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1.
Viruses ; 13(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34452405

RESUMO

Transcriptomics, proteomics and pathogen-host interactomics data are being explored for the in silico-informed selection of drugs, prior to their functional evaluation. The effectiveness of this kind of strategy has been put to the test in the current COVID-19 pandemic, and it has been paying off, leading to a few drugs being rapidly repurposed as treatment against SARS-CoV-2 infection. Several neglected tropical diseases, for which treatment remains unavailable, would benefit from informed in silico investigations of drugs, as performed in this work for Dengue fever disease. We analyzed transcriptomic data in the key tissues of liver, spleen and blood profiles and verified that despite transcriptomic differences due to tissue specialization, the common mechanisms of action, "Adrenergic receptor antagonist", "ATPase inhibitor", "NF-kB pathway inhibitor" and "Serotonin receptor antagonist", were identified as druggable (e.g., oxprenolol, digoxin, auranofin and palonosetron, respectively) to oppose the effects of severe Dengue infection in these tissues. These are good candidates for future functional evaluation and clinical trials.


Assuntos
Antivirais/uso terapêutico , Dengue/tratamento farmacológico , Transcriptoma , Adenosina Trifosfatases/antagonistas & inibidores , Antagonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/uso terapêutico , Antivirais/farmacologia , Encéfalo/metabolismo , Simulação por Computador , Dengue/sangue , Dengue/genética , Dengue/metabolismo , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Humanos , Fígado/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , NF-kappa B/metabolismo , Antagonistas da Serotonina/farmacologia , Antagonistas da Serotonina/uso terapêutico , Dengue Grave/sangue , Dengue Grave/tratamento farmacológico , Dengue Grave/genética , Dengue Grave/metabolismo , Baço/metabolismo
2.
Aging (Albany NY) ; 13(13): 17097-17117, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34252884

RESUMO

Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer's Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.


Assuntos
Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas de Choque Térmico/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Grupo com Ancestrais do Continente Africano , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína Beclina-1/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Grupo com Ancestrais do Continente Europeu , Técnicas de Silenciamento de Genes , Humanos , Quinolinas/farmacologia , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase-1/genética , Proteínas tau/biossíntese , Proteínas tau/genética
3.
Nat Commun ; 12(1): 3483, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108481

RESUMO

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , Adenosina Trifosfatases/química , Compostos de Boro/química , Proteínas de Saccharomyces cerevisiae/química , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Compostos de Boro/farmacologia , Resistência a Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Mutação , Nucleotídeos/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Int J Biol Macromol ; 182: 2130-2143, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087308

RESUMO

For centuries, dietary ginger has been known for its antioxidant, anticancer, and antibacterial properties. In the current study, we examined the link between antibacterial properties of 7 dietary ginger phenolics (DGPs)-gingerenone A, 6-gingerol, 8-gingerol, 10-gingerol, paradol, 6-shogaol, and zingerone-and inhibition of bacterial ATP synthase. DGPs caused complete (100%) inhibition of wild-type Escherichia coli membrane-bound F1Fo ATP synthase, but partial and variable (0%-87%) inhibition of phytochemical binding site mutant enzymes αR283D, αE284R, ßV265Q, and γT273A. The mutant enzyme ATPase activity was 16-fold to 100-fold lower than that of the wild-type enzyme. The growth of wild-type, null, and mutant strains in the presence of the 7 DGPs were abrogated to variable degrees on limiting glucose and succinate media. DGPs-caused variable inhibitory profiles of wild-type and mutant ATP synthase confirm that residues of α-, ß-, and γ-subunits are involved in the formation of phytochemical binding site. The variable degree of growth in the presence of DGPs also indicates the possibility of molecular targets other than ATP synthase. Our results establish that antibacterial properties of DGPs can be linked to the binding and inhibition of bacterial ATP synthase. Therefore, bacterial ATP synthase is a valuable molecular target for DGPs.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Dieta , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Gengibre/química , Fenóis/farmacologia , Adenosina Trifosfatases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Glucose/metabolismo , Mutação/genética , Fenóis/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Ácido Succínico/metabolismo
5.
J Virol ; 95(14): e0053121, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33952644

