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1.
Int J Mol Sci ; 25(13)2024 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-38999982

RESUMEN

G protein-coupled receptor (GPCR) transmembrane protein family members play essential roles in physiology. Numerous pharmaceuticals target GPCRs, and many drug discovery programs utilize virtual screening (VS) against GPCR targets. Improvements in the accuracy of predicting new molecules that bind to and either activate or inhibit GPCR function would accelerate such drug discovery programs. This work addresses two significant research questions. First, do ligand interaction fingerprints provide a substantial advantage over automated methods of binding site selection for classical docking? Second, can the functional status of prospective screening candidates be predicted from ligand interaction fingerprints using a random forest classifier? Ligand interaction fingerprints were found to offer modest advantages in sampling accurate poses, but no substantial advantage in the final set of top-ranked poses after scoring, and, thus, were not used in the generation of the ligand-receptor complexes used to train and test the random forest classifier. A binary classifier which treated agonists, antagonists, and inverse agonists as active and all other ligands as inactive proved highly effective in ligand function prediction in an external test set of GPR31 and TAAR2 candidate ligands with a hit rate of 82.6% actual actives within the set of predicted actives.


Asunto(s)
Simulación del Acoplamiento Molecular , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Ligandos , Sitios de Unión , Descubrimiento de Drogas/métodos , Humanos , Unión Proteica
2.
J Appl Microbiol ; 134(8)2023 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-37481696

RESUMEN

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.


Asunto(s)
Staphylococcus aureus , Titanio , Animales , Titanio/farmacología , Titanio/química , Escherichia coli , Biopelículas , Antibacterianos/farmacología , Mamíferos
3.
Phys Chem Chem Phys ; 24(35): 21216-21222, 2022 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-36040138

RESUMEN

Trimethylamine N-oxide (TMAO) and urea are small organic biological molecules. While TMAO is known as a protective osmolyte that promotes the native form of biomolecules, urea is a denaturant. An understanding of the impact of TMAO and urea on water structure may aid in uncovering the molecular mechanisms that underlie this activity. Here we investigate binary solutions of TMAO-water, urea-water and ternary solutions of TMAO-urea-water using NMR spectroscopy at 300 K. An enhancement of the total hydrogen bonding in water was found upon the addition of TMAO and this effect was neutralised by a mole ratio of 1-part TMAO to 4-parts urea. Urea was found to have little effect on the strength of water's hydrogen bonding network and the dynamics of water molecules. Evidence was found for a weak interaction between TMAO and urea. Taken together, these results suggest that TMAO's function as a protective osmolyte, and its counteraction of urea, may be driven by the strength of its hydrogen bond interactions with water, and by a secondary reinforcement of water's own hydrogen bond network. They also suggest that the TMAO-urea complex forms through the donation of a hydrogen bond by urea.


Asunto(s)
Urea , Agua , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Metilaminas/química , Urea/química , Agua/química
4.
Mar Drugs ; 19(10)2021 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-34677455

RESUMEN

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P.aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.


Asunto(s)
Antibacterianos/farmacología , Materiales Biocompatibles/farmacología , Quitosano/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Animales , Antibacterianos/química , Vendajes , Materiales Biocompatibles/química , Biopelículas/efectos de los fármacos , Quitosano/química , Ácidos Grasos Monoinsaturados/química , Humanos , Ratones , Células 3T3 NIH/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Ingeniería de Tejidos , Cicatrización de Heridas/efectos de los fármacos
5.
Langmuir ; 36(34): 10091-10102, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32787024

