RESUMO
On average, an approved drug currently costs US$2-3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Simulação de Acoplamento Molecular/métodos , Software , Interface Usuário-Computador , Acesso à Informação , Automação/métodos , Automação/normas , Computação em Nuvem , Simulação por Computador , Bases de Dados de Compostos Químicos , Descoberta de Drogas/normas , Avaliação Pré-Clínica de Medicamentos/normas , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Simulação de Acoplamento Molecular/normas , Terapia de Alvo Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Reprodutibilidade dos Testes , Software/normas , TermodinâmicaRESUMO
BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.
Assuntos
Éteres Fenílicos/farmacologia , Proteína X Associada a bcl-2/química , Apoptose , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Éteres Fenílicos/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Active bandwidth and global quality factor are the two main metrics used to quantitatively compare the performance of TOCSY mixing sequences. Active bandwidth refers to the spectral region over which at least 50 % of the magnetization is transferred via a coupling. Global quality factor scores mixing sequences according to the worst-case transfer over a range of possible mixing times and chemical shifts. Both metrics reward high transfer efficiency away from the main diagonal of a two-dimensional spectrum. They can therefore be used to design mixing sequences that will function favorably in experiments. Here, we develop optimization methods tailored to these two metrics, including precise control of off-diagonal cross peak buildup rates. These methods produce square shaped transfer efficiency profiles, directly matching the desirable properties that the metrics are intended to measure. The optimization methods are analytical, rather than numerical. The two resultant shaped pulses have significantly higher active bandwidth and quality factor, respectively, than all other known sequences. They are therefore highly suitable for use in NMR spectroscopy. We include experimental verification of these improved waveforms on small molecule and protein samples.
Assuntos
Modelos Teóricos , Algoritmos , Espectroscopia de Ressonância Magnética/métodosRESUMO
Broadband homonuclear mixing pulses with low radiofrequency power are essential for NMR spectroscopy of proteins and small molecules, especially for emerging applications in high field NMR. We have analytically designed a mixing pulse with high bandwidth-to-power ratio, using our recently developed multi-frame method. Here, we compare the new pulse, NF4 (mixing in the fourth nutating frame), to the best currently available sequence, focusing on the low-power regime. We use simulations and experiments to compare the two pulses' relaxation properties and bandwidth, and demonstrate that NF4 has approximately 1.35 times higher bandwidth, with similar effective relaxation. Therefore, NF4 is a good choice for broadband homonuclear mixing, particularly when the available radiofrequency power is limited.
Assuntos
Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Análise de Sequência de Proteína , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética/métodos , Ondas de RádioRESUMO
We present a method for designing radio-frequency (RF) pulses for broadband or multi-band isotropic mixing at low power, suitable for protein NMR spectroscopy. These mixing pulses are designed analytically, rather than by numerical optimization, by repeatedly constructing new rotating frames of reference. We show how pulse parameters can be chosen frame-by-frame to systematically reduce the effective chemical shift bandwidth, but maintain most of the effective J-coupling strength. The effective Hartmann-Hahn mixing condition is then satisfied in a multi-rotating frame of reference. This design method yields multi-band and broadband mixing pulses at low RF power. In particular, the ratio of RF power to mixing bandwidth for these pulses is lower than for existing mixing pulses, such as DIPSI and FLOPSY. Carbon-carbon TOCSY experiments at low RF power support our theoretical analysis.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Humanos , Modelos Teóricos , Peptídeos/química , Proteínas Recombinantes/químicaRESUMO
Establishing robust structure-activity relationships (SARs) is key to successful drug discovery campaigns, yet it often remains elusive due to screening and hit validation artifacts (false positives and false negatives), which frequently result in unproductive downstream expenditures of time and resources. To address this issue, we developed an integrative biophysics-driven strategy that expedites hit-to-lead discovery, mitigates false positives/negatives and common hit validation errors, and provides a robust approach to obtaining accurate binding and affinity measurements. The advantage of this method is that it vastly improves the clarity and reproducibility for affinity-driven SAR by monitoring and eliminating confounding factors. We demonstrate the ease at which high-quality micromolar binders can be generated from the initial millimolar fragment screening hits against an "undruggable" protein target, HRas.
Assuntos
Descoberta de Drogas , Imageamento por Ressonância Magnética , Reprodutibilidade dos Testes , Espectroscopia de Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
In NMR spectroscopy, many specialized shaped pulses are available for broadband excitation, beyond the bandwidth of conventional high-powered hard pulses. These shaped pulses typically have long duration. However, long-duration pulses are unsuitable for spectra containing significant homonuclear couplings, such as polyfluorinated compounds in 19F NMR. J-coupling evolution during the excitation pulse leads to spectral artifacts and incorrect peak integrals. Here, we report an approach to optimal control pulse design which significantly reduces the pulse length required to excite large bandwidths of chemical shift frequencies. The target state phase is not chosen beforehand but is instead only constrained to be linearly dependent on offset frequency. The first-order phase of the target state is then treated as a free-variable, to be optimized at the same time as the RF waveform itself. The resulting spectra are easily phased using standard NMR processing software. We observe that the required pulse length is significantly shorter than for currently available in-phase excitation schemes. Spectral artifacts from homonuclear couplings are avoided. We also demonstrate that pure in-phase excitation can be obtained over the same bandwidth by appending two inversion pulses, at the expense of increased overall duration.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Algoritmos , Artefatos , Simulação por Computador , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por ComputadorRESUMO
Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN's catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN's cellular functions.
