Screening Of New Drug Candidates Research Articles

REMD Simulations of Full-Length Alpha-Synuclein Together with Ligands Reveal Binding Region and Effect on Amyloid Conversion.

Alpha-synuclein is a key protein involved in the development and progression of Parkinson’s disease and other synucleinopathies. The intrinsically disordered nature of alpha-synuclein hinders the computational screening of new drug candidates for the treatment of these neurodegenerative diseases. In the present work, replica exchange molecular dynamics simulations of the full-length alpha-synuclein together with low-molecular ligands were utilized to predict the binding site and effect on the amyloid-like conversion of the protein. This approach enabled an accurate prediction of the binding sites for three tested compounds (fasudil, phthalocyanine tetrasulfonate, and spermine), giving good agreement with data from experiments by other groups. Lots of information about the binding and protein conformational ensemble enabled the suggestion of a putative effect of the ligands on the amyloid-like conversion of alpha-synuclein and the mechanism of anti- and pro-amyloid activity of the tested compounds. Therefore, this approach looks promising for testing new drug candidates for binding with alpha-synuclein or other intrinsically disordered proteins and at the same time the estimation of the effect on protein behavior, including amyloid-like aggregation.

Screening novel CNS drug candidates for P-glycoprotein interactions using the cell line iP-gp: In vitro efflux ratios from iP-gp and MDCK-MDR1 monolayers compared to brain distribution data from mice

Drug efflux by P-glycoprotein (P-gp, ABCB1) is considered as a major obstacle for brain drug delivery for small molecules. P-gp-expressing cell monolayers are used for screening of new drug candidates during early states of drug development. It is, however, uncertain how well the in vitro studies can predict the in vivo P-gp mediated efflux at the blood–brain barrier (BBB). We previously developed a novel cell line of porcine origin, the iP-gp cell line, with high transepithelial resistance and functional expression of human P-gp. The aim of the present study was to evaluate the applicability of the cell line for screening of P-gp interactions of novel drug candidates. For this purpose, bidirectional fluxes of 14 drug candidates were measured in iP-gp cells and in MDCK-MDR1 cells, and compared with pharmacokinetic data obtained in male C57BL/6 mice. The iP-gp cells formed extremely tight monolayers (>15 000 Ω∙cm2) as compared to the MDCK- MDR1 cells (>250 Ω∙cm2) and displayed lower Papp,a-b values. The efflux ratios obtained with iP-gp and MDCK-MDR1 monolayers correlated with Kp,uu,brain values from the in vivo studies, where compounds with the lowest Kp,uu,brain generally displayed the highest efflux ratios. 12 of the tested compounds displayed a poor BBB penetration in mice as judged by Kp,uu less than 1. Of these compounds, nine compounds were categorized as P-gp substrates in the iP-gp screening, whereas analysis of data estimated in MDCK-MDR1 cells indicated four compounds as potential substrates. The results suggest that the iP-gp cell model may be a sensitive and useful screening tool for drug screening purposes to identify possible substrates of human P-glycoprotein.

Development of a zebrafish screening model for diabetic retinopathy induced by hyperglycemia: Reproducibility verification in animal model

This study aimed at creating a zebrafish screening model for diabetic retinopathy, and evaluated the effects of aflibercept, which is being used to treated diabetic retinopathy. A morphological change occurred at 160 mM of glucose. The survival and hatching rate decreased in a dose-dependent manner. In the 130 mM glucose group, the retinal vessel diameter was more than double that in the normal group. The zebrafish embryo morphology changed in 200 μg/mL and 400 μg/mL at aflibercept. The survival and hatching rate decrease at 400 μg/mL. Aflibercept 100 μg/mL was a nontoxic and effective dose for the zebrafish diabetic retinopathy model. The expression of diabetic retinopathy inflammatory markers was increased in hyperglycemia. But the inflammation was improved by aflibercept in the zebrafish eye. In a zebrafish diabetic retinopathy model, the diameters of retinal vessels were reduced after treatment with aflibercept, and molecular biological and histopathological efficacy was confirmed. This model can serve for screening of new drug candidates for treatment of in diabetic retinopathy.

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction.

