RESUMO
Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.
Assuntos
Cisteína , Desenho de Fármacos , Bibliotecas de Moléculas Pequenas , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Cisteína/química , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Relação Estrutura-Atividade , Modelos Moleculares , Antígenos de Histocompatibilidade MenorRESUMO
Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.
RESUMO
Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.
RESUMO
The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Acriflavina , Animais , Antivirais/química , Antivirais/farmacologia , Humanos , Camundongos , Simulação de Acoplamento Molecular , PandemiasRESUMO
Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.
Assuntos
Peptídeo Hidrolases/metabolismo , Periodontite/microbiologia , Inibidores de Proteases/farmacologia , Tannerella forsythia/enzimologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Hidrolases/efeitos dos fármacos , Inibidores de Proteases/química , Tannerella forsythia/isolamento & purificação , TemperaturaRESUMO
Although the concepts of nonuniform sampling (NUSâââââââ) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago , it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection of closely spaced peaks (i.e., "resolution") or peaks of weak intensity (i.e., "sensitivity"). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the "Nonuniform Sampling Contest" (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform.
RESUMO
Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.
Assuntos
Lisina/química , Proteína NEDD8/química , Poliubiquitina/química , Processamento de Proteína Pós-Traducional , Ubiquitina/química , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Lisina/metabolismo , Modelos Moleculares , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. Video abstract.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Área Sob a Curva , Células COS , Chlorocebus aethiops , Simulação por Computador , Fluorescência , Hidrodinâmica , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The presence of an unpaired electron in paramagnetic molecules generates significant effects in NMR spectra, which can be exploited to provide restraints complementary to those used in standard structure-calculation protocols. NMR already occupies a central position in drug discovery for its use in fragment screening, structural biology and validation of ligand-target interactions. Paramagnetic restraints provide unique opportunities, for example, for more sensitive screening to identify weaker-binding fragments. A key application of paramagnetic NMR in drug discovery, however, is to provide new structural restraints in cases where crystallography proves intractable. This is particularly important at early stages in drug-discovery programs where crystal structures of weakly-binding fragments are difficult to obtain and crystallization artefacts are probable, but structural information about ligand poses is crucial to guide medicinal chemistry. Numerous applications show the value of paramagnetic restraints to filter computational docking poses and to generate interaction models. Paramagnetic relaxation enhancements (PREs) generate a distance-dependent effect, while pseudo-contact shift (PCS) restraints provide both distance and angular information. Here, we review strategies for introducing paramagnetic centers and discuss examples that illustrate the utility of paramagnetic restraints in drug discovery. Combined with standard approaches, such as chemical shift perturbation and NOE-derived distance information, paramagnetic NMR promises a valuable source of information for many challenging drug-discovery programs.
Assuntos
Descoberta de Drogas/métodos , Espectroscopia de Ressonância Magnética/métodos , Sítios de Ligação , Ligantes , Proteínas/química , Proteínas/metabolismo , Marcadores de SpinRESUMO
Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-ß-lactamases (MBLs) target the most widely used antibiotic class, the ß-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-ß-lactamase inhibitors, essential in the fight against antibiotic resistance.
Assuntos
beta-Lactamases , Antibacterianos/farmacologia , Inibidores de beta-Lactamases , beta-Lactamases/genética , beta-Lactamas , CarbapenêmicosRESUMO
G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using 19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the ß1-adrenergic receptor to agonist stimulation and coupling to a Gs-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed.
Assuntos
Imagem por Ressonância Magnética de Flúor-19 , Receptores Adrenérgicos beta 1/química , Receptores Acoplados a Proteínas G/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica , Receptores Adrenérgicos beta 1/isolamento & purificação , Receptores Adrenérgicos beta 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
We present a model-based method for estimation of relaxation parameters from time-domain NMR data specifically suitable for processing data in popular 2D phase-sensitive experiments. Our model is formulated in terms of commutative bicomplex algebra, which allows us to use the complete information available in an NMR signal acquired with principles of quadrature detection without disregarding any of its dimensions. Compared to the traditional intensity-analysis method, our model-based approach offers an important advantage for the analysis of overlapping peaks and is robust over a wide range of signal-to-noise ratios. We assess its performance with simulated experiments and then apply it for determination of [Formula: see text], [Formula: see text], and [Formula: see text] relaxation rates in datasets of a protein with more than 100 cross peaks.
