RESUMEN
Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.
Asunto(s)
Butiratos/farmacología , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , PPAR alfa/agonistas , Compuestos de Fenilurea/farmacología , Butiratos/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Ligandos , Modelos Moleculares , Mutación , Compuestos de Fenilurea/metabolismo , Conformación Proteica/efectos de los fármacosRESUMEN
The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteína Disulfuro Reductasa (Glutatión)/química , Animales , Sitios de Unión , Burkholderia pseudomallei/enzimología , Burkholderia pseudomallei/patogenicidad , Dominio Catalítico , Ligandos , Ratones , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Proteína Disulfuro Reductasa (Glutatión)/genética , Relación Estructura-Actividad Cuantitativa , Proteínas Recombinantes , Bibliotecas de Moléculas Pequeñas/química , Solubilidad , Tiazoles/químicaRESUMEN
Drugs in the chemical space beyond the rule of 5 (bRo5) can modulate targets with difficult binding sites while retaining cell permeability and oral absorption. Reviewing the syntheses of bRo5 drugs approved since 1990 highlights synthetic chemistry's contribution to drug discovery in this space. Initially, bRo5 drugs were mainly natural products and semi-synthetic derivatives. Later, peptidomimetics and de novo designed compounds, that include up to seven chiral centres and macrocyclic rings became dominant. These drugs are prepared by total synthesis, sometimes by routes of more than 25â steps with stereocentres originating from the chiral pool, or being installed by chiral induction or enzymatic resolution. Interestingly, ring-closing metathesis proved to be the method of choice for macrocyclisation in hepatitisâ C virus protease inhibitors. We conclude that structural simplification, planning of synthetic routes regarding incorporation of stereocentres and macrocyclisation, as well as incorporation of structural knowledge and consideration of chameleonic properties in design, should facilitate drug discovery in bRo5 space.
Asunto(s)
Descubrimiento de Drogas , Preparaciones Farmacéuticas/síntesis química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Hepacivirus/enzimología , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/metabolismo , Peptidomiméticos , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against Neisseria-specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among Neisseria spp. and that this enzyme is essential for survival of N. gonorrhoeae DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of Neisseria DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the Neisseria n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Disulfuros/metabolismo , Neisseria gonorrhoeae/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Disulfuros/química , Modelos Moleculares , Oxidación-Reducción , Dominios ProteicosRESUMEN
Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.
Asunto(s)
Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacocinética , Células CACO-2 , Humanos , Estructura Molecular , Permeabilidad , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met εCH3 assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-13C,15N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.
Asunto(s)
Ligandos , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Sitios de Unión , Marcaje Isotópico , Espectroscopía de Resonancia Magnética/métodos , Metales/química , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica , Protones , SolubilidadRESUMEN
The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Antibacterianos/química , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
Although multiple approaches for characterizing protein-ligand interactions are available in target-based drug discovery, their throughput for determining selectivity is quite limited. Herein, we describe the application of native mass spectrometry for rapid, multiplexed screening of the selectivity of eight small-molecule ligands for five fatty acid-binding protein isoforms. Using high-resolution mass spectrometry, we were able to identify and quantify up to 20 different protein species in a single spectrum. We show that selectivity profiles generated by native mass spectrometry are in good agreement with those of traditional solution-phase techniques such as isothermal titration calorimetry and fluorescence polarization. Furthermore, we propose strategies for effective investigation of selectivity by native mass spectrometry, thus highlighting the potential of this technique to be used as an orthogonal method to traditional biophysical approaches for rapid, multiplexed screening of protein-ligand complexes.
RESUMEN
Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Cetonas , Escherichia coli/enzimología , Escherichia coli/efectos de los fármacos , Cetonas/química , Cetonas/farmacología , Cetonas/síntesis química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Especificidad por Sustrato , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis químicaRESUMEN
Fragment-based drug design relies heavily on structural information for the elaboration and optimisation of hits. The ability to identify neighbouring binding hot spots, energetically favourable interactions and conserved binding motifs in protein structures through X-ray crystallography can inform the evolution of fragments into lead-like compounds through structure-based design. The composition of fragment libraries can be designed and curated to fit this purpose and herein, we describe and compare screening libraries containing compounds comprising between 2 and 18 heavy atoms. We evaluate the properties of the compounds in these libraries and assess their ability to probe protein surfaces for binding hot spots.
RESUMEN
The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes.
Asunto(s)
Diseño de Fármacos , Proteínas , Proteínas/metabolismo , Relación Estructura-Actividad , Dominios Proteicos , LigandosRESUMEN
Structures of protein-ligand complexes provide critical information for drug design. Most protein-ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein-ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein-ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein-ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein-ligand complexes.
Asunto(s)
Proteínas , Fenómenos Biofísicos , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas/químicaRESUMEN
DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V.â cholerae DsbA (VcDsbA) compared to E.â coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446±10â µM. The same benzimidazole compound has â¼8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD >3.5â mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.