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug–DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA–DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

Down-regulation of cathepsin S and matrix metalloproteinase-9 via Src, a non-receptor tyrosine kinase, suppresses triple-negative breast cancer growth and metastasis

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer with poor prognosis. In the present study, we demonstrated that Src, a non-receptor tyrosine kinase, might provide an effective therapeutic strategy to overcome TNBC invasion and metastasis, which are mediated via the synergistic action of the lysosomal enzyme cathepsin S (CTSS) and gelatinase MMP-9. Knock-down of MMP-9 and CTSS using siRNAs resulted in a synergistic suppression of MDA-MB-231 cell invasion, which was similarly observed with pharmacological inhibitors. During the screening of new drug candidates that suppress both CTSS and MMP-9, BJ-2302, a novel 7-azaindolin-2-one derivative, was discovered. Src, an upstream activator of both pathways (PI3K/Akt and Ras/Raf/ERK) responsible for the expression of CTSS and MMP-9, was identified as a high-affinity target of BJ-2302 (IC90: 3.23 µM) through a Src kinase assay and a drug affinity responsive target stability (DARTS) assay. BJ-2302 effectively suppressed MDA-MB-231 cell invasion (Matrigel invasion assay) and metastasis (chorioallantoic membrane assay xenografted with MDA-MB-231-luc2-tdTomato cancer cells). Unlike Z-FL-COCHO (potent CTSS inhibitor), BJ-2302 did not induce any cytotoxicity in MCF-10A normal breast epithelial cells. Additionally, BJ-2302 (1 mg/kg) strongly suppressed TNBC cell proliferation in vitro and tumor growth in a xenograft mouse tumor model. The anti-metastatic and anti-tumor effects of BJ-2302 were superior to those of Z-FL-COCHO (1 mg/kg) or batimastat (30 mg/kg), a pan-MMP inhibitor. In summary, inhibition of Src kinase suppressed TNBC tumor growth and metastasis, and Src inhibitors such as BJ-2302 may constitute a novel therapeutic tool to treat breast cancer that expresses high levels of CTSS and MMP-9.

An In Vitro Model for Candida albicans⁻Streptococcus gordonii Biofilms on Titanium Surfaces.

The oral cavity serves as a nutrient-rich haven for over 600 species of microorganisms. Although many are essential to maintaining the oral microbiota, some can cause oral infections such as caries, periodontitis, mucositis, and endodontic infections, and this is further exacerbated with dental implants. Most of these infections are mixed species in nature and associated with a biofilm mode of growth. Here, after optimization of different parameters including cell density, growth media, and incubation conditions, we have developed an in vitro model of C. albicans–S. gordonii mixed-species biofilms on titanium discs that is relevant to infections of peri-implant diseases. Our results indicate a synergistic effect for the development of biofilms when both microorganisms were seeded together, confirming the existence of beneficial, mutualistic cross-kingdom interactions for biofilm formation. The morphological and architectural features of these dual-species biofilms formed on titanium were determined using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Mixed biofilms formed on titanium discs showed a high level of resistance to combination therapy with antifungal and antibacterial drugs. This model can serve as a platform for further analyses of complex fungal/bacterial biofilms and can also be applied to screening of new drug candidates against mixed-species biofilms.

Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor

Monitoring the expression of cell-surface receptors, their interaction with extracellular ligands, and their fate upon ligand binding is important for understanding receptor function and developing new therapies. We describe a cell-based method that utilizes bioluminescent protein complementation technology to interrogate binding of a cellular receptor with its extracellular protein ligand, specifically LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Purified, full-length tagged PCSK9 is added to assay wells containing cells that stably express LDLR with an extracellular complementary tag. When the tagged PCSK9 binds the receptor, a bright luminescence signal is generated. The interaction is detected at the cell membrane with add-and-read simplicity, no wash steps, and flexibility, allowing data to be collected in endpoint format, kinetically, or with bioluminescent imaging. The assay is flexible, is rapid, and reports accurate biology. It is amenable to 96-well and 384-well formats, and the robustness allows for screening of new drug candidates (Z′ = 0.83). The assay reports correct potencies for antibody titrations across a 50%–150% potency range and detects potency changes due to heat stress, suggesting that it may be useful during drug development. This assay technology can be broadly applied when studying other receptors with their extracellular ligands, whether protein or small-molecule binding partners.

