoA Redutases/química , Hidroximetilglutaril-CoA Redutases/metabolismo , Testes de Sensibilidade Microbiana , Pandemias , Pirróis/metabolismo , Pirróis/farmacologia
10.
J Virol ; 96(2): e0132621, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34669461

RESUMO

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 µM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 µM) without prominent cytotoxicity (CC50 = 71.8 µM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 µM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 µM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.


Assuntos
Antivirais/farmacologia , Células Precursoras Eritroides/virologia , Ensaios de Triagem em Larga Escala/métodos , Parvovirus B19 Humano/efeitos dos fármacos , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Parvovirus B19 Humano/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
11.
Org Biomol Chem ; 20(2): 396-409, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34904608

RESUMO

A straightforward synthesis of indolizines and pyrrolo[1,2-a]pyrazines was performed through a cascade condensation/cyclization/aromatization reaction of substituted 2-formyl-N-propargylpyrroles with active methylene compounds such as nitromethane, alkyl malonates, methyl cyanoacetate and malononitrile. Under basic conditions, the reaction proceeded satisfactorily to provide the corresponding 6,7-disubstituted indolizines. The condensation of the pyrrolic analogues with ammonium acetate gave rise to pyrrolo[1,2-a]pyrazines in high yields. N-Allenyl-2-formylpyrroles behaved as more reactive substrates than 2-formyl-N-propargylpyrroles, furnishing the expected indolizines in higher yields. Hence, an allenyl-containing intermediate was probably generated as the reactive species in the reaction mechanism of some N-propargyl pyrroles prior to the cyclization reaction.

12.
Pharmaceuticals (Basel) ; 14(11)2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34832892

RESUMO

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a-o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a-h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a-o and 6a-h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a-o and 6a-h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.

13.
Cell Rep ; 36(2): 109376, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260931

RESUMO

eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.


Assuntos
Códon de Iniciação/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas/genética , Expansão das Repetições de Trinucleotídeos/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação a DNA/química , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Células HEK293 , Humanos , Masculino , Modelos Biológicos , Modelos Moleculares , Mutação/genética , Iniciação Traducional da Cadeia Peptídica , Ligação Proteica , Domínios Proteicos , Receptores Imunológicos/metabolismo
14.
Cureus ; 13(4): e14365, 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33972916

RESUMO

Pelvic renal ectopia is a congenital anomaly secondary to poor renal migration to the lower back. Usually, these pathologies are of asymptomatic course, therefore its finding is usually fortuitous during radiological examinations for other causes or in work-up of the infrequent symptomatic cases characterized by the occurrence of recurrent infections or symptoms of obstructive uropathy. The objective of this report is to present a case of a 37-year-old female with the unusual manifestation of left pelvic renal ectopia. She was intervened for acute lithiasic cholecystitis, and radiologic techniques diagnosed left pelvic renal ectopia. An updated review of the literature is performed. Despite the anomaly, the patient's renal function tests were normal, so only cholecystectomy was performed without complications.

16.
Nucleic Acids Res ; 48(22): 12983-12999, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33270897

RESUMO

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.


Assuntos
Trifosfato de Adenosina/análogos & derivados , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Proteínas Virais/genética , Trifosfato de Adenosina/genética , Microscopia Crioeletrônica , DNA Helicases/genética , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Dependovirus/ultraestrutura , Humanos , Proteínas Virais/ultraestrutura
17.
Beilstein J Org Chem ; 16: 1320-1334, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32595780

