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1.
Sci Rep ; 10(1): 3021, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080249

RESUMO

Chronic hepatitis B virus (HBV) infection is a major public health problem that affects millions of people worldwide. Nucleoside analogue reverse transcriptase (RT) inhibitors, such as entecavir (ETV) and lamivudine (3TC), serve as crucial anti-HBV drugs. However, structural studies of HBV RT have been hampered due to its unexpectedly poor solubility. Here, we show that human immunodeficiency virus type-1 (HIV-1) with HBV-associated amino acid substitutions Y115F/F116Y/Q151M in its RT (HIVY115F/F116Y/Q151M) is highly susceptible to ETV and 3TC. Additionally, we experimentally simulated previously reported ETV/3TC resistance for HBV using HIVY115F/F116Y/Q151M with F160M/M184V (L180M/M204V in HBV RT) substituted. We determined crystal structures for HIV-1 RTY115F/F116Y/Q151M:DNA complexed with 3TC-triphosphate (3TC-TP)/ETV-triphosphate (ETV-TP)/dCTP/dGTP. These structures revealed an atypically tight binding conformation of 3TC-TP, where the Met184 side-chain is pushed away by the oxathiolane of 3TC-TP and exocyclic methylene of ETV-TP. Structural analysis of RTY115F/F116Y/Q151M/F160M/M184V:DNA:3TC-TP also demonstrated that the loosely bound 3TC-TP is misaligned at the active site to prevent a steric clash with the side chain γ-methyl of Val184. These findings shed light on the common structural mechanism of HBV and HIV-1 resistance to 3TC and ETV and should aid in the design of new agents to overcome drug resistance to 3TC and ETV.

2.
Am J Physiol Heart Circ Physiol ; 318(1): H72-H77, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31729903

RESUMO

Preeclampsia is a hypertensive disorder of pregnancy characterized by systemic perturbations of nitric oxide function, reflective of generalized endothelial dysfunction. Therapies that target the nitric oxide pathway have shown promise in both clinical and preclinical studies of preeclampsia. The glucagon-like peptide 1 agonists have been shown to increase nitric oxide and lower blood pressure in patients with diabetes, in part, through activation of nitric oxide synthase (NOS). Therefore, we hypothesized that a direct acting glucagon-like peptide 1 receptor agonist would improve stigmata of the preeclampsia syndrome. Using the reduced uterine perfusion pressure rat model, we found that treatment with liraglutide significantly lowered blood pressure, improved renal function, and upregulated NOS3 protein expression in the mesenteric arterial bed. However, there were adverse effects on pup growth that were likely related to diminished food intake in the dams. Collectively, these data support the premise that the use of drugs that improve NOS abundance, including the glucagon-like peptide 1 agonists, is a rational therapeutic approach to the treatment of preeclampsia, but suggest cautious and careful study of their safety before potential clinical use in humans.NEW & NOTEWORTHY Drugs that target the glucagon-like peptide-1 pathway such as liraglutide are already used clinically, and it has been shown to promote endothelial nitric oxide synthase (NOS3) expression. We demonstrate that liraglutide, a glucagon-like peptide 1 receptor (GLP-1R) agonist, lowers blood pressure, improves renal function, and upregulates NOS3 in a rat model of placental ischemia. These data suggest that drugs that target the nitric oxide system, including GLP-1R agonists, are a potential therapeutic option for preeclampsia.

3.
Nihon Yakurigaku Zasshi ; 154(5): 275-287, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31735758

RESUMO

Brexpiprazole (Rexulti®) is the second antipsychotic agent in the world with dopamine D2 receptor partial agonist which was developed by Otsuka Pharmaceutical Co. Ltd. It is categorized as 〝Serotonin- dopamine Activity Modulator (SDAM)〟 that regulates both serotoninergic and dopaminergic systems by acting as a partial agonist for serotonin 5-HT1A receptors and D2 receptors and as an antagonist for 5-HT2A receptors. In preclinical pharmacological studies, brexpiprazole showed the equivalent antipsychotic-like effects to those of other atypical antipsychotics. And it was suggested that brexpiprazole has the low potentials to induce extrapyramidal symptoms, hyperprolactinemia and tardive dyskinesia, with improvement effects on cognitive dysfunction. Furthermore, brexpiprazole has the weak effects on histamine H1 receptors which are associated with sedation and weight gain in clinical. In the clinical trials in patients with schizophrenia in both acute and maintenance phase, brexpiprazole showed improvement of antipsychotic effects against placebo, and low incidence of adverse events, e.g., extrapyramidal symptoms, hyperprolactinemia, and weight gain, as suggested in preclinical studies. Furthermore, brexpiprazole showed low incidence of metabolic abnormalities. In particular, brexpiprazole showed relatively low incidences of akathisia, insomnia and agitation which has been commonly reported with aripiprazole. This would be based on the pharmacological features of brexpiprazole that is more potent antagonism at 5-HT2A receptors and D2 receptors partial agonism with lower intrinsic activity compared to those of aripiprazole. In conclusion, brexpiprazole could be one of the antipsychotics with the most rational mechanism of action, and the better efficacy and safety/tolerability profiles would contribute to the treatment of patients with schizophrenia.


