Analysis Of Drug-protein Interactions Research Articles

Prediction of the binding interactions between rosmarinic acid and cysteinyl leukotriene receptor type 1 by molecular docking and immobilized receptor chromatography.

Drug-protein interaction analysis is still at the center of research efforts to illustrate binding mechanisms and provide valuable information for selecting drug candidates with ideal properties in the early drug discovery stage. We present the prediction of the binding of rosmarinic acid (RA) to cysteinyl leukotriene receptor type1 (CysLTR1) by molecular docking. According to our findings, CysLTR1 is a potential anti-inflammatory target of RA. Under this assumption, we prepared the immobilized CysLTR1 column via a one-step method and characterized the immobilized CysLTR1 by fluorescent and chromatographic analyses. Furthermore, we used the immobilized CysLTR1 column to evaluate the binding interactions between RA and the immobilized receptor. Molecular docking showed that Tyr 249, Phe 174, Thr 280, Pro 177, and Thr 100 are the main sites for RA to interact with CysLTR1. The main forces that drive the findings are hydrogen bonds and hydrophobic interactions. Characterization results show that CysLTR1 is successfully immobilized with high specificity and stability. Almost no non-specific binding is observed on the immobilized CysLTR1 gels. The association constant and the binding sites are calculated to be 7.268 × 105 L mol-1 and 1.237 × 10-8 mol L-1 by injection amount-dependent method. These results, taken together, confirm the potential target of RA on the anti-inflammatory effect. We believe that it can provide valuable reference information on the in-depth exploration of drug-protein interaction mechanisms, and lead compound screening by this method.

Site-specific immobilization of Cysteinyl leukotriene receptor 1 through enzymatic DNA-protein conjugation strategy for lead screening

Immobilization of functional protein, especially G protein-coupled receptors (GPCRs), is particularly significant in various fields such as the development of assays for diagnosis, lead compound screening, as well as drug-protein interaction analysis. However, there are still some challenges with the immobilized proteins such as undefined loads, orientations, and the loss of activity. Herein, we introduced a DNA conjugation strategy into the immobilization of Cysteinyl leukotriene receptor 1(CysLTR1) which enables exquisite molecular control and higher activity of the receptor. We used the bacterial relaxases VirD2 as an immobilized tag fused at the C terminus of CysLTR1. Tyrosine residue(Y29) at the core binding site of the VirD2 tag can react with the single-strand piece of DNA(T-DNA) in the form of a covalent bond. Inspired by this strategy, we developed a new immobilization method by mixing the T-DNA-modified silica gel with the cell lysate containing the expressed VirD2-tagged CysLTR1 for 1 hour. We found that the successful formation of DNA-protein conjugate enables the immobilization of CysLTR1 fast, site-specific, and with minimal loss of activity. The feasibility of the immobilized CysLTR1 was evaluated in drug-protein binding interaction by frontal analysis and adsorption energy distribution analysis. The binding of pranlukast, zafirlukast, and MK571 to the immobilized CysLTR1 was realized, and the association constants presented good agreement between the two methods. Rosmarinic acid was retained in the immobilized CysLTR1 column, and the in-vitro test revealed that the compound binds to the receptor in one type of binding site mode. Despite these results, we concluded that the DNA-protein conjugate strategy will probably open up the possibilities for capturing other functional proteins in covalent and site-specific modes from the complex matrices and the immobilized receptor preserves the potential in fishing out lead compounds from natural products.

