Drug-receptor Interactions Research Articles

In Vitro Enzymatic and Kinetic Studies, and In Silico Drug-Receptor Interactions, and Drug-Like Profiling of the 5-Styrylbenzamide Derivatives as Potential Cholinesterase and β-Secretase Inhibitors with Antioxidant Properties.

The 5-(styryl)anthranilamides were transformed into the corresponding 5-styryl-2-(p-tolylsulfonamido)benzamide derivatives. These 5-styrylbenzamide derivatives were evaluated through enzymatic assays in vitro for their capability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase (BACE-1) activities as well as for antioxidant potential. An in vitro cell-based antioxidant activity assay involving lipopolysaccharides (LPS)-induced reactive oxygen species (ROS) production revealed that compounds 2a and 3b have the capability of scavenging free radicals. The potential of the most active compound, 5-styrylbenzamide (2a), to bind copper (II) or zinc (II) ions has also been evaluated spectrophotometrically. Kinetic studies of the most active derivatives from each series against the AChE, BChE, and β-secretase activities have been performed. The experimental results are complemented with molecular docking studies into the active sites of these enzymes to predict the hypothetical protein–ligand binding modes. Their drug likeness properties have also been predicted.

Potential antiviral activity of isorhamnetin against SARS-CoV-2 spike pseudotyped virus in vitro.

Coronavirus Disease 2019 (COVID‐19) cases and deaths are still rising worldwide, there is currently no effective treatment for severe inflammation and acute lung injury caused by new coronavirus (SARS‐COV‐2) infection. Therapies to prevent or treat COVID‐19, including antiviral drug and several vaccines, are still being development. Human angiotensin‐converting enzyme 2 (ACE2), expressing in lung, has been confirmed to be a receptor for SARS‐COV‐2 infection, interventions for attachment of spike protein of SARS‐CoV‐2 to ACE2 may be a potential approach to prevent viral infections and it is considered as a potential target for drug development. In this study, we observed that seabuckthorn and its flavonoid compounds quercetin and isorhamnetin were shown strong retention to ACE2 overexpression HEK293 (ACE2h) cells by CMC analysis. Based on drug receptor interaction analysis and viral entry studies in vitro, we evaluated the interaction of two flavonoid compounds and ACE2 as well as the inhibitory effect of the two compounds on viral entry. Surface plasmon resonance assay proved the effect that isorhamnetin bound to the ACE2, and its affinity (KD value) was at the micromolar level, that was, 2.51 ± 0.68 μM. Viral entry studies in vitro indicated that isorhamnetin inhibited SARS‐CoV‐2 spike pseudotyped virus entering ACE2h cells. Based on promising in vitro results, we proposed isorhamnetin to be a potential therapeutic candidate compound against COVID‐19.

Computational drug repurposing study elucidating simultaneous inhibition of entry and replication of novel corona virus by Grazoprevir

Outcomes of various clinical studies for the coronavirus disease 2019 (COVID-19) treatment indicated that the drug acts via inhibition of multiple pathways (targets) is likely to be more successful and promising. Keeping this hypothesis intact, the present study describes for the first-time, Grazoprevir, an FDA approved anti-viral drug primarily approved for Hepatitis C Virus (HCV), mediated multiple pathway control via synergistic inhibition of viral entry targeting host cell Angiotensin-Converting Enzyme 2 (ACE-2)/transmembrane serine protease 2 (TMPRSS2) and viral replication targeting RNA-dependent RNA polymerase (RdRP). Molecular modeling followed by in-depth structural analysis clearly demonstrated that Grazoprevir interacts with the key residues of these targets. Futher, Molecular Dynamics (MD) simulations showed stability and burial of key residues after the complex formation. Finally, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis identified the governing force of drug-receptor interactions and stability. Thus, we believe that Grazoprevir could be an effective therapeutics for the treatment of the COVID-19 pandemic with a promise of unlikely drug resistance owing to multiple inhibitions of eukaryotic and viral proteins, thus warrants further clinical studies.

