RESUMEN
Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.
Asunto(s)
Antirretrovirales , Descubrimiento de Drogas , Retrovirus Endógenos , ADN Polimerasa Dirigida por ARN , Inhibidores de la Transcriptasa Inversa , Antirretrovirales/química , Antirretrovirales/farmacología , Retrovirus Endógenos/enzimología , Retrovirus Endógenos/genética , Genes Virales , Transcriptasa Inversa del VIH/química , Humanos , Multimerización de Proteína , ADN Polimerasa Dirigida por ARN/química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
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
We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.
Asunto(s)
Imidazoles/farmacología , Janus Quinasa 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Imidazoles/síntesis química , Imidazoles/química , Janus Quinasa 1/metabolismo , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Factor de Transcripción STAT3/metabolismo , Relación Estructura-ActividadRESUMEN
The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.
Asunto(s)
Anilidas/química , Anilidas/farmacología , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Acetilación/efectos de los fármacos , Aminación , Animales , Células Cultivadas , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Histonas/metabolismo , Humanos , Cinética , Ratones , Simulación del Acoplamiento MolecularRESUMEN
Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.
Asunto(s)
Antivirales/farmacología , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Ensayos Analíticos de Alto Rendimiento , Hidrodinámica , Ratones , Modelos Moleculares , Nucleoproteínas/ultraestructura , Orthomyxoviridae/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Bibliotecas de Moléculas Pequeñas/uso terapéutico , SolucionesRESUMEN
The discovery of nitrile compound 4, a potent inhibitor of Cathepsin K (Cat K) with good bioavailability in dog is described. The compound was used to demonstrate target engagement and inhibition of Cat K in an in vivo dog PD model. The margin to hERG ion channel inhibition was deemed too low for a clinical candidate and an optimisation program to find isosteres or substitutions on benzothiazole group led to the discovery of 20, 24 and 27; all three free from hERG inhibition.
Asunto(s)
Benzotiazoles/química , Benzotiazoles/farmacología , Catepsina K/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Nitrilos/química , Nitrilos/farmacología , Animales , Benzotiazoles/metabolismo , Benzotiazoles/farmacocinética , Catepsina K/metabolismo , Perros , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Nitrilos/metabolismo , Nitrilos/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
With the ever-increasing number of synthesis-on-demand compounds for drug lead discovery, there is a great need for efficient search technologies. We present the successful application of a virtual screening method that combines two advances: (1) it avoids full library enumeration (2) products are evaluated by molecular docking, leveraging protein structural information. Crucially, these advances enable a structure-based technique that can efficiently explore libraries with billions of molecules and beyond. We apply this method to identify inhibitors of ROCK1 from almost one billion commercially available compounds. Out of 69 purchased compounds, 27 (39%) have Ki values < 10 µM. X-ray structures of two leads confirm their docked poses. This approach to docking scales roughly with the number of reagents that span a chemical space and is therefore multiple orders of magnitude faster than traditional docking.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas , Simulación del Acoplamiento Molecular , Ligandos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Unión ProteicaRESUMEN
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
A complex of RON(M1254T) with AMP-PNP and Mg(2+) reveals a substratelike positioning of Tyr1238 as well as likely catalysis-competent placement of the AMP-PNP and Mg(2+) components and indicates a tendency for cis phosphorylation. The structure shows how the oncogenic mutation may cause the constitutive activation and suggests a mechanistic hypothesis for the autophosphorylation of receptor tyrosine kinases.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Adenilil Imidodifosfato , Fosforilación , Fosfotransferasas , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
The displacement of probes that bind selectively to subdomains IIA or IIIA on human serum albumin (HSA) by competing compounds has been followed using fluorescence spectroscopy, and has therefore been used to assign a primary binding site for these compounds in the presence and absence of fatty acids. The crystal structures have also been solved for three compounds: a matched pair of carboxylic acids whose binding strength to HSA unexpectedly decreased as the lipophilicity increased; and a highly bound sulphonamide that appeared not to displace the probes in the displacement assay. The crystallography results support the findings from the fluorescence displacement assay. The results indicate that drug binding to subdomain IB might also be important location for certain compounds.
Asunto(s)
Preparaciones Farmacéuticas/química , Albúmina Sérica/química , Sitios de Unión , Cristalografía por Rayos X , Interacciones Farmacológicas , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Albúmina Sérica/metabolismo , Espectrometría de FluorescenciaRESUMEN
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.
RESUMEN
A number of molecular recognition features have been exploited in structure-based design of selective Cathepsin inhibitors.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Catepsinas/metabolismo , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.