Assuntos
Antipsicóticos/farmacologia , Quinolonas/farmacologia , Tiofenos/farmacologia , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Esquizofrenia/tratamento farmacológico , Comprimidos , Resultado do Tratamento
4.
Nat Commun ; 10(1): 4176, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519908

RESUMO

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.


Assuntos
Autofagia/fisiologia , Centríolos/metabolismo , Centrossomo/metabolismo , Mitose/fisiologia , Autofagia/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Immunoblotting , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microscopia de Fluorescência , Microtúbulos/metabolismo , Mitose/genética , Simulação de Dinâmica Molecular
5.
Neuropsychopharmacol Rep ; 39(4): 279-288, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31487433

RESUMO

BACKGROUND: Long-term antipsychotic treatment in patients with schizophrenia can induce supersensitivity psychosis and tardive dyskinesia which is thought to be caused by dopamine D2 receptor sensitization. We evaluated the effects of brexpiprazole on D2 receptor sensitivity after subchronic treatment in rats. We also evaluated whether brexpiprazole could suppress enhanced response to D2 receptors in rats subchronically dosed with another atypical antipsychotic. METHODS: The maximum D2 receptor density (Bmax ) and apomorphine (a D2 receptor agonist)-induced stereotypy were measured in rats orally dosed with vehicle, haloperidol (1 mg/kg), or brexpiprazole (4 or 30 mg/kg for Bmax , 6 or 30 mg/kg for stereotypy) for 21 days. Then, effects of oral administrations of brexpiprazole (3 mg/kg), aripiprazole (10 mg/kg), and olanzapine (3 mg/kg) against increases in apomorphine-induced hyperlocomotion and (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI: a 5-HT2A receptor agonist)-induced head twitches were evaluated in rats subcutaneously treated with risperidone (1.5 mg/kg/d) via minipumps for 21 days. RESULTS: Haloperidol and brexpiprazole (30 mg/kg: approximately tenfold ED50 of anti-apomorphine-induced stereotypy) but not brexpiprazole (4 or 6 mg/kg) significantly increased the Bmax and apomorphine-induced stereotypy. Brexpiprazole (3 mg/kg) and olanzapine (3 mg/kg) significantly suppressed both increases in apomorphine-induced hyperlocomotion and also DOI-induced head twitches in rats subchronically treated with risperidone, but aripiprazole (10 mg/kg) significantly suppressed only apomorphine-induced hyperlocomotion. CONCLUSION: Brexpiprazole has a low risk of D2 receptor sensitization after a repeated administration and suppresses the rebound phenomena related to D2 and 5-HT2A receptors after a repeated administration of risperidone.

6.
Curr Top Med Chem ; 19(18): 1621-1649, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424371

RESUMO

The history of the human immunodeficiency virus (HIV)/AIDS therapy, which spans over 30 years, is one of the most dramatic stories of science and medicine leading to the treatment of a disease. Since the advent of the first AIDS drug, AZT or zidovudine, a number of agents acting on different drug targets, such as HIV enzymes (e.g. reverse transcriptase, protease, and integrase) and host cell factors critical for HIV infection (e.g. CD4 and CCR5), have been added to our armamentarium to combat HIV/AIDS. In this review article, we first discuss the history of the development of anti-HIV drugs, during which several problems such as drug-induced side effects and the emergence of drug-resistant viruses became apparent and had to be overcome. Nowadays, the success of Combination Antiretroviral Therapy (cART), combined with recently-developed powerful but nonetheless less toxic drugs has transformed HIV/AIDS from an inevitably fatal disease into a manageable chronic infection. However, even with such potent cART, it is impossible to eradicate HIV because none of the currently available HIV drugs are effective in eliminating occult "dormant" HIV cell reservoirs. A number of novel unique treatment approaches that should drastically improve the quality of life (QOL) of patients or might actually be able to eliminate HIV altogether have also been discussed later in the review.