RESUMEN

Herein, we report a novel approach that involves Pickering stabilization of micometer-sized liquid crystal (LC) droplets with biocompatible soft materials such as a whey protein microgel (WPM) to facilitate the analysis of analyte-induced configurational transition of the LC droplets. The WPM particles were able to irreversibly adsorb at the LC-water interface, and the resulting WPM-stabilized LC droplets possessed a remarkable stability against coalescence over time. Although the LC droplets were successfully protected by a continuous network of the WPM layer, the LC-water interface was still accessible for small molecules such as sodium dodecyl sulfate (SDS) that could diffuse through the meshes of the adsorbed WPM network or through the interfacial pores and induce an LC response. This approach was exploited to investigate the dynamic range of the WPM-stabilized LC droplet response to SDS. Nevertheless, the presence of the unadsorbed WPM in the aqueous medium reduced the access of SDS molecules to the LC droplets, thus suppressing the configuration transition. An improved LC response to SDS with a lower detection limit was achieved after washing off the unadsorbed WPM. Interestingly, the LC exhibited a detection limit as low as ∼0.85 mM for SDS within the initial WPM concentration ranging from 0.005 to 0.1 wt %. Furthermore, we demonstrate that the dose-response behavior was strongly influenced by the number of droplets exposed to the aqueous analytes and the type of surfactants such as anionic SDS, cationic dodecyltrimethylammonium bromide (DTAB), and nonionic tetra(ethylene glycol)monododecyl ether (C12E4). Thus, our results address key issues associated with the quantification of aqueous analytes and provide a promising colloidal platform toward the development of new classes of biocompatible LC droplet-based optical sensors.

6.
J Comput Aided Mol Des ; 34(10): 1027-1044, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32737667

RESUMEN

G protein-coupled receptors (GPCR) comprise the largest family of membrane proteins and are of considerable interest as targets for drug development. However, many GPCR structures remain unsolved. To address the structural ambiguity of these receptors, computational tools such as homology modeling and loop modeling are often employed to generate predictive receptor structures. Here we combined both methods to benchmark a protocol incorporating homology modeling based on a locally selected template and extracellular loop modeling that additionally evaluates the presence of template ligands during these modeling steps. Ligands were also docked using three docking methods and two pose selection methods to elucidate an optimal ligand pose selection method. Results suggest that local template-based homology models followed by loop modeling produce more accurate and predictive receptor models than models produced without loop modeling, with decreases in average receptor and ligand RMSD of 0.54 Å and 2.91 Å, respectively. Ligand docking results showcased the ability of MOE induced fit docking to produce ligand poses with atom root-mean-square deviation (RMSD) values at least 0.20 Å lower (on average) than the other two methods benchmarked in this study. In addition, pose selection methods (software-based scoring, ligand complementation) selected lower RMSD poses with MOE induced fit docking than either of the other methods (averaging at least 1.57 Å lower), indicating that MOE induced fit docking is most suited for docking into GPCR homology models in our hands. In addition, target receptor models produced with a template ligand present throughout the modeling process most often produced target ligand poses with RMSD values ≤ 4.5 Å and Tanimoto coefficients > 0.6 after selection based on ligand complementation than target receptor models produced in the absence of template ligands. Overall, the findings produced by this study support the use of local template homology modeling in combination with de novo ECL2 modeling in the presence of a ligand from the template crystal structure to generate GPCR models intended to study ligand binding interactions.


Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Programas Informáticos , Benchmarking , Humanos , Ligandos , Unión Proteica , Conformación Proteica
7.
J Comput Aided Mol Des ; 33(6): 573-595, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31123958

RESUMEN

G protein-coupled receptors (GPCR) are important drug discovery targets. Despite progress, many GPCR structures have not yet been solved. For these targets, comparative modeling is used in virtual ligand screening to prioritize experimental efforts. However, the structure of extracellular loop 2 (ECL2) is often poorly predicted. This is significant due to involvement of ECL2 in ligand binding for many Class A GPCR. Here we examine the performance of loop modeling protocols available in the Rosetta (cyclic coordinate descent [CCD], KIC with fragments [KICF] and next generation KIC [NGK]) and Molecular Operating Environment (MOE) software suites (de novo search). ECL2 from GPCR crystal structures served as the structure prediction targets and were divided into four sets depending on loop length. Results suggest that KICF and NGK sampled and scored more loop models with sub-angstrom and near-atomic accuracy than CCD or de novo search for loops of 24 or fewer residues. None of the methods were able to sample loop conformations with near-atomic accuracy for the longest targets ranging from 25 to 32 residues based on 1000 models generated. For these long loop targets, increased conformational sampling is necessary. The strongly conserved disulfide bond between Cys3.25 and Cys45.50 in ECL2 proved an effective filter. Setting an upper limit of 5.1 Å on the S-S distance improved the lowest RMSD model included in the top 10 scored structures in Groups 1-4 on average between 0.33 and 1.27 Å. Disulfide bond formation and geometry optimization of ECL2 provided an additional incremental benefit in structure quality.


