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1.
Nat Chem Biol ; 19(1): 55-63, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36577875

RESUMO

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.


Assuntos
Proteínas Nucleares , Fatores de Transcrição , Proteínas Nucleares/metabolismo , Microscopia Crioeletrônica , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ligases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
2.
Nature ; 557(7704): 196-201, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29720648

RESUMO

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Quinolinas/química , Quinolinas/farmacologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Alostérica/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Escherichia coli/química , Hidrocarbonetos/química , Hidrocarbonetos/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos
3.
Bioconjug Chem ; 31(8): 1995-2007, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32701261

RESUMO

Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Lipoproteínas/química , Nanoestruturas/química , Sistemas de Liberação de Medicamentos , Fragmentos Fab das Imunoglobulinas/farmacologia , Maleimidas/química , Reologia
4.
Proc Natl Acad Sci U S A ; 114(30): E6044-E6053, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28698362

RESUMO

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein N-acyltransferase (Lnt) catalyzes the transfer of the sn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant of Escherichia coli Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon-nitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by E. coli Lnt.


Assuntos
Aciltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Lipoproteínas/metabolismo , Acilação , Aciltransferases/química , Sítios de Ligação , Domínio Catalítico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Mutação , Conformação Proteica
5.
J Biol Chem ; 288(6): 3753-67, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23277359

RESUMO

The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3ß to foster phosphorylation of the Wnt effector ß-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced ß-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteína Axina/metabolismo , Poliubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Via de Sinalização Wnt/fisiologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Proteína Axina/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Poliubiquitina/genética , Ligação Proteica , Ubiquitina-Proteína Ligases/genética
6.
J Biol Chem ; 288(37): 26926-43, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23897821

RESUMO

Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/química , Histonas/química , Acetilação , Benzamidas/química , Ligação Competitiva , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/química , Concentração Inibidora 50 , Cinética , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Piridinas/química , Transcrição Gênica , Vorinostat
7.
Curr Protoc ; 3(9): e872, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37671955

RESUMO

The drug discovery landscape is ever-evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher-throughput mid-scale semi-automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult-to-express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high-quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co-expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation-prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co-infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA-encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Baculovirus generation via homologous recombination Support Protocol 1: Anti-glycoprotein 64 antibody assay Basic Protocol 2: Generation of insect cell biomass expressing target protein(s) Basic Protocol 3: Mid-scale affinity purification Support Protocol 2: Automated method for affinity purification on Hamilton STAR Basic Protocol 4: Size exclusion chromatography Support Protocol 3: Chromeleon 7 operation on Vanquish Duo.


Assuntos
Acetaminofen , Aspirina , Animais , Baculoviridae , Bioensaio , Descoberta de Drogas , Escherichia coli , Mamíferos
8.
Nanoscale Adv ; 3(13): 3929-3941, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-36133017

RESUMO

Nanolipoprotein particles (NLPs) have been evaluated as an in vivo delivery vehicle for a variety of molecules of therapeutic interest. However, delivery of peptide-like drugs in combination with therapeutic Fabs has not yet been evaluated. In this study, we describe the development and characterization of cystine-knot peptide (CKP)-containing NLPs and Fab-CKP-NLP conjugates. CKPs were incorporated into NLPs using a self-assembly strategy. The trypsin inhibitor EETI-II, a model CKP, was produced with a C16 fatty acyl chain to enable incorporation of the CKP into the lipid bilayer core during NLP assembly. The CKP-NLP retained trypsin inhibitory function although the overall activity was reduced by ∼5 fold compared to free CKP, which was presumably due to steric hindrance. The NLP platform was also shown to accommodate up to ∼60 CKP molecules. Moreover, the stability of the CKP-NLP was comparable to the NLP control, displaying a relatively short half-life (∼1 h) in 50% serum at 37 °C. Therapeutic Fabs were also loaded onto the CKP-NLP by introducing thiol-reactive lipids that would undergo a covalent reaction with the Fab. Using this strategy, Fab loading could be reliably controlled from 1-50 Fabs per CKP-NLP and was found to be independent of CKP density. Surprisingly, Fab incorporation into CKP-NLPs led to a substantial improvement in NLP stability (half-life > 24 h) at 37 °C; also, there was no reduction in CKP activity in the Fab-CKP-NLP conjugates compared to CKP-NLPs. Altogether, our data demonstrate the potential of NLPs as a promising platform for the targeted or multidrug delivery of peptide-based drug candidates in combination with Fabs.

9.
Methods Mol Biol ; 2025: 51-68, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31267448

RESUMO

The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.


Assuntos
Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética
10.
Sci Rep ; 8(1): 7136, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29740124

RESUMO

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/imunologia , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Imunização , Conformação Proteica , Dobramento de Proteína , Transporte Proteico/genética , Transporte Proteico/imunologia , Vacinação
11.
J Med Chem ; 57(23): 10176-91, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25383627

RESUMO

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.


Assuntos
Aminopiridinas/síntese química , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Substituição de Aminoácidos , Aminopiridinas/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cristalografia por Raios X , Receptores ErbB/genética , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Metionina/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Treonina/genética
12.
J Mol Biol ; 408(3): 420-31, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21396940

RESUMO

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.


Assuntos
Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Enzimas de Conjugação de Ubiquitina/genética
13.
Structure ; 19(10): 1433-42, 2011 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-21944579

RESUMO

The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Domínios e Motivos de Interação entre Proteínas , Proteínas Adaptadoras de Transdução de Sinal , Anticorpos/metabolismo , Densidade Óssea , Proteínas Morfogenéticas Ósseas/química , Cromatografia de Afinidade , Cromatografia em Gel , Sequência Consenso , Marcadores Genéticos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mutação de Sentido Incorreto , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt1/metabolismo
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