Drug Screening Methods SK Gupta
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High Throughput Screening for High Speed Drug DiscoveryCHAPTER 1

 
HIGH THROUGHPUT SCREENING FOR HIGH SPEED DRUG DISCOVERY
Discovery of new lead compounds for novel therapeutic targets is a multi-step process involving drug design, synthesis and its pharmacological screening. As the first step towards drug development the biological target needs to be identified. In the next step, the potential compounds are screened against the identified target. Conventionally this has been a slow and tedious manual process requiring huge investment of manpower, time and money. Depending upon the steps involved in the screening protocol, like centrifugation, phase extraction, filtration, precipitation and subsequent signal amplification and detection, the evaluation of a few hundred compounds might take weeks to months. Many more years elapse in trying to understand the molecular mechanisms and for evaluating the kinetics and toxicity, before a lead compound is generated for investigation in clinical studies. Consequently, the conventional drug discovery program has been termed as Low Throughput Screening (LTS).
 
What is High Throughput Screening (HTS)?
The last two decades have seen innovations in technology that have helped to evolve LTS into an automated, microprocessor controlled robotic process called ‘High Throughput Screening (HTS)’. This qualitative leap in drug discovery paradigm has been achieved via a synergy of chemistry, biology, engineering and informatics. HTS has helped to speed up LTS and now over 50,000-100,000 compounds can be screened per week, against the validated biological target. Further advancements are making it possible to screen 10,000-100,000 compounds within 24 h time and are called as ultra high throughput screening (uHTS). High throughput synthesis of large number of test compounds in a lesser time is also a reality now. This is achieved by combinatorial chemistry using parallel synthesis technology. A similar strategy has also been adopted in studies towards molecular mechanisms of drug action, absorption, metabolism and toxicity studies to decrease the time spent in preclinical studies in the drug development program. Thus, valuable compounds can now be selected rapidly from a large number of samples with minimal human and cost involvement.4
 
HTS Pharmacodynamic Studies
After the fundamental biological research, the effective target is identified and validated for its function. Then the method development takes a few months to initiate screening process. In HTS, the trend is to replace radiolabeling by simple luminescence techniques. Since the yield of target protein or purified biological target is generally low (often obtained only in a few milligrams), technologies that permit screening with reduced volumes (3–20 µl) and reduced protein/ligand have played a pivotal role in facilitating HTS. Development of detection techniques having ultra high precisions are used in these assays to give more valuable information about the ligand-protein interaction. The conventional 96-well plates have been replaced with 384-well plates and subsequently by 1536-well plates with low internal volume to make the screening possible at a high speed.
In the pharmacodynamic screening Hit and Lead are two frequently used terminologies. Hit is explained as: “a molecule with confirmed activity from primary HTS assay with a good profile in secondary assays and with confirmed structure,” and Lead is explained as: “a hit series for which the Structure Activity Relationship (SAR) is shown and activities demonstrated both in vitro and in vivo.”
 
IN VITRO MATRIX-LIGAND INTERACTION STUDIES
In HTS the interaction of ligand with the biological compartment is elucidated by luminescence-based binding assays. In this manner several thousands of compounds from chemical library can be assessed for their binding in a few days period. Various fluorescence techniques like Fluorescence Anisotropy (FA), Fluorescence Correlation Spectroscopy (FCS), Fluorescence Intensity (FI), Fluorescence Lifetime Imaging Microscopy (FLIM), Fluorescence Resonance Energy Transfer (FRET), Total Internal Reflection Fluorescence (TIRF) and Time Resolved Resonance Anisotropy (TRRA) are used. Along with these techniques, certain specific nano bead-based techniques like Scintillation Proximity Assay (SPA), Amplified Luminescence Proximity Homogeneous Assay (ALPHA) are also used (Figure 1.1). Drugs that target ion channels or G protein-coupled receptors (GPCRs) can be screened in HTS mode by intracellular measurement of ions like calcium using fluorescence-based methods. Apart from this, following activation of receptors on the cell surface the subsequent opening of ion channels or secondary cellular messenger pathway leads to the alteration in the intracellular calcium concentration of mammalian cells. To evaluate this process, high-throughput intracellular calcium monitoring assay has also been developed to find out the functional importance of receptors in cell-based assays.
 
