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1.
Antioxidants (Basel) ; 13(6)2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38929122

RESUMO

Oxidation-reduction post-translational modifications (redox-PTMs) are chemical alterations to amino acids of proteins. Redox-PTMs participate in the regulation of protein conformation, localization and function, acting as signalling effectors that impact many essential biochemical processes in the cells. Crucially, the dysregulation of redox-PTMs of proteins has been implicated in the pathophysiology of numerous human diseases, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. This review aims to highlight the current gaps in knowledge in the field of redox-PTMs biology and to explore new methodological advances in proteomics and computational modelling that will pave the way for a better understanding of the role and therapeutic potential of redox-PTMs of proteins in neurodegenerative diseases. Here, we summarize the main types of redox-PTMs of proteins while providing examples of their occurrence in neurodegenerative diseases and an overview of the state-of-the-art methods used for their detection. We explore the potential of novel computational modelling approaches as essential tools to obtain insights into the precise role of redox-PTMs in regulating protein structure and function. We also discuss the complex crosstalk between various PTMs that occur in living cells. Finally, we argue that redox-PTMs of proteins could be used in the future as diagnosis and prognosis biomarkers for neurodegenerative diseases.

2.
Nucleic Acids Res ; 2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38813824

RESUMO

We present CGeNArate, a new model for molecular dynamics simulations of very long segments of B-DNA in the context of biotechnological or chromatin studies. The developed method uses a coarse-grained Hamiltonian with trajectories that are back-mapped to the atomistic resolution level with extreme accuracy by means of Machine Learning Approaches. The method is sequence-dependent and reproduces very well not only local, but also global physical properties of DNA. The efficiency of the method allows us to recover with a reduced computational effort high-quality atomic-resolution ensembles of segments containing many kilobases of DNA, entering into the gene range or even the entire DNA of certain cellular organelles.

3.
J Chem Inf Model ; 64(1): 265-275, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38113509

RESUMO

Excipients are included within protein biotherapeutic solution formulations to improve colloidal and conformational stability but are generally not designed for the specific purpose of preventing aggregation and improving cryoprotection in solution. In this work, we have explored the relationship between the structure and antiaggregation activity of excipients by utilizing coarse-grained molecular dynamics modeling of protein-excipient interaction. We have studied human serum albumin as a model protein, and we report the interaction of 41 excipients (polysorbates, fatty alcohol ethoxylates, fatty acid ethoxylates, phospholipids, glucosides, amino acids, and others) in terms of the reduction of solvent accessible surface area of aggregation-prone regions, proposed as a mechanism of aggregation prevention. Polyoxyethylene sorbitan had the greatest degree of interaction with aggregation-prone regions, decreasing the solvent accessible surface area of APRs by 20.7 nm2 (40.1%). Physicochemical descriptors generated by Mordred are employed to probe the structure-property relationship using partial least-squares regression. A leave-one-out cross-validated model had a root-mean-square error of prediction of 4.1 nm2 and a mean relative error of prediction of 0.077. Generally, longer molecules with a large number of alcohol-terminated PEG units tended to interact more, with qualitatively different protein interactions, wrapping around the protein. Shorter or less ethoxylated compounds tend to form hemimicellar clusters at the protein surface. We propose that an improved design would feature many short chains of 5 to 10 PEG units in many distinct branches and at least some hydrophobic content in the form of medium-length or greater aliphatic chains (i.e., six or more carbon atoms). The combination of molecular dynamics simulation and quantitative modeling is an important first step in an all-purpose protein-independent model for the computer-aided design of stabilizing excipients.


Assuntos
Produtos Biológicos , Excipientes , Humanos , Excipientes/química , Excipientes/metabolismo , Proteínas , Aminoácidos/química , Solventes
4.
J Med Chem ; 66(18): 12911-12930, 2023 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-37523859

RESUMO

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.


