RESUMEN
All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.
Asunto(s)
Factores de Transcripción , Ubiquitina-Proteína Ligasas , Humanos , Expresión Génica , Células HEK293 , Células HeLa , Mutación , Transducción de Señal , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1ß-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.
Asunto(s)
Retroelementos , Animales , Humanos , Ratones , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Retrovirus Endógenos/genética , Regulación de la Expresión Génica/genética , Histonas/metabolismo , Histonas/genética , Elementos de Nucleótido Esparcido Largo/genética , Unión Proteica , Retroelementos/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Complejos Multiproteicos/metabolismoRESUMEN
Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.
Asunto(s)
Cromatina , Factores de Transcripción , Humanos , Ligandos , Cromatina/genética , Factores de Transcripción/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Ubiquitinas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , ADN , Histonas , Factores de Transcripción ARNTL/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , ADN/genética , ADN/metabolismo , Secuencias Hélice-Asa-Hélice/genética , Histonas/química , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Unión Proteica , Proteínas CLOCK/química , Proteínas CLOCK/metabolismo , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/metabolismo , Regulación Alostérica , Leucina Zippers , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Multimerización de ProteínaRESUMEN
Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4DCAF12 ) E3 ligase preferentially ubiquitinates proteins that carry a C-terminal double glutamate (di-Glu) motif. Reported CRL4DCAF12 di-Glu-containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4DCAF12 and CCT5/TRiC is currently unknown. Here, we present the cryo-EM structure of the DDB1-DCAF12-CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the ß-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4DCAF12 E3 ligase in overseeing the assembly of a key cellular complex.
Asunto(s)
Proteínas Portadoras , Ubiquitina-Proteína Ligasas , Proteínas Portadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Chaperonina con TCP-1/metabolismoRESUMEN
Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.
Asunto(s)
Ciclinas , Ubiquitina-Proteína Ligasas , Ciclinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteolisis , Relación Estructura-ActividadRESUMEN
The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons 2-7. cGAS is also present inside the cell nucleus, which is replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.
Asunto(s)
Microscopía por Crioelectrón , Nucleosomas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Células HeLa , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutación , Nucleosomas/química , Nucleosomas/ultraestructura , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/ultraestructuraRESUMEN
The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the Ë29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.
Asunto(s)
Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas Cullin/metabolismo , Humanos , Modelos Moleculares , Mutación , Dominios Proteicos , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.
RESUMEN
Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Schizosaccharomyces/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Alelos , Sustitución de Aminoácidos , Centrómero/química , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Perfilación de la Expresión Génica , Heterocromatina/química , Heterocromatina/metabolismo , Cinética , Modelos Genéticos , Anotación de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Schizosaccharomyces/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Péptidos Cíclicos/química , Péptidos/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Diseño de Fármacos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Simulación de Dinámica Molecular , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Péptidos/metabolismo , Péptidos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Co-translational protein targeting to membranes relies on the signal recognition particle (SRP) system consisting of a cytosolic ribonucleoprotein complex and its membrane-associated receptor. SRP recognizes N-terminal cleavable signals or signal anchor sequences, retards translation, and delivers ribosome-nascent chain complexes (RNCs) to vacant translocation channels in the target membrane. While our mechanistic understanding is well advanced for the small bacterial systems it lags behind for the large bacterial, archaeal and eukaryotic SRP variants including an Alu and an S domain. Here we describe recent advances on structural and functional insights in domain architecture, particle dynamics and interplay with RNCs and translocon and GTP-dependent regulation of co-translational protein targeting stimulated by SRP RNA.