Fluorescence Techniques in Drug Screening
The recently developed fluorescence-based assays are good enough to elucidate the molecular mechanism of receptor function and signal transduction processes, as well as for applications in the field of screening for novel therapeutic compounds.1
 
Fluorescence Anisotropy (FA)
This procedure is suitable to detect receptor-ligand binding reactions. The binding restricts the rotational mobility of the fluorophore; therefore, a bright fluorescence is exhibited which reflects the ligand binding (Figure 1.2). Moreover, it is independent of total fluorescence intensity. Specific binding of fluorescein-Trp25-exendin-4, a biologically active fluorescent ligand to human 5glucagon-like peptide 1 receptor was reported using FA.2 The change in anisotropy changes the intrinsic fluorescence of the insulin receptor following insulin binding. This property was exploited and the results were interpreted to explain conformational changes in the receptor.3
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Figure 1.1: Amplified luminescence proximity assay
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Figure 1.2: The binding restricts the rotational mobility of the fluorophore; therefore, a bright fluorescence is exhibited which reflects the ligand binding
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Fluorescence Correlation Spectroscopy (FCS)
The molecular interactions give rise to measurable traces of fluctuations in the fluorescence intensity. These fluctuations can be observed subsequently and analyzed by FCS. Depending on the environment, the fluorescent intensity of the fluoroprobe varies. The main advantage of FCS relies on the involvement of very less amount of material (nM-µM/1 µl) and relatively short measurement times.1
 
Fluorescence Resonance Energy Transfer (FRET)
FRET occurs when two different chromophores interact via dipole-dipole mechanism, where the excited, so-called donor, chromophore transfers its excitation energy nonradiatively to a closely located acceptor chromophore (Figure 1.3). This way, the original fluorescence decreases and the later one becomes dominant. To enable FRET, both donor and acceptor should be in immediate proximity. The rate of monoclonal antibody (tagged with Cy5) binding with interleukin 1 receptor (tagged with highly fluorescent europium chelate), to form a stable complex with the interleukin 1 receptor, has been monitored by FRET.4
 
Fluorescence Lifetime Imaging Microscopy (FLIM)
Using these techniques the sample is illuminated with a modulated continuous light wave like pulsed laser and the lifetime of the fluorescent probe is determined from the phase shift between the modulation of the excitation light and the emission of fluorescence. This method is useful to measure the lifetime of a fluorophore. The fluorophore gives more precise information about the environment around it as a function of time.
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Figure 1.3: Fluorescence resonance energy transfer (FRET) occurs when two different chromophores interact via dipole-dipole mechanism, where the excited, so-called donor, chromophore transfers its excitation energy nonradiatively to a closely located acceptor chromophore
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FLIM along with FRET allows imaging of molecular interactions on cell surfaces. Oligomerization of labeled epidermal growth factor receptors was studied using FLIM-FRET.5 It has been used also for the measurement of receptor tyrosine kinase activity and protein kinase C regulation mechanisms.6
 
Surface-sensitive Fluorescence Detection Technique
In this method, a membrane receptor can be immobilized on a sensitive surface through adsorption of native membrane fragments via affinity-tags or by physiosorption. An exciting light beam is coupled to the optical transducer like fibreoptic cable or glass plate. This creates an evanescent field that decays exponentially a few 100 nm into the medium. The receptors attached on the surface would not be excited as much; but if the fluorophore-labeled ligand comes in contact, then it emits fluorescence. This method has been reported to detect fluorescein-labeled antagonist on immobilized 5-HT3a receptor amounting to 1 attomol.7
 
Time Resolved Fluorescence Anisotropy (TRFA)
TRFA combines the advantages of steady-state anisotropy with those of total intensity time-resolved spectroscopy. The method exploits the change in the rotational diffusion of phosphorescent-labeled membrane receptor when they bind a ligand. This technique also delivers information on protein structural fluctuations that might be of functional importance. Ho and coworkers studied the binding of an activating fluorescent phorbol ester to the C1 domain of recombinant protein kinase Cα.8 It has enabled the authors to simultaneously determine the concentration of free ligand in solution, bound to lipids and bound to the kinase. TRFA also senses the different ligand's specific motions without requiring the comparison of absolute fluorescence intensities, which is often technically difficult to perform.
 