Assuntos
Fármacos Anti-HIV/farmacologia , Descoberta de Drogas , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/química , Farmacorresistência Viral/efeitos dos fármacos , Humanos
7.
Sci Rep ; 9(1): 12326, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444406

RESUMO

Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5' LTR to the end of the 3' LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.

8.
Mol Cancer Ther ; 18(9): 1602-1614, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31285280

RESUMO

Repurposing cationic amphiphilic drugs (CAD) for cancer treatment is emerging as an attractive means to enhance the efficacy of chemotherapy. Many commonly used CADs, including several cation amphiphilic antihistamines and antidepressants, induce cancer-specific, lysosome-dependent cell death and sensitize cancer cells to chemotherapy. CAD-induced inhibition of lysosomal acid sphingomyelinase is necessary, but not sufficient, for the subsequent lysosomal membrane permeabilization and cell death, while other pathways regulating this cell death pathway are largely unknown. Prompted by significant changes in the expression of genes involved in Ca2+ and cyclic AMP (cAMP) signaling pathways in CAD-resistant MCF7 breast cancer cells, we identified here an early lysosomal Ca2+ release through P2X purinergic receptor 4 (P2RX4) and subsequent Ca2+- and adenylyl cyclase 1 (ADCY1)-dependent synthesis of cAMP as a signaling route mediating CAD-induced lysosomal membrane permeabilization and cell death. Importantly, pharmacologic and genetic means to increase cellular cAMP levels either by activating cAMP-inducing G-protein-coupled receptors (GPR3 or ß2 adrenergic receptor) or ADCY1, or by inhibiting cAMP-reducing guanine nucleotide-binding protein G(i) subunit α2, C-X-C motif chemokine receptor type 4, or cAMP phosphodiesterases, sensitized cancer cells to CADs. These data reveal a previously unrecognized lysosomal P2RX4- and ADCY1-dependent signaling cascade as a pathway essential for CAD-induced lysosome-dependent cell death and encourage further investigations to find the most potent combinations of CADs and cAMP-inducing drugs for cancer therapy.

9.
Sci Rep ; 9(1): 6726, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040365

RESUMO

The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca2+ - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca2+ flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 - enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane.

10.
Physiol Genomics ; 51(8): 342-355, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125289

RESUMO

Preeclampsia (PE), a multifactorial pregnancy-specific syndrome accounting for up to 8% of pregnancy complications, is a leading cause of maternal and fetal morbidity and mortality. PE is also associated with long-term risk of hypertension and stroke for both mother and fetus. Currently, the only "cure" is delivery of the baby and placenta, largely because the pathogenesis of PE is not yet fully understood. PE is associated with impaired vascular remodeling at the maternal-fetal interface and placental insufficiency; however, specific factors contributing to this impairment have not been identified. To identify molecular pathways involved in PE, we examined temporal transcriptomic changes occurring within the uterus, uterine implantation sites, and placentae from the Dahl salt-sensitive (Dahl S) rat model of superimposed PE compared with Sprague Dawley (SD) rats. We hypothesized that targeted gene analysis and whole transcriptome analysis would identify genetic factors that contribute to development of the preeclamptic phenotype in the Dahl S rat and unveil novel biomarkers, therapeutic targets, and mechanistic pathways in PE. Quantitative real-time PCR (qRT-PCR) and whole genome microarray analysis were performed on isolated total RNA from uterus (day 0), uterine implantation sites (days 7 and 10), and placenta (days 14 and 20). We found 624, 332, 185, and 366 genes to be differentially expressed between Dahl S (PE) and SD (normal pregnancy) on days 0, 7, 10, and 14, respectively. Our data revealed numerous pathways that may play a role in the pathophysiology of spontaneous superimposed PE and allow for further investigation of novel therapeutic targets and biomarker development.

