RESUMEN
Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.
RESUMEN
Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.
Asunto(s)
Factor D del Complemento/antagonistas & inhibidores , Vía Alternativa del Complemento/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Factor D del Complemento/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Human interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that plays a critical role in the regulation of the immune response and the development of various inflammatory diseases. In this publication, we disclose our efforts toward the discovery of IL-1ß binders that interfere with IL-1ß signaling. To this end, several technologies were used in parallel, including fragment-based screening (FBS), DNA-encoded library (DEL) technology, peptide discovery platform (PDP), and virtual screening. The utilization of distinct technologies resulted in the identification of new chemical entities exploiting three different sites on IL-1ß, all of them also inhibiting the interaction with the IL-1R1 receptor. Moreover, we identified lysine 103 of IL-1ß as a target residue suitable for the development of covalent, low-molecular-weight IL-1ß antagonists.
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Interleucina-1beta , Humanos , Descubrimiento de Drogas , Interleucina-1beta/metabolismo , Ligandos , Receptores Tipo I de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , ADN/química , Biblioteca de GenesRESUMEN
WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple processes. It is also a prominent target for pharmacological inhibition in diseases such as cancer, aging, and neurodegenerative disorders. Interactions between WDR5 and various partners are essential for sustaining its function. Most drug discovery efforts center on the WIN (WDR5 interaction motif) site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe the discovery of novel WDR5 inhibitors for the other WBM (WDR5 binding motif) pocket on this scaffold protein, to disrupt WDR5 interaction with its binding partner MYC by high-throughput biochemical screening, subsequent molecule optimization, and biological assessment. These new WDR5 inhibitors provide useful probes for future investigations of WDR5 and an avenue for targeting WDR5 as a therapeutic strategy.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Neoplasias , Humanos , Unión Proteica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cromatina , Descubrimiento de DrogasRESUMEN
Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.
Asunto(s)
Citocinas , Delgadez , Humanos , Interleucina-1beta , Peso Molecular , Sitios de Unión , BiofisicaRESUMEN
The replication of flaviviruses requires the correct processing of their polyprotein by the viral NS3 protease (NS3pro). Essential for the activation of NS3pro is a 47-residue region of NS2B. Here we report the crystal structures of a dengue NS2B-NS3pro complex and a West Nile virus NS2B-NS3pro complex with a substrate-based inhibitor. These structures identify key residues for NS3pro substrate recognition and clarify the mechanism of NS3pro activation.
Asunto(s)
Virus del Dengue/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Sitios de Unión , Activación Enzimática , Enlace de Hidrógeno , Sustancias Macromoleculares , Modelos Moleculares , Oligopéptidos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismoRESUMEN
A novel strategy for the design of a fluorinated fragment library that takes into account the local environment of fluorine is described. The procedure, based on a fluorine fingerprints descriptor, and the criteria used in the design, selection, and construction of the library are presented. The library, named LEF (Local Environment of Fluorine), combined with (19)F NMR ligand-based screening experiments represents an efficient and sensitive approach for the initial fragment identification within a fragment-based drug discovery project and for probing the presence of fluorophilic protein environments. Proper setup of the method, according to described theoretical simulations, allows the detection of very weak-affinity ligands and the detection of multiple ligands present within the same tested mixture, thus capturing all the potential fragments interacting with the receptor. These NMR hits are then used in the FAXS experiments for the fragment optimization process and for the follow-up screening aimed at identifying other chemical scaffolds relevant for the binding to the receptor.
Asunto(s)
Flúor/análisis , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/análisis , Bibliotecas de Moléculas Pequeñas/análisis , Descubrimiento de Drogas , LigandosRESUMEN
In this work we explore the possibilities of using fragment-based screening data to prioritize compounds from a full HTS library, a method we call virtual fragment linking (VFL). The ability of VFL to identify compounds of nanomolar potency based on micromolar fragment binding data was tested on 75 target classes from the WOMBAT database and succeeded in 57 cases. Further, the method was demonstrated for seven drug targets from in-house screening programs that performed both FBS of 8800 fragments and screens of the full library. VFL captured between 28% and 67% of the hits (IC 50 < 10microM) in the top 5% of the ranked library for four of the targets (enrichment between 5-fold and 13-fold). Our findings lead us to conclude that proper coverage of chemical space by the fragment library is crucial for the VFL methodology to be successful in prioritizing HTS libraries from fragment-based screening data.
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Evaluación Preclínica de Medicamentos , Sistemas de Administración de Bases de Datos , Peso MolecularRESUMEN
BACE2 is a membrane-bound aspartic protease of the A1 family with a high level of sequence homology to BACE1. While BACE1 is involved in the generation of amyloid plaques in Alzheimer's disease by cleaving Abeta-peptides from the amyloid precursor protein, the physiological function of BACE2 is not well understood. BACE2 appears to be associated with the early onset of dementia in patients with Down's syndrome, and it has been shown to be highly expressed in breast cancers. Further, it may participate in the function of normal and abnormal processes of human muscle biology. Similar to other aspartic proteases, BACE2 is expressed as an inactive zymogen requiring the cleavage of its pro-sequence during the maturation process. We have produced mature BACE2 by expression of pro-BACE2 in Escherichia coli as inclusion bodies, followed by refolding and autocatalytic activation at pH 3.4. Using a C and N-terminally truncated BACE2 variant, we were able to crystallize and determine the crystal structure of mature BACE2 in complex with a hydroxyethylamine transition-state mimetic inhibitor at 3.1 angstroms resolution. The structure of BACE2 follows the general fold of A1 aspartic proteases. However, similar to BACE1, its C-terminal domain is significantly larger than that of the other family members. Furthermore, the structure of BACE2 reveals differences in the S3, S2, S1' and S2' active site substrate pockets as compared to BACE1, and allows, therefore, for a deeper understanding of the structural features that may facilitate the design of selective BACE1 or BACE2 inhibitors.
