RESUMO
Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC50 of 72.25 nM. We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositol-requiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Proteínas de Ciclo Celular/química , Linhagem Celular , Endorribonucleases/metabolismo , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Modelos Moleculares , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/química , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The C-aryl-tetrahydropyran motif is prevalent in nature in a number of natural products with biological activity; however, this challenging architecture still requires novel synthetic approaches. We demonstrate the application of a stereoselective Heck redox-relay strategy for the synthesis of functionalized 2,6-trans-tetrahydropyrans in excellent selectivity in a single step from an enantiopure dihydropyranyl alcohol, proceeding through a novel exo-cyclic migration. The strategy has also been applied to the total synthesis of a trans-epimer of the natural product centrolobine in excellent yield and stereoselectivity.
RESUMO
We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.
Assuntos
Ácidos Borônicos/síntese química , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Domínio Catalítico , Desenho de Fármacos , Hepacivirus/enzimologia , Estrutura Molecular , Serina/química , Relação Estrutura-AtividadeRESUMO
One of the factors preventing the general application of free energy methods in rational drug design remains the lack of sufficient computational resources. Many nonequilibrium (NE) free energy methods, however, are easily made embarrassingly parallel in comparison to equilibrium methods and may be conveniently run on desktop computers using distributed computing software. In recent years, there has been a proliferation of NE methods, but the general applicability of these approaches has not been determined. In this study, a subset including only those NE methods which are easily parallelised were considered for examination, with a view to their application to the prediction of protein-ligand binding affinities. A number of test systems were examined, including harmonic oscillator (HO) systems and the calculation of relative free energies of hydration of water-methane. The latter system uses identical potentials to the protein ligand case and is therefore an appropriate model system on which methods may be tested. As well as investigating existing protocols, a replica exchange NE approach was developed, which was found to offer advantages over conventional methods. It was found that Rosenbluth-based approaches to optimizing the NE work values used in NE free energy estimates were not consistent in the improvements in accuracy achieved and that, given their computational cost, the simple approach of taking each work value in an unbiased way is to be preferred. Of the two free energy estimators examined, Bennett's acceptance ratio was the most consistent and is, therefore, to be preferred over the Jarzynski estimator. The recommended protocols may be run very efficiently within a distributed computing environment and are of similar accuracy and precision to equilibrium free energy methods.
Assuntos
Termodinâmica , Simulação por Computador , Ligantes , Metano/química , Oscilometria , Proteínas/química , Água/químicaRESUMO
Nonequilibrium (NE) free energy methods are embarrassingly parallel and may be very conveniently run on desktop computers using distributed computing software. In recent years there has been a proliferation of NE methods, but these approaches have barely, if at all, been used in the context of calculating protein-ligand binding free energies. In a recent study by these authors, different combinations of NE methods with various test systems were compared and protocols identified which yielded results as accurate as replica exchange thermodynamic integration (RETI). The NE approaches, however, lend themselves to extensive parallelization through the use of distributed computing. Here the best performing of those NE protocols, a replica exchange method using Bennett's acceptance ratio as the free energy estimator (RENE), is applied to two sets of congeneric inhibitors bound to neuraminidase and cyclooxygenase-2. These protein-ligand systems were originally studied with RETI, giving results to which NE and RENE simulations are compared. These NE calculations were carried out on a large, highly distributed group of low-performance desktop computers which are part of a Condor pool. RENE was found to produce results of a predictive quality at least as good as RETI in less than half the wall clock time. However, non-RE NE results were found to be far less predictive. In addition, the RENE method successfully identified a localized region of rapidly changing free energy gradients without the need for prior investigation. These results suggest that the RENE protocol is appropriate for use in the context of predicting protein-ligand binding free energies and that it can offer advantages over conventional, equilibrium approaches.