11.
Am J Physiol Renal Physiol ; 317(5): F1274-F1284, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892934

RESUMO

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder that is characterized by prevalent hypertension, renal injury, and cardiovascular disease. Numerous studies have reported a low prevalence and/or impaired function of regulatory T (TREG) cells in both patients with SLE and murine models of the disease. Evidence suggests that TREG cell dysfunction in SLE results from a deficiency in IL-2. Recent studies have reported that low-dose IL-2 therapy expands TREG cells in mouse models of SLE, but whether expanding TREG cells protects against hypertension and renal injury during SLE is unclear. To examine this question, female SLE (NZBWF1) and control (NZW) mice were injected with vehicle or recombinant mouse IL-2 three times in 24 h followed by single maintenance doses every 5 days for 4 wk. Treatment with IL-2 effectively expanded TREG cell populations in the peripheral blood, spleen, and kidneys. Circulating levels of anti-dsDNA IgG autoantibodies, a marker of SLE disease activity, were higher in SLE mice compared with control mice but were unaffected by IL-2 treatment. As previously reported by our laboratory, mean arterial pressure, measured in conscious mice by a carotid catheter, was higher in SLE mice than in control mice. Mean arterial pressure was significantly lower in IL-2-treated SLE mice compared with vehicle-treated SLE mice, suggesting that expanding TREG cells using low-dose IL-2 attenuates the development of hypertension. While the mechanism for the protection against hypertension is unclear, it does not appear to be related to the delay of SLE disease progression.

12.
Sci Rep ; 9(1): 4828, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886166

RESUMO

CCR5 is a member of the G-protein coupled receptor family that serves as an essential co-receptor for cellular entry of R5-tropic HIV-1, and is a validated target for therapeutics against HIV-1 infections. In the present study, we designed and synthesized a series of novel small CCR5 inhibitors and evaluated their antiviral activity. GRL-117C inhibited the replication of wild-type R5-HIV-1 with a sub-nanomolar IC50 value. These derivatives retained activity against vicriviroc-resistant HIV-1s, but did not show activity against maraviroc (MVC)-resistant HIV-1. Structural modeling indicated that the binding of compounds to CCR5 occurs in the hydrophobic cavity of CCR5 under the second extracellular loop, and amino acids critical for their binding were almost similar with those of MVC, which explains viral cross-resistance with MVC. On the other hand, one derivative, GRL-10018C, less potent against HIV-1, but more potent in inhibiting CC-chemokine binding, occupied the upper region of the binding cavity with its bis-THF moiety, presumably causing greater steric hindrance with CC-chemokines. Recent studies have shown additional unique features of certain CCR5 inhibitors such as immunomodulating properties and HIV-1 latency reversal properties, and thus, continuous efforts in developing new CCR5 inhibitors with unique binding profiles is necessary.

13.
Biochem Biophys Res Commun ; 509(4): 943-948, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30648556

RESUMO

Nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs) are major antiviral agents against hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1). However, the notorious insoluble property of HBV RT has prevented atomic-resolution structural studies and rational anti-HBV drug design. Here, we created HIV-1 RT mutants containing HBV-mimicking sextuple or septuple amino acid substitutions at the nucleoside-binding site (N-site) and verified that these mutants retained the RT activity. The most active RT mutant, HIV-1 RT7MC, carrying Q151M/G112S/D113A/Y115F/F116Y/F160L/I159L was successfully crystallized, and its three-dimensional structure was determined in complex with DNA:dGTP/entecavir-triphosphate (ETV-TP), a potent anti-HBV guanosine analogue RT inhibitor, at a resolution of 2.43 Šand 2.60 Å, respectively. The structures reveal significant positional rearrangements of the amino acid side-chains at the N-site, elucidating the mechanism underlying the differential susceptibility of HIV-1 and HBV against recently reported 4'-modified NRTIs.


Assuntos
Transcriptase Reversa do HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Substituição de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação/genética , Domínio Catalítico , Cristalografia por Raios X , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Humanos , Proteínas Mutantes/química , Conformação Proteica , Inibidores da Transcriptase Reversa/química
14.
J Biol Chem ; 294(1): 116-129, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30413535

RESUMO

Latency-reversing agents (LRAs) are considered a potential strategy for curing cells of HIV-1 infection. Certain protein kinase C (PKC) activators have been previously reported to be LRAs because they can reverse HIV latency. In the present study, we examined the activities of a panel of benzolactam derivatives against cells latently infected with HIV. Using determination of p24 antigen in cell supernatants or altered intracellular GFP expression to measure HIV reactivation from latently infected cells along with a cytotoxicity assay, we found that some of the compounds exhibited latency-reversing activity, which was followed by enhanced release of HIV particles from the cells. One derivative, BL-V8-310, displayed activity in ACH-2 and J-Lat cells latently infected with HIV at a concentration of 10 nm or higher, which was superior to the activity of another highly active PKC activator, prostratin. These results were confirmed with peripheral blood cells from HIV-infected patients. We also found that these drugs up-regulate the expression of caspase 3 and enhance apoptosis specifically in latently HIV-infected cells. Moreover, combining BL-V8-310 with a bromodomain-containing 4 (BRD4) inhibitor, JQ1, not only enhanced HIV latency-reversing activity, but also reduced the effect on cytotoxic cytokine secretion from CD4+ T-cells induced by BL-V8-310 alone. Our results suggest that BL-V8-310 and its related benzolactam derivatives are potential LRA lead compounds that are effective in reversing HIV latency and reducing viral reservoirs in HIV-positive individuals with few adverse effects.


