RESUMEN
Clamp loaders load sliding clamps onto primer-template DNA. The structure of the E. coli clamp loader bound to DNA reveals the formation of an ATP-dependent spiral of ATPase domains that tracks only the template strand, allowing recognition of both RNA and DNA primers. Unlike hexameric helicases, in which DNA translocation requires distinct conformations of the ATPase domains, the clamp loader spiral is symmetric and is set up to trigger release upon DNA recognition. Specificity for primed DNA arises from blockage of the end of the primer and accommodation of the emerging template along a surface groove. A related structure reveals how the psi protein, essential for coupling the clamp loader to single-stranded DNA-binding protein (SSB), binds to the clamp loader. By stabilizing a conformation of the clamp loader that is consistent with the ATPase spiral observed upon DNA binding, psi binding promotes the clamp-loading activity of the complex.
Asunto(s)
Adenosina Trifosfato/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Catálisis , Cristalografía por Rayos X , ADN/metabolismo , ADN Polimerasa III/química , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , ARN/metabolismoRESUMEN
OBJECTIVES: Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease. METHODS: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker. RESULTS: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy. CONCLUSION: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Antiinflamatorios/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Antirreumáticos/farmacología , Línea Celular , Humanos , Ratas , Ratas Endogámicas Lew , SpodopteraRESUMEN
The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/química , Janus Quinasa 3/metabolismo , Inhibidores de Proteínas Quinasas , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/genética , Dominio Catalítico , Línea Celular , Humanos , Janus Quinasa 3/genética , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.
Asunto(s)
ADN , Descubrimiento de Drogas , Interleucina-17 , Bibliotecas de Moléculas Pequeñas , Interleucina-17/metabolismo , Interleucina-17/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , ADN/metabolismo , ADN/química , Humanos , Animales , Relación Estructura-Actividad , Unión Proteica , RatonesRESUMEN
Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.
RESUMEN
BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
Asunto(s)
Diseño de Fármacos , Indazoles/química , Indazoles/farmacología , TYK2 Quinasa/antagonistas & inhibidores , TYK2 Quinasa/química , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Estabilidad de Enzimas/efectos de los fármacos , Humanos , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Ratones , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de Proteína , Proteolisis/efectos de los fármacos , Relación Estructura-Actividad , TYK2 Quinasa/aislamiento & purificaciónRESUMEN
Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.
Asunto(s)
Artritis Psoriásica , Espondiloartritis Axial , Interleucina-17 , Psoriasis , Citocinas , Diseño de Fármacos , Humanos , Interleucina-17/antagonistas & inhibidoresRESUMEN
Acidic mammalian chitinase (AMCase) is an enzyme that selectively degrades the biopolymer chitin. Several chitinase enzymes are utilized by mammals to hydrolyze chitin encountered by inhalation and ingestion. AMCase is distinct from other mammalian chitinases as its activity is retained in strongly acidic conditions (pH <2.0). AMCase expression is induced by antigen-induced mouse models of allergic lung inflammation. This protein has also been implicated in the pathogenesis of asthma although its precise role is poorly defined. We describe a novel way to express and purify active murine AMCase. This material retains properties observed in mouse bronchoalveolar lavage (BAL) fluid with regard to pH preference of activity and its inhibition by cyclic peptide inhibitors argifin and argadin. We found that chitinase in BAL from both antigen-challenged and control animals have similar properties in this regard. This strongly supports the notion the same enzyme (AMCase) gives rise to chitinase activity in both challenged and unchallenged animals. We also describe expression of active human AMCase. The methods described in this paper provide a reliable source of recombinant AMCase that can be utilized to expand understanding of AMCase's role in regulating allergic inflammation.
Asunto(s)
Quitinasas/genética , Quitinasas/metabolismo , Clonación Molecular/métodos , Secuencia de Aminoácidos , Animales , Líquido del Lavado Bronquioalveolar , Células COS , Línea Celular , Quitinasas/aislamiento & purificación , Chlorocebus aethiops , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Péptidos Cíclicos/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Interleucina-23/inmunología , Psoriasis/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Quimiocina CXCL1/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Humanos , Interleucina-17/administración & dosificación , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucina-23/administración & dosificación , Interleucina-23/metabolismo , Masculino , Ratones , Ratones Noqueados , Psoriasis/patología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Transducción de Señal/inmunología , Piel/inmunología , Piel/patologíaRESUMEN
BACKGROUND: Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA. RESULTS: We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 A resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved. CONCLUSION: Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.