Cell-based Assays to Study Intracellular Events
Opening of voltage-gated, ligand-gated or store-operated calcium channels, or the release of calcium from intracellular stores, will result in a rapid increase in calcium concentration. Activation of GPCRs and receptor tyrosine kinases stimulates the production of inositol 1,4,5-trisphosphate (IP3), that binds to IP3 receptors located on the endoplasmic reticulum to release stored calcium into the cytosol.
Fluorescent indicators of calcium were originally developed to study changes in intracellular calcium levels in studies using fluorescence microscopy, flow cytometry or fluorescence spectroscopy. These fluorescent probes are structurally related to the calcium chelators like ethylene glycol-bis (β-aminoethyl ether) tetraacetic acid (EGTA) and 1,2-bis (2-aminophenoxy) ethane tetraacetic acid (BAPTA), and show a rapid spectral change on binding of calcium, a feature that makes them ideal for kinetic calcium measurements. The calcium binding dyes are classified functionally as single-wavelength or dual wavelength radiometric dyes. Variation in calcium concentration changes the fluorescent intensity in single-wavelength detection at same excitation and emission wavelength. In contrast, dual wavelength radiometric dyes exhibit a shift in fluorescence intensity and peak maxima in the presence of calcium. The calcium binding in the presence of Fura-2 shifts the excitation peak shifts from 335 nm to 362 nm.9 Some of the single wavelength dyes used successfully for calcium binding assays are Fura-3, Calcium Green-1 and Oregon Green 488 BAPTA. Newly developed Fluorometric Imaging 8Plate Reader (FLIPR) (Molecular Device, Sunnyvale, CA, USA) is capable of reading fluorescent calcium signals of all 96 wells simultaneously.10 The laser light is directed to the distance of 200 µm from the bottom of the well and excitation is captured by a charge-coupled device (CCD) camera from all the wells and data are stored on a computer.
Further improvement in FLIPR scans using 384 wells is being developed for mass screening. Apart from fluorescent readers, many Chemiluminescence Imaging Plate Readers (CLIPRs) have been developed to detect intracellular mechanisms. For this, the photoprotein aequorin, which is a 21 KDa apoaequorin protein bound to the prosthetic group coelenterazine and molecular oxygen is used. Aequorin is a calcium binding protein, which undergoes conformational change resulting in the oxidation of coelenterazine to coelenteramide. The relaxation of coelenteramide from the excited state to the ground state results in the emission of blue light (470 nm).11, 12 In the jellyfish, the blue light is transferred to green fluorescent protein (GFP) by FRET, and green light is emitted as GFP relaxes back to its ground state. Cloning of the apoaequorin gene has made it possible to express GFP in many types of cells and to reconstitute the aequorin complex by incubating the cells with coelenterazine. The cloned gene is targeted to specific intracellular locations, such as cytoplasm, nucleus, mitochondria and endoplasmic reticulum, by fusing the gene to specific target sequences. This aequorin assays can be validated for many GPCRs and calcium channels and the dose responses created by these assays are similar to the values obtained using fluorescent calcium dyes.13
The second type of intracellular calcium tracking assay is enabled through calcium regulated reporter genes. The nuclear factor of activated T cells (NFAT) and cAMP Response Element Binding Protein (CREB) are examples of transcription factors that become activated when there is a rapid rise in the intracellular calcium concentration.14,15 When levels of calcium increase, these factors bind to unique sites on promoters and stimulate transcription. Reporter genes are constructed by fusing calcium-regulated promoter to the coding sequence of an enzyme that catalyzes a luminescent or fluorescent reaction such as firefly luciferase, β-lactamase or β-galactosidase.16,17 Calcium-receptor gene assays convert a transient change in calcium concentration into a sustained luminescent readout. The use of NFAT reporter genes for assaying GPCRs has been studied in lymphoid and nonlymphoid cells.18 To detect small and rapid change in calcium concentration, aequorin and fluorescent dye assays might be preferred. The reporter gene assays might be used in primary screening applications, followed by the information-rich kinetic assays as a secondary screening tool.
 
