RESUMO
BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
Assuntos
Apoptose , Proteína X Associada a bcl-2 , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Humanos , Apoptose/efeitos dos fármacos , Cisteína/química , Cisteína/metabolismo , Animais , Aldeídos/química , Aldeídos/farmacologia , Modelos Moleculares , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fosRESUMO
The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.
Assuntos
Apoptose/efeitos dos fármacos , Dissulfetos/farmacologia , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Dissulfetos/química , Relação Dose-Resposta a Droga , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Moleculares , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-Atividade , Triptofano/análogos & derivados , Triptofano/química , Triptofano/farmacologiaRESUMO
BCL-2 family proteins converge at the mitochondrial outer membrane to regulate apoptosis and maintain the critical balance between cellular life and death. This physiologic process is essential to organism homeostasis and relies on protein-protein and protein-lipid interactions among BCL-2 family proteins in the mitochondrial lipid environment. Here, we find that trans-2-hexadecenal (t-2-hex), previously implicated in regulating BAX-mediated apoptosis, does so by direct covalent reaction with C126, which is located on the surface of BAX at the junction of its α5/α6 core hydrophobic hairpin. The application of nuclear magnetic resonance spectroscopy, hydrogen-deuterium exchange mass spectrometry, specialized t-2-hex-containing liposomes, and BAX mutational studies in mitochondria and cells reveals structure-function insights into the mechanism by which covalent lipidation at the mitochondria sensitizes direct BAX activation. The functional role of BAX lipidation as a control point of mitochondrial apoptosis could provide a therapeutic strategy for BAX modulation by chemical modification of C126.
Assuntos
Apoptose , Cisteína/metabolismo , Lipídeos/química , Proteína X Associada a bcl-2/metabolismo , Aldeídos , Animais , Humanos , Lipossomos , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Camundongos , Mitocôndrias/metabolismo , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Proteína X Associada a bcl-2/químicaRESUMO
Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide-vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity.
Assuntos
Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Alcenos/química , Alcenos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/química , Bactérias/metabolismo , Bioquímica/métodos , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Oxirredução , Selenocisteína/química , Selenocisteína/metabolismo , Vancomicina/química , Vancomicina/metabolismoRESUMO
The facile rearrangement of "S-acyl isopeptides" to native peptide bonds via S,N-acyl shift is central to the success of native chemical ligation, the widely used approach for protein total synthesis. Proximity-driven amide bond formation via acyl transfer reactions in other contexts has proven generally less effective. Here, we show that under neutral aqueous conditions, "O-acyl isopeptides" derived from hydroxy-asparagine [aspartic acid-ß-hydroxamic acid; Asp(ß-HA)] rearrange to form native peptide bonds via an O,N-acyl shift. This process constitutes a rare example of an O,N-acyl shift that proceeds rapidly across a medium-size ring (t1/2 â¼ 15 min), and takes place in water with minimal interference from hydrolysis. In contrast to serine/threonine or tyrosine, which form O-acyl isopeptides only by the use of highly activated acyl donors and appropriate protecting groups in organic solvent, Asp(ß-HA) is sufficiently reactive to form O-acyl isopeptides by treatment with an unprotected peptide-αthioester, at low mM concentration, in water. These findings were applied to an acyl transfer-based chemical ligation strategy, in which an unprotected N-terminal Asp(ß-HA)-peptide and peptide-αthioester react under aqueous conditions to give a ligation product ultimately linked by a native peptide bond.
RESUMO
BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.
Assuntos
Éteres Fenílicos/farmacologia , Proteína X Associada a bcl-2/química , Apoptose , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Éteres Fenílicos/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
The Armillaria and Lactarius genera of fungi produce the antimicrobial and cytotoxic mellolide, protoilludane, and marasmane sesquiterpenoids. We report a unified synthetic strategy to access the protoilludane, mellolide, and marasmane families of natural products. The key features of these syntheses are 1)â the organocatalytic, enantioselective construction of key chiral intermediates from a simple achiral precursor, 2)â the utility of a key 1,2-cyclobutanediol intermediate to serve as a precursor to each natural product class, and 3)â a direct chemical conversion of a protoilludane to a marasmane through serendipitous ring contraction, which provides experimental support for their proposed biosynthetic relationships.