RESUMO

Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endoplasmic reticulum (ER) membrane into a functional N-terminal signal peptide (SP) and the C terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, and yet it was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug brefeldin A (BFA), which interferes with the ER-to-Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with endoglycosidase H indicated that Rem-CT does not traffic through the Golgi apparatus. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was colocalized with Rab5-positive (Rab5+) early endosomes. The expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (containing a mutation of threonine to asparagine at position 31 [T31N]) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase p97/VCP rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes. IMPORTANCE Mouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi apparatus. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host antiviral immunity.


Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Endossomos/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Proteínas Nucleares/antagonistas & inibidores , Precursores de Proteínas/metabolismo , Proteínas do Envelope Viral/metabolismo
6.
ACS Chem Biol ; 16(4): 775-785, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33739813

RESUMO

ClpB is a tightly regulated AAA+ disaggregation machine. Each ClpB molecule is composed of a flexibly attached N-terminal domain (NTD), an essential middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains. The NTD is not well-characterized structurally and is commonly considered to serve as a dispensable substrate-binding domain. Here, we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB's NTD and reveal its unexpected autoinhibitory function. We find that the NTD fluctuates on the microsecond time scale, and these dynamics result in steric hindrance that limits the conformational space of the MD to restrict its tilting. This leads to significantly inhibited ATPase and disaggregation activities of ClpB, an effect that is alleviated upon binding of a substrate protein or the cochaperone DnaK. This entropic inhibition mechanism, which is mediated by ultrafast motions of the NTD and is not dependent on any strong interactions, might be common in related ATP-dependent proteases and other multidomain proteins to ensure their fast and reversible activation.


Assuntos
Endopeptidase Clp/química , Adenosina Trifosfatases/antagonistas & inibidores , Transferência Ressonante de Energia de Fluorescência , Conformação Proteica , Especificidade por Substrato
7.
J Biochem ; 169(6): 731-745, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-33576821

RESUMO

Plasma membrane tubulin is an endogenous regulator of P-ATPases and the unusual accumulation of tubulin in the erythrocyte membrane results in a partial inhibition of some their activities, causing hemorheological disorders like reduced cell deformability and osmotic resistance. These disorders are of particular interest in hypertension and diabetes, where the abnormal increase in membrane tubulin may be related to the disease development. Phosphatidylserine (PS) is more exposed on the membrane of diabetic erythrocytes than in healthy cells. In most cells, PS is transported from the exoplasmic to the cytoplasmic leaflet of the membrane by lipid flippases. Here, we report that PS is more exposed in erythrocytes from both hypertensive and diabetic patients than in healthy erythrocytes, which could be attributed to the inhibition of flippase activity by tubulin. This is supported by: (i) the translocation rate of a fluorescent PS analog in hypertensive and diabetic erythrocytes was slower than in healthy cells, (ii) the pharmacological variation of membrane tubulin in erythrocytes and K562 cells was linked to changes in PS translocation and (iii) the P-ATPase-dependent PS translocation in inside-out vesicles (IOVs) from human erythrocytes was inhibited by tubulin. These results suggest that tubulin regulates flippase activity and hence, the membrane phospholipid asymmetry.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Diabetes Mellitus/patologia , Eritrócitos/metabolismo , Hipertensão/patologia , Fosfatidilserinas/metabolismo , Tubulina (Proteína)/metabolismo , Adenosina Trifosfatases/metabolismo , Adulto , Estudos de Casos e Controles , Diabetes Mellitus/metabolismo , Feminino , Humanos , Hipertensão/metabolismo , Masculino , Pessoa de Meia-Idade
8.
Eur J Med Chem ; 213: 113148, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33476933