A Survey of Inhibitors for the Main Protease of Coronaviruses with the Potential for Development of Broad-Spectrum Therapeutics

The coronaviruses plaguing humanity in the 21st century share much in common: a spontaneous route of origin from wild animals, a propensity to take human life, and, importantly, a highly conserved set of biological machinery necessary for viral replication. Most recently, the SARS-CoV-2 is decimating economies around the world and has claimed over two million human lives, reminding the world of a need for an effective drug against present and future coronaviruses. To date, attempts to repurpose clinically approved antiviral medications show minimal promise, highlighting the need for development of new antiviral drugs. Nucleotide analog inhibitors are a promising therapeutic candidate, but early data from clinical studies suggests these compounds have limited efficacy. However, novel compounds targeting the main protease responsible for critical steps in viral assembly are gaining considerable interest because they offer the potential for broad-spectrum coronavirus therapy. Here, we review the literature regarding potential inhibitors for the main protease of coronaviruses, especially SARS-CoV-2, analyze receptor-drug interactions, and draw conclusions about candidate inhibitors for future outbreaks. Promising candidates for development of a broad-spectrum coronavirus protease inhibitor include the neuraminidase inhibitor 3K, the peptidomimetic inhibitor 11a and 11b, the α-ketoamide inhibitor 13b, the aldehyde prodrug, and the phosphate prodrug developed by Pfizer. In silico and in vitro analyses have shown that these inhibitors strongly interact with the active site of the main protease, and to varying degrees, prevent viral replication via interactions with the largely conserved active site pockets. KEYWORDS: Severe Acute Respiratory Syndrome Coronavirus; Middle East Respiratory Syndrome Coronavirus; Severe Acute Respiratory Syndrome Coronavirus 2; Replicase Polypeptide; Protease; Neuraminidase Inhibitor; Peptidomimetic Inhibitor; α-Ketoamide Inhibitor; Molecular Docking

Unveiling the Molecular Structure of Antimalarial Drugs Chloroquine and Hydroxychloroquine in Solution through Analysis of 1H NMR Chemical Shifts

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been standard antimalarial drugs since the early 1950s, and very recently, the possibility of their use for the treatment of COVID-19 patients has been considered. To understand the drug mode of action at the submicroscopic level (atoms and molecules), molecular modeling studies with the aid of computational chemistry methods have been of great help. A fundamental step in such theoretical investigations is the knowledge of the predominant drug molecular structure in solution, which is the real environment for the interaction with biological targets. Our strategy to access this valuable information is to perform density functional theory (DFT) calculations of 1H NMR chemical shifts for several plausible molecular conformers and then find the best match with experimental NMR profile in solution (since it is extremely sensitive to conformational changes). Through this procedure, after optimizing 30 trial distinct molecular structures (ωB97x-D/6-31G(d,p)-PCM level of calculation), which may be considered representative conformations, we concluded that the global minimum (named M24), stabilized by an intramolecular N-H hydrogen bond, is not likely to be observed in water, chloroform, and dimethyl sulfoxide (DMSO) solution. Among fully optimized conformations (named M1 to M30, and MD1 and MD2), we found M12 (having no intramolecular H-bond) as the most probable structure of CQ and HCQ in water solution, which is a good approximate starting geometry in drug-receptor interaction simulations. On the other hand, the preferred CQ and HCQ structure in chloroform (and CQ in DMSO-d6) solution was assigned as M8, showing the solvent effects on conformational preferences. We believe that the analysis of 1H NMR data in solution can establish the connection between the macro level (experimental) and the sub-micro level (theoretical), which is not so apparent to us and appears to be more appropriate than the thermodynamic stability criterion in conformational analysis studies.

Drug Repurposing Strategy (DRS): Emerging Approach to Identify Potential Therapeutics for Treatment of Novel Coronavirus Infection.