Bioinformatics approach to analyse COVID-19 biomarkers accountable for generation of intracranial aneurysm in COVID-19 patients

COVID-19 became a health emergency on January 30, 2020. SARS-CoV-2 is the causative agent of the coronavirus disease known as COVID-19 and can develop cardiometabolic and neurological disorders. Intracranial aneurysm (IA) is considered the most significant reason for hemorrhagic stroke,and it accounts for approximately 85% of all subarachnoid hemorrhages (SAH). Retinoid signaling abnormalities may explain COVID-19's pathogenesis with inhibition of AEH2, from which COVID-19 infection may enhance aneurysm formation and rupture due to abrupt blood pressure changes, endothelial cell injury, and systemic inflammation. The objective of this study was to investigate the potential biomarkers, differentially expressed genes (DEGs), and metabolic pathways associated with both COVID-19 and intracranial aneurysm (IA) using simulation databases like DIsGeNET. The purpose was to confirm prior findings and gain a comprehensive understanding of the underlying mechanisms that contribute to the development of these conditions. We combined the regulated genes to describe intracranial aneurysm formation in COVID-19. To determine DEGs in COVID-19 and IA patient tissues, we compared gene expression transcriptomic datasets from healthy and diseased individuals. There were 41 differentially expressed genes (DEGs) shared by both the COVID-19 and IA datasets (27 up-regulated genes and 14 down-regulated genes). Using protein-protein interaction analysis, we were able to identify hub proteins (C3, NCR1, IL10RA, OXTR, RSAD2, CD38, IL10RB, MX1, IL10, GFAP, IFIT3, XAF1, USP18, OASL, IFI6, EPSTI1, CMPK2, and ISG15), which were not described as key proteins for both COVID-19 and IA before. We also used Gene Ontology analysis (6 significant ontologies were validated), Pathway analysis (the top 20 were validated), TF-Gene interaction analysis, Gene miRNA analysis, and Drug-Protein interaction analysis methods to comprehend the extensive connection between COVID-19 and IA. In Drug-Protein interaction analysis, we have gotten the following three drugs: LLL-3348, CRx139, and AV41 against IL10 which was both common for COVID-19 and IA disease. Our study with different cabalistic methods has showed the interaction between the proteins and pathways with drug analysis which may direct further treatment development for certain diseases.

Covalent immobilization of beta2 adrenergic receptor through trans-methylation reaction by SNAP-tag and its application in anti-asthmatic compound screening from Raphani Semen

Beta2-adrenergic receptor (β2-AR) is believed as an attractive target for anti-asthmatic drugs. Its crystal structure and pharmacological activity have been clearly investigated. Yet the number of the approved anti-asthmatic drugs has declined in recent years. This work reports on the preparation of an immobilized β2-AR column through the specific trans-methylation reaction between SNAP tag and the benzyl-guanine derivative and application in anti-asthmatic compound screening from Raphani Semen. The characterization of the immobilized β2-AR was performed by scanning electron microscopy (SEM) and receptor-ligand interaction analysis by chromatographic methods. SEM analysis showed that the receptor has been successfully coated on the surface of PEGA amino microspheres. Binding constants of salbutamol and terbutaline calculated from frontal analysis within the temperature range of 10–30 ℃ confirmed the feasibility of the method in a thermodynamic viewpoint. Hydrogen bond was verified as the main driving force for drug-receptor interaction analysis. Sinapine was identified as the potential bioactive compound in Raphani Semen that specifically bind with β2-AR with a specific binding site of Ser 207. Taking together, the immobilized β2-AR column is promising in exploring drug-protein interaction analysis and anti-asthmatic drug screening.

Human miRNAs to Identify Potential Regions of SARS-CoV-2.

It is two years now but the world is still struggling against COVID-19 due to the havoc created by the SARS-CoV-2 virus and its multiple variants. Considering this perspective, in this work, we have hypothesized a new approach in order to identify potential regions in SARS-CoV-2 similar to the human miRNAs. Thus, they may have similar consequences as caused by the human miRNAs in human body. Therefore, the same way by which human miRNAs are inhibited can be applied for such potential regions of virus as well by administering drugs to the interacting human proteins. In this regard, the multiple sequence alignment technique Clustal Omega is used to align 2656 human miRNAs with the SARS-CoV-2 reference genome to identify the potential regions within the virus reference genome which have high similarities with the human miRNAs. The potential regions in virus genome are identified based on the highest number of nucleotide match, greater than or equal to 5 at a genomic position, for the aligned miRNAs. As a result, 38 potential SARS-CoV-2 regions are identified consisting of 249 human miRNAs. Among these 38 potential regions, some top regions belong to nucleocapsid, RdRp, helicase, and ORF8. To understand the biological significance of these potential regions, the targets of the human miRNAs are considered for KEGG pathways and protein–protein and drug–protein interaction analysis as the human miRNAs are similar to the potential regions of SARS-CoV-2. Significant pathways are found which lead to comorbidities. Subsequently, drugs like emodin, bicalutamide, vorinostat, etc. are identified that may be used for clinical trials.