Assuntos
Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Algoritmos , Espectroscopia de Ressonância Magnética/métodos , Modelos Teóricos , Ressonância Magnética Nuclear Biomolecular/métodos , Reprodutibilidade dos TestesRESUMO
Over recent years, nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful mechanistic tool for the investigation of G protein-coupled receptors (GPCRs). NMR provides insights which underpin the dynamic nature of these important receptors and reveals experimental evidence for a complex conformational energy landscape that is explored during receptor activation resulting in signalling. NMR studies have highlighted both the dynamic properties of different receptor states as well as the exchange pathways and intermediates formed during activation, extending the static view of GPCRs obtained from other techniques. NMR studies can be undertaken in realistic membrane-like phospholipid environments and an ever-increasing choice of labelling strategies provides comprehensive, receptor-wide information. Combined with other structural methods, NMR is contributing to our understanding of allosteric signal propagation and the interaction of GPCRs with intracellular binding partners (IBP), crucial to explaining cellular signalling.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Receptores Acoplados a Proteínas G/química , Animais , Humanos , Ligantes , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Sparse lipid fluorination enhances the lipids' 1H signal dispersion, enables clean molecular distinction by 19F NMR, and evinces micelle insertion of proteins via fluorine-induced signal shifts. We present a minimal fluorination scheme, and illustrate the concept on di-(4-fluoro)-heptanoylphosphatidylcholine micelles and solubilised seven-helix transmembrane pSRII protein.
Assuntos
Proteínas Arqueais/química , Carotenoides/química , Lipídeos/química , Ressonância Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Halogenação , Micelas , Modelos MolecularesRESUMO
A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and ß-arrestin. Using 13C methyl methionine NMR for the ß1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive µs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Anticorpos de Domínio Único/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animais , Cristalografia por Raios X , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Ligantes , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Células Sf9 , Anticorpos de Domínio Único/química , SpodopteraRESUMO
Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy.
Assuntos
Biomimética , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membranas Artificiais , Fosfolipídeos/química , Dimiristoilfosfatidilcolina/química , Concentração de Íons de Hidrogênio , Nanopartículas/química , Nanopartículas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Saposinas/química , Saposinas/metabolismoRESUMO
The Drosophila and plant (maize) functional counterparts of the abundant vertebrate chromosomal protein HMGB1 (HMG-D and ZmHMGB1, respectively) differ from HMGB1 in having a single HMG box, as well as basic and acidic flanking regions that vary greatly in length and charge. We show that despite these variations, HMG-D and ZmHMGB1 exist in dynamic assemblies in which the basic HMG boxes and linkers associate with their intrinsically disordered, predominantly acidic, tails in a manner analogous to that observed previously for HMGB1. The DNA-binding surfaces of the boxes and linkers are occluded in "auto-inhibited" forms of the protein, which are in equilibrium with transient, more open structures that are "binding-competent." This strongly suggests that the mechanism of auto-inhibition may be a general one. HMG-D and ZmHMGB1 differ from HMGB1 in having phosphorylation sites in their tail and linker regions. In both cases, in vitro phosphorylation of serine residues within the acidic tail stabilizes the assembled form, suggesting another level of regulation for interaction with DNA, chromatin, and other proteins that is not possible for the uniformly acidic (hence unphosphorylatable) tail of HMGB1.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Animais , Drosophila melanogaster , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Zea maysRESUMO
We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.
Assuntos
Detergentes/química , Lipídeos/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Micelas , Diacilglicerol Quinase/química , Difusão , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Fluorescência Verde/química , Halobacteriaceae/química , Espectroscopia de Ressonância Magnética/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas de Transporte de Monossacarídeos/química , Nanopartículas/química , Plasmídeos/metabolismo , Rodopsinas Sensoriais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Simportadores/químicaRESUMO
Central to structural studies of biomolecules are multidimensional experiments. These are lengthy to record due to the requirement to sample the full Nyquist grid. Time savings can be achieved through undersampling the indirectly-detected dimensions combined with non-Fourier Transform (FT) processing, provided the experimental signal-to-noise ratio is sufficient. Alternatively, resolution and signal-to-noise can be improved within a given experiment time. However, non-FT based reconstruction of undersampled spectra that encompass a wide signal dynamic range is strongly impeded by the non-linear behaviour of many methods, which further compromises the detection of weak peaks. Here we show, through an application to a larger α-helical membrane protein under crowded spectral conditions, the potential use of compressed sensing (CS) l (1)-norm minimization to reconstruct undersampled 3D NOESY spectra. Substantial signal overlap and low sensitivity make this a demanding application, which strongly benefits from the improvements in signal-to-noise and resolution per unit time achieved through the undersampling approach. The quality of the reconstructions is assessed under varying conditions. We show that the CS approach is robust to noise and, despite significant spectral overlap, is able to reconstruct high quality spectra from data sets recorded in far less than half the amount of time required for regular sampling.