Drug repurposing is also termed as drug repositioning or therapeutic switching. This method is applied to identify the novel therapeutic agents from the existing FDA approved clinically used drug molecules. It is considered as an efficient approach to develop drug candidates with new pharmacological activities or therapeutic properties. As the drug discovery is a costly, time-consuming, laborious, and highly risk process, the novel approach of drug repositioning is employed to increases the success rate of drug development. This strategy is more advantageous over traditional drug discovery process in terms of reducing duration of drug development, low-cost, highly efficient and minimum risk of failure. In addition to this, World health organization declared Coronavirus disease (COVID-19) as pandemic globally on February 11, 2020. Currently, there is an urgent need to develop suitable therapeutic agents for the prevention of the outbreak of COVID-19. So, various investigations were carried out to design novel drug molecules by utilizing different approaches of drug repurposing to identify drug substances for treatment of COVID-19, which can act as significant inhibitors against viral proteins. It has been reported that COVID-19 can infect human respiratory system by entering into the alveoli of lung via respiratory tract. So, the infection occurs due to specific interaction or binding of spike protein with angiotensin converting enzyme-2 (ACE-2) receptor. Hence, drug repurposing strategy is utilized to identify suitable drugs by virtual screening of drug libraries. This approach helps to determine the binding interaction of drug candidates with target protein of coronavirus by using computational tools such as molecular similarity and homology modeling etc. For predicting the drug-receptor interactions and binding affinity, molecular docking study and binding free energy calculations are also performed. The methodologies involved in drug repurposing can be categorized into three groups such as drug-oriented, target-oriented and disease or therapy-oriented depending on the information available related to quality and quantity of the physico-chemical, biological, pharmacological, toxicological and pharmacokinetic property of drug molecules. This review focuses on drug repurposing strategy applied for existing drugs including Remdesivir, Favipiravir, Ribavirin, Baraticinib, Tocilizumab, Chloroquine, Hydroxychloroquine, Prulifloxacin, Carfilzomib, Bictegravir, Nelfinavir, Tegobuvir and Glucocorticoids etc to determine their effectiveness toward the treatment of COVID-19.

Photochemical control of drug efficacy - a comparison of uncaging and photoswitching ifenprodil on NMDA receptors.

Ifenprodil is an important negative allosteric modulator of the N-methyl-D-aspartate (NMDA) receptors. We have synthesized caged and photoswitchable derivatives of this small molecule drug. Caged ifenprodil was biologically inert before photolysis, UV irradiation efficiently released the drug allowing selective inhibition of GluN2B-containing NMDA receptors. Azobenzene-modified ifenprodil, on the other hand, is inert in both its trans and cis configurations, although in silico modeling predicted the trans form to be able to bind to the receptor. The disparity in effectiveness between the two compounds reflects, in part, the inherent ability of each method in manipulating the binding properties of drugs. With appropriate structure-activity relationship uncaging enables binary control of effector binding, whereas photoswitching using feely diffusable chromophores shifts the dose-response curve of drug-receptor interaction. Our data suggest that the efficacy of pharmacophores having a confined binding site such as ifenprodil can be controlled more easily by uncaging in comparison to photoswitching.

In silico Molecular Docking, DFT Analysis and ADMET Studies of Carbazole Alkaloid and Coumarins from Roots of Clausena anisata: A Potent Inhibitor for Quorum Sensing.