Assuntos
Receptores de Interleucina-8B , Sítio Alostérico , Ligantes , Sítios de Ligação , Regulação Alostérica
5.
Artigo em Inglês | MEDLINE | ID: mdl-36881023

RESUMO

Bacterial infections are increasingly problematic due to the rise of antimicrobial resistance. Consequently, the rational design of materials naturally resistant to biofilm formation is an important strategy for preventing medical device-associated infections. Machine learning (ML) is a powerful method to find useful patterns in complex data from a wide range of fields. Recent reports showed how ML can reveal strong relationships between bacterial adhesion and the physicochemical properties of polyacrylate libraries. These studies used robust and predictive nonlinear regression methods that had better quantitative prediction power than linear models. However, as nonlinear models' feature importance is a local rather than global property, these models were hard to interpret and provided limited insight into the molecular details of material-bacteria interactions. Here, we show that the use of interpretable mass spectral molecular ions and chemoinformatic descriptors and a linear binary classification model of attachment of three common nosocomial pathogens to a library of polyacrylates can provide improved guidance for the design of more effective pathogen-resistant coatings. Relevant features from each model were analyzed and correlated with easily interpretable chemoinformatic descriptors to derive a small set of rules that give model features tangible meaning that elucidate relationships between the structure and function. The results show that the attachment of Pseudomonas aeruginosa and Staphylococcus aureus can be robustly predicted by chemoinformatic descriptors, suggesting that the obtained models can predict the attachment response to polyacrylates to identify anti-attachment materials to synthesize and test in the future.

6.
FASEB J ; 36(11): e22576, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36183332

RESUMO

G protein-coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at these receptors offer further potential for exploitation. Intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g., G protein) and inhibit agonist responses noncompetitively. This potentially offers greater selectivity between receptor subtypes compared to classical orthosteric ligands. However, while examples of IAM ligands are well described, a more general methodology for assessing compound interactions at the IAM site is lacking. Here, fluorescent labeled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR-IAM site. In TR-FRET binding studies, unlabeled peptides derived from the Gαs subunit were first characterized for their ability to positively modulate agonist affinity at the ß2 -adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid Gαs peptide (TMR-Gαs19cha18, where cha = cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-Gαs19cha18 was recruited to Gs coupled ß2 -adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-Gαs19cha18 enabled direct assessment of the affinity of unlabeled ligands for ß2 -adrenoceptor IAM site. Thus, the NanoBRET platform using fluorescent-labeled G protein peptide mimetics offers novel potential for medium-throughput screens to identify IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.


Assuntos
Técnicas Biossensoriais , Receptores de Interleucina-8B , Regulação Alostérica , Sítio Alostérico , Aminoácidos , Proteínas de Ligação ao GTP/metabolismo , Ligantes , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina-8B/metabolismo
7.
Curr Issues Mol Biol ; 45(1): 175-196, 2022 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-36661500

RESUMO

Here, we describe the anticancer activity of our novel bis-triazoles MS47 and MS49, developed previously as G-quadruplex stabilizers, focusing specifically upon the human melanoma MDA-MB-435 cell line. At the National Cancer Institute (NCI), USA, bis-triazole MS47 (NCS 778438) was evaluated against a panel of sixty human cancer cell lines, and showed selective, distinct multi-log differential patterns of activity, with GI50 and LC50 values in the sub-micromolar range against human cancer cells. MS47 showed highly selective cytotoxicity towards human melanoma, ovarian, CNS and colon cancer cell lines; in contrast, the leukemia cell lines interestingly showed resistance to MS47 cytotoxic activity. Further studies revealed the potent cell growth inhibiting properties of MS47 and MS49 against the human melanoma MDA-MB-435 cell line, as verified by MTT assays; both ligands were more potent against cancer cells than MRC-5 fetal lung fibroblasts (SI > 9). Melanoma colony formation was significantly suppressed by MS47 and MS49, and time- and dose-dependent apoptosis induction was also observed. Furthermore, MS47 significantly arrested melanoma cells at the G0/G1 cell cycle phase. While the expression levels of Hsp90 protein in melanoma cells were significantly decreased by MS49, corroborating its binding to the G4-DNA promoter of the Hsp90 gene. Both ligands failed to induce senescence in the human melanoma cells after 72 h of treatment, corroborating their weak stabilization of the telomeric G4-DNA.

8.
J Chem Theory Comput ; 17(12): 7930-7937, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34852200

RESUMO

We describe a general approach to transforming molecular models between different levels of resolution, based on machine learning methods. The approach uses a matched set of models at both levels of resolution for training, but requires only the coordinates of their particles and no side information (e.g., templates for substructures, defined mappings, or molecular mechanics force fields). Once trained, the approach can transform further molecular models of the system between the two levels of resolution in either direction with equal facility.