Assuntos
Proteínas/metabolismo , Ligantes , Neuraminidase/metabolismo , TermodinâmicaRESUMO
Aldose reductase is the first enzyme of the polyol pathway in which glucose is converted to fructose via sorbitol. The understanding of this key enzyme is important as it has been linked to some diabetes mellitus complications. The mechanism of the enzyme was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. It was found that depending on the protonation state of His110 the mechanism can be concerted or stepwise and the proton donor can be either Tyr48 or His110. These findings are different from the previous theoretical studies based on QM/MM calculations using either AM1 or HF/4-31G, in which the reduction is, respectively, a stepwise or one-step process. The QM/MM energy barriers for the reduction of d-glyceraldehyde were evaluated at a B3LYP/6-31G* level for both HIP and HIE protonation states of His110. These were, respectively, 6.5 ± 2.2 and 16.7 ± 1.0 kcal/mol, which makes only the HIE protonation state consistent with the experimental value of 14.8 kcal/mol derived from kinetics experiments and makes Tyr48 the most probable proton donor.
RESUMO
Over the last decade, a number of computational methods have been developed, which attempt to evaluate the thermodynamic properties of individual water molecules at the solute-solvent interface, in order to assess contributions to protein-ligand binding. In some cases, these tools tell us what we already know, e.g. that hydrophobic pockets prefer lipophilic substituents, and in other cases the methods only seem to add clarity when retrospectively applied. Hence we have grappled with how to utilize such approaches to understand non-intuitive results and to generate chemistry ideas that otherwise would not have been developed. Here we provide our perspective on these methods and describe how results have been interpreted and applied. We include examples from GSK and elsewhere that highlight how water methods have been (1) utilized retrospectively to explain non-intuitive structure- activity relationships and (2) applied prospectively for chemistry design. Finally, we discuss where this field of study could lead to maximal impact in drug discovery research.
Assuntos
Desenho de Fármacos , Água/química , Ligantes , Estrutura Molecular , Proteínas/química , TermodinâmicaRESUMO
An ability to organize and encapsulate multiple active proteins into defined objects and spaces at the nanoscale has potential applications in biotechnology, nanotechnology, and synthetic biology. Previously, we have described the design, assembly, and characterization of peptide-based self-assembled cages (SAGEs). These ≈100 nm particles comprise thousands of copies of de novo designed peptide-based hubs that array into a hexagonal network and close to give caged structures. Here, we show that, when fused to the designed peptides, various natural proteins can be co-assembled into SAGE particles. We call these constructs pSAGE for protein-SAGE. These particles tolerate the incorporation of multiple copies of folded proteins fused to either the N or the C termini of the hubs, which modeling indicates form the external and internal surfaces of the particles, respectively. Up to 15% of the hubs can be functionalized without compromising the integrity of the pSAGEs. This corresponds to hundreds of copies giving mM local concentrations of protein in the particles. Moreover, and illustrating the modularity of the SAGE system, we show that multiple different proteins can be assembled simultaneously into the same particle. As the peptide-protein fusions are made via recombinant expression of synthetic genes, we envisage that pSAGE systems could be developed modularly to actively encapsulate or to present a wide variety of functional proteins, allowing them to be developed as nanoreactors through the immobilization of enzyme cascades or as vehicles for presenting whole antigenic proteins as synthetic vaccine platforms.
Assuntos
Peptídeos/química , Proteínas/química , Biologia Sintética/métodos , Biotecnologia , Nanotecnologia/métodos , Dobramento de ProteínaRESUMO
Reversible Digitally Filtered Molecular Dynamics (RDFMD) is a method of amplifying or suppressing motions in a molecular dynamics simulation, through the application of a digital filter to the simulation velocities. RDFMD and its derivatives have been previously used to promote conformational motions in liquid-phase butane, the Syrian hamster prion protein, alanine dipeptide, and the pentapeptide, YPGDV. The RDFMD method has associated with it a number of parameters that require specification to optimize the desired response. In this paper methods for the systematic analysis of these parameters are presented and applied to YPGDV with the specific emphasis of ensuring a gentle and progressive method that produces maximum conformation change from the energy put into the system. The portability of the new parameter set is then shown with an application to the M20 loop of E-coli dihydrofolate reductase. A conformational change is induced from a closed to an open structure similar to that seen in the DHFR-NADP(+) complex.