IntroductionIn modern drug design, in silico methods are largely used to understand drug-receptor interactions and quantum chemical properties. In the present study, a computational de novo design approach was used to confirm mode of binding for antibacterial activity, elucidating quantum chemical properties and ADMET-drug-likeness of carbazole alkaloid (1) and three coumarins (2–4) isolated from roots of Clausena anisata.MethodsDocking studies were performed with DNA-Gyrase (6F86) and LasR binding domain (2UV0) employing a flexible ligand docking approach using AutoDock Vina. SwissADME prediction and toxicological predictions were performed by ADMET. The optimized structures and molecular electrostatic potential surface of the isolated compounds were predicted by DFT analysis using B3LYP/6-31G basis levels.Results and DiscussionThe docking results revealed that compound 3 showed better docking scores against both DNA gyrase B and LasR binding domain compared with ciprofloxacin with potential as an inhibitor of bacterial DNA gyrase and quorum sensing LasR binding domain. The SwissADME prediction results showed that all the isolated compounds (1–4) satisfy Lipinski’s rule of five with zero violations. Toxicological prediction results suggested that all compounds and ciprofloxacin are non-hepatotoxic, non-carcinogenic, non-irritant, immunogenic, and non-cytotoxic. The DFT analysis results revealed that compound 3 has large electronegativity (χeV), global softness (σ eV−1), global electrophilicity (ωeV), and mutagenicity value closer to ciprofloxacin (with LD50 value of 480 mg/kg) suggesting better bioactivity and chemical reactivity with considerable intra-molecular charge transfer between electron-donor to electron-acceptor groups.ConclusionOverall, compound 3 may serve as a lead molecule that could be developed into a potent E. coli DNA gyrase B inhibitor and efficient inhibitor for quorum sensing auto-inducer LasR binding domain of Pseudomonas aeruginosa.

Halo-tagged protein immobilization: Effect of halide linkers on peak profile and drug-protein interaction

In previous work, we have established a one-step method to immobilize halo-tagged proteins onto microspheres through the covalent bond formed between the halo-tag and the halide linkers on the support surface. We observe extremely tailed peaks of most of drugs on the immobilized proteins, which is reasoned by the nonspecific interaction between the linkers and the drugs. To prove this, the current work designed five different halide linkers for the immobilization of beta2-adrenoceptor (β2-AR). We applied the immobilized receptor to systematically realize the effects of these halide linkers on drug-receptor interaction by analyzing peak profiles of five drugs. The retention times and the half-widths of the drugs appeared to be negatively correlated to the atom numbers of the linkers in the range of 6–13 atoms. Subsequent increase of linker atoms resulted in reduced retention times and wider peaks of the drugs. Applying identical linker length, we observed clear reduced retention times and half-widths of the five drugs than the linker in the absence of oxygen atom. Such improvement was dominated by the number of oxygen atoms. These indicated that linker S-4 (2-(2-(2-(2-chloroethoxy) ethoxy) ethoxy) acetic acid) was optimal to eliminate the unwanted non-specific interactions. In comparison with the columns prepared by linker S-1 (6-chlorocaproic acid) and histidine tagged β2-AR, the drugs on the linker S-4 column gave greater dissociation rate constants (e.g. 60.3±0.3 s-1 for salbutamol), which is closer to the data in literatures. Taking together, we concluded that optimization of the linker structure plays particular role in reducing the non-specific interaction between the immobilized protein and the drugs, thereby making the determination of drug-protein interaction more reliable.

Advances in cell membrane chromatography

Cell membrane chromatography (CMC) is a biomimetic chromatographic method based on the ability of membrane receptors to selectively interact with their ligands in vivo. Using membrane receptors as a stationary phase, the CMC method helps in determining the binding characteristics between ligands and membrane receptors and in efficiently identifying specific target components in a complex sample that produce the cellular biological effects of ligands (drugs, antibodies, enzymes, cytokines, etc.). CMC is an analytical tool for revealing characteristics of ligand–receptor interactions, screening and discovering target substances, and accurately controlling the quality of drugs.Since establishment of CMC in the early 1990s, with the rapid development of cell biology, significant progress has been made in the development of high-expression receptors, engineered cell cultures, and standardized preparations, which allowed in vitro immobilization of cell membrane receptors and miniaturization of binding assays. A variety of CMC models have been established using different membrane receptors as a stationary phase, and many new methods have been developed by combining CMC with high-performance liquid chromatography (HPLC)/mass spectrometry or HPLC-IT-TOF technologies. CMC methods have been widely used to study drug–receptor interactions and to screen complex samples for effective or harmful components.