9.
PLoS One ; 14(12): e0222055, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856175

RESUMO

Cruzain, a cysteine protease of Trypanosoma cruzi, is a validated target for the treatment of Chagas disease. Due to its high similarity in three-dimensional structure with human cathepsins and their sequence identity above 70% in the active site regions, identifying potent but selective cruzain inhibitors with low side effects on the host organism represents a significant challenge. Here a panel of nitrile ligands with varying potencies against cathepsin K, cathepsin L and cruzain, are studied by molecular dynamics simulations as both non-covalent and covalent complexes. Principal component analysis (PCA), identifies and quantifies patterns of ligand-induced conformational selection that enable the construction of a decision tree which can predict with high confidence a low-nanomolar inhibitor of each of three proteins, and determine the selectivity for one against others.


Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Domínio Catalítico , Catepsina L/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Doença de Chagas , Cisteína Endopeptidases/metabolismo , Cisteína Proteases/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Análise de Componente Principal/métodos , Ligação Proteica , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Trypanosoma cruzi/metabolismo
10.
Exp Parasitol ; 200: 84-91, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30954455

RESUMO

Cysteine proteases are involved in critical cell processes to the protozoa from Leishmania genus, and their inhibition is a therapeutic alternative to treat the disease. In this work, derivatives of dipeptidyl nitriles acting as reversible covalent inhibitors of cysteine proteases were studied as cytostatic agents. The proteolytic activity inside the living and lysed parasite cells was quantified using a selective substrate for cysteine proteases (Z-FR-MCA) from Leishmania amazonensis and L. infantum. The overall proteolytic activity of intact cells and even cell extracts was only marginally affected at high concentrations, with the observation of cytostatic activity and cell cycle arrest of promastigotes. However, the cytotoxic effects were only observed for infected J774 macrophages, which impaired further analysis of the amastigote infection. Therefore, the proteolytic inhibition in intact L. amazonensis and L. infantum promastigotes had no relationship to the cytostatic activity, which emphasizes that these dipeptidyl nitriles act through another mechanism of action.


Assuntos
Antiprotozoários/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Citostáticos/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Nitrilas/farmacologia , Animais , Antiprotozoários/química , Linhagem Celular , Inibidores de Cisteína Proteinase/química , Citostáticos/química , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Leishmania infantum/enzimologia , Leishmania mexicana/enzimologia , Macrófagos/efeitos dos fármacos , Camundongos , Nitrilas/química
11.
J Chem Theory Comput ; 15(4): 2587-2596, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30620585

RESUMO

CoCo ("complementary coordinates") is a method for ensemble enrichment based on principal component analysis (PCA) that was developed originally for the investigation of NMR data. Here we investigate the potential of the CoCo method, in combination with molecular dynamics simulations (CoCo-MD), to be used more generally for the enhanced sampling of conformational space. Using the alanine penta-peptide as a model system, we find that an iterative workflow, interleaving short multiple-walker MD simulations with long-range jumps through conformational space informed by CoCo analysis, can increase the rate of sampling of conformational space up to 10 times for the same computational effort (total number of MD timesteps). Combined with the reservoir-REMD method, free energies can be readily calculated. An alternative, approximate but fast and practically useful, alternative approach to unbiasing CoCo-MD generated data is also described. Applied to cyclosporine A, we can achieve far greater conformational sampling than has been reported previously, using a fraction of the computational resource. Simulations of the maltose binding protein, begun from the "open" state, effectively sample the "closed" conformation associated with ligand binding. The PCA-based approach means that optimal collective variables to enhance sampling need not be defined in advance by the user but are identified automatically and are adaptive, responding to the characteristics of the developing ensemble. In addition, the approach does not require any adaptations to the associated MD code and is compatible with any conventional MD package.

12.
Phys Chem Chem Phys ; 20(37): 24317-24328, 2018 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-30211406

RESUMO

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases.


Assuntos
Cisteína Endopeptidases/química , Cisteína Proteases/química , Inibidores de Cisteína Proteinase/química , Nitrilas/síntese química , Proteínas de Protozoários/química , Tripanossomicidas/química , Trypanosoma cruzi/enzimologia , Desenho de Fármacos , Simulação de Dinâmica Molecular , Ligação Proteica , Teoria Quântica , Termodinâmica
13.
Mol Pharm ; 15(10): 4654-4667, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30142269