Assuntos
Apoptose/efeitos dos fármacos , Benzodiazepinonas/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteína Quinase C/metabolismo , Latência Viral/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Caspase 3/biossíntese , Caspase 3/genética , Proteínas de Ciclo Celular , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Masculino , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Quinase C/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Front Microbiol ; 9: 2022, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30283406

RESUMO

Latency-reversing agents (LRAs) are considered a potential tool to cure human immunodeficiency virus type 1 (HIV-1) infection, but when they are taken alone, virus production by reactivated cells and subsequent infection will occur. Hence, it is crucial to simultaneously take appropriate measures to prevent such secondary HIV-1 infection. In this regard, a strategy to minimize the production of infectious viruses from LRA-reactivated cells is worth pursuing. Here, we focused on a second mitochondria-derived activator of caspases (Smac) mimetic, birinapant, to induce apoptosis in latent HIV-1-infected cells. When birinapant was administered alone, it only slightly increased the expression of caspase-3. However, in combination with an LRA (e.g., PEP005), it strongly induced the expression of caspase-3 followed by enhanced apoptosis. Importantly, the combination eliminated reactivated cells and drastically reduced HIV-1 production. Finally, we found that birinapant decreased the mRNA expression of HIV-1 that was induced by PEP005 in the primary CD4+ T-cells from HIV-1-carrying patients as well. These results suggest that the combination of an LRA and an "apoptosis-inducing" agent, such as a Smac mimetic, is a possible treatment option to decrease HIV-1 reservoirs without the occurrence of HIV-1 production by reactivated cells.

16.
Cell Chem Biol ; 25(10): 1268-1278.e3, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30174310

RESUMO

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA/MK-8591), a nucleoside reverse transcriptase inhibitor (NRTI) under clinical trials, is a potent and promising long-acting anti-HIV type 1 (HIV-1) agent. EFdA and its derivatives possess a modified 4'-moiety and potently inhibit the replication of a wide spectrum of HIV-1 strains resistant to existing NRTIs. Here, we report that EFdA and NRTIs with a 4'-ethynyl- or 4'-cyano-moiety exerted activity against HIV-1 with an M184V mutation and multiple NRTI-resistant HIV-1s, whereas NRTIs with other moieties (e.g., 4'-methyl) did not show this activity. Structural analysis indicated that EFdA and 4'-ethynyl-NRTIs (but not other 4'-modified NRTIs), formed strong van der Waals interactions with critical amino acid residues of reverse transcriptase. Such interactions were maintained even in the presence of a broad resistance-endowing M184V substitution, thus potently inhibiting drug-resistant HIV-1 strains. These findings also explain the mechanism for the potency of EFdA and provide insights for further design of anti-HIV-1 therapeutics.


Assuntos
Domínio Catalítico/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Mutação Puntual , Inibidores da Transcriptase Reversa/farmacologia , Linhagem Celular , Desoxiadenosinas/química , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Humanos , Simulação de Acoplamento Molecular , Inibidores da Transcriptase Reversa/química
17.
Cell Res ; 28(10): 996-1012, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30127373

RESUMO

Dysregulated intracellular pH is emerging as a hallmark of cancer. In spite of their acidic environment and increased acid production, cancer cells maintain alkaline intracellular pH that promotes cancer progression by inhibiting apoptosis and increasing glycolysis, cell growth, migration, and invasion. Here we identify signal transducer and activator of transcription-3 (STAT3) as a key factor in the preservation of alkaline cytosol. STAT3 associates with the vacuolar H+-ATPase in a coiled-coil domain-dependent manner and increases its activity in living cells and in vitro. Accordingly, STAT3 depletion disrupts intracellular proton equilibrium by decreasing cytosolic pH and increasing lysosomal pH, respectively. This dysregulation can be reverted by reconstitution with wild-type STAT3 or STAT3 mutants unable to activate target genes (Tyr705Phe and DNA-binding mutant) or to regulate mitochondrial respiration (Ser727Ala). Upon cytosolic acidification, STAT3 is transcriptionally inactivated and further recruited to lysosomal membranes to reestablish intracellular proton equilibrium. These data reveal STAT3 as a regulator of intracellular pH and, vice versa, intracellular pH as a regulator of STAT3 localization and activity.