Site-selective covalently immobilized alpha 1A adrenergic receptor for thermodynamic and extra-thermodynamic study of four ligands binding to the receptor by chromatographic methods

Immobilized G protein-coupled receptor is a versatile tool to study ligand-receptor interactions. In this work, we synthesized the immobilized alpha 1A adrenergic receptor (α1A-AR), a GPCR subtype mediating smooth muscle contraction, through a site-selective covalent method that relies on the reaction between haloalkane dehalogenase tagged α1A-AR and macroporous silica gel coated with 6-chlorohexanoic acid. To investigate thermodynamic and extra-thermodynamic parameters for ligand binding, we utilized the covalently immobilized receptor as stationary phase to perform frontal analysis and injection-amount dependent analysis as well as compared with the random immobilization method. Terazosin gave the association constant of 1.48 × 105 M−1 to α1A-AR, indicating that the oriented immobilization of α1A-AR enhances the ligand-binding activity by one order of magnitude in comparison with the random immobilization method (7.9 × 104 M−1). The binding of phentolamine and tamsulosin to the receptor was accompanied by a large absolute heat capacity (ΔCp) of 1.28 ± 0.23 kJ mol−1, demonstrating that the binding enthalpy and entropy appear to compensate for one another. These results indicated that the covalent immobilization of the receptor onto solid support has a profound impact on the ligand-binding activity of the receptor and the determination of ligand-receptor binding parameters. The receptor immobilized through the site-selective method will act as a benchmark for chromatographic determination of binding parameters in ligand-receptor interactions and can be used as an effective approach for rapid analysis of drug-protein interactions with high accuracy.

Advances in chromatography in the study of drug-plasma protein interactions

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α1 -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。

Protein network exploration prioritizes targets for modulating neuroinflammation in Parkinson’s disease

Parkinson's disease is a progressive neurodegenerative disease associated with a loss of dopaminergic neurons in the substantia nigra of the brain. Neuroinflammation, another hallmark of the disease, is thought to play an important role in the neurodegenerative process. While mitigating neuroinflammation could prove beneficial for Parkinson's disease, identifying the most relevant biological processes and pharmacological targets as well as drugs to modulate them remains highly challenging. The present study aimed to better understand the protein network behind neuroinflammation in Parkinson's disease and to prioritize possible targets for its pharmacological modulation. We first used text-mining to systematically collect the proteins significantly associated to Parkinson's disease neuroinflammation over the scientific literature. The functional interaction network formed by these proteins was then analyzed by integrating functional enrichment, network topology analysis and drug–protein interaction analysis. We identified 57 proteins significantly associated to neuroinflammation in Parkinson's disease. Toll-like Receptor Cascades as well as Interleukin 4, Interleukin 10 and Interleukin 13 signaling appeared as the most significantly enriched biological processes. Protein network analysis using STRING and CentiScaPe identified 8 proteins with the highest ability to control these biological processes underlying neuroinflammation, namely caspase 1, heme oxygenase 1, interleukin 1beta, interleukin 4, interleukin 6, interleukin 10, tumor necrosis factor alpha and toll-like receptor 4. These key proteins were indexed to be targetable by a total of 38 drugs including 27 small compounds 11 protein-based therapies. In conclusion, our study highlights key proteins in Parkinson's disease neuroinflammation as well as pharmacological compounds acting on them. As such, it may facilitate the prioritization of biomarkers for the development of diagnostic, target-engagement assessment and therapeutic tools against Parkinson's disease.