Asunto(s)
Receptores Acoplados a Proteínas G/química , Animales , Bases de Datos de Proteínas , Humanos , Modelos Moleculares , Conformación Proteica , Programas Informáticos
8.
Mol Cell ; 39(3): 421-32, 2010 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-20705243

RESUMEN

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.


Asunto(s)
Adipocitos/metabolismo , Macrófagos/metabolismo , PPAR gamma/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Células 3T3-L1 , Animales , Diferenciación Celular/fisiología , Ratones , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , PPAR gamma/genética , Ácidos Fosfatidicos/genética , Fosfolipasa D/genética , Ratas , Transcripción Genética/fisiología
9.
Bioorg Med Chem ; 24(19): 4660-4674, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27544588

RESUMEN

Autotaxin (ATX) is a ubiquitous ectoenzyme that hydrolyzes lysophosphatidylcholine (LPC) to form the bioactive lipid mediator lysophosphatidic acid (LPA). LPA activates specific G-protein coupled receptors to elicit downstream effects leading to cellular motility, survival, and invasion. Through these pathways, upregulation of ATX is linked to diseases such as cancer and cardiovascular disease. Recent crystal structures confirm that the catalytic domain of ATX contains multiple binding regions including a polar active site, hydrophobic tunnel, and a hydrophobic pocket. This finding is consistent with the promiscuous nature of ATX hydrolysis of multiple and diverse substrates and prior investigations of inhibitor impacts on ATX enzyme kinetics. The current study used virtual screening methods to guide experimental identification and characterization of inhibitors targeting the hydrophobic region of ATX. An initially discovered inhibitor, GRI392104 (IC50 4µM) was used as a lead for synthetic optimization. In total twelve newly synthesized inhibitors of ATX were more potent than GRI392104 and were selective for ATX as they had no effect on other LPC-specific NPP family members or on LPA1-5 GPCR.


Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lisofosfatidilcolinas/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Hidrolasas Diéster Fosfóricas/química , Relación Estructura-Actividad Cuantitativa
10.
Biochim Biophys Acta ; 1831(1): 139-46, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23000541

RESUMEN

Sphingosine kinases 1 and 2 (SK1 and SK2) generate the bioactive lipid mediator sphingosine 1-phosphate and as such play a significant role in cell fate and in human health and disease. Despite significant interest in and examination of the role played by SK enzymes in disease, comparatively little is currently known about the three-dimensional structure and catalytic mechanisms of these enzymes. To date, limited numbers of studies have used site directed mutagenesis and activity determinations to examine the roles of individual SK residues in substrate, calmodulin, and membrane binding, as well as activation via phosphorylation. Assays are currently available that allow for both single and bisubstrate kinetic analysis of mutant proteins that show normal, lowered and enhanced activity as compared to wild type controls. Additional studies will be required to build on this foundation to completely understand SK mediated substrate binding and phosphoryl group transfer. A deeper understanding of the SK catalytic mechanism, as well as SK interactions with potential small molecule inhibitors will be invaluable to the future design and identification of SK activity modulators as research tools and potential therapeutics. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.


Asunto(s)
Biocatálisis , Mutagénesis Sitio-Dirigida , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Enfermedad , Humanos , Cinética , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Relación Estructura-Actividad
11.
Mol Pharmacol ; 84(3): 415-24, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23793291

RESUMEN

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.