Assuntos
Produtos Biológicos/síntese química , Compostos Heterocíclicos/química , Metano/análogos & derivados , Sesquiterpenos/síntese química , Produtos Biológicos/química , Catálise , Metano/química , Modelos Moleculares , Estrutura Molecular , Sesquiterpenos Policíclicos , Sesquiterpenos/químicaRESUMO
The NHC-catalyzed transformation of unsaturated aldehydes into saturated esters through an organocatalytic homoenolate process has been thoroughly studied. Leveraging a unique "Umpolung"-mediated ß-protonation, this process has evolved from a test bed for homoenolate reactivity to a broader platform for asymmetric catalysis. Inspired by our success in using the ß-protonation process to generate enals from ynals with good E/Z selectivity, our early studies found that an asymmetric variation of this reaction was not only feasible, but also adaptable to a kinetic resolution of secondary alcohols through NHC-catalyzed acylation. In-depth analysis of this process determined that careful catalyst and solvent pairing is critical for optimal yield and selectivity; proper choice of nonpolar solvent provided improved yield through suppression of an oxidative side reaction, while employment of a cooperative catalytic approach through inclusion of a hydrogen bond donor cocatalyst significantly improved enantioselectivity.
Assuntos
Aldeídos/química , Catálise , Oxirredução , Prótons , EstereoisomerismoRESUMO
MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure-function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutagênese , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias/genética , Neoplasias/metabolismo , Mutação Puntual , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismoRESUMO
Herein we report an umpolung strategy for the bioconjugation of selenocysteine in unprotected peptides. This mild and operationally simple approach takes advantage of the electrophilic character of an oxidized selenocysteine (Se-S bond) to react with a nucleophilic arylboronic acid to provide the arylated selenocysteine within hours. This reaction is amenable to a wide range of boronic acids with different biorelevant functional groups and is unique to selenocysteine. Experimental evidence indicates that under oxidative conditions the arylated derivatives are more stable than the corresponding alkylated selenocysteine.
Assuntos
Hidrocarbonetos Aromáticos/química , Peptídeos/química , Selenocisteína/química , Ácidos Borônicos/químicaRESUMO
An enantioselective N-heterocyclic carbene (NHC)-catalyzed ß-protonation through the orchestration of three distinct organocatalysts has been developed. This cooperative catalyst system enhances both yield and selectivity, compared to only the NHC-catalyzed process. This new method allows for the efficient conversion of a large scope of aryl-oxobutenoates to highly enantioenriched succinate derivatives and demonstrates the benefits of combining different activation modes in organocatalysis.
Assuntos
Acroleína/análogos & derivados , Ésteres/síntese química , Compostos Heterocíclicos/química , Metano/análogos & derivados , Prótons , Acroleína/química , Catálise , Ésteres/química , Metano/química , Modelos Moleculares , Conformação Molecular , Teoria Quântica , EstereoisomerismoRESUMO
A mild, efficient, and low-temperature palladium-catalyzed cyanation of (hetero)aryl halides and triflates is reported. Previous palladium-catalyzed cyanations of (hetero)aryl halides have required higher temperatures to achieve good catalytic activity. This current reaction allows the cyanation of a general scope of (hetero)aryl halides and triflates at 2-5 mol % catalyst loadings with temperatures ranging from rt to 40 °C. This mild method was applied to the synthesis of lersivirine, a reverse transcriptase inhibitor.
Assuntos
Hidrocarbonetos Halogenados/química , Mesilatos/química , Nitrilas/química , Paládio/química , Pirazóis/síntese química , Inibidores da Transcriptase Reversa/síntese química , Estrutura Molecular , Nitrilas/síntese química , Pirazóis/química , Inibidores da Transcriptase Reversa/químicaRESUMO
An unusual room temperature ß-lactone decarboxylation facilitated a five-step enantioselective formal synthesis of the cyclopentane core of an estrogen receptor ß-agonist. A computational study probed the underlying factors facilitating unprecedented, rapid decarboxylation. Aryl substitution promotes faster reaction in the retro-[2+2] as a result of conjugative stabilization with the forming olefin. Additionally, the configuration of the α-ester in these fused ß-lactones leads to differential decarboxylation rates.