Scutellariabarbata D. Don extraction selectively targets stemness-prone NSCLC cells by attenuating SOX2/SMO/GLI1 network loop

Ethnopharmacological relevanceScutellariabarbata D. Don extraction (SBE), a traditional Chinese medicine, has been proved effective against various malignant disorders in clinics with tolerable side-effects when administered alone or in combination with conventional chemotherapeutic regimens. Aim of this studyMulti-drug resistance of cancer is attributed to existence of cancer stemness-prone cells that harbor aberrantly high activation of Sonic Hedgehog (SHH) cascade. Our previous study has demonstrated that SBE sensitized non-small cell lung cancer (NSCLC) cells to Cisplatin (DDP) treatment by downregulating SHH pathway. Yet, whether SBE could prohibit proliferation of cancer stemness-prone cells and its underlying molecular mechanisms remain to be investigated. In this article, we further investigated intervention of SBE on NSCLC cell stemness-associated phenotypes and its potential mode of action. Materials and methodsCCK-8 and clonal formation detection were used to measure the anti-proliferative potency of SBE against NSCLC and normal epithelial cells. Sphere formation assay and RQ-PCR were used to detect proliferation of cancer stemness cells and associated marker expression upon SBE incubation. Mechanistically, DARTS-WB and SPR were used to unveil binding target of SBE. Immunodeficient mice were implanted with patient derived tumor bulk for in vivo validation of anti-cancer effect of SBE. ResultsSBE selectively attenuated proliferation and stemness-like phenotypes of NSCLC cells rather than bronchial normal epithelial cells. Drug-protein interaction analysis revealed that SBE could directly bind with stem cell-specific transcription factor sex determining region Y-box 2 (SOX2) and interfere with the SOX2/SMO/GLI1 positive loop. In vivo assay using patient-derived xenografts (PDXs) model further proved that SBE diminished tumor growth and SOX2 expression in vivo. ConclusionOur data indicate that SBE represses stemness-related features of NSCLC cells via targeting SOX2 and may serve as an alternative therapeutic option for clinic treatment.

Potent Natural Antioxidant Carveol Attenuates MCAO-Stress Induced Oxidative, Neurodegeneration by Regulating the Nrf-2 Pathway.

Ischemic stroke is a severe neurological disorder with a high prevalence rate in developed countries. It is characterized by permanent or transient cerebral ischemia and it activates syndrome of pathological events such as membrane depolarization, glutamate excitotoxicity, and intracellular calcium buildup. Carveol is widely employed as anti-inflammatory and antioxidant in traditional Chinese medicine. In the present study, the neuroprotective effects of post-treated carveol were demonstrated against transient middle cerebral artery occlusion (MCAO) induced focal ischemic cerebral injury. Male Sprague Dawley (SD) rats were subjected to two different experimental protocols to determine the dose and effects of carveol, and to demonstrate the underlying role of the nuclear factor E2-related factor (Nrf2) pathway. Our results showed that MCAO induced marked neuronal injury in the ipsilateral cortex and striatum associated with higher inflammatory cytokines expression, along with apoptotic markers such as caspase-3 and the phosphorylated c-Jun N-terminal kinase (JNK). Furthermore, MCAO induced a marked increase in oxidative stress as evidenced by high lipid peroxidase (LPO) content accompanied by the depressed antioxidant system. Carveol significantly reversed the oxidative stress and downregulated inflammatory cascades by enhancing endogenous antioxidant mechanisms including the Nrf2 gene, which critically regulates the expression of several downstream antioxidants. Further, to determine the possible involvement of Nrf2 in carveol mediated neuroprotection, we antagonized Nrf2 by all-trans retinoic acid (ATRA), and such treatment abrogated the protective effects of carveol accompanied with exaggerated neuronal toxicity as demonstrated by higher infarction area. The target effects of carveol were further supported by molecular docking analysis of drug–protein interactions. Together, our findings suggest that carveol could activate endogenous master anti-oxidant Nrf2, which further regulates the expression of downstream antioxidants, eventually ameliorating MCAO-induced neuroinflammation and neurodegeneration.