RESUMO

A highly regio-, chemo- and stereoselective divergent synthesis of isoindolo- and pyrrolo-fused polycyclic indoles is herein described, starting from 2-formylpyrrole and employing Diels-Alder and Heck arylation reactions. 3-(N-Benzyl-2-pyrrolyl)acrylates and 4-(pyrrol-2-yl)butenones underwent a highly endo-Diels-Alder cycloaddition with maleimides to furnish octahydropyrrolo[3,4-e]indoles, which served as precursors in the regioselective synthesis of aza-polycyclic skeletons via an intramolecular Heck arylation reaction. Through the latter reaction, the 3-(N-benzyl-2-pyrrolyl)acrylates give rise to 3-(pyrrolo[2,1-a]isoindol-3-yl)acrylates. A further oxidative aromatization of the polycyclic intermediates provides the corresponding polycyclic pyrrolo-isoindoles and isoindolo-pyrrolo-indoles. A theoretical study on the stereoselective Diels-Alder reactions, carried out by calculating the endo/exo transition states, revealed the assistance of non-covalent interactions in governing the endo stereocontrol.

18.
J Exp Med ; 217(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31816635

RESUMO

Type I interferon (IFN-I) provides effective antiviral immunity but can exacerbate harmful inflammatory reactions and cause hematopoietic stem cell (HSC) exhaustion; therefore, IFN-I expression must be tightly controlled. While signaling mechanisms that limit IFN-I induction and function have been extensively studied, less is known about transcriptional repressors acting directly on IFN-I regulatory regions. We show that NFAT5, an activator of macrophage pro-inflammatory responses, represses Toll-like receptor 3 and virus-induced expression of IFN-I in macrophages and dendritic cells. Mice lacking NFAT5 exhibit increased IFN-I production and better control of viral burden upon LCMV infection but show exacerbated HSC activation under systemic poly(I:C)-induced inflammation. We identify IFNß as a primary target repressed by NFAT5, which opposes the master IFN-I inducer IRF3 by binding to an evolutionarily conserved sequence in the IFNB1 enhanceosome that overlaps a key IRF site. These findings illustrate how IFN-I responses are balanced by simultaneously opposing transcription factors.


Assuntos
Interferon Tipo I/imunologia , Fatores de Transcrição/imunologia , Animais , Células Dendríticas/imunologia , Feminino , Inflamação/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon gama/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli I-C/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Transcrição Gênica/imunologia
19.
Bioorg Med Chem ; 27(20): 115053, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31471100

RESUMO

Since NSAIDs are commonly used anti-inflammatory agents that produce adverse effects, there have been ongoing efforts to develop more effective and less toxic compounds. Based on the structure of the anti-inflammatory pyrrolizines licofelone and ketorolac, a series of 1-arylpyrrolizin-3-ones was synthesized. Also prepared was a series of substituted pyrroles, mimicking similar known anti-inflammatory agents. The anti-inflammatory activity of the test compounds was determined with a phorbol ester (TPA)-induced murine ear edema protocol. For the most active derivatives, 19b-c/20b-c, the anti-inflammatory effect was the same as that of the reference compound (indomethacin) and was dose-dependent. These compounds have an aryl ring at the C-1 position and a methoxycarbonyl group at the C-2 position of the pyrrolizine framework, which represent plausible pharmacophore groups with anti-inflammatory activity. The anti-inflammatory activity of 1-substituted analogs containing a five- or six-membered heterocycles was lower but still good, while that of the pyrroles was only moderate. Although the docking studies suggests that the effect of analogs 19a-c/20a-c is associated with the inhibition of cyclooxygenase-2, experimental assays did not corroborate this idea. Indeed, a significant inhibition of NO was found experimentally as a plausible mechanism of action.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Edema/tratamento farmacológico , Cetorolaco/farmacologia , Pirróis/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Cetorolaco/química , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Acetato de Tetradecanoilforbol
20.
Chirality ; 31(9): 719-749, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31318100

RESUMO

Enantiopure 3-((R)- and 3-((S)-1-phenylethyl)-4-oxazoline-2-ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N-(R)- or N-(S)-1-phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4- and 5-positions of the 4-oxazolin-2-one ring through thermal and MW-promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six-membered carbo- and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4-methylene-2-oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero-Diels-Alder cycloaddition.

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