Drug-Receptor Interaction Kinetics: A Case Study of Immobilized Endothelin Receptor A and Its Ligands

The kinetic study in drug-receptor interaction analysis is of great significance for the efficacy and safety of a drug. In previous work, we established a one-step method to immobilize the endothelin receptor A (ETA) on the surface of macroporous silica gel through a covalent interaction between the epidermal growth factor tyrosine kinase (EGFR) at the C terminal of ETA and the covalent inhibitor ibrutinib. The affinity stationary phase was used for lead screening and investigating the thermodynamic binding of ligands and ETA. Herein, we intend to introduce the ETA column in the kinetic study of the receptor and three specific ligands (bosentan, macitentan, and ambrisentan) as well as three leads (ferulic acid, berberine, and palmatine) from herbal medicines. Three classical methods in affinity chromatography, nonlinear chromatography, peak decay, and peak profiling, were performed to determine the dissociation rate constants of the ligands to ETA. A comparison of the three methods was made and the binding parameters were discussed. The three methods gave the same order of the dissociation constants between the six ligands and ETA. The results indicated that the immobilized ETA can be used for not only thermodynamic studies but also kinetic investigations, which provides a reliable and fast evaluation for drug-receptor interaction analysis.

Cell-based 3D bionic screening by mimicking the drug-receptor interaction environment in vivo.

Most small-molecule drugs influence cell behavior through their interaction with one or more cellular proteins. The efficacy is unanticipated in the later stages of drug development if small-molecule drugs are discovered in the absence of a biological context. Bionic screening is an in vivo drug-receptor interaction platform that can identify small molecules with recognized activity, improving the likelihood of drug efficacy in the clinic. Here, we report the design of an innovative cell-based bionic screening system using 3D microcarrier cultures to simulate in vivo conditions and facilitate small-molecule drug discovery. Through its combination with HPLC/MS, the method can comprehensively identify small-molecule lead compounds in arbitrarily complex systems in an unbiased manner. In particular, cell-covered microcarriers provide a high-density of cells for affinity performance assessments in the absence of appreciable cell damage and maintain immunogenicity, the 3D structure of which is similar to tissue morphology in vivo, thereby mimicking in vivo drug-receptor interactions. The method is scalable, easy to handle, and requires minimal optimization across a range of different cell lines to realize high-throughput drug screening for the corresponding diseases. This provides a valuable tool for lead compound discovery in more physiologically relevant systems and may address the lack of clinically available drugs.

Molecular Modeling and Bioinformatics Analysis of Drug-Receptor Interactions in the System Formed by Glargine, Its Metabolite M1, the Insulin Receptor, and the IGF1 Receptor

Introduction:Insulin and insulin-like growth factor type 1 (IGF1) regulate multiple physiological functions by acting on the insulin receptor (IR) and insulin-like growth factor type 1 receptor (IGF1R). The insulin analog glargine differs from insulin in three residues (GlyA21, ArgB31, ArgB32), and it is converted to metabolite M1 (lacks residues ArgB31 and ArgB32) by in vivo processing. It is known that activation of these receptors modulates pathways related to metabolism, cell division, and growth. Though, the structures and structural basis of the glargine interaction with these receptors are not known.Aim:To generate predictive structural models, and to analyze the drug/receptor interactions in the system formed by glargine, its metabolite M1, IR, and IGF1R by using bioinformatics tools.Methods:Ligand/receptor models were built by homology modeling using SWISSMODEL, and surface interactions were analyzed using Discovery Studio® Visualizer. Target and hetero target sequences and appropriate template structures were used for modeling.Results:Our glargine/IR and metabolite M1/IR models showed an overall symmetric T-shaped conformation and full occupancy with four ligand molecules. The glargine/IR model revealed that the glargine residues ArgB31 and ArgB32 fit in a hydrophilic region formed by the α-chain C-terminal helix (αCT) and the cysteine-rich region (CR) domain of this receptor, close to the CR residues Arg270-Arg271-Gln272 and αCT residue Arg717. Regarding IGF1R, homologous ligand/receptor models were further built assuming that the receptor is in a symmetrical T-shaped conformation and is fully occupied with four ligand molecules, similar to what we described for IR. Our glargine/IGF1R model showed the interaction of the glargine residues ArgB31 and ArgB32 with Glu264 and Glu305 in the CR domain of IGF1R.Conclusion:Using bioinformatics tools and predictive modeling, our study provides a better understanding of the glargine/receptor interactions.