Immobilized angiotensin II type I receptor: A powerful method of high throughput screening for antihypertensive compound identification through binding interaction analysis

The enormous growth in drug discovery paradigm has necessitated continuous exploration of new methods for drug-protein interaction analysis. To enhance the role of these methodologies in designing rational drugs, this work extended an immobilized angiotensin II type I receptor (AT1R) based affinity chromatography in antihypertensive compound identification. We fused haloalkane dehalogenase at C-terminus of AT1R and expressed the fusion receptor in E. coli. The expressed receptor was covalently immobilized onto 8.0 μm microspheres by mixing the cell lysate with 6-chlorocaproic acid-modified amino polystyrene microspheres. The immobilized AT1R was utilized for thermodynamic and kinetic interaction analysis between the receptor and four specific ligands. Following confirmation of these interactions by molecular docking, we identified puerarin and rosmarinic acid by determining their binding to the receptor. Azilsartan, candesartan, valsartan and olmesartan displayed two kinds of binding sites to AT1R by injection amount-dependent method. By molecular docking, we recognize the driving forces of the interaction as electrostatic interaction, hydrogen bonds and van der Waals force. The dissociation rate constants (kd) of azilsartan, candesartan, valsartan and olmesartan to AT1R were 0.01138 ± 0.003, 0.05142 ± 0.003, 0.07547 ± 0.004 and 0.01310 ± 0.005 min−1 by peak profiling assay. Comparing with these parameters, puerarin and rosmarinic acid presented lower affinity (KA: 0.12 × 104 and 1.5 × 104/M) and slower kinetics (kd: 0.6864 ± 0.03 and 0.3005 ± 0.01 min−1) to the receptor. These results, taking together, indicated that the immobilized AT1R has the capacity to probe antihypertensive compounds.

Characterization of the heterogeneous adsorption of three drugs on immobilized bovine serum albumin by adsorption energy distribution

Characterization of the heterogeneity of a protein surface is important for understanding the binding mechanism of a drug to the protein. A systematic methodology of integrated adsorption energy distribution (AED) calculation with the Scatchard plot was used to characterize the heterogeneous binding of drugs to bovine serum albumin (BSA). Frontal affinity chromatography (FAC) was applied to generate the adsorption data of three drugs on the immobilized BSA column. The concave Scatchard plots cannot distinguish the heterogeneous model between Toth and bi-Langmuir. The calculation of AED profiles allowed the accurate selection of adsorption models to reveal the physical sense of the drugs. Warfarin-BSA complex proved to be the bi-Langmuir model with association constants of 6.6 × 105 M−1 and 2.4 × 103 M−1, respectively. These results were highly consistent with traditional frontal analysis, ultrafiltration, and dynamic dialysis. Applying the AED related method, we achieved multiple and single kinds of binding sites for ephedrine-BSA and L-tryptophan-BSA complexes. Site-specific competitive studies exhibited non-competitive binding of ephedrine with warfarin or L-tryptophan on BSA, underlining that the location of ephedrine binding sites in the subdomain IIIB of BSA. This method permits an accurate heterogeneous characterization of drugs on a protein surface and has great potential in the reliable drug-protein interaction analysis.

Metformin Results in Diametrically Opposed Effects by Targeting Non-Stem Cancer Cells but Protecting Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been shown as a distinct population of cancer cells strongly implicated with resistance to conventional chemotherapy. Metformin, the most widely prescribed drug for diabetes, was reported to target cancer stem cells in various cancers. In this study, we sought to determine the effects of metformin on head and neck squamous cell carcinoma (HNSCC). CSCs and non-stem HNSCC cells were treated with metformin and cisplatin alone, and in combination, and cell proliferation levels were measured through MTS assays. Next, potential targets of metformin were explored through computational small molecule binding analysis. In contrast to the reported effects of metformin on CSCs in other cancers, our data suggests that metformin protects HNSCC CSCs against cisplatin in vitro. Treatment with metformin resulted in a dose-dependent induction of the stem cell genes CD44, BMI-1, OCT-4, and NANOG. On the other hand, we observed that metformin successfully decreased the proliferation of non-stem HNSCC cells. Computational drug–protein interaction analysis revealed mitochondrial complex III to be a likely target of metformin. Based on our results, we present the novel hypothesis that metformin targets complex III to reduce reactive oxygen species (ROS) levels, leading to the differential effects observed on non-stem cancer cells and CSCs.

