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1.
Circulation ; 150(4): 302-316, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38695173

RESUMO

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.


Assuntos
Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt , Ubiquitina Tiolesterase , Animais , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/metabolismo , Humanos , Camundongos , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Masculino , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Ratos Sprague-Dawley , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/etiologia , Remodelação Vascular , Células Cultivadas , Proliferação de Células , Camundongos Endogâmicos C57BL , Indóis , Oximas
2.
Bioorg Med Chem Lett ; 94: 129458, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37634761

RESUMO

Malaria continues to be a major burden on global health, responsible for 619,000 deaths in 2021. The causative agent of malaria is the eukaryotic parasite Plasmodium. Resistance to artemisinin-based combination therapies (ACTs), the current first-line treatment for malaria, has emerged in Asia, South America, and more recently Africa, where >90% of all malaria-related deaths occur. This has necessitated the identification and investigation of novel parasite proteins and pathways as antimalarial targets, including components of the ubiquitin proteasome system. Here, we investigate Plasmodium falciparum deubiquitinase ubiquitin C-terminal hydrolase L3 (PfUCHL3) as one such target. We carried out a high-throughput screen with covalent fragments and identified seven scaffolds that selectively inhibit the plasmodial UCHL3, but not human UCHL3 or the closely related human UCHL1. After assessing toxicity in human cells, we identified four promising hits and demonstrated their efficacy against asexual P. falciparum blood stages and P. berghei sporozoite stages.


Assuntos
Antimaláricos , Enzimas Desubiquitinantes , Antagonistas do Ácido Fólico , Antimaláricos/farmacologia , Eucariotos , Plasmodium falciparum , Complexo de Endopeptidases do Proteassoma , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/química , Proteínas de Protozoários
3.
J Enzyme Inhib Med Chem ; 38(1): 2284119, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37994421

RESUMO

Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195-0.959 µM and of 0.149-1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.


Assuntos
Anidrases Carbônicas , Enterococos Resistentes à Vancomicina , Bactérias , Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia
4.
J Enzyme Inhib Med Chem ; 37(1): 333-338, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34979838

RESUMO

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with KIs in the range of 28.6-469.5 µM, whereas VchCAα with KIs in the range of 39.8-438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with KIs of 137-948.9 µM for hCA I and of 296.5-961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.


Assuntos
Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Cumarínicos/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Vibrio cholerae/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neisseria gonorrhoeae/enzimologia , Relação Estrutura-Atividade , Vibrio cholerae/enzimologia
5.
J Enzyme Inhib Med Chem ; 37(1): 666-671, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35139743

RESUMO

The α-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) were investigated for their inhibition by a panel of phenols and phenolic acids. Mono-, di- and tri-substituted phenols incorporating additional hydroxyl/hydroxymethyl, amino, acetamido, carboxyl, halogeno and carboxyethenyl moieties were included in the study. The best NgCAα inhibitrs were phenol, 3-aminophenol, 4-hydroxy-benzylalcohol, 3-amino-4-chlorophenol and paracetamol, with KI values of 0.6-1.7 µM. The most effective VchCAα inhibitrs were phenol, 3-amino-4-chlorophenol and 4-hydroxy-benzyl-alcohol, with KI values of 0.7-1.2 µM. Small changes in the phenol scaffold led to drastic effects on the bacterial CA inhibitory activity. This class of underinvestigated bacterial CA inhibitors may thus lead to effective compounds for fighting drug resistant bacteria.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Neisseria gonorrhoeae/enzimologia , Fenóis/farmacologia , Vibrio cholerae/enzimologia , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Fenóis/química , Relação Estrutura-Atividade
6.
J Enzyme Inhib Med Chem ; 37(1): 1-8, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34894954

RESUMO

Recently, inorganic anions and sulphonamides, two of the main classes of zinc-binding carbonic anhydrase inhibitors (CAIs), were investigated for inhibition of the α-class carbonic anhydrase (CA, EC 4.2.1.1) from Neisseria gonorrhoeae, NgCA. As an extension to our previous studies, we report that dithiocarbamates (DTCs) derived from primary or secondary amines constitute a class of efficient inhibitors of NgCA. KIs ranging between 83.7 and 827 nM were measured for a series of 31 DTCs that incorporated various aliphatic, aromatic, and heterocyclic scaffolds. A subset of DTCs were selected for antimicrobial testing against N. gonorrhoeae, and three molecules displayed minimum inhibitory concentration (MIC) values less than or equal to 8 µg/mL. As NgCA was recently validated as an antibacterial drug target, the DTCs may lead to development of novel antigonococcal agents.


