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1.
Biochem Biophys Res Commun ; 503(3): 1868-1873, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30060950

RESUMO

SPAK and OSR1 are two protein kinases that play important roles in regulating the function of numerous ion co-transporters. They are activated by two distinct mechanisms that involve initial phosphorylation at their T-loops by WNK kinases and subsequent binding to a scaffolding protein termed MO25. To understand this latter SPAK and OSR1 regulation mechanism, we herein show that MO25 binding to these two kinases is enhanced by serine phosphorylation in their highly conserved WEWS motif, which is located in their C-terminal domains. Furthermore, we show that this C-terminal phosphorylation is carried out by WNK kinases in vitro and involves WNK kinases in cells. Mutagenesis studies revealed key MO25 residues that are important for MO25 binding and activation of SPAK and OSR1 kinases. Collectively, this study provides new insights into the MO25-mediated activation of SPAK and OSR1 kinases, which are emerging as important players in regulating ion homeostasis.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Células HEK293 , Humanos , Fosforilação
2.
Chembiochem ; 19(19): 2072-2080, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-29999233

RESUMO

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative-stress-responsive kinase 1 (OSR1) are two serine/threonine protein kinases that play key roles in regulating ion homeostasis. Various SPAK and OSR1 mouse models exhibited reduced blood pressure. Herein, the discovery of verteporfin, a photosensitising agent used in photodynamic therapy, as a potent inhibitor of SPAK and OSR1 kinases is reported. It is shown that verteporfin binds the kinase domains of SPAK and OSR1 and inhibits their catalytic activity in an adenosine triphosphate (ATP)-independent manner. In cells, verteporfin was able to suppress the phosphorylation of the ion co-transporter NKCC1; a downstream physiological substrate of SPAK and OSR1 kinases. Kinase panel screening indicated that verteporfin inhibited a further eight protein kinases more potently than that of SPAK and OSR1. Although verteporfin has largely been studied as a modifier of the Hippo signalling pathway, this work indicates that the WNK-SPAK/OSR1 signalling cascade is also a target of this clinical agent. This finding could explain the fluctuation in blood pressure noted in patients and animals treated with this drug.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/efeitos dos fármacos , Verteporfina/farmacologia , Células HEK293 , Homeostase , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo
3.
Chembiochem ; 19(5): 425-429, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29226533

RESUMO

Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early-onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.


Assuntos
Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Niclosamida/análogos & derivados , Niclosamida/farmacologia , Proteínas Quinases/metabolismo , Descoberta de Drogas , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/enzimologia
4.
Chembiochem ; 18(5): 460-465, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28004876

RESUMO

The binding of the scaffolding protein MO25 to SPAK and OSR1 protein kinases, which regulate ion homeostasis, causes increases of up to 100-fold in their catalytic activity. Various animal models have shown that the inhibition of SPAK and OSR1 lowers blood pressure, and so here we present a new indirect approach to inhibiting SPAK and OSR1 kinases by targeting their protein partner MO25. To explore this approach, we developed a fluorescent polarisation assay and used it in screening of a small in-house library of ≈4000 compounds. This led to the identification of one compound-HK01-as the first small-molecule inhibitor of the MO25-dependent activation of SPAK and OSR1 in vitro. Our data confirm the feasibility of targeting this protein-protein interaction by small-molecule compounds and highlights their potential to modulate ion co-transporters and thus cellular electrolyte balance.


Assuntos
Fenilalanina/análogos & derivados , Ftalimidas/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Bioensaio , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Immunoblotting , Camundongos , Fenilalanina/química , Fenilalanina/metabolismo , Ftalimidas/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacos
5.
Bioorg Med Chem Lett ; 23(9): 2671-4, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23540647

RESUMO

In this study, we report the design, synthesis and antioxidant activity of a series of substituted 2-(4-aminophenyl)-1H-indoles and 2-(methoxyphenyl)-1H-indoles. The new compounds are structurally related to the known indole-based antioxidant lead compound melatonin (MLT), and the antitumour 2-(4-aminophenyl)benzothiazole and 2-(3,4-dimethoxyphenyl)benzothiazole series. Efficient access to the target 2-phenylindoles was achieved via Fischer indole synthesis between substituted phenylhydrazines and acetophenones. 2-(4-Aminophenyl)indoles (such as the 6-fluoro analogue 3b) in particular showed potent antioxidant activity in the DPPH and superoxide radical scavenging assays (80% and 81% inhibition at 1mM concentration of 3b, respectively), at a level comparable with the reference standard MLT (98% and 75% at 1 mM).