Asunto(s)
ADN/química , Antígeno Nuclear de Célula en Proliferación/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cristalografía por Rayos X , ADN/fisiología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/fisiología , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Human immunoglobulin G1 (IgG1) contains 12 domains, and each has an intrachain disulfide bond that connects the two layers of antiparallel beta-sheets. These intrachain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with [(12)C]iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alkylation with [(13)C]iodoacetic acid. The percentage of modification by [(12)C]iodoacetic acid of each cysteine residue was calculated using mass spectra of the cysteine-containing tryptic peptides and used to follow the unfolding of each domain. It demonstrated that the CH2 domain was the least stable domain of the antibody, whereas the CH3 domain was the most stable domain of the antibody. Other domains showed intermediate resistance to the denaturant concentration, similar to the overall unfolding transition monitored by the intrinsic tryptophan fluorescence wavelength shift.
Asunto(s)
Anticuerpos Monoclonales/química , Espectrometría de Masas/métodos , Alquilación , Anticuerpos Monoclonales/genética , Cromatografía Líquida de Alta Presión , Disulfuros/química , Guanidina/química , Humanos , Inmunoglobulina G/química , Ácido Yodoacético/química , Oxidación-Reducción , Desnaturalización Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Sliding clamps are loaded onto DNA by ATP-dependent clamp loader complexes. A recent crystal structure of a clamp loader-clamp complex suggested an unexpected mechanism for DNA recognition, in which the ATPase subunits of the loader spiral around primed DNA. We report the results of fluorescence-based assays that probe the mechanism of the Escherichia coli clamp loader and show that conserved residues clustered within the inner surface of the modeled clamp loader spiral are critical for DNA recognition, DNA-dependent ATPase activity and clamp release. Duplex DNA with a 5'-overhang single-stranded region (corresponding to correctly primed DNA) stimulates clamp release, as does blunt-ended duplex DNA, whereas duplex DNA with a 3' overhang and single-stranded DNA are ineffective. These results provide evidence for the recognition of DNA within an inner chamber formed by the spiral organization of the ATPase domains of the clamp loader.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Escherichia coli/enzimología , Secuencia de Bases , Sitios de Unión , ADN/química , ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Especificidad por SustratoRESUMEN
Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of â¼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Inflamación/metabolismo , Interleucina-17/metabolismo , Queratinocitos/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Homeostasis , Humanos , Queratinocitos/citología , ARN Interferente Pequeño/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes the formation of prostaglandin E2 (PGE2) from the endoperoxide prostaglandin H2 (PGH2). Expression of this enzyme is induced during the inflammatory response, and mouse knockout experiments suggest it may be an attractive target for antiarthritic therapies. Assaying the activity of this enzyme in vitro is challenging because of the unstable nature of the PGH2 substrate. Here, the authors present an mPGES-1 activity assay suitable for characterization of enzyme preparations and for determining the potency of inhibitor compounds. This plate-based competition assay uses homogenous time-resolved fluorescence to measure PGE2 produced by the enzyme. The assay is insensitive to DMSO concentration up to 10% and does not require extensive washes after the initial enzyme reaction is concluded, making it a simple and convenient way to assess mPGES-1 inhibition.
Asunto(s)
Oxidorreductasas Intramoleculares/metabolismo , Microsomas/metabolismo , Espectrometría de Fluorescencia/métodos , Animales , Artritis/tratamiento farmacológico , Baculoviridae/metabolismo , Unión Competitiva , Regulación Enzimológica de la Expresión Génica , Humanos , Inflamación , Concentración 50 Inhibidora , Insectos , Microsomas/enzimología , Peróxidos/metabolismo , Prostaglandina H2/metabolismo , Prostaglandina-E Sintasas , Factores de TiempoRESUMEN
In vitro analysis of covalent inhibitors requires special consideration, due to the time-dependent and typically irreversible nature of their target interaction. While many analyses are reported for the characterization of a final candidate, it is less clear which are most useful in the lead optimization phase of drug discovery. In the context of identifying covalent inhibitors of Bruton's tyrosine kinase (BTK), we evaluated multiple techniques for characterizing covalent inhibitors. Several methods qualitatively support the covalent mechanism of action or support a particular aspect of interaction but were not otherwise informative to differentiate inhibitors. These include the time dependence of IC50, substrate competition, mass spectrometry, and recovery of function after inhibitor removal at the biochemical and cellular level. A change in IC50 upon mutation of the targeted BTK C481 nucleophile or upon removal of the electrophilic moiety of the inhibitor was not always a reliable indicator of covalent inhibition. Determination of kinact and KI provides a quantitative description of covalent interactions but was challenging at scale and frequently failed to provide more than the ratio of the two values, kinact/KI. Overall, a combination of approaches is required to assess time-dependent, covalent, and irreversible inhibitors in a manner suitable to reliably advance drug candidates.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Agammaglobulinemia Tirosina Quinasa/química , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Activación Enzimática/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Espectrometría de Masas , Inhibidores de Proteínas Quinasas/química , Proteínas RecombinantesRESUMEN
