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The fragment molecular orbital (FMO) method is an efficient quantum chemical calculation technique for large biomolecules, dividing each into smaller fragments and providing interfragment interaction energies (IFIEs) that support our understanding of molecular recognition. The ab initio fragment MO method (ABINIT-MP), an FMO processing program, can automatically divide typical proteins and nucleic acids. In contrast, small molecules such as ligands and heterosystems must be manually divided. Thus, we developed a graphical user interface to easily handle such manual fragmentation as a library for the Molecular Operating Environment (MOE) that preprocesses and visualizes FMO calculations. We demonstrated fragmentation with IFIE analyses for the two following cases: (1) covalent cysteine-ligand bonding inside the SARS-CoV-2 main protease (Mpro) and nirmatrelvir (Paxlovid) complex and (2) the metal coordination inside a zinc-bound cyclic peptide. IFIE analysis successfully identified the key amino acid residues for the molecular recognition of nirmatrelvir with Mpro and the details of their interactions (e.g., hydrogen bonds and CH/π interactions) via ligand fragmentation of functional group units. In metalloproteins, we found an efficient and accurate scheme for the fragmentation of Zn2+ ions with four histidines coordinated to the ion. FMOe simplifies manual fragmentation, allowing users to experiment with various fragmentation patterns and perform in-depth IFIE analysis with high accuracy. In the future, our findings will provide valuable insight into complicated cases, such as ligand fragmentation in modality drug discovery, especially for medium-sized molecules and metalloprotein fragmentation around metals.
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Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only a limited number of examples have been reported to date. Here, we show that desmethyl desamino pateamine A (DMDA-PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, onto GNG motifs. By systematically comparing multiple eIF4A inhibitors by ribosome profiling, we found that DMDA-PatA has unique mRNA selectivity for translation repression. Unbiased Bind-n-Seq reveals that DMDA-PatA-targeted eIF4A exhibits a preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. A combination of classical molecular dynamics simulations and quantum chemical calculations, and the subsequent development of an inactive DMDA-PatA derivative reveals that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified that DDX3, another DEAD-box protein, is an alternative DMDA-PatA target with the same effects on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and the mRNA-selective inhibition of protein synthesis by small-molecule compounds.
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ARN Helicasas DEAD-box , Factor 4A Eucariótico de Iniciación , Biosíntesis de Proteínas , ARN Mensajero , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Simulación de Dinámica Molecular , Ribosomas/metabolismo , Motivos de Nucleótidos , Unión Proteica , Células HEK293 , Compuestos Epoxi , Tiazoles , MacrólidosRESUMEN
Autophagy induction in cancer is involved in cancer progression and the acquisition of resistance to anticancer agents. Therefore, autophagy is considered a potential therapeutic target in cancer therapy. In this study, we found that long-chain fatty acids (LCFAs) have inhibitory effects on Atg4B, which is essential for autophagosome formation, through screening based on the pharmacophore of 21f, a recently developed Atg4B inhibitor. Among these fatty acids, docosahexaenoic acid (DHA), a polyunsaturated fatty acid, exhibited the most potent Atg4B inhibitory activity. DHA inhibited autophagy induced by androgen receptor signaling inhibitors (ARSI) in LNCaP and 22Rv1 prostate cancer cells and significantly increased apoptotic cell death. Furthermore, we investigated the effect of DHA on resistance to ARSI by establishing darolutamide-resistant prostate cancer 22Rv1 (22Rv1/Dar) cells, which had developed resistance to darolutamide, a novel ARSI. At baseline, 22Rv1/Dar cells showed a higher autophagy level than parental 22Rv1 cells. DHA significantly suppressed Dar-induced autophagy and sensitized 22Rv1/Dar cells by inducing apoptotic cell death through mitochondrial dysfunction. These results suggest that DHA supplementation may improve prostate cancer therapy with ARSI.