Predicting the Absorption Potential of Chemical Compounds Through a Deep Learning Approach

The human colorectal carcinoma cell line (Caco-2) is a commonly used in-vitro test that predicts the absorption potential of orally administered drugs. In-silico prediction methods, based on the Caco-2 assay data, may increase the effectiveness of the high-throughput screening of new drug candidates. However, previously developed in-silico models that predict the Caco-2 cellular permeability of chemical compounds use handcrafted features that may be dataset-specific and induce over-fitting problems. Deep Neural Network (DNN) generates high-level features based on non-linear transformations for raw features, which provides high discriminant power and, therefore, creates a good generalized model. We present a DNN-based binary Caco-2 permeability classifier. Our model was constructed based on 663 chemical compounds with in-vitro Caco-2 apparent permeability data. Two hundred nine molecular descriptors are used for generating the high-level features during DNN model generation. Dropout regularization is applied to solve the over-fitting problem and the non-linear activation. The Rectified Linear Unit (ReLU) is adopted to reduce the vanishing gradient problem. The results demonstrate that the high-level features generated by the DNN are more robust than handcrafted features for predicting the cellular permeability of structurally diverse chemical compounds in Caco-2 cell lines.

Integrating multiple omics to unravel mechanisms of Cyclosporin A induced hepatotoxicity in vitro

In order to improve attrition rates of candidate-drugs there is a need for a better understanding of the mechanisms underlying drug-induced hepatotoxicity. We aim to further unravel the toxicological response of hepatocytes to a prototypical cholestatic compound by integrating transcriptomic and metabonomic profiling of HepG2 cells exposed to Cyclosporin A. Cyclosporin A exposure induced intracellular cholesterol accumulation and diminished intracellular bile acid levels. Performing pathway analyses of significant mRNAs and metabolites separately and integrated, resulted in more relevant pathways for the latter. Integrated analyses showed pathways involved in cell cycle and cellular metabolism to be significantly changed. Moreover, pathways involved in protein processing of the endoplasmic reticulum, bile acid biosynthesis and cholesterol metabolism were significantly affected. Our findings indicate that an integrated approach combining metabonomics and transcriptomics data derived from representative in vitro models, with bioinformatics can improve our understanding of the mechanisms of action underlying drug-induced hepatotoxicity. Furthermore, we showed that integrating multiple omics and thereby analyzing genes, microRNAs and metabolites of the opposed model for drug-induced cholestasis can give valuable information about mechanisms of drug-induced cholestasis in vitro and therefore could be used in toxicity screening of new drug candidates at an early stage of drug discovery.

ToxDBScan: Large-scale similarity screening of toxicological databases for drug candidates.

We present a new tool for hepatocarcinogenicity evaluation of drug candidates in rodents. ToxDBScan is a web tool offering quick and easy similarity screening of new drug candidates against two large-scale public databases, which contain expression profiles for substances with known carcinogenic profiles: TG-GATEs and DrugMatrix. ToxDBScan uses a set similarity score that computes the putative similarity based on similar expression of genes to identify chemicals with similar genotoxic and hepatocarcinogenic potential. We propose using a discretized representation of expression profiles, which use only information on up- or down-regulation of genes as relevant features. Therefore, only the deregulated genes are required as input. ToxDBScan provides an extensive report on similar compounds, which includes additional information on compounds, differential genes and pathway enrichments. We evaluated ToxDBScan with expression data from 15 chemicals with known hepatocarcinogenic potential and observed a sensitivity of 88%. Based on the identified chemicals, we achieved perfect classification of the independent test set. ToxDBScan is publicly available from the ZBIT Bioinformatics Toolbox.

Bestimmung der Inhibition der mitochondrialen Biogenese

Mitochondria are produced from the transcription and translation of genes both in the nuclear genome and in the mitochondrial genome, termed mitobiogenesis. Measuring mitobiogenesis is useful for early toxicity screening of new drug candidates. This requires an assay that is quantitative, specific, and reproducible and is also flexible enough to be applied in a variety of cell types.