RESUMO

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a ß-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 µM, 300 µM, and 100 µM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 µM, 6.1 µM, and 3.4 µM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/metabolismo , Relação Estrutura-Atividade
9.
Bioorg Chem ; 104: 104316, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33022549

RESUMO

Herein, molecular hybridization strategy was utilized in the design of new benzosuberone-thiazole derivatives. The structures of the synthesized hybrids were determined on the basis of elemental and spectral analyses. These compounds were evaluated for their antibacterial activities against five bronchitis causing bacteria in addition to their anti-tubercular activities. Most compounds revealed promising activities. Amongst active compounds, benzosuberone-dithiazole derivatives 22a and 28 with MIC value = 1.95 µg/ml against H. influenza, M. pneumonia, and B. pertussis displayed four times the activity of ciprofloxacin (MIC = 7.81 µg/ml) against H. influenza, twice the activity of ciprofloxacin (MIC = 3.9 µg/ml) against M. pneumonia and were equipotent to ciprofloxacin against B. pertussis (MIC = 1.95 µg/ml). Additionally, benzosuberone-dithiazole derivatives 22a and 27 were the most promising anti-tubercular among the tested compounds with MIC values of 0.12 and 0.24 µg/ml, respectively against sensitive M. tuberculosis in addition to high activity against resistant strain of M. tuberculosis (MIC = 0.98 and 1.95 µg/ml, respectively) compared to isoniazid (MIC = 0.12 µg/ml against sensitive M. tuberculosis and no activity against resistant M. tuberculosis). Cytotoxicity study of the active dithiazole derivatives 22a, 27 and 28 against normal human lung cells (WI-38) indicated their high safety profile as showed from their high IC50 values (IC50 = 107, 74.8, and 117 µM, respectively). Furthermore, DNA gyrase supercoiling and ATPase activity assays showed that 22a, 27 and 28 have the potential to inhibit DNA gyrase at low micromolar levels (IC50 = 3.29-15.64 µM). Molecular docking analysis was also carried out to understand the binding profiles of the synthesized compounds into the ATPase binding sites of bacterial and mycobacterial DNA gyraseB.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Cumarínicos/farmacologia , DNA Girase/metabolismo , Tiazóis/farmacologia , Inibidores da Topoisomerase II/farmacologia , Adenosina Trifosfatases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Bordetella pertussis/efeitos dos fármacos , Linhagem Celular , Cumarínicos/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycoplasma pneumoniae/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazóis/química , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química
10.
J Med Chem ; 63(19): 11131-11148, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32894018

RESUMO

Inhibitors of muscle myosin ATPases are needed to treat conditions that could be improved by promoting muscle relaxation. The lead compound for this study ((3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one; BHC) was previously discovered to inhibit skeletal myosin II. BHC and 34 analogues were synthesized to explore structure-activity relationships. The properties of analogues, including solubility, stability, and toxicity, suggest that the BHC scaffold may be useful for developing therapeutics. Inhibition of actin-activated ATPase activity of fast skeletal and cardiac muscle myosin II, inhibition of skeletal muscle contractility ex vivo, and slowing of in vitro actin-sliding velocity were measured. Several analogues with aromatic side arms showed improved potency (half-maximal inhibitory concentration (IC50) <1 µM) and selectivity (≥12-fold) for skeletal myosin versus cardiac myosin compared to BHC. Several analogues blocked neurotransmission, suggesting that they are selective for nonmuscle myosin II over skeletal myosin. Competition and molecular docking studies suggest that BHC and blebbistatin bind to the same site on myosin.