Asunto(s)
Antineoplásicos/química , Benzamidas/química , Bencenoacetamidas/química , Hidrazinas/química , Indoles/química , Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/química , Sulfonamidas/química , Tetrazoles/química , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Bencenoacetamidas/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrazinas/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Mutación , Invasividad Neoplásica , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Relación Estructura-Actividad , Sulfonamidas/farmacología , Tetrazoles/farmacología
12.
Bioconjug Chem ; 24(3): 376-86, 2013 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-23360450

RESUMEN

Nanotechnology-based photothermal therapy has emerged as a promising treatment for cancer during the past decade. However, heterogeneous laser heating and limited light penetration can lead to incomplete tumor cell eradication. Here, we developed a method to overcome these limitations by combining chemotherapy with photothermal therapy using paclitaxel-loaded gold nanorods. Paclitaxel was loaded to gold nanorods with high density (2.0 × 10(4) paclitaxel per gold nanorod) via nonspecific adsorption, followed by stabilization with poly(ethylene glycol) linked with 11-mercaptoundecanoic acid. Paclitaxel was entrapped in the hydrophobic pocket of the polymeric monolayer on the surface of gold nanorods, which allows direct cellular delivery of the hydrophobic drugs via the lipophilic plasma membrane. Highly efficient drug release was demonstrated in a cell membrane mimicking two-phase solution. Combined photothermal therapy and chemotherapy with the paclitaxel-loaded gold nanorods was shown to be highly effective in killing head and neck cancer cells and lung cancer cells, superior to photothermal therapy or chemotherapy alone due to a synergistic effect. The paclitaxel-gold nanorod enabled photothermal chemotherapy has the potential of preventing tumor reoccurrence and metastasis and may have an important impact on the treatment of head and neck cancer and other malignancies in the clinic.


Asunto(s)
Portadores de Fármacos/administración & dosificación , Oro/administración & dosificación , Nanotubos , Neoplasias , Paclitaxel/administración & dosificación , Fototerapia/métodos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Oro/química , Oro/metabolismo , Humanos , Nanotubos/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Paclitaxel/química , Paclitaxel/metabolismo
13.
Bioorg Med Chem ; 21(17): 5548-60, 2013 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-23816044

RESUMEN

Autotaxin (ATX) catalyzes the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). Both ATX and LPA have been linked to pathophysiologies ranging from cancer to neuropathic pain. Inhibition of LPA production by ATX is therefore of therapeutic interest. Here we report the application of previously-developed, subsite-targeted pharmacophore models in a screening workflow that involves either docking or binary QSAR as secondary filters to identify ATX inhibitors from previously unreported structural types, four of which have sub-micromolar inhibition constants. Cell-based assays demonstrate that ATX inhibition and cytotoxicity structure-activity-relationships (SAR) exhibit selectivity cliffs, characterized by structurally similar compounds exhibiting similar biological activities with respect to ATX inhibition but very different biological activities with respect to cytotoxicity. Thus, general cytotoxicity should not be used as an early filter to eliminate candidate ATX inhibitor scaffolds from further SAR studies. Assays using two substrates of vastly different sizes demonstrate that the tools developed to identify compounds binding outside the central core of the active site did identify compounds acting at an allosteric site. In contrast, tools developed to identify active-site directed compounds did not identify active-site directed compounds. The stronger volume overlap imposed when selecting screening candidates expected to bind outside the active site is likely responsible for the stronger match between intended and actual target site.


Asunto(s)
Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/química , Animales , Sitios de Unión , Dominio Catalítico , Línea Celular Tumoral , Bases de Datos de Compuestos Químicos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Simulación del Acoplamiento Molecular , Inhibidores de Fosfodiesterasa/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-Actividad
14.
J Mol Graph Model ; 121: 108434, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36841204

RESUMEN

G protein-coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for drug development due to their role in transmitting cellular signals in a multitude of biological processes. Of the six classes categorizing GPCR (A, B, C, D, E, and F), class A contains the largest number of therapeutically relevant GPCR. Despite their importance as drug targets, many challenges exist for the discovery of novel class A GPCR ligands serving as drug precursors. Though knowledge of the structural and functional characteristics of GPCR has grown significantly over the past 20 years, a large portion of GPCR lack reported, experimentally determined structures. Furthermore, many GPCR have no known endogenous and/or synthetic ligands, limiting further exploration of their biochemical, cellular, and physiological roles. While many successes in GPCR ligand discovery have resulted from experimental high-throughput screening, computational methods have played an increasingly important role in GPCR ligand identification in the past decade. Here we discuss computational techniques applied to GPCR ligand discovery. This review summarizes class A GPCR structure/function and provides an overview of many obstacles currently faced in GPCR ligand discovery. Furthermore, we discuss applications and recent successes of computational techniques used to predict GPCR structure as well as present a summary of ligand- and structure-based methods used to identify potential GPCR ligands. Finally, we discuss computational hit list generation and refinement and provide comprehensive workflows for GPCR ligand identification.