RESUMO

The problem of predicting small molecule-polymer compatibility is relevant to many areas of chemistry and pharmaceutical science but particularly drug delivery. Computational methods based on Hildebrand and Hansen solubility parameters, and the estimation of the Flory-Huggins parameter, χ, have proliferated across the literature. Focusing on the need to develop amorphous solid dispersions to improve the bioavailability of poorly soluble drug candidates, an innovative, high-throughput 2D printing method has been employed to rapidly assess the compatibility of 54 drug-polymer pairings (nine drug compounds in six polymers). In this study, the first systematic assessment of the in silico methods for this application, neither the solubility parameter approach nor the calculated χ, correctly predicted drug-polymer compatibility. The theoretical limitations of the solubility parameter approach are discussed and used to explain why this approach is fundamentally unsuitable for predicting polymer-drug interactions. Examination of the original sources describing the method for calculating χ shows that only the enthalpic contributions to the term have been included, and the corrective entropic term is absent. The development and application of new in silico techniques, that consider all parts of the free energy of mixing, are needed in order to usefully predict small molecule-polymer compatibility and to realize the ambition of a drug-polymer screening method.


Assuntos
Polímeros/química , Estabilidade de Medicamentos , Simulação de Dinâmica Molecular , Preparações Farmacêuticas/química , Solubilidade , Termodinâmica
15.
PLoS One ; 12(5): e0177683, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28505196

RESUMO

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer's disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments-huprine and rhein-that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8-14, 154-169 and 307-318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable "floppy" pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors.


Assuntos
Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/química , Sítios de Ligação , Inibidores Enzimáticos/química , Conformação Molecular , Simulação de Dinâmica Molecular , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Ligação Proteica , Relação Estrutura-Atividade
16.
Mol Pharm ; 14(6): 2079-2087, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28502181

RESUMO

A miniaturized, high-throughput assay was optimized to screen polymer-drug solid dispersions using a 2-D Inkjet printer. By simply printing nanoliter amounts of polymer and drug solutions onto an inert surface, drug/polymer microdots of tunable composition were produced in an easily addressable microarray format. The amount of material printed for each dried spot ranged from 25 ng to 650 ng. These arrays were used to assess the stability of drug/polymer dispersions with respect to recrystallization, using polarized light microscopy. One array with a panel of 6 drugs formulated at different ratios with a poly(vinylpyrrolidone-vinyl acetate) (PVPVA) copolymer was developed to estimate a possible bulk (gram-scale) approximation threshold from the final printed nanoamount of formulation. Another array was printed at a fixed final amount of material to establish a literature comparison of one drug formulated with different commercial polymers for validation. This new approach may offer significant efficiency in pharmaceutical formulation screening, with each experiment in the nanomicro-array format requiring from 3 up to 6 orders of magnitude lower amounts of sample than conventional screening methods.


Assuntos
Composição de Medicamentos/métodos , Polímeros/química , Povidona/análogos & derivados , Portadores de Fármacos/química , Microscopia de Polarização , Povidona/química
17.
J Biomol Struct Dyn ; 35(12): 2604-2619, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27532213

RESUMO

G protein-coupled receptors (GPCRs) are proteins of pharmaceutical importance, with over 30% of all drugs in clinical use targeting them. Increasing numbers of X-ray crystal (XRC) structures of GPCRs offer a wealth of data relating to ligand binding. For the ß-adrenoceptors (ß-ARs), XRC structures are available for human ß2- and turkey ß1-subtypes, in complexes with a range of ligands. While these structures provide insight into the origins of ligand structure-activity relationships (SARs), questions remain. The ligands in all published complexed XRC structures lack extensive substitution, with no obvious way the ligand-binding site can accommodate ß1-AR-selective antagonists with extended side-chains para- to the common aryloxypropanolamine pharmacophore. Using standard computational docking tools with such ligands generally returns poses that fail to explain known SARs. Application of our Active Site Pressurisation modelling method to ß-AR XRC structures and homology models, however, reveals a dynamic area in the ligand-binding pocket that, through minor changes in amino acid side chain orientations, opens a fissure between transmembrane helices H4 and H5, exposing intra-membrane space. This fissure, which we term the "keyhole", is ideally located to accommodate extended moieties present in many high-affinity ß1-AR-selective ligands, allowing the rest of the ligand structure to adopt a canonical pose in the orthosteric binding site. We propose the keyhole may be a feature of both ß1- and ß2-ARs, but that subtle structural differences exist between the two, contributing to subtype-selectivity. This has consequences for the rational design of future generations of subtype-selective ligands for these therapeutically important targets.