Assuntos
4-Hidroxicumarinas/química , 4-Hidroxicumarinas/farmacologia , Iminas/química , Miosinas/antagonistas & inibidores , 4-Hidroxicumarinas/síntese química , Adenosina Trifosfatases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Relação Estrutura-Atividade
11.
Biochem Pharmacol ; 180: 114200, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32805211

RESUMO

The eukaryotic genetic material is packaged in the form of chromatin by wrapping DNA around nucleosomes. Cells maintain chromatin in a dynamic state by utilising various ATP-dependent chromatin remodelling complexes which can induce structural transformations in the chromatin. All chromatin remodelers contain an ATP hydrolysing-DNA translocase motor which facilitates nucleosomal DNA translocation. By DNA translocation ISWI and CHD subfamily remodelers slide nucleosomes and arrange them in a regularly spaced array. While SWI/SNF subfamily remodelers evict or displace nucleosomes from chromatin, which promotes recruitment of transcription machinery and DNA repair factors on the DNA. Besides DNA translocation, ISWI, CHD and INO80 subfamily remodelers escort nucleosome organisation and editing. In this review; we discuss different mechanisms by which chromatin remodelers regulate chromatin accessibility, nucleosome assembly and nucleosome editing. We attempt to elucidate how their action mediates various cellular and developmental processes, and their deregulation leads to disease pathogenesis. We emphasised on their role in cancer progression and potential therapeutic implications of these complexes. We also described the drugs and strategies which are being developed to target different subunits of remodelling complexes, histone modifying enzymes and polycomb repressive complex. This includes ATPase inhibitors, EZH2 (enhancer of zeste homolog 2) inhibitors, BET (bromodomain and extra terminal) inhibitors, PROTAC (proteolysis targeting chimaera) and inhibitors of protein-protein interaction.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Animais , Montagem e Desmontagem da Cromatina/fisiologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Estrutura Secundária de Proteína , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo
12.
Mol Biochem Parasitol ; 239: 111313, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32735998

RESUMO

Misfolded proteins trapped in the endoplasmic reticulum (ER) are specifically recognized and retrotranslocated to the cytosol by the ER-Associated Degradation (ERAD) system and delivered to the proteasome for destruction. This process was recently described in Trypanosoma brucei (T. brucei) using the misfolded epitope tagged Transferrin Receptor subunits ESAG7:Ty and HA:ESAG6 (HA:E6). Critical to this work was the proteasomal inhibitor MG132. However, MG132 has off-target inhibitory effects on lysosomal Cathepsin L that could cause misinterpretation of turnover results. Here, we evaluate an orally bioavailable p97 inhibitor, CB-5083, for use in T. brucei. p97 is a ubiquitous protein involved in many cellular events including the membrane extraction step of ERAD. CB-5083 strongly inhibits turnover of HA:E6, with comparable protein recovery to MG132 treatment. Interestingly, little deglycosylated cytoplasmic species accumulates, though it normally emerges with MG132 treatment. This suggests that CB-5083 blocks ERAD upstream of the proteasome, as expected for inhibition of the trypanosomal p97 orthologue TbVCP. Under CB-5083 treatment, HA:E6 is also strongly membrane-associated, suggesting ER localization. Finally, we provide an experimental example where CB-5083 treatment offers clarity to the off-target effects of MG132 treatment.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Indóis/farmacologia , Leupeptinas/farmacologia , Pirimidinas/farmacologia , Trypanosoma brucei brucei , Adenosina Trifosfatases/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Dobramento de Proteína , Transporte Proteico , Proteólise/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo
13.
Sci Rep ; 10(1): 11391, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647350

RESUMO

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human ß-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Resveratrol/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Adenosina Trifosfatases/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Quimioterapia Combinada/métodos , Histatinas/imunologia , Histatinas/metabolismo , Humanos , Imunidade Inata , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Polimixinas/farmacologia , Polimixinas/uso terapêutico , Resveratrol/uso terapêutico , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , beta-Defensinas/imunologia , beta-Defensinas/metabolismo
14.
Sci Rep ; 10(1): 9175, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514052