Asunto(s)
Membrana Celular/genética , Proteínas de la Membrana/genética , Transporte de Proteínas/genética , Partícula de Reconocimiento de Señal/genética , Elementos Alu/genética , Archaea/genética , Bacterias/genética , Membrana Celular/ultraestructura , Células Eucariotas/metabolismo , Proteínas de la Membrana/ultraestructura , Dominios Proteicos/genética , Modificación Traduccional de las Proteínas/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Partícula de Reconocimiento de Señal/ultraestructuraRESUMEN
The Alu domain of the signal recognition particle (SRP) arrests protein biosynthesis by competition with elongation factor binding on the ribosome. The mammalian Alu domain is a protein-RNA complex, while prokaryotic Alu domains are protein-free with significant extensions of the RNA. Here we report the crystal structure of the complete Alu domain of Bacillus subtilis SRP RNA at 2.5 Å resolution. The bacterial Alu RNA reveals a compact fold, which is stabilized by prokaryote-specific extensions and interactions. In this 'closed' conformation, the 5' and 3' regions are clamped together by the additional helix 1, the connecting 3-way junction and a novel minor groove interaction, which we term the 'minor-saddle motif' (MSM). The 5' region includes an extended loop-loop pseudoknot made of five consecutive Watson-Crick base pairs. Homology modeling with the human Alu domain in context of the ribosome shows that an additional lobe in the pseudoknot approaches the large subunit, while the absence of protein results in the detachment from the small subunit. Our findings provide the structural basis for purely RNA-driven elongation arrest in prokaryotes, and give insights into the structural adaption of SRP RNA during evolution.
Asunto(s)
Elementos Alu , ARN Bacteriano/química , Partícula de Reconocimiento de Señal/química , Bacillus subtilis/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos , ARN de Transferencia/químicaRESUMEN
Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.
Asunto(s)
Lisina , Pez Cebra , Animales , Histona Desacetilasa 6/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Dominio Catalítico , Cristalización , Lisina/metabolismo , Ubiquitinas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , AcetilaciónRESUMEN
Dysfunctional cilia cause pleiotropic human diseases termed ciliopathies. These hereditary maladies are often caused by defects in cilia assembly, a complex event that is regulated by the ciliogenesis and planar polarity effector (CPLANE) proteins Wdpcp, Inturned, and Fuzzy. CPLANE proteins are essential for building the cilium and are mutated in multiple ciliopathies, yet their structure and molecular functions remain elusive. Here, we show that mammalian CPLANE proteins comprise a bona fide complex and report the near-atomic resolution structures of the human Wdpcp-Inturned-Fuzzy complex and of the mouse Wdpcp-Inturned-Fuzzy complex bound to the small guanosine triphosphatase Rsg1. Notably, the crescent-shaped CPLANE complex binds phospholipids such as phosphatidylinositol 3-phosphate via multiple modules and a CPLANE ciliopathy mutant exhibits aberrant lipid binding. Our study provides critical structural and functional insights into an enigmatic ciliogenesis-associated complex as well as unexpected molecular rationales for ciliopathies.
RESUMEN
The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.
Asunto(s)
Virus de la Influenza A , Gripe Humana , Infección por el Virus Zika , Virus Zika , Histona Desacetilasa 6/metabolismo , Humanos , Virus de la Influenza A/metabolismo , Ubiquitina/metabolismo , Virus Zika/metabolismoRESUMEN
The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.
Asunto(s)
Enfermedad de Huntington , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Ratones , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasas Asociadas a rhoRESUMEN
The eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly 'closed' conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an 'open' Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.
Asunto(s)
Elementos Alu , Plasmodium falciparum/metabolismo , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Ribosomas/genética , Dispersión del Ángulo PequeñoRESUMEN
The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative stress. Inhibition of the interaction between NRF2 and its main negative regulator KEAP1 is an attractive strategy toward neuroprotective agents. We report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue is utilized as a tool to probe structural changes within a specific pocket of the KEAP1 binding site. We used structural information from peptide screening at the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an explorative SAR at position 5 of THIQs to identify a series of neutral THIQ analogs that bind to KEAP1 in the low micromolar range. These analogs establish new H-bond interactions at the P3 and P2 pockets allowing the replacement of the carboxylic acid functionality by a neutral primary carboxamide. X-ray crystallographic studies reveal the novel binding mode of these molecules to KEAP1.