BACKGROUND: Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously. RESULTS: We have determined the crystal structure of the dimeric beta subunit of the DNA polymerase III holoenzyme of Streptococcus pyogenes. The sliding clamp from this Gram-positive organism forms a ring-shaped dimeric assembly that is similar in overall structure to that of the sliding clamps from Gram-negative bacteria, bacteriophage T4, eukaryotes and archaea. The dimer has overall dimensions of approximately 90 A x approximately 70 A x approximately 25 A with a central chamber that is large enough to accommodate duplex DNA. In comparison to the circular shape of other assemblies, the S. pyogenes clamp adopts a more elliptical structure. CONCLUSION: The sequences of sliding clamps from S. pyogenes and E. coli are only 23% identical, making the generation of structural models for the S. pyogenes clamp difficult in the absence of direct experimental information. Our structure of the S. pyogenes beta subunit completes the catalog of clamp structures from all the major sequence grouping of sliding clamps. The more elliptical rather than circular structure of the S. pyogenes clamp implies that the topological nature of encircling DNA, rather than a precise geometric shape, is the most conserved aspect for this family of proteins.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Bacterias Grampositivas/metabolismo , Secuencia de Aminoácidos , Bacteriófago T4/metabolismo , Cristalografía por Rayos X , ADN/química , Dimerización , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Streptococcus pyogenes/metabolismoRESUMEN
Clamp loaders are heteropentameric ATPase assemblies that load sliding clamps onto DNA and are critical for processive DNA replication. The DNA targets for clamp loading are double-stranded/single-stranded junctions with recessed 3' ends (primer-template junctions). Here, we briefly review the crystal structures of clamp loader complexes and the insights they have provided into the mechanism of the clamp loading process.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Adenosina Trifosfato/metabolismo , ADN/metabolismo , ADN Polimerasa III/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Sliding clamps are ring-shaped proteins that tether DNA polymerases to their templates during processive DNA replication. The action of ATP-dependent clamp loader complexes is required to open the circular clamps and to load them onto DNA. The crystal structure of the pentameric clamp loader complex from Escherichia coli (the gamma complex), determined in the absence of nucleotides, revealed a highly asymmetric and extended form of the clamp loader. Consideration of this structure suggested that a compact and more symmetrical inactive form may predominate in solution in the absence of crystal packing forces. This model has the N-terminal domains of the delta and delta' subunits of the clamp loader close to each other in the inactive state, with the clamp loader opening in a crab-claw-like fashion upon ATP-binding. We have used fluorescence resonance energy transfer (FRET) to investigate the structural changes in the E.coli clamp loader complex that result from ATP-binding and interactions between the clamp loader and the beta clamp. FRET measurements using fluorophores placed in the N-terminal domains of the delta and delta' subunits indicate that the distances between these subunits in solution are consistent with the previously crystallized extended form of the clamp loader. Furthermore, the addition of nucleotide and clamp to the labeled clamp loader does not appreciably alter these FRET distances. Our results suggest that the changes that occur in the relative positioning of the delta and delta' subunits when ATP binds to and activates the complex are subtle, and that crab-claw-like movements are not a significant component of the clamp loader mechanism.
Asunto(s)
ADN Polimerasa III/química , Proteínas de Unión al ADN/química , Adenosina Trifosfato , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Colorantes Fluorescentes , Sustancias Macromoleculares , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Soluciones , Espectrometría de Fluorescencia/métodosRESUMEN
Development of inhibitor compounds selective against undesirable targets is critical in drug discovery. Selectivity ratios for candidate compounds are evaluated by dividing potencies from two assays assessing the off-target and target. Because all potency measurements have underlying uncertainty, understanding error propagation is essential to interpreting selectivity data. Assay noise introduces ambiguity in the statistical significance of selectivity ratios, particularly at low replicate numbers when compounds are often prioritized for subsequent testing. The ability to differentiate potency results for any pair of compounds in one assay is evaluated using a metric called minimum significant ratio (MSR). Potency results of one compound tested in a pair of assays can be differentiated by the minimum significant selectivity ratio (MSSR). To differentiate selectivity ratios for any pair of compounds, we extend this concept by proposing two new parameters called the minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR). Importantly, these tools can be used after a single selectivity measurement. We describe these methods and illustrate their usefulness using structure-activity relationship data from a Janus kinase inhibitor project, in which these tools informed a cogent retesting strategy and enabled rapid and objective decision making.
Asunto(s)
Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Quinasas Janus/antagonistas & inhibidores , Preparaciones Farmacéuticas/análisis , Fenómenos Fisiológicos Celulares , Interpretación Estadística de Datos , Inhibidores Enzimáticos/química , Quinasas Janus/metabolismo , Relación Estructura-ActividadRESUMEN
Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.