Assuntos
Conformação Proteica , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 2/química , Receptores Acoplados a Proteínas G/química , Relação Estrutura-Atividade
18.
Nat Methods ; 13(1): 55-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26569599

RESUMO

We present parmbsc1, a force field for DNA atomistic simulation, which has been parameterized from high-level quantum mechanical data and tested for nearly 100 systems (representing a total simulation time of ∼ 140 µs) covering most of DNA structural space. Parmbsc1 provides high-quality results in diverse systems. Parameters and trajectories are available at http://mmb.irbbarcelona.org/ParmBSC1/.


Assuntos
DNA/química , Teoria Quântica
19.
Mol Pharmacol ; 87(1): 18-30, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25316768

RESUMO

ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate; Rigosertib, Estybon], a styryl benzylsulfone, is a phase III stage anticancer agent. This non-ATP competitive kinase inhibitor has multitargeted activity, promoting mitotic arrest and apoptosis. Extensive phase I/II studies with ON01910.Na, conducted in patients with solid tumors and hematologic cancers, demonstrate excellent efficacy. However, issues remain affecting its development. These include incomplete understanding of antitumor mechanisms, low oral bioavailability, and unpredictable pharmacokinetics. We have identified a novel (E)-styrylsulfonyl methylpyridine [(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide (TL-77)] which has shown improved oral bioavailability compared with ON01910.Na. Here, we present detailed cellular mechanisms of TL-77 in comparison with ON01910.Na. TL-77 displays potent growth inhibitory activity in vitro (GI50 < 1µM against HCT-116 cells), demonstrating 3- to 10-fold greater potency against tumor cell lines when compared with normal cells. Cell-cycle analyses reveal that TL-77 causes significant G2/M arrest in cancer cells, followed by the onset of apoptosis. In cell-free conditions, TL-77 potently inhibits tubulin polymerization. Mitotically arrested cells display multipolar spindles and misalignment of chromosomes, indicating that TL-77 interferes with mitotic spindle assembly in cancer cells. These effects are accompanied by induction of DNA damage, inhibition of Cdc25C phosphorylation [indicative of Plk1 inhibition], and downstream inhibition of cyclin B1. However, kinase assays failed to confirm inhibition of Plk1. Nonsignificant effects on phosphoinositide 3-kinase/Akt signal transduction were observed after TL-77 treatment. Analysis of apoptotic signaling pathways reveals that TL-77 downregulates expression of B-cell lymphoma 2 family proteins (Bid, Bcl-xl, and Mcl-1) and stimulates caspase activation. Taken together, TL-77 represents a promising anticancer agent worthy of further evaluation.


Assuntos
Antineoplásicos/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Neoplasias/patologia , Estirenos/farmacologia , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Antineoplásicos/síntese química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Células MCF-7 , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Estirenos/síntese química , Sulfonamidas/síntese química , Tubulina (Proteína)/metabolismo
20.
Molecules ; 19(7): 9134-59, 2014 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-24983862

RESUMO

The pathological characteristics of Alzheimer's Disease (AD) have been linked to the activity of three particular kinases--Glycogen Synthase Kinase 3ß (GSK3ß), Cyclin-Dependent Kinase 5 (CDK5) and Extracellular-signal Regulated Kinase 2 (ERK2). As a consequence, the design of selective, potent and drug-like inhibitors of these kinases is of particular interest. Structure-based design methods are well-established in the development of kinase inhibitors. However, progress in this field is limited by the difficulty in obtaining X-ray crystal structures suitable for drug design and by the inability of this method to resolve highly flexible regions of the protein that are crucial for ligand binding. To address this issue, we have undertaken a study of human protein kinases CDK5/p25, CDK5, ERK2 and GSK3ß using both conventional molecular dynamics (MD) and the new Active Site Pressurisation (ASP) methodology, to look for kinase-specific patterns of flexibility that could be leveraged for the design of selective inhibitors. ASP was used to examine the intrinsic flexibility of the ATP-binding pocket for CDK5/p25, CDK5 and GSK3ß where it is shown to be capable of inducing significant conformational changes when compared with X-ray crystal structures. The results from these experiments were used to quantify the dynamics of each protein, which supported the observations made from the conventional MD simulations. Additional information was also derived from the ASP simulations, including the shape of the ATP-binding site and the rigidity of the ATP-binding pocket. These observations may be exploited in the design of selective inhibitors of GSK3ß, CDK5 and ERK2.


Assuntos
Doença de Alzheimer/enzimologia , Proteínas Quinases/química , Trifosfato de Adenosina/química , Domínio Catalítico , Desenho de Fármacos , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Análise de Componente Principal , Ligação Proteica , Inibidores de Proteínas Quinases/química , Estrutura Secundária de Proteína
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