The action of aripiprazole and brexpiprazole at the receptor level in singultus.

The hiccup (Latin singultus) is an involuntary periodic contraction of the diaphragm followed by glottic closure, which can be a rare side effect of aripiprazole. In contrast to the structurally closely related aripiprazole, brexpiprazole was not associated with this particular adverse drug reaction. Having two very similar drugs that differ in their ability to induce hiccups represents a unique opportunity to gain insight into the receptors involved in the pathophysiology of the symptom and differences in clinical effects between aripiprazole and brexpiprazole. The overlap between maneuvers used to terminate paroxysmal supraventricular tachycardia and those employed to terminate bouts of hiccups suggests that activation of efferent vagal fibers can be therapeutic in both instances. Recent work seems to support a pivotal role for serotonin receptors in such vagal activation. It is unlikely that a unique receptor-drug interaction could explain the different effects of the examined drugs on hiccup. The different effect is most likely the consequence of several smaller effects at more than one receptor. Brexpiprazole is a highly affine (potent) α2C antagonist and, therefore, also an indirect 5-HT1A agonist. In contrast, aripiprazole is a partial 5-HT1A agonist (weak antagonist) and an HT3 antagonist. Activation of 5-HT1A receptors enhances vagal activity while HT3 blockade reduces it. Vagus nerve activation is therapeutic for hiccups. A definitive answer continues to be elusive.

G protein-coupled receptor-in-paper, a versatile chromatographic platform to study receptor-drug interaction

High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (β2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that β2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to β2-AR were calculated to be 2.02 × 104 M−1, 1.15 × 104 M−1, 1.75 × 104 M−1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.

IN SILICO ADMET PROFILING AND MOLECULAR DOCKING OF NOVEL SUBSTITUTED THIENO[3,2-d] PYRIMIDINES AGAINST LIGAND BINDING DOMAIN OF THE HUMAN PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR GAMMA IN COMPLEX WITH SYNTHETIC AGONIST

Pyrimidine ring system has wide range of pharmacological activities in the form of substituted and fused ring system and its derivatives. In this study we use some new computational tools for predicting ADMET, Pharmacological profile and Molecular docking of some novel substituted Thieno[3,2-d]pyrimidine. The side effect during the investigation is Carcinogenicity, Hepatotoxicity, etc., Molecular docking by using QSAR Software (Drug-receptor Interaction) it also focuses.

Pharmacokinetics and pharmacodynamics

Pharmacokinetics and pharmacodynamics are vast and complex topics with medical textbooks dedicating entire chapters to explain the details. Due to this, only some of the many relevant concepts have been explained. Compartment models have not been discussed despite their importance to the anaesthetist. These two concepts have been described as mathematical expressions of the relationship between drug dose and concentration for pharmacokinetics, and drug concentration and effect for pharmacodynamics. Concentration provides the link between pharmacokinetics and pharmacodynamics and will enable rational dosing. Pharmacokinetics consists of four linked processes namely absorption, distribution, biotransformation and excretion. Clinical pharmacokinetics including bioavailability, bioequivalence, first-order and zero-order kinetics as well as context-sensitive half-life have been discussed. A basic explanation of drug-receptor interactions, dose-response relationships and therapeutic index is given for pharmacodynamics. The purpose of understanding these concepts is to enable doctors to simultaneously achieve the desired therapeutic effects while minimising adverse effects. This is particularly relevant to the practising anaesthetist where multiple agents are required to produce optimal anaesthesia.