RESUMO

Cilostazol, a phosphodiesterase 3 inhibitor, reduces the amyloid-beta (Aß) burden in mouse models of Alzheimer disease by as yet unidentified mechanisms. In the present study, we examined the possibility that cilostazol ameliorates lysosomal dysfunction. Astrocytes treated with bafilomycin A1 (BafA1) exhibited markedly reduced DND-189 and acridine orange (AO) fluorescence, indicating reduced lysosomal acidity. In both cases, BafA1-induced alkalization was reversed by addition of cilostazol, dibutyryl cAMP or forskolin. All three agents significantly increased free zinc levels in lysosomes, and addition of the zinc chelator TPEN abrogated lysosomal reacidification. These treatments did not raise free zinc levels or reverse BafA1-mediated lysosomal alkalization in metallothionein 3 (Mt3)-null astrocytes, indicating that the increases in zinc in astrocytes were derived mainly from Mt3. Lastly, in FITC-Aß-treated astrocytes, cilostazol reversed lysosomal alkalization, increased cathepsin D activity, and reduced Aß accumulation in astrocytes. Cilostazol also reduced mHtt aggregate formation in GFP-mHttQ74-expressing astrocytes. Collectively, our results present the novel finding that cAMP/PKA can overcome the v-ATPase blocking effect of BafA1 in a zinc- and Mt3-dependent manner.


Assuntos
Astrócitos/citologia , Cilostazol/farmacologia , AMP Cíclico/metabolismo , Lisossomos/metabolismo , Macrolídeos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Inibidores da Fosfodiesterase 3/farmacologia , Zinco/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Animais , Autofagia , Catepsina D/metabolismo , Células Cultivadas , Inibidores Enzimáticos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Camundongos
15.
Toxicol Appl Pharmacol ; 401: 115080, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32497533

RESUMO

Upregulation of ABCB1/MDR1 (P-gp) and BIRC5/Survivin promotes multidrug resistance in a variety of human cancers. LCL161 is an anti-cancer DIABLO/SMAC mimetic currently being tested in patients with solid tumors, but the molecular mechanism of action of LCL161 in cancer cells is still incompletely understood. It is still unclear whether LCL161 is therapeutically applicable for patients with ABCB1-overexpressing multidrug resistant tumors. In this study, we found that the potency of LCL161 is not affected by the expression of ABCB1 in KB-TAX50, KB-VIN10, and NTU0.017 cancer cells. Besides, LCL161 is equally potent towards the parental MCF7 breast cancer cells and its BIRC5 overexpressing, hormone therapy resistance subline MCF7-TamC3 in vitro. Mechanistically, we found that LCL161 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multi-drug efflux activity at low cytotoxic concentrations (i.e. 0.5xIC50 or less). Further analysis revealed that LCL161 also decreases intracellular ATP levels in part through BIRC5 downregulation. Therapeutically, co-treatment with LCL161 at low cytotoxic concentrations restored the sensitivity to the known ABCB1 substrate, paclitaxel, in ABCB1-expressing cancer cells and increased the sensitivity to tamoxifen in MCF7-TamC3 cells. In conclusion, LCL161 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide important information to physicians for designing a more "patient-specific" LCL161 clinical trial program in the future.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas Mitocondriais/farmacologia , Survivina/antagonistas & inibidores , Tiazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antineoplásicos/química , Proteínas Reguladoras de Apoptose/química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Mitocondriais/química , Estrutura Secundária de Proteína , Survivina/biossíntese , Survivina/genética , Tiazóis/química
16.
Biochemistry ; 59(28): 2667-2678, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32567308