Asunto(s)
Diseño de Fármacos , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/química , Sitios de Unión , Ligandos , Transducción de Señal
15.
J Mol Graph Model ; 121: 108429, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36804368

RESUMEN

Pharmacophores are three-dimensional arrangements of molecular features required for biological activity that are often used in virtual screening efforts to prioritize ligands for experimental testing. G protein-coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for ligand discovery and drug development. Ligand-based pharmacophore models can be constructed to identify structural commonalities between known bioactive ligands for targets including GPCR. However, structure-based pharmacophores (which only require an experimentally determined or modeled structure for a protein target) have gained more attention to aid in virtual screening efforts as the number of publicly available experimentally determined GPCR structures have increased (140 unique GPCR represented as of October 24, 2022). Thus, the goal of this study was to develop a method of structure-based pharmacophore model generation applicable to ligand discovery for GPCR that have few known ligands. Pharmacophore models were generated within the active sites of 8 class A GPCR crystal structures via automated annotation of 5 randomly selected functional group fragments to sample diverse combinations of pharmacophore features. Each of the 5000 generated pharmacophores was then used to search a database containing active and decoy/inactive compounds for 30 class A GPCR and scored using enrichment factor and goodness-of-hit metrics to assess performance. Application of this method to the set of 8 class A GPCR produced pharmacophore models possessing the theoretical maximum enrichment factor value in both resolved structures (8 of 8 cases) and homology models (7 of 8 cases), indicating that generated pharmacophore models can prove useful in the context of virtual screening.


Asunto(s)
Farmacóforo , Receptores Acoplados a Proteínas G , Ligandos , Receptores Acoplados a Proteínas G/química , Dominio Catalítico , Simulación del Acoplamiento Molecular
16.
J Mol Graph Model ; 122: 108488, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37121167

RESUMEN

Pharmacophore models are three-dimensional arrangements of molecular features required for biological activity that are used in ligand identification efforts for many biological targets, including G protein-coupled receptors (GPCR). Though GPCR are integral membrane proteins of considerable interest as targets for drug development, many of these receptors lack known ligands or experimentally determined structures necessary for ligand- or structure-based pharmacophore model generation, respectively. Thus, we here present a structure-based pharmacophore modeling approach that uses fragments placed with Multiple Copy Simultaneous Search (MCSS) to generate high-performing pharmacophore models in the context of experimentally determined, as well as modeled GPCR structures. Moreover, we have addressed the oft-neglected topic of pharmacophore model selection via development of a cluster-then-predict machine learning workflow. Herein score-based pharmacophore models were generated in experimentally determined and modeled structures of 13 class A GPCR and resulted in pharmacophore models exhibiting high enrichment factors when used to search a database containing 569 class A GPCR ligands. In addition, classification of pharmacophore models with the best performing cluster-then-predict logistic regression classifier resulted in positive predictive values (PPV) of 0.88 and 0.76 for selecting high enrichment pharmacophore models from among those generated in experimentally determined and modeled structures, respectively.


Asunto(s)
Farmacóforo , Receptores Acoplados a Proteínas G , Ligandos , Receptores Acoplados a Proteínas G/química , Transducción de Señal , Unión Proteica
17.
Pharmaceutics ; 15(10)2023 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-37896236

RESUMEN

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

18.
Nat Commun ; 14(1): 5593, 2023 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-37696784

RESUMEN

Fibrous networks constructed from high aspect ratio protein building blocks are ubiquitous in nature. Despite this ubiquity, the functional advantage of such building blocks over globular proteins is not understood. To answer this question, we engineered hydrogel network building blocks with varying numbers of protein L domains to control the aspect ratio. The mechanical and structural properties of photochemically crosslinked protein L networks were then characterised using shear rheology and small angle neutron scattering. We show that aspect ratio is a crucial property that defines network architecture and mechanics, by shifting the formation from translationally diffusion dominated to rotationally diffusion dominated. Additionally, we demonstrate that a similar transition is observed in the model living system: fibrin blood clot networks. The functional advantages of this transition are increased mechanical strength and the rapid assembly of homogenous networks above a critical protein concentration, crucial for in vivo biological processes such as blood clotting. In addition, manipulating aspect ratio also provides a parameter in the design of future bio-mimetic and bio-inspired materials.