Assuntos
Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Tiocarbamatos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neisseria gonorrhoeae/enzimologia , Relação Estrutura-Atividade , Tiocarbamatos/síntese química , Tiocarbamatos/química
7.
J Enzyme Inhib Med Chem ; 37(1): 51-61, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34894972

RESUMO

Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide's Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.


Assuntos
Acetazolamida/farmacologia , Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Etoxzolamida/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Acetazolamida/síntese química , Acetazolamida/química , Antibacterianos/síntese química , Antibacterianos/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Etoxzolamida/síntese química , Etoxzolamida/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neisseria gonorrhoeae/enzimologia , Relação Estrutura-Atividade , Estados Unidos , United States Food and Drug Administration
8.
J Enzyme Inhib Med Chem ; 37(1): 1838-1844, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35758212

RESUMO

Vancomycin-resistant enterococci (VRE), consisting of pathogenic Enterococcus faecalis and E. faecium, is a leading cause of hospital-acquired infections (HAIs). We recently repurposed the FDA-approved human carbonic anhydrase (CA) inhibitor acetazolamide (AZM) against VRE agent with the likely mechanism of action for the molecules being inhibition of one, or both, of the bacterial CA isoforms expressed in VRE. To elucidate how inhibitor binding to the enzymes relates to MIC, we further characterised the inhibition constants (Ki) against the E. faecium α-CA (Efα-CA) and γ-CA (Efγ-CA), as well as against human CA I (hCAI) and human CA II (hCAII) to assess selectivity. We have also utilised homology modelling and molecular dynamics (MD) simulations to gain a better understanding of the potential interactions the molecules are making with the targets. In this paper, we elaborate on the SAR for the AZM analogs as it pertains to MIC and Ki for each CA.


Assuntos
Anidrases Carbônicas , Infecções por Bactérias Gram-Positivas , Enterococos Resistentes à Vancomicina , Acetazolamida , Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Enterococcus faecalis , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Vancomicina/farmacologia
9.
Biochemistry ; 60(8): 584-596, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33583181

RESUMO

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ubiquitinas/metabolismo , Catálise , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Conformação Proteica , Ubiquitinação
10.
Artigo em Inglês | MEDLINE | ID: mdl-33495225

RESUMO

Vancomycin-resistant enterococci (VRE) represent a major public health threat that requires the development of new therapeutics. In the present study, acetazolamide (AZM) was evaluated against enterococci. It inhibited different enterococcal strains tested at clinically achievable concentrations. Moreover, AZM outperformed linezolid, the drug of choice for VRE infections, in two in vivo VRE mouse models-murine colonization-reduction and VRE septicemia. Collectively, these results indicate that AZM warrants consideration as a promising treatment option for VRE infections.


Assuntos
Infecções por Bactérias Gram-Positivas , Enterococos Resistentes à Vancomicina , Acetazolamida/farmacologia , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Linezolida/farmacologia , Camundongos
11.
J Enzyme Inhib Med Chem ; 36(1): 1061-1066, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34030562

RESUMO

The bacterial pathogen Neisseria gonorrhoeae encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), NgCA, which was investigated for its inhibition with a series of inorganic and organic anions. Perchlorate and hexafluorophosphate did not significantly inhibit NgCA CO2 hydrase activity, whereas the halides, azide, bicarbonate, carbonate, stannate, perosmate, diphosphate, divanadate, perruthenate, and trifluoromethanesulfonate showed inhibition constants in the range of 1.3-9.6 mM. Anions/small molecules such as cyanate, thiocyanate, nitrite, nitrate, bisulphite, sulphate, hydrogensulfide, phenylboronic acid, phenylarsonic acid, selenate, tellurate, tetraborate, perrhenate, peroxydisulfate, selenocyanate, iminodisulfonate, and fluorosulfonate showed KIs in the range of 0.15-1.0 mM. The most effective inhibitors detected in this study were sulfamide, sulfamate, trithiocarbonate and N,N-diethyldithiocarbamate, which had KIs in the range of 5.1-88 µM. These last compounds incorporating the CS2- zinc-binding group may be used as leads for developing even more effective NgCA inhibitors in addition to the aromatic/heterocyclic sulphonamides, as this enzyme was recently validated as an antibacterial drug target for obtaining novel antigonococcal agents.


Assuntos
Antibacterianos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Ânions/síntese química , Ânions/química , Ânions/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Neisseria gonorrhoeae/enzimologia , Relação Estrutura-Atividade
12.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668938

RESUMO

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteases/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Ubiquitina Tiolesterase/metabolismo
13.
Biochemistry ; 59(37): 3447-3462, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32865982

RESUMO

Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors.