Assuntos
Antineoplásicos/síntese química , Antioxidantes/síntese química , Indóis/química , Antineoplásicos/química , Antioxidantes/química , Benzotiazóis/química , Desenho de Fármacos , Indóis/síntese química , Melatonina/química , Relação Estrutura-Atividade , Superóxidos/química , Superóxidos/metabolismo
6.
J Med Chem ; 63(19): 11258-11270, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32930595

RESUMO

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC50 ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.


Assuntos
Antígenos/imunologia , Imunidade Celular , Compostos Organofosforados/química , Pró-Fármacos/farmacologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/efeitos dos fármacos , Antígenos/química , Humanos , Pró-Fármacos/química , Linfócitos T/imunologia
7.
ChemMedChem ; 15(8): 671-674, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32162793

RESUMO

The specific targeting of protein-protein interactions by phosphoserine-containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinder its uptake into cells. To address these issues, we herein report the synthesis of phosphoserine aryloxy triester phosphoramidates as phosphoserine prodrugs that are enzymatically metabolized to release phosphoserine. This phosphoserine-masking approach was applied to a phosphoserine-containing inhibitor of 14-3-3 dimerization, and the generated prodrugs exhibited improved pharmacological activity. Collectively, this provided a proof of concept that the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups is a viable intracellular delivery system for phosphoserine-containing molecules. Ultimately, this will facilitate the discovery of phosphoserine-containing small-molecule therapeutics.


Assuntos
Amidas/farmacologia , Ácidos Fosfóricos/farmacologia , Fosfosserina/antagonistas & inibidores , Pró-Fármacos/farmacologia , Amidas/síntese química , Amidas/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Ácidos Fosfóricos/síntese química , Ácidos Fosfóricos/química , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/química
8.
Mini Rev Med Chem ; 8(7): 711-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18537726

RESUMO

Resistance to apoptosis is one of the fundamental hallmarks of cancer. Recently, the selective induction of apoptosis in cancer cells has emerged as an exciting possibility for the development of future selective cancer therapy. This review will summarise the development of the major classes of small molecule "pro-apoptotic" antitumour agents.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Antineoplásicos/química , Antineoplásicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
9.
J Enzyme Inhib Med Chem ; 23(5): 641-7, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18821253

RESUMO

A new series of fluorinated and non-fluorinated 2-phenylbenzimidazoles bearing oxygenated substituents on the phenyl ring has been synthesized. Synthesis of the new series was based on our previous discovery of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) as a potent and selective antitumour agent in vitro (sub-nanomolar GI(50) in sensitive human cancer cell lines), but with poor aqueous solubility and lack of a definitive cellular target limiting further development. In this study we test the hypothesis that 2-phenylbenzimidazoles with similar substitution patterns to PMX 610 would retain potent antitumour activity but with potentially superior pharmaceutical properties. In general the new compounds were less active than the former benzothiazole series in vitro when tested against the breast cancer cell lines MCF-7 and MDA 468; however the two most active compounds in the present series (3j and 3k) exhibit low micromolar GI(50) values in both cell lines and provide the opportunity for further chemical derivatization with a view to target identification.


Assuntos
Antineoplásicos/síntese química , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Hidrocarbonetos Fluorados , Relação Estrutura-Atividade
10.
ChemMedChem ; 13(11): 1088-1091, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29603892

RESUMO

Niclosamide is an anthelmintic drug that has been used for over 50 years mainly to treat tapeworm infections. However, with the increase in drug repurposing initiatives, niclosamide has emerged as a true hit in many screens against various diseases. Indeed, from being an anthelmintic drug, it has now shown potential in treating Parkinson's disease, diabetes, viral and microbial infections, as well as various cancers. Such diverse pharmacological activities are a result of niclosamide's ability to uncouple mitochondrial phosphorylation and modulate a selection of signaling pathways, such as Wnt/ß-catenin, mTOR and JAK/STAT3, which are implicated in many diseases. In this highlight, we discuss the plethora of diseases that niclosamide has shown promise in treating.


Assuntos
Reposicionamento de Medicamentos , Niclosamida/farmacologia , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
11.
ChemMedChem ; 12(20): 1677-1686, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28881465

RESUMO

Since the discovery of WNK mutations that cause an inherited form of hypertension in humans, there has been increasing interest in targeting WNK signaling as a novel strategy for modulating blood pressure. This notion is now supported by numerous mouse models with impaired WNK signaling that exhibit reduced blood pressure. Biochemical analyses of the various protein components that make up this signaling pathway have identified a number of plausible molecular targets that are amenable to targeting by small molecules. To date, a selection of small-molecule WNK signaling inhibitors have been identified and have shown promise in suppressing the activity of WNK signaling in cells and in animals. In this Minireview, we briefly discuss the WNK signaling pathway and provide an overview of the various druggable targets within this cascade, as well as the different WNK signaling inhibitors discovered to date.