Application of a stepwise frontal analysis method in cell membrane chromatography

Bio-affinity chromatography is used in the study of drug-receptor interactions. A stepwise frontal analysis (SFA) method was developed based on frontal analysis (FA). A high expression alpha 1A adrenergic receptor (α1A AR) cell membrane chromatography (CMC) method was then developed and combined with SFA to investigate the affinity of three model α1A AR-binding drugs towards α1A AR. Equilibrium dissociation constant (Kd) values for drug-receptor interactions were determined by FA and SFA; results showed that these methods were highly consistent. The results demonstrate that the CMC/SFA method is a time-saving and less wasteful method than traditional method for the evaluation of drug-receptor binding characteristics, and could be used to study the interactions between drugs and membrane receptors.

Alkylated monoterpene indole alkaloid derivatives as potent P-glycoprotein inhibitors in resistant cancer cells.

Aiming at generating a series of monoterpene indole alkaloids with enhanced multidrug resistance (MDR) reversing activity in cancer, two major epimeric alkaloids isolated from Tabernaemontana elegans, tabernaemontanine (1) and dregamine (2), were derivatized by alkylation of the indole nitrogen. Twenty-six new derivatives (3-28) were prepared by reaction with different aliphatic and aromatic halides, whose structures were elucidated mainly by NMR, including 2D NMR experiments. Their MDR reversal ability was evaluated through a functional assay, using as models resistant human colon adenocarcinoma and human ABCB1-gene transfected L5178Y mouse lymphoma cells, overexpressing P-glycoprotein (P-gp), by flow cytometry. A considerable increase of activity was found for most of the derivatives, being the strongest P-gp inhibitors those sharing N-phenethyl moieties, displaying outstanding inhibitory activity, associated with weak cytotoxicity. Chemosensitivity assays were also performed in a model of combination chemotherapy in the same cell lines, by studying the invitro interactions between the compounds and the antineoplastic drug doxorubicin. Most of the compounds have shown strong synergistic interactions with doxorubicin, highlighting their potential as MDR reversers. QSAR models were also explored for insights on drug-receptor interaction, and it was found that lipophilicity and bulkiness features were associated with inhibitory activity, although linear correlations were not observed.

Microtiter plate-based antibody-competition assay to determine binding affinities and plasma/blood stability of CXCR4 ligands

C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Thus, CXCR4 represents a promising drug target and several CXCR4 antagonizing agents are in preclinical or clinical development. Important parameters in drug lead evaluation are determination of binding affinities to the receptor and assessment of their stability and activity in plasma or blood of animals and humans. Here, we designed a microtiter plate-based CXCR4 antibody competition assay that enables to measure inhibitory concentrations (IC50 values) and affinity constants (Ki values) of CXCR4 targeting drugs. The assay is based on the observation that most if not all CXCR4 antagonists compete with binding of the fluorescence-tagged CXCR4 antibody 12G5 to the receptor. We demonstrate that this antibody-competition assay allows a convenient and cheap determination of binding affinities of various CXCR4 antagonists in living cells within just 3 h. Moreover, the assay can be performed in the presence of high concentrations of physiologically relevant body fluids, and thus is a useful readout to evaluate stability (i.e. half-life) of CXCR4 ligands in serum/plasma, and even whole human and mouse blood ex vivo. Thus, this optimized 12G5 antibody-competition assay allows a robust and convenient determination and calculation of various important pharmacological parameters of CXCR4 receptor-drug interaction and may not only foster future drug development but also animal welfare by reducing the number of experimental animals.