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1.
Nat Struct Mol Biol ; 30(12): 1958-1969, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38049566

RESUMO

Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Receptores Androgênicos/química , Androgênios/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Domínios Proteicos , Fatores de Transcrição , Linhagem Celular Tumoral
2.
Nat Struct Mol Biol ; 28(12): 1009-1019, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34887560

RESUMO

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.


Assuntos
Metabolismo Energético/fisiologia , Histonas/genética , Histonas/metabolismo , NAD/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Reparo do DNA/genética , Eucariotos/metabolismo , Humanos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores
3.
Protein Sci ; 30(7): 1427-1437, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33978290

RESUMO

The phase equilibria of intrinsically disordered proteins are exquisitely sensitive to changes in solution conditions and this can be used to investigate the driving forces of phase separation in vitro as well as the biological roles of phase transitions in live cells. Here we investigate how using D2 O as co-solvent in an aqueous buffer changes the phase equilibrium of a fragment of the activation domain of the androgen receptor, a transcription factor that plays a role in the development of the male phenotype and is a therapeutic target for castration resistant prostate cancer. We show how replacing even small fractions of H2 O with D2 O increases the propensity of this fragment to undergo liquid-liquid phase separation, likely reflecting a stabilization of the hydrophobic interactions that drive condensation. Our results indicate that it is necessary to take this effect into consideration when studying phase separation phenomena with biophysical methods that require using D2 O as a co-solvent. In addition, they suggest that additions of D2 O may be used to enhance phase separation phenomena in cells, facilitating their observation.


Assuntos
Óxido de Deutério/química , Proteínas Intrinsicamente Desordenadas/química , Receptores Androgênicos/química , Humanos , Domínios Proteicos
4.
Curr Opin Chem Biol ; 62: 90-100, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33812316

RESUMO

Intrinsically disordered domains represent attractive therapeutic targets because they play key roles in cancer, as well as in neurodegenerative and infectious diseases. They are, however, considered undruggable because they do not form stable binding pockets for small molecules and, therefore, have not been prioritized in drug discovery. Under physiological solution conditions many biomedically relevant intrinsically disordered proteins undergo phase separation processes leading to the formation of mesoscopic highly dynamic assemblies, generally known as biomolecular condensates that define environments that can be quite different from the solutions surrounding them. In what follows, we review key recent findings in this area and show how biomolecular condensation can offer opportunities for modulating the activities of intrinsically disordered targets.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Preparações Farmacêuticas/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Descoberta de Drogas , Humanos , Transição de Fase/efeitos dos fármacos , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Termodinâmica , Temperatura de Transição
5.
Structure ; 26(1): 145-152.e3, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29225078

RESUMO

The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.


Assuntos
DNA/química , Proteínas Intrinsicamente Desordenadas/química , Receptores Androgênicos/química , Fatores de Transcrição TFII/química , Motivos de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Masculino , Modelos Moleculares , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Ativação Transcricional
6.
Eur J Med Chem ; 96: 318-29, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25899336

RESUMO

Two new polycyclic scaffolds were synthesized and evaluated as anti-influenza A compounds. The 5-azapentacyclo[6.4.0.0(2,10).0(3,7).0(9,11)]dodecane derivatives were only active against the wild-type M2 channel in the low-micromolar range. However, some of the 14-azaheptacyclo[8.6.1.0(2,5).0(3,11).0(4,9).0(6,17).0(12,16)]heptadecane derivatives were dual inhibitors of the wild-type and the V27A mutant M2 channels. The antiviral activity of these molecules was confirmed by cell culture assays. Their binding mode was analysed through molecular dynamics simulations, which showed the existence of distinct binding modes in the wild type M2 channel and its V27A variant.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Compostos Policíclicos/farmacologia , Proteínas da Matriz Viral/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/química , Sítios de Ligação/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Vírus da Influenza A/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mutação , Compostos Policíclicos/síntese química , Compostos Policíclicos/química , Relação Estrutura-Atividade , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética
7.
ACS Med Chem Lett ; 5(7): 831-6, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25050174

RESUMO

The synthesis of several [4,4,3], [4,3,3], and [3,3,3]azapropellanes is reported. Several of the novel amines displayed low-micromolar activities against an amantadine-resistant H1N1 strain, but they did not show activity against an amantadine-sensitive H3N2 strain. None of the tested compounds inhibit the influenza A/M2 proton channel function. Most of the compounds did not show cytotoxicity for MDCK cells.

8.
J Med Chem ; 57(13): 5738-47, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-24941437

RESUMO

Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.


Assuntos
Amantadina/análogos & derivados , Aminas/síntese química , Vírus da Influenza A/efeitos dos fármacos , Pirrolidinas/síntese química , Proteínas da Matriz Viral/antagonistas & inibidores , Aminas/farmacologia , Animais , Cães , Farmacorresistência Viral , Canais Iônicos/efeitos dos fármacos , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação , Infecções por Orthomyxoviridae/tratamento farmacológico , Técnicas de Patch-Clamp , Pirrolidinas/farmacologia , Relação Estrutura-Atividade , Proteínas da Matriz Viral/genética , Xenopus
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