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Ionic liquids (ILs) exhibit very diverse physicochemical properties, such as non-volatility, stability, and miscibility, which render them excellent candidate excipients for multi-purpose use. Six novel arginine (Arg)-based ILs were obtained using a one-step ultrasound method. Salt formation was confirmed by Fourier-transform infrared (FTIR), Raman, and nuclear magnetic resonance (NMR) spectroscopies. Moreover, the effects of anions and molar ratio on the molecular states and thermal properties of Arg-ILs were investigated. In addition, the solubilization of drugs with different pKa and LogP values was attempted using Arg-ILs consisting of asparagine, proline, octanoic acid, and malic acid, respectively, and a comparative study was performed. Furthermore, the interaction mode between the drugs and ILs was determined by FTIR and Raman spectroscopy. Presumably, partial interaction between the component of ILs and drugs such as ofloxacin and valsartan occurred, whereas flurbiprofen and isosorbide mononitrate were dispersed in the viscous IL. The development of strategies for the application of ILs as solubilizers or carriers of active pharmaceutical ingredients is an extremely promising and wide avenue of research.
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Arginina , Líquidos Iónicos , Solubilidad , Arginina/química , Líquidos Iónicos/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Excipientes/química , Espectrometría Raman , Espectroscopía de Resonancia Magnética/métodos , Iones/químicaRESUMEN
Our previous findings indicated that many respiratory syncytial virus (RSV) isolates are unstable at 4 °C compared to 20 °C. Some of the strains completely lose infectivity after 24 h at 4 °C. This study analyzed the inactivation process at 4 °C using a representative strain, RSV/Sendai/851/13. After 24 h of storage at 4 °C, the virus was completely inactivated but retained its ability to attach to and to be taken into host cells. It suggested a reduced fusion ability between the viral and cellular membranes. During storage at 4 °C, the RSV fusion (F) protein underwent a conformational change and was no longer recognized by pre-fusion form-specific antibodies. When the RSV/Sendai/851/13 strain was passaged at 4 °C, a variant with an amino acid substitution, I148T, in the F protein fusion peptide was selected. Also, an amino acid change in G protein demonstrating stability at low temperatures was obtained. These results show that the inactivation of RSV at 4 °C is due to the loss of membrane fusion activity in the F protein, which cannot maintain its pre-fusion state at 4 °C.
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Frío , Virus Sincitial Respiratorio Humano , Proteínas Virales de Fusión , Inactivación de Virus , Proteínas Virales de Fusión/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/química , Humanos , Virus Sincitial Respiratorio Humano/fisiología , Animales , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales RespiratoriosRESUMEN
The formulation of active pharmaceutical ingredients involves discovering stable crystal packing arrangements or polymorphs, each of which has distinct pharmaceutically relevant properties. Traditional experimental screening techniques utilizing various conditions are commonly supplemented with in silico crystal structure prediction (CSP) to inform the crystallization process and mitigate risk. Predictions are often based on advanced classical force fields or quantum mechanical calculations that model the crystal potential energy landscape but do not fully incorporate temperature, pressure, or solution conditions during the search procedure. This study proposes an innovative alchemical path that utilizes an advanced polarizable atomic multipole force field to predict crystal structures based on direct sampling of the NPT ensemble. The use of alchemical (i.e., nonphysical) intermediates, a novel Monte Carlo barostat, and an orthogonal space tempering bias combine to enhance the sampling efficiency of the deposition/sublimation phase transition. The proposed algorithm was applied to 2-((4-(2-(3,4-dichlorophenyl)ethyl)phenyl)amino)benzoic acid (Cambridge Crystallography Database Centre ID: XAFPAY) as a case study to showcase the algorithm. Each experimentally determined polymorph with one molecule in the asymmetric unit was successfully reproduced via approximately 1000 short 1 ns simulations per space group where each simulation was initiated from random rigid body coordinates and unit cell parameters. Utilizing two threads of a recent Intel CPU (a Xeon Gold 6330 CPU at 2.00 GHz), 1 ns of sampling using the polarizable AMOEBA force field can be acquired in 4 h (equating to more than 300 ns/day using all 112 threads/56 cores of a dual CPU node) within the Force Field X software (https://ffx.biochem.uiowa.edu). These results demonstrate a step forward in the rigorous use of the NPT ensemble during the CSP search process and open the door to future algorithms that incorporate solution conditions using continuum solvation methods.