Asunto(s)
Catepsina L/antagonistas & inhibidores , Catepsina L/química , Nitrilos/síntesis química , Animales , Cartílago Articular/patología , Catálisis , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Humanos , Ligandos , Ratones , Ratones Transgénicos , Nitrilos/química , Nitrilos/farmacología , Péptidos/síntesis química , Péptidos/farmacología , Proteínas/química , Relación Estructura-ActividadRESUMEN
The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against nonimmune cells, which limits its use for autoimmune diseases. An attractive alternative is to selectively inhibit the immune cell-specific immunoproteasome to deplete pathogenic immune cells and spare nonhematopoietic cells. Here, we disclose the development of highly subunit-selective immunoproteasome inhibitors using insights obtained from the first bona fide human immunoproteasome cocrystal structures. Evaluation of these inhibitors revealed that immunoproteasome-specific inhibition does not lead to immune cell death as anticipated and that targeting viability requires inhibition of both immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knockout experiments confirmed upregulation of the constitutive proteasome upon disruption of the immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition alone is not a suitable approach to deplete immune cells.
Asunto(s)
Diseño de Fármacos , Inmunidad Celular/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/síntesis química , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Humanos , Inmunidad Celular/fisiología , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Estructura Terciaria de ProteínaRESUMEN
New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation.IMPORTANCEPseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the function of MvfR domains that may benefit the optimization of the chemical, pharmacological, and safety properties of MvfR antagonist series.
Asunto(s)
Proteínas Bacterianas/química , Inhibidores Enzimáticos/química , Pseudomonas aeruginosa/enzimología , Factores de Virulencia/química , Proteínas Bacterianas/metabolismo , Bencimidazoles/química , Bencimidazoles/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Resonancia por Plasmón de Superficie , Factores de Virulencia/metabolismoRESUMEN
Aggregation of tau protein and spreading of tau aggregates are pivotal pathological processes in a range of neurological disorders. Accumulating evidence suggests that immunotherapy targeting tau may be a viable therapeutic strategy. We have previously described the isolation of antibody CBTAU-22.1 from the memory B-cell repertoire of healthy human donors. CBTAU-22.1 was shown to specifically bind a disease-associated phosphorylated epitope in the C-terminus of tau (Ser422) and to be able to inhibit the spreading of pathological tau aggregates from P301S spinal cord lysates in vitro, albeit with limited potency. Using a combination of rational design and random mutagenesis we have derived a variant antibody with improved affinity while maintaining the specificity of the parental antibody. This affinity improved antibody showed greatly enhanced potency in a cell-based immunodepletion assay using paired helical filaments (PHFs) derived from human Alzheimer's disease (AD) brain tissue. Moreover, the affinity improved antibody limits the in vitro aggregation propensity of full length tau species specifically phosphorylated at position 422 produced by employing a native chemical ligation approach. Together, these results indicate that in addition to being able to inhibit the spreading of pathological tau aggregates, the matured antibody can potentially also interfere with the nucleation of tau which is believed to be the first step of the pathogenic process. Finally, the functionality in a P301L transgenic mice co-injection model highlights the therapeutic potential of human antibody dmCBTAU-22.1.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Anticuerpos/farmacología , Encéfalo/metabolismo , Serina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Afinidad de Anticuerpos/efectos de los fármacos , Autopsia , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Epítopos/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Modelos Moleculares , Mutagénesis , Mutación/genética , Fosforilación/fisiología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/terapiaRESUMEN
Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.
Asunto(s)
Catepsina B/metabolismo , Descubrimiento de Drogas , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Humanos , Espacio Intracelular/metabolismo , Especificidad por SustratoRESUMEN
The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/farmacocinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piperazinas/farmacología , Piperazinas/farmacocinética , Piperidinas/farmacología , Trasplante Homólogo , Administración Oral , Animales , Disponibilidad Biológica , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/metabolismo , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/metabolismo , Inmunosupresores/farmacocinética , Inmunosupresores/farmacología , Ratones , Simulación del Acoplamiento Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Piperazinas/administración & dosificación , Piperazinas/metabolismo , Piperidinas/administración & dosificación , Piperidinas/metabolismo , Conformación ProteicaRESUMEN
Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5-51/VL4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5-51 and VL4-1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.
Asunto(s)
Linfocitos B/metabolismo , Inmunoglobulina G/farmacología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Adolescente , Adulto , Anciano , Especificidad de Anticuerpos , Linfocitos B/efectos de los fármacos , Cristalización , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Epítopos Inmunodominantes/metabolismo , Masculino , Microglía/metabolismo , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Agregado de Proteínas , Adulto JovenRESUMEN
A prevalent observation in high-throughput screening and drug discovery programs is the inhibition of protein function by small-molecule compound aggregation. Here, we present the X-ray structural description of aggregation-based inhibition of a protein-protein interaction involving tumor necrosis factor α (TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor (JNJ525) induces a quaternary structure switch of TNFα that inhibits the protein-protein interaction between TNFα and TNFα receptors. SPD-304 may employ a similar mechanism of inhibition.