Confirming therapeutic target of protopine using immobilized β2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β2 -adrenoceptor (β2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β2 -AR by the 2 methods were (1.00±0.06)×105 M-1 and (1.52±0.14)×104 M-1 . The numbers of binding sites were (1.23±0.07)×10-7 M and (9.09±0.06)×10-7 M, respectively. These results indicated that β2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.

Rapid analysis of interaction between six drugs and β2 -adrenergic receptor by injection amount-dependent method.

Drug-protein interaction analysis has become a considerable topic in life science which includes clarifying protein functions, explaining drug action mechanisms and uncovering novel drug candidates. This work was to determine the association constants (KA ) of six drugs to β2 -adrenergic receptor by injection amount-dependent method using stationary phase containing the immobilized receptor. The values of KA were calculated to be (25.85 ± 0.035) × 104 m-1 for clorprenaline, (42.51 ± 0.054) × 104 m-1 for clenbuterol, (6.67 ± 0.008) × 104 m-1 for terbutaline, (33.99 ± 0.025) × 104 m-1 for tulobuterol, (7.59 ± 0.011) × 104 m-1 for salbutamol and (78.52 ± 0.087) × 104 m-1 for bambuterol. This rank order agreed well with the data determined by zonal elution, frontal analysis and nonlinear chromatography, even using different batches of β2 -AR column. A good correlation was found between the association constants by the current method and radio-ligand binding assay. Our data indicates that the injection amount-dependent method is a powerful alternative for rapid analysis of ligand-receptor interactions.

Recent Advances in Biosensing With Photonic Crystal Surfaces: A Review

Photonic crystal surfaces that are designed to function as wavelength-selective optical resonators have become a widely adopted platform for label-free biosensing, and for enhancement of the output of photon-emitting tags used throughout life science research and in vitro diagnostics. While some applications, such as analysis of drug-protein interactions, require extremely high resolution and the ability to accurately correct for measurement artifacts, others require sensitivity that is high enough for detection of disease biomarkers in serum with concentrations less than 1 pg/ml. As the analysis of cells becomes increasingly important for studying the behavior of stem cells, cancer cells, and biofilms under a variety of conditions, approaches that enable high resolution imaging of live cells without cytotoxic stains or photobleachable fluorescent dyes are providing new tools to biologists who seek to observe individual cells over extended time periods. This paper will review several recent advances in photonic crystal biosensor detection instrumentation and device structures that are being applied towards direct detection of small molecules in the context of high throughput drug screening, photonic crystal fluorescence enhancement as utilized for high sensitivity multiplexed cancer biomarker detection, and label-free high resolution imaging of cells and individual nanoparticles as a new tool for life science research and single-molecule diagnostics.

Physiological concentrations of albumin favor drug binding.

The ability to track drug binding and release makes electron paramagnetic resonance (EPR) spectroscopy well suited for drug delivery studies. Using the continuous wave (cw) EPR technique to extract information about the dynamics of the spin labeled drugs we can simultaneously determine the bound and unbound drugs. Here, spin labeled salicylic acid (SLSA) binding to and release from bovine serum albumin (BSA) is investigated, as a model for drug-transport protein interaction. We studied SLSA-BSA binding in a wide concentration range and found that the stoichiometry of the drug-protein increases significantly when the physiological range of BSA concentration is reached. Our EPR results explicitly reveal that up to ∼7 SLSA can bind to one albumin at the physiological concentration, whereas at lower BSA concentrations (<0.125 mM) the SLSA-BSA stoichiometry is maximum 2. Moreover, we studied drug release and showed that the ratio of bound to unbound SLSA concentrations remains relatively stable during dialysis. This indicates that the binding equilibrium of SLSA is not altered through the process of dialysis. This study demonstrates that cw EPR spectroscopy in combination with spin labeled drugs is an effective technique for binding and release studies and stoichiometric analysis of drug-protein interactions.