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In this study, we synthesized a family of novel ionic liquids (ILs) with meglumine (MGM) as cations and tartaric acid (TA), azelaic acid (AA), geranic acid (GA), and capric acid (CPA) as anions, using pharmaceutical additives via simple acid-base neutralization reactions. The successful synthesis was validated by attenuated total reflection-Fourier transform infrared (ATR-FTIR) and powder X-ray diffraction (PXRD). Thermal analysis using differential scanning calorimetry confirmed the glass transition temperature of MGM-ILs to be within the range of -43.4 °C--13.8 °C. We investigated the solubilization of 15 drugs with varying pKa and partition coefficient (log P) values using these ILs and performed a comparative analysis. Furthermore, we present MGM-IL as a new skin permeation enhancer for the drug model flurbiprofen (FRP). We confirmed that AA/MGM-IL improves the skin permeation of FRP through hairless mouse skin. Moreover, AA/MGM-IL enhanced drug skin permeability by affecting keratin rather than stratum corneum lipids, as confirmed by ATR-FTIR. To conclude, MGM-ILs exhibited potential as drug solubilizer and skin permeation enhancers of drugs.
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A novel in silico drug design procedure is described targeting the Main protease (Mpro) of the SARS-CoV-2 virus. The procedure combines molecular docking, molecular dynamics (MD), and fragment molecular orbital (FMO) calculations. The binding structure and properties of Mpro were predicted for Nelfinavir (NFV), which had been identified as a candidate compound through drug repositioning, targeting Mpro. Several poses of the Mpro and NFV complexes were generated by docking, from which four docking poses were selected by scoring with FMO energy. Then, each pose was subjected to MD simulation, 100 snapshot structures were sampled from each of the generated MD trajectories, and the structures were evaluated by FMO calculations to rank the pose based on binding energy. Several residues were found to be important in ligand recognition, including Glu47, Asp48, Glu166, Asp187, and Gln189, all of which interacted strongly with NFV. Asn142 is presumably regarded to form hydrogen bonds or CH/π interaction with NFV; however, in the present calculation, their interactions were transient. Moreover, the tert-butyl group of NFV had no interaction with Mpro. Identifying such strong and weak interactions provides candidates for maintaining and substituting ligand functional groups and important suggestions for drug discovery using drug repositioning. Besides the interaction between NFV and the amino acid residues of Mpro, the desolvation effect of the binding pocket also affected the ranking order. A similar procedure of drug design was applied to Lopinavir, and the calculated interaction energy and experimental inhibitory activity value trends were consistent. Our approach provides a new guideline for structure-based drug design starting from a candidate compound whose complex crystal structure has not been obtained.
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COVID-19 , Proteasas 3C de Coronavirus , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Nelfinavir/farmacología , SARS-CoV-2 , Simulación de Dinámica MolecularRESUMEN
Interest in small molecules that target RNA is flourishing, and the expectation set on them to treat diseases with unmet medical needs is high. However, several challenges remain, including difficulties in selecting suitable tools and establishing workflows for their discovery. In this context, we optimized experimental and computational approaches that were previously employed for the protein targets. Here, we demonstrate that a fluorescence-based assay can be effectively used to screen small molecule libraries for their ability to bind and stabilize an RNA stem-loop. Our screen identified several fluoroquinolones that bind to the target stem-loop. We further probed their interactions with the target using biolayer interferometry, isothermal titration calorimetry (ITC), and nuclear magnetic resonance spectroscopy. The results of these biophysical assays suggest that the fluoroquinolones bind the target in a similar manner. Armed with this knowledge, we built models for the complexes of the fluoroquinolones and the RNA target. Then, we performed fragment molecular orbital (FMO) calculations to dissect the interactions between the fluoroquinolones and the RNA. We found that the binding free energies obtained from the ITC experiments correlated strongly with the interaction energies calculated by FMO. Finally, we designed fluoroquinolone analogues and performed FMO calculations to predict their binding free energies. Taken together, the results of this study support the importance of conducting orthogonal assays in binding confirmation and compound selection and demonstrate the usefulness of FMO calculations in the rational design of RNA-targeted small molecules.
