RESUMEN
Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.
Asunto(s)
Autofagia , Proteínas Asociadas a Microtúbulos , Humanos , Animales , Proteínas Asociadas a Microtúbulos/metabolismo , Ligandos , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Autofagosomas/metabolismo , Mamíferos/metabolismoRESUMEN
New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.
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Anticuerpos Biespecíficos , Neoplasias Colorrectales , Inmunoglobulinas , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoglobulinas/inmunología , Ratones , Inestabilidad de Microsatélites , Linfocitos T/inmunologíaRESUMEN
Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.
RESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.
Asunto(s)
Asma/tratamiento farmacológico , Furanos/uso terapéutico , Purinas/uso terapéutico , Canal Catiónico TRPA1/antagonistas & inhibidores , Animales , Asma/inducido químicamente , Asma/complicaciones , Células CHO , Cricetulus , Furanos/síntesis química , Furanos/metabolismo , Cobayas , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Ligandos , Masculino , Estructura Molecular , Ovalbúmina , Oxadiazoles/síntesis química , Oxadiazoles/metabolismo , Oxadiazoles/uso terapéutico , Unión Proteica , Purinas/síntesis química , Purinas/metabolismo , Ratas Sprague-Dawley , Relación Estructura-Actividad , Canal Catiónico TRPA1/metabolismoRESUMEN
Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.
Asunto(s)
Asma/tratamiento farmacológico , Inflamación Neurogénica/tratamiento farmacológico , Dolor/tratamiento farmacológico , Prurito/tratamiento farmacológico , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Canal Catiónico TRPA1/antagonistas & inhibidores , Adolescente , Adulto , Animales , Estudios de Cohortes , Modelos Animales de Enfermedad , Perros , Método Doble Ciego , Femenino , Cobayas , Voluntarios Sanos , Humanos , Isotiocianatos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Dolor/inducido químicamente , Prurito/inducido químicamente , Ratas , Ratas Sprague-Dawley , Canal Catiónico TRPA1/deficiencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.
Asunto(s)
Citomegalovirus/química , Glicoproteínas de Membrana/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Microscopía por Crioelectrón , Citomegalovirus/fisiología , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Proteoglicanos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/química , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.
Asunto(s)
Dimensión del Dolor/métodos , Canal Catiónico TRPA1/agonistas , Canal Catiónico TRPA1/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Células HEK293 , Humanos , Ligandos , Masculino , Dimensión del Dolor/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Canal Catiónico TRPA1/químicaRESUMEN
Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.
Asunto(s)
Antígenos CD20/química , Rituximab/química , Antígenos CD20/inmunología , Proteínas del Sistema Complemento/inmunología , Microscopía por Crioelectrón , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Conformación Proteica , Multimerización de Proteína , Rituximab/inmunologíaRESUMEN
Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
Asunto(s)
Antibacterianos/clasificación , Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Biocatálisis/efectos de los fármacos , Productos Biológicos/clasificación , Productos Biológicos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Escherichia coli/enzimología , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/patogenicidad , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/patogenicidad , Lisina/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/química , Porinas , Unión Proteica , Dominios Proteicos , Serina Endopeptidasas , Especificidad por SustratoRESUMEN
Ubiquitin-specific protease 7 (USP7) deubiquitinase activity is controlled by a number of regulatory factors, including stimulation by intramolecular accessory domains. Alone, the USP7 catalytic domain (USP7cd) shows limited activity and apo USP7cd crystal structures reveal a disrupted catalytic triad. By contrast, ubiquitin-conjugated USP7cd structures demonstrate the canonical cysteine protease active-site geometry; however, the structural features of the USP7cd that stabilize the inactive conformation and the mechanism of transition between inactive and active states remain unclear. Here we use comparative structural analyses, molecular dynamics simulations, and in silico sequence re-engineering via directed sampling by RosettaDesign to identify key molecular determinants of USP7cd activation and successfully engineer USP7cd for improved activity. Full kinetic analysis and multiple X-ray crystal structures of our designs indicate that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate USP7cd activation.