Discovering patterns in drug-protein interactions based on their fingerprints

BackgroundThe discovering of interesting patterns in drug-protein interaction data at molecular level can reveal hidden relationship among drugs and proteins and can therefore be of paramount importance for such application as drug design. To discover such patterns, we propose here a computational approach to analyze the molecular data of drugs and proteins that are known to have interactions with each other. Specifically, we propose to use a data mining technique called Drug-Protein Interaction Analysis (D-PIA) to determine if there are any commonalities in the fingerprints of the substructures of interacting drug and protein molecules and if so, whether or not any patterns can be generalized from them.MethodGiven a database of drug-protein interactions, D-PIA performs its tasks in several steps. First, for each drug in the database, the fingerprints of its molecular substructures are first obtained. Second, for each protein in the database, the fingerprints of its protein domains are obtained. Third, based on known interactions between drugs and proteins, an interdependency measure between the fingerprint of each drug substructure and protein domain is then computed. Fourth, based on the interdependency measure, drug substructures and protein domains that are significantly interdependent are identified. Fifth, the existence of interaction relationship between a previously unknown drug-protein pairs is then predicted based on their constituent substructures that are significantly interdependent.ResultsTo evaluate the effectiveness of D-PIA, we have tested it with real drug-protein interaction data. D-PIA has been tested with real drug-protein interaction data including enzymes, ion channels, and protein-coupled receptors. Experimental results show that there are indeed patterns that one can discover in the interdependency relationship between drug substructures and protein domains of interacting drugs and proteins. Based on these relationships, a testing set of drug-protein data are used to see if D-PIA can correctly predict the existence of interaction between drug-protein pairs. The results show that the prediction accuracy can be very high. An AUC score of a ROC plot could reach as high as 75% which shows the effectiveness of this classifier.ConclusionsD-PIA has the advantage that it is able to perform its tasks effectively based on the fingerprints of drug and protein molecules without requiring any 3D information about their structures and D-PIA is therefore very fast to compute. D-PIA has been tested with real drug-protein interaction data and experimental results show that it can be very useful for predicting previously unknown drug-protein as well as protein-ligand interactions. It can also be used to tackle problems such as ligand specificity which is related directly and indirectly to drug design and discovery.

Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.

Saccharomyces cerevisiae strain PP5 has a phosphodiesterase (PDE) deficiency that results in heat-shock sensitivity due to the intracellular accumulation of cAMP. This strain also carries the cam mutation, which confers permeability to cAMP and, as shown here, to other compounds. Expression of rat type IV PDE in these cells caused them to revert to heat-shock resistance. Treatment of the transformed PP5 cells with rolipram, an antidepressant in humans and a potent inhibitor of type IV PDEs, reinstated sensitivity to heat shock. The biochemical properties of deletion mutants of this PDE were determined, and an active enzyme of minimum length was created. Reversion to heat-shock resistance was then used to select for PDE mutants refractory to the inhibitory effects of rolipram. Four mutants (A1, A2, A3, and A5) were isolated. Each carries a single point mutation; two have mutations in the same codon. Each mutant showed distinct properties, based on analysis of their substrate kinetics and IC50 values for a variety of inhibitors. Mutant A5 had a reduced activity for substrate, mutants A1 and A3 showed no change in substrate kinetics, and mutant A2 displayed an increase in activity. For most mutants, the drug resistance was confined to the class of drug used in the selection. This study shows that it is possible to recreate in yeast cells the susceptibility of mammalian enzymes to pharmacological agents. Our study also demonstrates that such systems can be used to select rare mutants useful in the analysis of drug-protein interactions.