RESUMO

Shigella is the causative agent of bacillary dysentery and is responsible for an estimated 165 million infections and 600,000 deaths annually. Like many Gram-negative pathogens, Shigella relies on a type three secretion system (T3SS) to initiate and sustain infection by directly injecting effector proteins into host cells. Protein secretion through the needle-like injectisome and overall Shigella virulence rely on the T3SS ATPase Spa47, making it a likely means for T3SS regulation and an attractive target for therapeutic small molecule inhibitors. Here, we utilize a recently solved 2.15 Å crystal structure of Spa47 to computationally screen 7.6 million drug-like compounds for candidates which avoid the highly conserved active site by targeting a distal, but critical, interface between adjacent protomers of the Spa47 homohexamer. Ten of the top inhibitor candidates were characterized, identifying novel Spa47 inhibitors that reduce in vitro ATPase activity by as much as 87.9 ± 10.5% with IC50's as low as 25 ± 20 µM and reduce in vivo Shigella T3SS protein secretion by as much as 94.7 ± 3.0%. Kinetic analyses show that the inhibitors operate through a noncompetitive mechanism that likely supports the inhibitors' low cytotoxicity, as they avoid off-target ATPases involved in either Shigella or mammalian cell metabolism. Interestingly, the inhibitors display nearly identical inhibition profiles for Spa47 and the T3SS ATPases EscN from E. coli and FliI from Salmonella. Together, the results of this study provide much-needed insight into T3SS ATPase inhibition mechanisms and a strong platform for developing broadly effective cross-pathogen T3SS ATPase inhibitors.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Shigella flexneri/efeitos dos fármacos , Sistemas de Secreção Tipo III/antagonistas & inibidores , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Disenteria Bacilar/tratamento farmacológico , Disenteria Bacilar/microbiologia , Humanos , Simulação de Acoplamento Molecular , Shigella flexneri/química , Shigella flexneri/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/metabolismo
17.
ChemMedChem ; 15(8): 685-694, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32162487

RESUMO

A major challenge of targeted cancer therapy is the selection for drug-resistant mutations in tumor cells leading to loss of treatment effectiveness. p97/VCP is central regulator of protein homeostasis and a promising anticancer target because of its vital role in cell growth and survival. One ATP-competitive p97 inhibitor, CB-5083, has entered clinical trials. Selective pressure on HCT116 cells dosed with CB-5083 identified five different resistant mutants. Identification of p97 inhibitors with different mechanisms of action would offer the potential to overcome this class of resistance mutations. Our results demonstrate that two CB-5083 resistant p97 mutants, N660 K and T688 A, were also resistant to several other ATP-competitive p97 inhibitors, whereas inhibition by two allosteric p97 inhibitors NMS-873 and UPCDC-30245 were unaffected by these mutations. We also established a CB-5083 resistant cell line that harbors a new p97 double mutation (D649 A/T688 A). While CB-5083, NMS-873, and UPCDC-30245 all effectively inhibited proliferation of the parental HCT116 cell line, NMS-873 and UPCDC-30245 were 30-fold more potent in inhibiting the CB-5083 resistant D649 A/T688 A double mutant than CB-5083. Our results suggest that allosteric p97 inhibitors are promising alternatives when resistance to ATP-competitive p97 inhibitors arises during anticancer treatment.


Assuntos
Acetanilidas/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/farmacologia , Acetanilidas/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Antineoplásicos/química , Benzotiazóis/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Células HCT116 , Humanos , Indóis/química , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/química
18.
J Enzyme Inhib Med Chem ; 35(1): 639-649, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32048531

RESUMO

Leishmaniasis is a neglected disease caused by the protozoa Leishmania ssp. Environmental differences found by the parasites in the vector and the host are translated into cellular stress, leading to the production of heat shock proteins (Hsp). These are molecular chaperones involved in the folding of nascent proteins as well as in the regulation of gene expression, signalling events and proteostasis. Since Leishmania spp. use Hsp90 to trigger important transitions between their different stages of the life cycle, this protein family becomes a profitable target in anti-parasite drug discovery. In this work, we implemented a multidisciplinary strategy coupling molecular modelling with in vitro assays to identify small molecules able to inhibit Hsp90 from L. braziliensis (LbHsp90). Overall, we identified some compounds able to kill the promastigote form of the L. braziliensis, and to inhibit LbHsp90 ATPase activity.


Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Leishmania braziliensis/efeitos dos fármacos , Chaperonas Moleculares/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas de Choque Térmico HSP90/metabolismo , Leishmania braziliensis/química , Modelos Moleculares , Chaperonas Moleculares/síntese química , Chaperonas Moleculares/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
19.
J Recept Signal Transduct Res ; 40(2): 148-156, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32009493

RESUMO

Schizophrenia is a devastating illness and displays a wide range of psychotic symptoms. Accumulating evidence indicate impairment of bioenergetic pathways including energy storage and usage in the pathogenesis of schizophrenia. Although well-established synthetic drugs are being used for the management of schizophrenia, most of them have several adverse effects. Hence, natural products derived from medicinal plants represent a continuous major source for ethnomedicine-derived pharmaceuticals for different neurological disorders including schizophrenia. In the present study, we have investigated the neuroprotective effect of the novel bioactive compound i.e. "3-(3,4-dimethoxy phenyl) -1- (4-methoxyphenyl) prop-2-en-1-one" of Celastrus paniculata against ketamine-induced schizophrenia with particular reference to the activities of ATPase using in vivo and in silico methods. Ketamine-induced schizophrenia caused significant reduction in the activities of all three ATPases (Na+/K+, Ca2+ and Mg2+) in different regions of brain which reflects the decreased turnover of ATP, presumably due to the inhibition of oxidoreductase system and uncoupling of the same from the electron transport system. On par with the reference compound, clozapine, the activity levels of all three ATPases were restored to normal after pretreatment with the compound suggesting recovery of energy loss that was occurred during ketamine-induced schizophrenia. Besides, the compound has shown strong interaction and exhibited highest binding energies against all the three ATPases with a lowest inhibition constant value than the clozapine. The results of the present study clearly imply that the compound exhibit significant neuroprotective and antischizophrenic effect by modulating bioenergietic pathways that were altered during induced schizophrenia.


Assuntos
Adenosina Trifosfatases/genética , Antipsicóticos/farmacologia , Celastrus/química , Propano/farmacologia , Esquizofrenia/tratamento farmacológico , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Antipsicóticos/química , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Clozapina/farmacologia , Simulação por Computador , Modelos Animais de Doenças , Humanos , Ketamina/toxicidade , Propano/análogos & derivados , Ratos , Esquizofrenia/induzido quimicamente , Esquizofrenia/genética , Esquizofrenia/patologia
20.
Int J Biol Macromol ; 150: 23-30, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32035966

RESUMO

Insect venom peptides (IVPs) eumenitin, lasiocepsin, lycosin1, mastoparanB, panurgine1, and protonectin possess antibacterial properties, and the ubiquitous enzyme ATP synthase has a peptide-binding site. In the present study, we studied the effect of IVPs on binding and inhibition of three Escherichia coli strains (wild type, mutant, and null) and isolated E. coli ATP synthase. IVPs and their C-terminal amide (-NH2) analogs caused variable inhibition of membrane-bound F1Fo ATP synthase. While wild type E. coli growth was substantially hampered, null E. coli growth was near normal in the presence of IVPs and their C-terminal-NH2 analogs. The presence of C-terminal-NH2 groups on IVPs resulted in increased inhibition of ATP synthase and reduced growth of E. coli strains. Insignificant inhibition of the ßDELSEED-motif mutant enzyme with the ßAAAAAAA-motif confirmed that IVPs interact with the ßDELSEED-motif, also known as the peptide-binding site. The higher level of growth loss in E. coli strains by eumenitin, lasiocepsin, lycosin1, mastoparanB, panurgine1, and protonectin and their C-terminal-NH2 analogs suggested the likelihood of additional cellular or molecular targets. IVPs caused inhibition of E. coli strains, which demonstrates an association between antimicrobial traits of IVPs and bacterial ATP synthase.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Venenos de Artrópodes/química , Escherichia coli/enzimologia , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica
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