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Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/química , Inhibidores Enzimáticos/química , Etanolaminas/química , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión , Cristalización , Diseño de Fármacos , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Cuerpos de Inclusión/enzimología , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Misdirected catalytic activity of histone methyltransferase Dot1L is believed to be causative for a subset of highly aggressive acute leukemias. Targeting the catalytic domain of Dot1L represents a potential therapeutic approach for these leukemias. In the context of a comprehensive Dot1L hit finding strategy, a knowledge-based virtual screen of the Dot1L SAM binding pocket led to the discovery of 2, a non-nucleoside fragment mimicking key interactions of SAM bound to Dot1L. Fragment linking of 2 and 3, an induced back pocket binder identified in earlier studies, followed by careful ligand optimization led to the identification of 7, a highly potent, selective and structurally novel Dot1L inhibitor.
RESUMEN
Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.
Asunto(s)
Inhibidores de Proteasas/farmacología , Dominio Catalítico , Factor D del Complemento/química , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Inhibidores de Proteasas/químicaRESUMEN
The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.
Asunto(s)
Factor D del Complemento/antagonistas & inhibidores , Vía Alternativa del Complemento/efectos de los fármacos , Prolina/análogos & derivados , Prolina/farmacología , Administración Oral , Animales , Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Síndrome Hemolítico Urémico Atípico/inmunología , Factor D del Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/antagonistas & inhibidores , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Femenino , Haplorrinos , Humanos , Macaca fascicularis , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/inmunología , Masculino , Ratones , Prolina/administración & dosificación , Prolina/farmacocinéticaRESUMEN
We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1-P1') but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors--namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.
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Biología Computacional/métodos , Péptido Hidrolasas/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Teorema de Bayes , Simulación por Computador , Humanos , Oligopéptidos/química , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Ratas , Reproducibilidad de los Resultados , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when (13)C(beta) and (13)C' shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using (13)C(alpha) connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs.
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Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Butiratos/química , Hemiterpenos , Cetoácidos/química , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Proteínas/químicaRESUMEN
Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin. Nevertheless, several inhibitors including those targeting renin, BACE1 and gamma-secretase are in clinical or preclinical development, and some other aspartic proteases are discussed as potential drug target. The crystal structures of seven human aspartic proteases have now been solved and, together with a detailed kinetic understanding of their catalytic mechanism, this has greatly contributed to the design and discovery of novel inhibitors for this protease class. This review describes current aspartic protease drug targets and summarizes the drug discovery efforts in this field. In addition, it highlights recent developments which may lead to a new generation of aspartic protease inhibitors.
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Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/química , Diseño de Fármacos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Diseño Asistido por Computadora , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Molecular , Presenilinas/antagonistas & inhibidores , Presenilinas/química , Conformación Proteica , Renina/antagonistas & inhibidores , Renina/química , Relación Estructura-ActividadRESUMEN
To facilitate NMR spectroscopy studies of interactions with various ligands and potential inhibitors, we report the NMR backbone resonance assignments for the 26 kD human enzyme UCH-L3, a member of the ubiquitin C-hydrolase family of ubiquitin-specific cysteine proteases.
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Cisteína Endopeptidasas/química , Dominio Catalítico , Cisteína Endopeptidasas/genética , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ubiquitina TiolesterasaRESUMEN
1,4-Diazepane-2,5-diones (2) are found to be a new class of potent LFA-1 inhibitors. The synthesis, structure, and biological evaluation of these 1,4-diazepine-2,5-diones and related derivatives are described.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Cetonas/síntesis química , Antígeno-1 Asociado a Función de Linfocito/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Cetonas/farmacología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Unión Proteica/efectos de los fármacos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible alpha/beta heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the alpha(L) chain but also in the I-like domain of the beta(2) chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role.
Asunto(s)
Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/química , Sitio Alostérico , Sitios de Unión , Biotinilación , Adhesión Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Ácido Edético/farmacología , Ensayo de Inmunoadsorción Enzimática , Epítopos , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Leucocitos/metabolismo , Ligandos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The integrin alpha X beta 2 (CD11c/CD18, p150,95) binds ligands through the I domain of the alpha X subunit. Ligands include the complement factor fragment iC3b, a key component in the innate immune defense, which, together with the expression of alpha X beta 2 on dendritic cells and on other leukocytes, suggests a role in the immune response. We now report the structure of the alpha X I domain resolved at 1.65 A by x-ray crystallography. To analyze structural requirements for ligand binding we made a mutation in the alpha X I domain C-terminal helix, which increased the affinity for iC3b approximately 200-fold to 2.4 microM compared with the wild-type domain affinity of approximately 400 microM. Gel permeation chromatography supported a conformational change between the wild-type and mutated domains. Conservation of allosteric regulation in the alpha X I domain points to the functional importance of this phenomenon.
Asunto(s)
Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , Cartilla de ADN , Integrina alfaXbeta2/genética , Datos de Secuencia Molecular , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
The design, synthesis, and biological evaluation of 1,4-diazepane-2-ones as novel LFA-1 antagonists from a scaffold-based combinatorial library are described. Initial optimization of the library lead has resulted in high-affinity antagonists of the LFA-1/ICAM-1 interaction, such as compounds 18d and 18e with IC(50) values of 110 and 70 nM, respectively.