Assuntos
Mutação , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Processamento de Proteína Pós-Traducional , Ubiquitina/genética , Ubiquitina Tiolesterase/genética
14.
Chembiochem ; 21(5): 712-722, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31449350

RESUMO

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.


Assuntos
Inibidores Enzimáticos , Ubiquitina Tiolesterase , Sítios de Ligação , Linhagem Celular , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Estrutura Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo
15.
Mol Pharm ; 17(4): 1261-1275, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32134677

RESUMO

Drug loading is an important parameter known to impact the release rate of a poorly soluble drug from an amorphous solid dispersion (ASD). Recent studies have shown that small increases in drug loading can dramatically reduce the drug release rate from ASDs prepared with poly(vinylpyrrolidone-co-vinyl acetate) (PVPVA). However, the link between drug physicochemical properties and the drug loading where the release is abruptly compromised is not well understood. This study probes the role of different factors on the relative dissolution rates of drug and polymer from PVPVA-based ASDs as a function of drug loading: (1) the impact of drug-polymer hydrogen bonding interactions on the initial dissolution rate of ASDs, investigated using two structural analogues, indomethacin (IND) and indomethacin methyl ester (INDester), (2) the influence of surface drug crystallization, observed for INDester ASDs, and (3) by changing temperature, the impact of the "wet" glass transition temperature (Tg). Scanning electron microscopy (SEM), with or without energy dispersive X-ray (EDX) analysis, Fourier transform infrared spectroscopy (FTIR), and powder X-ray diffraction (PXRD) were utilized to study the solid-state phase behavior and/or drug enrichment on the partially dissolved ASD tablet surfaces. Nanoparticle tracking analysis (NTA) was utilized to study the solution-state phase behavior. It was found that, contrary to expectations, ASDs with drug-polymer hydrogen bonding exhibited poorer initial release at moderate drug loadings (15-25%) as compared to the non-hydrogen bonding analogue ASDs. Surface crystallization led to the deterioration of dissolution performance. Lastly, Tg relative to experimental temperatures also appeared to play a role in the observed dissolution behavior as a function of drug loading. These findings shed light on potential mechanisms governing ASD dissolution performance and will aid in the development of optimized ASD formulations with enhanced dissolution performance.


Assuntos
Preparações Farmacêuticas/química , Polímeros/química , Pirrolidinas/química , Comprimidos/química , Compostos de Vinila/química , Cristalização/métodos , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Ligação de Hidrogênio , Nanopartículas/química , Solubilidade , Temperatura de Transição
16.
Nat Chem Biol ; 12(12): 1023-1030, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27694802

RESUMO

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.


Assuntos
Benzamidas/farmacologia , Senescência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hexosiltransferases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Benzamidas/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Hexosiltransferases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Membrana/metabolismo , Estrutura Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química
17.
Bioorg Med Chem Lett ; 27(4): 755-758, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28119024

RESUMO

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC50=0.010µM) while several compounds showed ∼10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC50>25µM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.


Assuntos
Antiparasitários/química , Benzamidas/química , Sulfonamidas/química , Antiparasitários/farmacologia , Antiparasitários/toxicidade , Benzamidas/farmacologia , Benzamidas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leishmania/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Sulfonamidas/toxicidade , Trypanosoma brucei brucei/efeitos dos fármacos
19.
Bioorg Med Chem Lett ; 24(16): 3968-73, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25017033

RESUMO

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC50=16 nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.


Assuntos
Amidas/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Relação Estrutura-Atividade
20.
Enzymes ; 55: 283-311, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39222994

RESUMO

Carbonic anhydrase metalloenzymes are encoded in genomes throughout all kingdoms of life with a conserved function catalyzing the reversible conversion of CO2 to bicarbonate. Carbonic anhydrases have been well-investigated in humans, but are still relatively understudied in bacterial organisms, including Enterococci. Studies over the past decade have presented bacterial carbonic anhydrases as potential drug targets, with some chemical scaffolds potently inhibiting the Enterococcus carbonic anhydrases in vitro and displaying antimicrobial efficacy against Enterococcus organisms. While carbonic anhydrases in Enterococci still have much to be explored, hypotheses may be drawn from similar Gram-positive organisms for which known information exists about carbonic anhydrase function and relevance. Within this chapter is reported information and rational hypotheses regarding the subcellar locations, potential physiological roles, essentiality, structures, and kinetics of carbonic anhydrases in Enterococci.


Assuntos
Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Enterococcus , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Enterococcus/efeitos dos fármacos , Enterococcus/enzimologia , Humanos , Antibacterianos/farmacologia
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