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ARN , Bibliotecas de Moléculas Pequeñas , Bibliotecas de Moléculas Pequeñas/química , Fluoroquinolonas , Unión ProteicaRESUMEN
A third-generation inhibitor of catechol O-methyltransferase (COMT), opicapone (1), has the 3-nitrocatechol scaffold as do the second-generation inhibitors such as entacapone (2) and tolcapone (3), but only 1 can sustainably inhibit COMT activity making it suitable for a once-daily regimen. These improvements should be attributed to the optimized sidechain moiety (oxidopyridyloxadiazolyl group) of 1 substituted at the 5-position of the 3-nitrocatechol ring. We analyzed the role of the sidechain moiety by solving the crystal structures of COMT/S-adenosylmethionine (SAM)/Mg/1 and COMT/S-adenosylhomocysteine (SAH)/Mg/1 complexes. Fragment molecular orbital (FMO) calculations elucidated that the dispersion interaction between the sidechains of Leu 198 and Met 201 on the ß6ß7-loop and the oxidopyridine ring of 1 were unique and important in both complexes. In contrast, the catechol binding site made a remarkable difference in the sidechain conformation of Lys 144. The ε-amino group of Lys 144 was outside of the catalytic pocket and was replaced by a water molecule in the COMT/SAH/Mg/1 complex. No nitrocatechol inhibitor has ever been reported to make a complex with COMT and SAH. Thus, the conformational change of Lys 144 found in the COMT/SAH/Mg/1 complex is the first crystallographic evidence that supports the role of Lys 144 as a catalytic base to take out a proton ion from the reaction site to the outside of the enzyme. The fact that 1 generated a complex with SAH and COMT also suggests that 1 could inhibit COMT twofold, as a typical substrate mimic competitive inhibitor and as a product-inhibition enhancer.
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Inhibidores de Catecol O-Metiltransferasa , Catecol O-Metiltransferasa , Inhibidores de Catecol O-Metiltransferasa/farmacología , Inhibidores de Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/metabolismo , Tolcapona , Oxadiazoles/farmacologíaRESUMEN
A non-covalent oral drug targeting SARS-CoV-2 main protease (Mpro), ensitrelvir (Xocova), has been developed using structure-based drug design (SBDD). To elucidate the factors responsible for enhanced inhibitory activities from an in silico screening hit compound to ensitrelvir, we analyzed the interaction energies of the inhibitors with each residue of Mpro using fragment molecular orbital (FMO) calculations. This analysis reveals that functional group conversion for P1' and P1 parts in the inhibitors increases the strength of existing interactions with Mpro and also provides novel interactions for ensitrelvir; the associated changes in the conformation of Mpro induce further interactions for ensitrelvir in other parts, including hydrogen bonds, a halogen bond, and π-orbital interactions. Thus, we illuminate the promising strategies of SBDD for leading ensitrelvir to get higher activity against Mpro by elucidating microscopic interactions through FMO-based analysis. These detailed mechanism findings, including water cross-linkings, will help to design novel inhibitors in SBDD.
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COVID-19 , Humanos , SARS-CoV-2 , Proteasas 3C de Coronavirus , Inhibidores de Proteasas/farmacología , Antivirales/farmacología , Simulación del Acoplamiento MolecularRESUMEN
Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high-order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) evolved into a GTP sensor from ATP-utilizing kinase. Mechanistically, PI5P4Kß has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP-dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations-the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kß is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell.
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Adenosina Trifosfato , Evolución Molecular , Animales , Guanosina Trifosfato/metabolismo , Adenosina Trifosfato/metabolismo , Mamíferos/metabolismoRESUMEN
The aim of this study was to enhance the solubility and stability of the water-insoluble drug carvedilol (CAR) with maleic acid (MLE) to create a co-amorphous system by a solvent evaporation method. Phase diagrams of co-amorphous CAR-MLE, constructed from peak height in the Fourier-transform infrared (FTIR) spectra and the glass transition temperature (Tg) from differential scanning calorimetry (DSC) measurements, revealed that the optimal molar ratio of CAR to MLE was 2:1. The FTIR spectra indicated that the secondary amine-derived peak of CAR and the carboxy group-derived peak of MLE disappeared in the CAR:MLE (2:1) co-amorphous system. DSC measurements showed that the endothermic peaks associated with the melting of CAR and MLE disappeared and a Tg at 43 °C was apparent. Furthermore, the solubility of CAR tested using the shaking flask method for 24 h at 37 °C was 1.2 µg/mL, whereas that of the co-amorphous system was approximately three times higher, at 3.5 µg/mL. Finally, the stability was evaluated by powder- X-ray diffraction at 40 °C; no clear diffraction peaks originating from crystals were observed in the amorphous state until after approximately three months of storage. These results indicate that co-amorphization of CAR with MLE improved the solubility of CAR while maintaining its stability in an amorphous form.