Assuntos
Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Humanos
12.
ChemMedChem ; 12(9): 639-645, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28371477

RESUMO

SPAK and OSR1 are two protein kinases that have emerged as attractive targets in the discovery of novel antihypertensive agents due to their role in regulating electrolyte balance in vivo. Herein we report the identification of an allosteric pocket on the highly conserved C-terminal domains of these two kinases, which influences their activity. We also show that some known WNK signaling inhibitors bind to this allosteric site. Using in silico screening, we identified the antiparasitic agent rafoxanide as a novel allosteric inhibitor of SPAK and OSR1. Collectively, this work will facilitate the rational design of novel SPAK and OSR1 kinase inhibitors that could be useful antihypertensive agents.


Assuntos
Colesterol/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Rafoxanida/farmacologia , Sítio Alostérico , Sequência de Aminoácidos , Polarização de Fluorescência , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo
13.
ACS Chem Biol ; 12(10): 2631-2643, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28862425

RESUMO

Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag's are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P-Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.


Assuntos
Antígenos CD/metabolismo , Butirofilinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Antígenos , Antígenos CD/genética , Butirofilinas/genética , Clonagem Molecular , Técnicas de Cocultura , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Mutação , Fosfoproteínas , Conformação Proteica , Domínios Proteicos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/metabolismo
14.
J Med Chem ; 60(8): 3518-3524, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28323427

RESUMO

Since loss of function mutations of PINK1 lead to early onset Parkinson's disease, there has been growing interest in the discovery of small molecules that amplify the kinase activity of PINK1. We herein report the design, synthesis, serum stability, and hydrolysis of four kinetin riboside ProTides. These ProTides, along with kinetin riboside, activated PINK1 in cells independent of mitochondrial depolarization. This highlights the potential of modified nucleosides and their phosphate prodrugs as treatments for neurodegenerative diseases.


Assuntos
Adenosina/farmacologia , Cinetina/farmacologia , Mitocôndrias/enzimologia , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray
15.
J Med Chem ; 59(23): 10400-10410, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27559756

RESUMO

Nucleoside monophosphates and monophosphonates have been known for a long time to exert favorable pharmacological effects upon intracellular delivery. However, their development as drug molecules has been hindered by the inherent poor druglike properties of the monophosphate and monophosphonate groups. These include inefficient cellular uptake and poor in vivo stability, with this latter drawback being most relevant to monophosphates than monophosphonates. To address these limitations, numerous monophosphate and monophosphonate prodrug strategies have been developed and applied in the discovery of nucleoside monophosphate and monophosphonate prodrugs that can treat viral infections and cancer. The approval of sofosbuvir, a nucleoside monophosphate prodrug, highlighted the success to be had by employing these prodrug technologies in the discovery of nucleotide therapeutics. In this Miniperspective, we discuss the different key monophosphate and monophosphonate nucleoside prodrugs that entered clinical development, some of which may in the future be approved to treat various human diseases.


Assuntos
Neoplasias/tratamento farmacológico , Nucleosídeos/uso terapêutico , Fosfatos/uso terapêutico , Pró-Fármacos/uso terapêutico , Viroses/tratamento farmacológico , Humanos , Nucleosídeos/química , Fosfatos/química , Pró-Fármacos/química
16.
J Med Chem ; 51(16): 5135-9, 2008 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-18666770

RESUMO

New fluorinated 2-aryl-benzothiazoles, -benzoxazoles, and -chromen-4-ones have been synthesized and their activity against MCF-7 and MDA 468 breast cancer cell lines compared with the potent antitumor benzothiazole 5. Analogues such as 9a, b and 12a, d yielded submicromolar GI50 values in both cell lines; however, none of the new compounds approached 5 in terms of antitumor potency. For 5, binding to the aryl hydrocarbon receptor appeared to be necessary but not sufficient for growth inhibition.


Assuntos
Antineoplásicos/síntese química , Benzopiranos/síntese química , Benzotiazóis/síntese química , Benzoxazóis/síntese química , Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Benzotiazóis/farmacologia , Benzoxazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Relação Estrutura-Atividade
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