Assuntos
Ciclopentanos/química , Compostos Heterocíclicos/química , Metano/análogos & derivados , Simulação de Dinâmica Molecular , Temperatura , Catálise , Ciclopentanos/síntese química , Descarboxilação , Cinética , Metano/química , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
This study describes the combined experimental and computational elucidation of the mechanism and origins of stereoselectivities in the NHC-catalyzed dynamic kinetic resolution (DKR) of α-substituted-ß-ketoesters. Density functional theory computations reveal that the NHC-catalyzed DKR proceeds by two mechanisms, depending on the stereochemistry around the forming bond: 1) a concerted, asynchronous formal (2+2) aldol-lactonization process, or 2) a stepwise spiro-lactonization mechanism where the alkoxide is trapped by the NHC-catalyst. These mechanisms contrast significantly from mechanisms found and postulated in other related transformations. Conjugative stabilization of the electrophile and non-classical hydrogen bonds are key in controlling the stereoselectivity. This reaction constitutes an interesting class of DKRs in which the catalyst is responsible for the kinetic resolution to selectively and irreversibly capture an enantiomer of a substrate undergoing rapid racemization with the help of an exogenous base.
RESUMO
Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative (31)P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays.
Assuntos
Conformação de Ácido Nucleico , RNA/análise , Riboswitch , Aptâmeros de Nucleotídeos/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , RNA/química , RNA/metabolismo , Espectrofotometria Ultravioleta , TemperaturaRESUMO
Several (+)- and (-)-α-pinene derivatives were synthesized and evaluated for their antimicrobial activity toward Gram-positive bacteria Micrococcus luteus and Staphylococcus aureus, Gram-negative bacterium Escherichia coli, and the unicellular fungus Candida albicans using bioautographic assays. (+)-α-Pinene 1a showed modest activity against the test organisms, whereas (-)-α-pinene 1b showed no activity at the tested concentration. Of all the α-pinene derivatives evaluated, the ß-lactam derivatives (10a and 10b) were the most antimicrobial. The increase in the antimicrobial activity of 10a compared to 1a ranged from nearly 3.5-fold (C. albicans) to 43-fold (S. aureus). The mean ± standard deviation for the zone of inhibition (mm) for 10a (C. albicans) was 31.9 ± 4.3 and that for S. aureus was 51.1 ± 2.9. Although (-)-α-pinene 1b was not active toward the test microorganisms, the corresponding ß-lactam 10b, amino ester 13b, and amino alcohol 14b showed antimicrobial activity toward the test microorganisms. The increase in the antimicrobial activity of 10b compared to 1b ranged from 32-fold (S. aureus) to 73-fold (M. luteus). The mean ± standard deviation for the zone of inhibition (mm) for 10b (S. aureus) was 32.0 ± 0.60 and that for M. luteus was 73.2 ± 0.30.
Assuntos
Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Monoterpenos/síntese química , Monoterpenos/farmacologia , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Monoterpenos Bicíclicos , Candida albicans/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Monoterpenos/química , Relação Estrutura-AtividadeRESUMO
N-Heterocyclic carbene (NHC) catalyzed transformations have emerged as powerful tactics for the construction of complex molecules. Since Stetter's report in 1975 of the total synthesis of cis-jasmon and dihydrojasmon by using carbene catalysis, the use of NHCs in total synthesis has grown rapidly, particularly over the last decade. This renaissance is undoubtedly due to the recent developments in NHC-catalyzed reactions, including new benzoin, Stetter, homoenolate, and aroylation processes. These transformations employ typical as well as Umpolung types of bond disconnections and have served as the key step in several new total syntheses. This Minireview highlights these reports and captures the excitement and emerging synthetic utility of carbene catalysis in total synthesis.
Assuntos
Produtos Biológicos/síntese química , Técnicas de Química Sintética/métodos , Metano/análogos & derivados , Aldeídos/química , Benzoína/química , Catálise , Cetonas/química , Metano/química , Estrutura Molecular , EstereoisomerismoRESUMO
New DKR type: An N-heterocyclic carbene (NHC)-catalyzed dynamic kinetic resolution of racemic α-substituted ß-keto esters has been developed. This method relies on the epimerization of an NHC-enol intermediate before subsequent aldol/acylation events. Highly substituted ß-lactones are produced in good yield with good to excellent selectivities (see scheme).
Assuntos
Lactonas/química , Metano/análogos & derivados , Aldeídos/síntese química , Aldeídos/química , Catálise , Ciclopentanos/síntese química , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Cinética , Lactonas/síntese química , Metano/síntese química , Metano/química , EstereoisomerismoRESUMO
A cooperative catalysis approach for the enantioselective formal [3+2] addition of α,ß-unsaturated aldehydes to isatins has been developed. Homoenolate annulations of ß-aryl enals catalyzed by an N-heterocyclic carbene (NHC) require the addition of lithium chloride for high levels of enantioselectivity. This NHC-catalyzed annulation has been used for the total synthesis of maremycin B.