Increased consistency and efficiency in routine potency testing by bioassay with direct use of cryopreserved (ready-to-plate) cells

The use of cells as a cryopreserved, readily available reagent has facilitated high-throughput screening of new drug candidates by bioassay. This practice is considerably less labor intensive and allows more flexibility in laboratory testing than traditional continuous cell culture. We have shown that this practice can be adapted to cell proliferation and reporter gene assay formats used in routine sample testing for determination of relative potency of commercial product in a Quality Control Laboratory. The ability to use the same, optimized population of cells provides consistency in an assay over time. Measures of assay performance to indicate maintenance of the validated state of a method and to determine benefit on variation in potency results were compared between cultured and cryopreserved (frozen ready-to-plate) cells. Control of the cellular component, which is the most variable aspect of most cell based potency assays, allowed detection of more minor contributors to variability. In a cell proliferation assay format, the final result was a highly precise bioassay (repeatability CV of 2%). An improvement in process capability (Cp) was noted when ready-to-plate cells were used in the studies completed over long periods of time.

Pharmaceutical physical form characterisation with fast (>200 °C min−1) DSC heating rates

Differential scanning calorimetry (DSC) has many applications during preformulation screening of new drug candidates, but definitive assignment of peaks to specific events in the sample is difficult without supplementary data from other techniques. To some extent these problems can be overcome by running multiple experiments at different heating rates. Typically 2 and 20 °C min−1 are indicated. However, modern instruments are capable of achieving much faster heating rates (up to 750 °C commercially); with this extended range comes a new capacity for investigating the physical form of materials. Here, the use of fast DSC heating rates for materials characterisation is explored, focussing on determination of melting temperatures, glass formation and polymorph screening.

Current In Vitro High Throughput Screening Approaches to Assess Nuclear Receptor Activation

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening.

Analysis of drug–protein binding by ultrafast affinity chromatography using immobilized human serum albumin

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug–protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug-binding studies and in the high-throughput screening of new drug candidates.

Blockade of the I Ks potassium channel: An overlooked cardiovascular liability in drug safety screening?

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I Ks channel, which along with the transient outward current (I to) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I Ks blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I Ks channel blockade and describe the discovery of a potent I Ks blocker whose activity was not detected by standard hERG or in vitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) in vivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I Ks blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I Ks blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I Ks) activity. Assay Drug Development Technology 5, 617–627]). Because of the presence of I Ks channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I Ks blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I Ks liability before testing in humans. The possibility of undetected I Ks blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427–1438] in the ICH S7B regulatory guidelines.

A System for LogD Screening of New Drug Candidates Using a Water-Plug Injection Method and Automated Liquid Handler

An efficient system for screening for the octanol/water distribution coefficient (logD) was developed. We previously developed a 96-well—based high-speed logD assay system (HSlogD) using a water-plug aspiration/injection method by auto liquid sampler (HTS-PAL) to analyze the water phase without contamination of the upper octanol phase. In this report, we introduce an automated liquid-handling system (Biomek2000) into the previously reported HSlogD system to dilute the octanol phase automatically. As a result, throughput is increased, allowing the analysis of 400 compounds per week with a range of 0 < logD < 5.

UV-Light Exposed Prion Protein Fails to Form Amyloid Fibrils

Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.

Capillary Electrophoresis with Laser-Induced Fluorescence Detection as a Tool for Enzyme Characterization and Inhibitor Screening

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.

Prediction of human absorption of natural compounds by the non-everted rat intestinal sac model

A major concern in natural drug research is that many substances with potent biological activity in vitro are unable to generate good activity in vivo owing to their poor water-solubility, poor permeability and/or poor stability. The permeability of drug candidates across the intestinal mucosa is one of the most important factors in defining drug bioavailability and biological activity. In order to screen promising compounds for further investigation, a non-everted rat intestinal sac model has been developed successfully to assay the permeability of natural compounds and to predict their human absorption. In this system, the drug solution was placed in non-everted intestinal sacs (NEIS), which were placed in an acceptor solution and the permeability of drug across intestine walls was determined. The feasibility of this method has been validated and demonstrated for 11 model compounds chosen from currently marketed drugs whose human fraction absorbed ( F a) data have been reported. The results of the studies indicate that a good relationship exists between the permeability of the model drugs and their corresponding F a data. The permeability of 13 natural compounds was evaluated using this system. Only fraxinellone and vitexin-7-glucoside exhibited high intestinal permeability, and predictive of excellent human absorption, which awaits confirmation from further investigation in vivo. This model provides an alternative method to everted intestinal sacs for the evaluation of in vitro permeability in rats, and for estimating human absorption of drugs. It may therefore hold great promise for oral absorption screening of new drug candidates.