Asunto(s)
Materiales Biomiméticos , Coagulación Sanguínea , Difusión , Hidrogeles , Modelos Biológicos
19.
J Mol Graph Model ; 112: 108119, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34979368

RESUMEN

G protein-coupled receptors (GPCR) are the largest family of cell surface receptors in vertebrates. Their abundance and role in nearly all physiological systems make GPCR the largest protein family targeted for development of pharmaceuticals. Ligand discovery aimed at identification of chemical tools and drug leads is aided by molecular docking simulations that allow critical analysis of the potential interactions between small molecules and proteins in resulting complexes. However, blind assessments of ligand pose quality and affinity prediction have thus far not provided broadly generalizable performance expectations for docking into experimentally-characterized GPCR targets. Likewise, the relative importance of receptor activation state and ligand function differences have also not been systematically assessed. This study compares performance when docking ligands of varied function into varied GPCR activation states in the absence of extensive resampling of the input GPCR structure, and only limited sidechain flexibility after ligand placement. Simulations were performed using 37 experimental structures of 11 Class A GPCR crystallized in multiple activation states (giving rise to 37 self-docking and 68 cross docking simulations). Our results show that one specific subset of cross-docking simulations gave results of similar quality to self-docking. Median ligand RMSD values for top-scored poses were 1.2 Å and 2.0 Å for self-docking and StateMatch/FunctionMatch cross-docking, respectively. The distributions of ligand RMSD values were not statistically different for these two conditions, according to a Kolmogorov-Smirnov test. Therefore, docking performance against GPCR targets can be estimated in advance based on docking target structure activation states, with higher accuracy expected when docking agonists into active state structures and inverse agonists or antagonists into inactive state structures. Receptor conformational sampling in advance of docking or receptor conformational adjustment after docking are more likely to produce substantial improvements for other pairings of receptor activation state and ligand function.


Asunto(s)
Receptores Acoplados a Proteínas G , Sitios de Unión , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Receptores Acoplados a Proteínas G/química
20.
Cryst Growth Des ; 22(10): 6120-6130, 2022 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-36217415

RESUMEN

Milk fat has more than 200 triacylglycerols (TAGs), which play a pivotal role in its crystallization behavior. Asymmetrical TAGs containing short butyryl chains contribute to a significant portion of milk fat TAGs. This work aims to elucidate the crystallization behavior of asymmetrical milk fat TAGs by employing the pure compound of 1-butyryl 2-stearoyl 3-palmitoyl-glycerol (BuSP). The structural evolution of BuSP after being cooled down to 20 °C from the melt is evaluated by small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC). The temporal structural observation shows that BuSP crystallizes into the α-form with short and long spacings of 4.10 and 56.9 Å, respectively, during the first hour of isothermal hold at 20 °C. The polymorphic transformation of the α to ß' phase occurred after 4 h of isothermal hold, and the ß'- to α-form fraction ratio was about 70:30 at the end of the isothermal experiment (18 h). Pure ß'-form X-ray patterns are obtained from the BuSP powder with short spacings of 4.33, 4.14, and 3.80 Å, while the long spacing of 51.2 Å depicts a three-chain-length lamellar structure with a tilt angle of 32°. Corresponding DSC measurements display that BuSP crystallizes from the melt at 29.1 °C, whereas the melting of α- and ß'-forms was recorded at 30.3 and 47.8 °C, respectively. In the absence of the ß-form, the ß'-polymorph is the most stable observed form in BuSP. This work exemplarily explains the crystallization behavior of asymmetrical milk fat TAGs and thus provides new insights into their role in overall milk fat crystallization.

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