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Carvedilol , Solventes/química , Estabilidad de Medicamentos , Temperatura de Transición , Difracción de Rayos X , Solubilidad , Rastreo Diferencial de Calorimetría , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Famotidine (FMT) is a competitive histamine-2 (H2) receptor antagonist that inhibits gastric acid secretion for the treatment of Gastroesophageal reflux disease. To study the promoting effect and mechanism of terpenes, including l-menthol, borneol, and geraniol, as chemical enhancers, FMT was used as a model drug. Attenuated total reflectance-Fourier transform IR spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC) were used to explore the effects of terpenes on the skin. Hairless mouse skin was mounted on Franz-type diffusion cell, and skin permeation experiment of FMT hydrogel was carried out. The results suggested that the thermodynamic activity influenced the permeability of the drug, and the main mechanism of terpenes to enhance skin permeation of the drug was based on increasing the fluidity of the intercellular lipids. Moreover, it was revealed that l-menthol simultaneously relaxed the packing structure and lamellar structure, whereas geraniol had a great influence on the lamellar structure only. Collectively, all terpenes had a promoting effect on skin permeation of FMT, indicating their potential as chemical enhancers to change the microstructure of stratum corneum and improve the permeation of FMT through the skin, and it has great potential to be used in transdermal formulations of FMT.
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Famotidina , Terpenos , Ratones , Animales , Terpenos/farmacología , Terpenos/metabolismo , Famotidina/farmacología , Famotidina/metabolismo , Absorción Cutánea , Mentol/farmacología , Mentol/química , Mentol/metabolismo , Piel , Administración Cutánea , PermeabilidadRESUMEN
Amorphous drug formulations exploiting drug-drug interactions have been extensively studied. This study aims to develop a transdermal system containing an amorphous complex of the nonsteroidal anti-inflammatory drug (NSAID) flurbiprofen (FLU) and lidocaine (LDC) for alleviating chronic pain. The high-viscosity complex between FLU and LDC (Complex) was obtained by heating in ethanol. For the complex, attenuated total reflection-Fourier transform infrared spectroscopy showed a shift in the carboxy-group-derived peak of FLU, and differential scanning calorimetry indicated the endothermic peaks associated with the melting of FLU and LDC disappeared. 13C dipolar decoupling and 15N cross-polarization magic-angle spinning nuclear magnetic resonance measurement suggested the interaction between the carboxyl group of FLU and the secondary amine of LDC. The interaction between the aromatic rings of FLU and LDC contributed to the molecular complex formation. The solubility of FLU from the complex was about 100 times greater than FLU alone. The skin permeation flux of FLU from the complex through the hairless mouse skin was 3.8 times higher than FLU alone in hypromellose gel. Thus, adding LDC to the formulation can be an effective method for enhancing the skin permeation of NSAIDs, which can prove useful for treating chronic pain and inflammatory diseases.