Assuntos
Citosol/química , Lisossomos/química , Fator de Transcrição STAT3/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Edição de Genes , Complexo de Golgi/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Mutagênese , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genética
18.
Sci Rep ; 8(1): 10309, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29985397

RESUMO

Annexins are a family of proteins characterized by their ability to bind anionic membranes in response to Ca2+-activation. They are involved in a multitude of cellular functions including vesiculation and membrane repair. Here, we investigate the effect of nine annexins (ANXA1-ANXA7, ANXA11, ANXA13) on negatively charged double supported membrane patches with free edges. We find that annexin members can be classified according to the membrane morphology they induce and matching a dendrogam of the annexin family based on full amino acid sequences. ANXA1 and ANXA2 induce membrane folding and blebbing initiated from membrane structural defects inside patches while ANXA6 induces membrane folding originating both from defects and from the membrane edges. ANXA4 and ANXA5 induce cooperative roll-up of the membrane starting from free edges, producing large rolls. In contrast, ANXA3 and ANXA13 roll the membrane in a fragmented manner producing multiple thin rolls. In addition to rolling, ANXA7 and ANXA11 are characterized by their ability to form fluid lenses localized between the membrane leaflets. A shared feature necessary for generating these morphologies is the ability to induce membrane curvature on free edged anionic membranes. Consequently, induction of membrane curvature may be a significant property of the annexin protein family that is important for their function.


Assuntos
Anexinas/metabolismo , Bicamadas Lipídicas/química , Silicatos de Alumínio/química , Anexinas/química , Anexinas/genética , Humanos , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Moleculares , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
19.
Glob Pediatr Health ; 5: 2333794X18785540, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30014010

RESUMO

Objectives. Document American football, National Football League (NFL), Lean State (LS) or Heavy State (FS) Public High School (PHS), sets similar player position mean body mass indexes (BMI). Review health risks related to BMI. Methods. Public accessible 2014-2015 football rosters were used to calculate individual player's BMI for four PHS teams about each LS and FS Capital City and 32 NFL teams. Mean BMI were compared for male player positions: quarterback (Q), backfield (B), and line (L) players. Results. Q, B, and L mean BMI were not significantly different for LS and FS PHS and NFL, but mean BMI was significantly (P < .01) different for Q or B versus L. Conclusion. Football sets similar BMI for player positions with PHS line prone to obese BMI (considered healthy for NFL players) regardless of regional BMI trends. We propose PHS football set player BMI upper limit 30 to support public health and sports safety goals.

20.
Sci Rep ; 8(1): 1624, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29374261

RESUMO

Hepatitis B virus (HBV) reverse transcriptase (RT) is essential for viral replication and is an important drug target. Nonetheless, the notorious insolubility of HBV RT has hindered experimental structural studies and structure-based drug design. Here, we demonstrate that a Q151M substitution alone at the nucleotide-binding site (N-site) of human immunodeficiency virus type-1 (HIV-1) RT renders HIV-1 highly sensitive to entecavir (ETV), a potent nucleoside analogue RT inhibitor (NRTI) against HBV. The results suggest that Met151 forms a transient hydrophobic interaction with the cyclopentyl methylene of ETV, a characteristic hydrophobic moiety of ETV. We thus solved the crystal structures of HIV-1 RTQ151M:DNA complex with bound dGTP or ETV-triphosphate (ETV-TP). The structures revealed that ETV-TP is accommodated at the N-site slightly apart from the ribose ring of the 3'-end nucleotide, compared to the position of bound dGTP and previously reported NRTI/dNTP. In addition, the protruding methylene group of bound ETV-TP directly pushes the side-chain of Met184 backward. Met184 is a key residue that confers ETV resistance upon substitution with smaller Ile/Val. These results provide novel insights into NRTI binding to the N-site and further provide important clues for the development of novel anti-HBV/HIV-1 RT inhibitors to overcome critical drug resistance.


Assuntos
Antivirais/farmacologia , Guanina/análogos & derivados , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Vírus da Hepatite B/enzimologia , Mutação de Sentido Incorreto , Antivirais/química , Antivirais/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Guanina/química , Guanina/metabolismo , Guanina/farmacologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , Vírus da Hepatite B/genética , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica
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