Assuntos
PTEN Fosfo-Hidrolase/química , Animais , Ciona intestinalis/química , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , FosforilaçãoRESUMO
Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution. However, resonance assignment remains challenging. The problem is to combine existing structural informational with sparse distance restraints and search for the most compatible assignment among the permutations. Prior classification of peaks as either from isoleucine, leucine, or valine reduces the search space by many orders of magnitude. However, this is hindered by overlapped leucine and valine frequencies. In contrast, the nearest-neighbor nuclei, coupled to the methyl carbons, resonate in distinct frequency bands. Here, we develop a framework to imprint additional information about passively coupled resonances onto the observed peaks. This depends on simultaneously orchestrating closely spaced bands of resonances along different magnetization trajectories, using principles from control theory. For methyl-NMR, the method is implemented as a modification to the standard fingerprint spectrum (the 2D-HMQC). The amino acid type is immediately apparent in the fingerprint spectrum. There is no additional relaxation loss or an increase in experimental time. The method is validated on biologically relevant proteins. The idea of generating new spectral information using passive, adjacent resonances is applicable to other contexts in NMR spectroscopy.
Assuntos
Espectroscopia de Ressonância Magnética , Aminoácidos/química , Simulação por Computador , Humanos , Proteínas Ligantes de Maltose/química , Metilação , Reprodutibilidade dos Testes , Streptococcus pyogenes/metabolismoRESUMO
Small molecules can self-assemble in aqueous solution into a wide range of nanoentity types and sizes (dimers, n-mers, micelles, colloids, etc.), each having their own unique properties. This has important consequences in the context of drug discovery including issues related to nonspecific binding, off-target effects, and false positives and negatives. Here, we demonstrate the use of the spin-spin relaxation Carr-Purcell-Meiboom-Gill NMR experiment, which is sensitive to molecular tumbling rates and can expose larger aggregate species that have slower rotational correlations. The strategy easily distinguishes lone-tumbling molecules versus nanoentities of various sizes. The technique is highly sensitive to chemical exchange between single-molecule and aggregate states and can therefore be used as a reporter when direct measurement of aggregates is not possible by NMR. Interestingly, we found differences in solution behavior for compounds within structurally related series, demonstrating structure-nanoentity relationships. This practical experiment is a valuable tool to support drug discovery efforts.
Assuntos
Nanopartículas/química , Bibliotecas de Moléculas Pequenas/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese químicaRESUMO
Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαß) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Proteínas de Escherichia coli/análise , Marcação por Isótopo/métodos , Proteínas Ligantes de Maltose/análise , Ácido Pirúvico/química , Escherichia coli , Espectroscopia de Ressonância MagnéticaRESUMO
The Bloch-Siegert shift is a phenomenon in NMR spectroscopy and atomic physics in which the observed resonance frequency is changed by the presence of an off-resonance applied field. In NMR, it occurs especially in the context of homonuclear decoupling. Here we develop a practical method for homonuclear decoupling that avoids inducing Bloch-Siegert shifts. This approach enables accurate observation of the resonance frequencies of decoupled nuclear spins. We apply this method to increase the resolution of the HNCA experiment. We also observe a doubling in sensitivity for a 30 kDa protein. We demonstrate the use of band-selective Cß decoupling to produce amino acid-specific line shapes, which are valuable for assigning resonances to the protein sequence. Finally, we assign the backbone of a 30 kDa protein, Human Carbonic Anhydrase II, using only HNCA experiments acquired with band-selective decoupling schemes, and instrument time of one week.
Assuntos
Espectroscopia de Ressonância Magnética , Modelos Teóricos , Aminoácidos/química , Isótopos de Carbono , Simulação por Computador , Humanos , Proteínas/química , Ondas de Rádio , Reprodutibilidade dos TestesRESUMO
Selective activation of peripheral cannabinoid CB1 receptors has the potential to become a valuable therapy for chronic pain conditions as long as central nervous system effects are attenuated. A new class of cannabinoid ligands was rationally designed from known aminoalkylindole agonists and showed good binding and functional activities at human CB1 and CB2 receptors. This has led to the discovery of a novel CB1/CB2 dual agonist, naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (13), which displays good oral bioavailability, potent antihyperalgesic activity in animal models, and limited brain penetration.
Assuntos
Analgésicos/síntese química , Encéfalo/metabolismo , Hiperalgesia/tratamento farmacológico , Naftalenos/síntese química , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Administração Oral , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Disponibilidade Biológica , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Humanos , Técnicas In Vitro , Microssomos Hepáticos/metabolismo , Naftalenos/farmacocinética , Naftalenos/farmacologia , Ensaio Radioligante , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
We present a numerical method for rapidly solving the Bloch equation for an arbitrary time-varying spin-1/2 Hamiltonian. The method relies on fast, vectorized computations such as summation and quaternion multiplication, rather than slow computations such as matrix exponentiation. A toggling frame is constructed in which the Hamiltonian is time-invariant, and therefore has a simple analytical solution. The key insight is that constructing this frame is faster than solving the system dynamics in the original frame. Rapidly solving the Bloch equations for an arbitrary Hamiltonian is particularly useful in the context of NMR optimal control. Optimal control theory can be used to design pulse shapes for a range of tasks in NMR spectroscopy. However, it requires multiple simulations of the Bloch equations at each stage of the algorithm, and for each relevant set of parameters (e.g. chemical shift frequencies). This is typically time consuming. We demonstrate that by working in an appropriate toggling frame, optimal control pulses can be generated much faster. We present a new alternative to the well-known GRAPE algorithm to continuously update the toggling-frame as the optimal pulse is generated, and demonstrate that this approach is extremely fast. The use and benefit of rapid optimal pulse generation is demonstrated for 19F fragment screening experiments.