Asunto(s)
Proteínas de Escherichia coli , Vibrio cholerae , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Bencimidazoles , Cristalografía por Rayos X , Escherichia coli , Humanos , Ligandos , Proteína Disulfuro IsomerasasRESUMEN
A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Conformación Molecular , Bibliotecas de Moléculas Pequeñas/farmacología , SolubilidadRESUMEN
Targets that have large and groove-shaped binding sites, such as cyclophilin, are difficult to drug with small molecules. Macrocycles of natural product origin can be ideal starting points for such targets as illustrated by the transformation of sanglifehrin A into an orally bioavailable potential candidate drug. Optimization benefits from development of convergent, modular synthetic routes in combination with structure and property based methods for lead optimization.
Asunto(s)
Ciclofilinas/metabolismo , Descubrimiento de Drogas , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Terapia Molecular Dirigida , Ciclofilinas/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Recent years have seen a dramatic increase in the number of drugs approved in chemical space outside of Lipinski's rule of 5, that is in what has been termed beyond rule of 5 (bRo5) space. The development of three major classes of oral drugs that treat HIV and HCV infections and the growing evidence that novel, difficult targets can be accessed has prompted research into understanding design of drugs displaying cell permeability, solubility and ultimately oral bioavailability in bRo5 space. Studies have found a consistent outer property limit for a reasonable chance of de novo designing oral bioavailability. In addition, several property-based guidelines, along with incorporation of chameleonic features, have emerged as strategies to aid design in bRo5 space. A more detailed understanding of the complex and environment dependent conformational landscape will likely be the focus of the next generation of guidelines allowing property predictions of ever more complex compounds. By pushing the boundaries of current orally designable chemical space we hope that discoveries will be made for fundamental science and also for discovery of novel therapeutics.
Asunto(s)
Descubrimiento de Drogas/métodos , Administración Oral , Animales , Disponibilidad Biológica , Humanos , Boca/metabolismo , Permeabilidad , SolubilidadRESUMEN
Herein we describe a method for the design, purchase, and assembly of a fragment-screening library from a list of commercially available compounds. The computational tools used in assessment of compound properties as well as the workflow for compound selection are provided for reference as implemented in commercially available software that is free and accessible to most academic users. The workflow can be modified as necessary to generate a fit-for-purpose fragment library with the desired compound property profiles. An analytical process for assessing the quality, identity, and suitability of a purchased fragment for inclusion in a screening collection is described. Results from our in-house library are presented as an example of compound progression through this quality control process.
Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas/métodos , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Bases de Datos Farmacéuticas , Humanos , Espectroscopía de Protones por Resonancia Magnética/métodos , Flujo de TrabajoRESUMEN
Conformational flexibility has been proposed to significantly affect drug properties outside rule-of-5 (Ro5) chemical space. Here, we investigated the influence of dynamically exposed polarity on cell permeability and aqueous solubility for a structurally diverse set of drugs and clinical candidates far beyond the Ro5, all of which populated multiple distinct conformations as revealed by X-ray crystallography. Efflux-inhibited (passive) Caco-2 cell permeability correlated strongly with the compounds' minimum solvent-accessible 3D polar surface areas (PSA), whereas aqueous solubility depended less on the specific 3D conformation. Inspection of the crystal structures highlighted flexibly linked aromatic side chains and dynamically forming intramolecular hydrogen bonds as particularly effective in providing "chameleonic" properties that allow compounds to display both high cell permeability and aqueous solubility. These structural features, in combination with permeability predictions based on the correlation to solvent-accessible 3D PSA, should inspire drug design in the challenging chemical space far beyond the Ro5.
Asunto(s)
Descubrimiento de Drogas , Células CACO-2 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Molecular , Permeabilidad , Solubilidad , Propiedades de Superficie , Agua/químicaRESUMEN
To improve discovery of drugs for difficult targets, the opportunities of chemical space beyond the rule of 5 (bRo5) were examined by retrospective analysis of a comprehensive set of structures for complexes between drugs and clinical candidates and their targets. The analysis illustrates the potential of compounds far beyond rule of 5 space to modulate novel and difficult target classes that have large, flat, and groove-shaped binding sites. However, ligand efficiencies are significantly reduced for flat- and groove-shape binding sites, suggesting that adjustments of how to use such metrics are required. Ligands bRo5 appear to benefit from an appropriate balance between rigidity and flexibility to bind with sufficient affinity to their targets, with macrocycles and nonmacrocycles being found to have similar flexibility. However, macrocycles were more disk- and spherelike, which may contribute to their superior binding to flat sites, while rigidification of nonmacrocycles lead to rodlike ligands that bind well to groove-shaped binding sites. These insights should contribute to altering perceptions of what targets are considered "druggable" and provide support for drug design in beyond rule of 5 space.