Asunto(s)
Inhibidores Enzimáticos/química , Mutación , Peptidomiméticos/química , Peptidasa Específica de Ubiquitina 7/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Activación Enzimática , Inhibidores Enzimáticos/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación de Dinámica Molecular , Peptidomiméticos/síntesis química , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Especificidad por Sustrato , Termodinámica , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Peptidasa Específica de Ubiquitina 7/genética , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Aminopiridinas/química , Sitios de Unión , Dominio Catalítico , Línea Celular , Simulación por Computador , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Espectroscopía de Resonancia Magnética/métodos , Oxadiazoles/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
Asunto(s)
Aminopiridinas/química , Aminopiridinas/farmacología , Indazoles/química , Indazoles/farmacología , Fenoles/química , Fenoles/farmacología , Piridinas/química , Piridinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Ubiquitina/metabolismo , Animales , Unión Competitiva , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/química , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/deficiencia , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
The deubiquitinase Ubiquitin Specific Protease 7 (USP7) is part of the regulatory cascade of proteins that modulates the activity of the tumor suppressor protein p53. Deubiquitination of its target Murine Double Minute 2 (MDM2) leads to increased proteosomal degradation of p53. Consequently, USP7 has emerged as an attractive oncology target because its inhibition stabilizes p53, thereby promoting p53-dependent apoptosis in cancer cells. Here we report the backbone resonance assignment for the 40.5 kDa catalytic domain of USP7.
Asunto(s)
Dominio Catalítico , Resonancia Magnética Nuclear Biomolecular , Ubiquitina Tiolesterasa/química , Secuencia de Aminoácidos , Humanos , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs.
Asunto(s)
Ubiquitina Tiolesterasa/química , Ubiquitina/química , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Termodinámica , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
A series of 4-azaindole-containing p21-activated kinase-1 (PAK1) inhibitors was prepared with the goal of improving physicochemical properties relative to an indole starting point. Indole 1 represented an attractive, non-basic scaffold with good PAK1 affinity and cellular potency but was compromised by high lipophilicity (clogD=4.4). Azaindole 5 was designed as an indole surrogate with the goal of lowering logD and resulted in equipotent PAK1 inhibition with a 2-fold improvement in cellular potency over 1. Structure-activity relationship studies around 5 identified additional 4-azaindole analogs with superior PAK1 biochemical activity (Ki <10nM) and up to 24-fold selectivity for group I over group II PAKs. Compounds from this series showed enhanced permeability, improved aqueous solubility, and lower plasma protein binding over indole 1. The improvement in physicochemical properties translated to a 20-fold decrease in unbound clearance in mouse PK studies for azaindole 5 relative to indole 1.
Asunto(s)
Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Perros , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Células de Riñón Canino Madin Darby , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Quinasas p21 Activadas/metabolismoRESUMEN
The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.
Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirazoles/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Humanos , Ratones , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/farmacocinética , Pirazoles/farmacocinética , Ratas , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismoRESUMEN
To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases.
RESUMEN
Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Fragmentos de Inmunoglobulinas/farmacología , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/inmunología , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/inmunología , Sitios de Unión , Fragmentos de Inmunoglobulinas/química , Datos de Secuencia Molecular , Unión Proteica , Staphylococcus aureus/enzimologíaRESUMEN
Structure-based methods were used to design a potent and highly selective group II p21-activated kinase (PAK) inhibitor with a novel binding mode, compound 17. Hydrophobic interactions within a lipophilic pocket past the methionine gatekeeper of group II PAKs approached by these type I 1/2 binders were found to be important for improving potency. A structure-based hypothesis and strategy for achieving selectivity over group I PAKs, and the broad kinome, based on unique flexibility of this lipophilic pocket, is presented. A concentration-dependent decrease in tumor cell migration and invasion in two triple-negative breast cancer cell lines was observed with compound 17.
Asunto(s)
Alquinos/síntesis química , Antineoplásicos/síntesis química , Bencimidazoles/síntesis química , Pirimidinas/síntesis química , Quinasas p21 Activadas/antagonistas & inhibidores , Alquinos/química , Alquinos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Pirimidinas/química , Pirimidinas/farmacología , Relación Estructura-Actividad , Neoplasias de la Mama Triple Negativas , Quinasas p21 Activadas/químicaRESUMEN
A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepin class of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was identified that increased selectivity over PI3Kß. PI3Kß-sparing compound 27 (PI3Kß Ki,app/PI3Kα Ki,app=57) demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differential effects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new structure-based hypothesis for reducing inhibition of the PI3K ß isoform while maintaining activity against α, δ and γ isoforms is presented.