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Moisture-activated dry granulation (MADG) is an eco-friendly granulation method that uses a small amount of water and insoluble excipients to absorb moisture. MADG is expected to improve productivity and reduce costs. Erythritol, an excipient used for preparing orally disintegrating tablets (ODTs), has poor tabletability and is difficult to form into tablets by conventional methods, such as high-shear granulation (HSG) and direct compression. In this study, we optimized the manufacturing conditions for ODTs to improve the tabletability of erythritol using MADG. The disintegration time of tablets made using the MADG method was approximately one-tenth that of those made using the HSG method, and the hardness was approximately 1.4 times higher. Moreover, MADG could delay disintegration and improve tabletability. We further attempted to optimize the manufacturing conditions using MADG, particularly in terms of the amount of water used. The disintegration time increased as the amount of added water increased. Moreover, water absorption tests revealed that capillary wetting decreased as the amount of water added increased, but the initial wetting did not change. These results suggested that the disintegration time was prolonged because of the increase in granule density and decrease in capillary wetting with the increase in the amount of added water. The hardness of the tablets increased because of the easy deformation of the granules after the addition of up to 3% water; however, when more than 3% water was added, the hardness decreased because of the aggregation of the granules with the excess water. Finally, two-dimensional maps of the effect of the amount of added water and water activity indicated that tablets with a hardness of ≥80 N and a disintegration time of ≤15 s could be produced by adjusting the amount of added water to within the range of 2.2-3.3% and water activity to 0.3-0.53. These results indicate that MADG can improve the tabletability of erythritol and be used for the granulation of ODTs. Tablets with appropriate hardness and disintegration properties can be produced by adjusting the water content to approximately 2.7% and the water activity to approximately 0.4 when producing ODTs with MADG.
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Estrogen receptors (ERs) are ligand-activated transcription factors, with two subtypes ERα and ERß. The endogenous ligand of ERs is the common 17ß-estradiol, and the ligand-binding pocket of ERα and ERß is very similar. Nevertheless, some ERß-selective agonist ligands have been reported. DPN (diarylpropionitrile) is a widely used ERß-selective agonist; however, the structure of the ERß-DPN complex has not been solved. Therefore, the bound-state conformation of DPN and its enantioselectivity remain unresolved. In this report, we present the structures of the complexes of ERß with DPN or its derivatives that include a chlorine atom by the X-ray crystallography. Additionally, we measured the binding affinity between ERß and DPN or derivatives by isothermal titration calorimetry (ITC) and estimated the binding affinity by fragment molecular orbital (FMO) calculations. We also examined the correlation between the ITC data and results from the FMO calculations. FMO calculations showed that S-DPN interacts strongly with three amino acids (Glu305, Phe356, and His475) of ERß, and ITC measurements confirmed that the chlorine atom of the DPN derivatives enhances binding affinity. The enthalpy change by ITC correlated strongly with the interaction energy (total IFIEs; inter-fragment interaction energies) calculated by FMO (R = 0.870). We propose that FMO calculations are a valuable approach for enhancing enthalpy contributions in drug design, and its scope of applications includes halogen atoms such as chlorine. This study is the first quantitative comparison of thermodynamic parameters obtained from ITC measurements and FMO calculations, providing new insights for future precise drug design.
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Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Calorimetría , Cloro , Estradiol , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Ligandos , Nitrilos , PropionatosRESUMEN
Fragment molecular orbital (FMO) method is a powerful computational tool for structure-based drug design, in which protein-ligand interactions can be described by the inter-fragment interaction energy (IFIE) and its pair interaction energy decomposition analysis (PIEDA). Here, we introduced a dynamically averaged (DA) FMO-based approach in which molecular dynamics simulations were used to generate multiple protein-ligand complex structures for FMO calculations. To assess this approach, we examined the correlation between the experimental binding free energies and DA-IFIEs of six CDK2 inhibitors whose net charges are zero. The correlation between the experimental binding free energies and snapshot IFIEs for X-ray crystal structures was R2 = 0.75. Using the DA-IFIEs, the correlation significantly improved to 0.99. When an additional CDK2 inhibitor with net charge of -1 was added, the DA FMO-based scheme with the dispersion energies still achieved R2 = 0.99, whereas R2 decreased to 0.32 employing all the energy terms of PIEDA.
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Simulación de Dinámica Molecular , Proteínas , Quinasa 2 Dependiente de la Ciclina , Diseño de Fármacos , Ligandos , Unión ProteicaRESUMEN
Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.
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Proteínas de Unión al GTP , Inosina Trifosfato , Proteínas de Unión al GTP/metabolismo , Guanina , Guanosina Trifosfato/metabolismo , Inosina Trifosfato/metabolismoRESUMEN
We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.