Virtual Screening
It is totally an indirect approach using advanced computer technology to screen newer compounds based on virtual coordinates of receptor and ligands. Computer-Aided Molecular Design (CAMD) approach involves computational analysis of large data set in order to highlight those compounds most likely to be active in the actual assay, so that a focused subset of compounds can be selected. CAMD covers a wide range of technologies leading to very fast property predictions through more computationally elaborate modeling of drug-receptor binding. Using receptor-based properties, such as binding affinity and receptor selectivity, CAMD calculates to propose a broad range of properties that are likely to be useful in drug design—from physical properties like molecular size and solubility to indicators of developmental issues like metabolic fate and toxicity, etc. Therefore, virtual screening can filter out undesirable compounds on the basis of a wide 9variety of criteria, depending on the problem in hand. Virtual screening needs the 3D molecular structure of the receptor along with the 3D structure of ligands to perform docking. However, this approach is highly applicable for the lead optimization process since for many newly synthesized compounds conformational data may not be available. Moreover, the techniques like X-ray crystallography and NMR studies are necessary for the information about spatial arrangement and virtual coordinates of targets and ligands.19
The applicability of computer models has also used completely empirical and statistical model like the Rule of Five or Lipinski's rule. According to this rule, a drug like compound looks like a molecule with a molecular weight less than 500, OH and NH groups less than 5, the sum of N and O atoms less than 10 and log P value less than 5 for a better absorption in the intestine.
 
HTS-Scintillation Methods in Drug Screening
Apart from fluorescent techniques, the advancements in utilizing the radioactive compounds to understand molecular mechanisms are also very interesting. One of such attempts is named as Scintillation Proximity Assay (SPA). The basis of this system uses the principle that an antibody or a receptor molecule, which is bound to a bead, emits light when beta emission from an isotope occurs in close proximity, i.e. when a radiolabelled ligand binds to the bead with receptor or antibody (Figure 1.4). Amersham has introduced several advancements in these techniques leading to make it as a successful high throughput screen. The basic technology of the SPA is based on the fluorescent signal produced by a scintillant-dyed polystyrene or poly vinyl toluene microsphere that could be excited by the proximity of a radiolabeled molecule. The scintillant-dyed microspheres are in the size range of < 2 µm in diameter.
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Figure 1.4: Figure showing the emission of light when beta-emitter labeled ligand in contact with receptor coupled scintillant bead
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The recent improvements in this technique are yttrium silicate and yttrium oxide beads which enable the use of higher number of wells in which the signal can be picked up by highly sensitive readers containing CCD camera instead of classical photomultiplier tubes. So far, this technology has been utilized in several attempts in drug discovery processes like evaluation of kinetics of protein kinase inhibitors, to evaluate neurotransmitter transporter inhibitors, to evaluate novel farnesyltransferase inhibitors, to identify poly (ADP-ribose) polymerase-1 inhibitors (enzyme involved in DNA repair), evaluation of HIV-I reverse transcriptase inhibitors, etc. SPA is a homogeneous, rapid, versatile and amenable to automation, which can also simplify the screening protocol in the drug discovery process.
 
HTS Pharmacokinetic Studies
 
HTS-absorption Studies
Oral route is a preferred way of drug administration. In the drug development process it is very important to screen the drugs for gastrointestinal absorption. Conventionally, it is a very lengthy and time-consuming process. Moreover, this process also requires a large number of animals. Intestinal absorption is mainly due to the concentration dependent diffusion. If the drug passes through paracellular route, then there is not much of a hurdle. Some compounds, like cyclosporine and digoxin, are substrates of well recognized P-gp transporters, which belong to the ATP Binding Cassette (ABC) transporters having Walker's motives. P-gp substrates were found to have variable bioavailability. Therefore, it is necessary to screen the drug candidates during the developmental stage to ensure that they are not substrates for P-gp. Colon cancer cell lines (CaCo) grow confluently and form a monolayer upon polycarbonate support or collagen coated polycarbonate support.20 They are quite suitable for performing intestinal permeation studies and to elucidate the drug candidates susceptible for P-gp efflux mechanism in the intestine. Moreover, they also express CYP3A4 enzyme allowing prediction of intestinal metabolism. This process can be automated to examine the intestinal absorption characteristics of drugs in a shortest possible period to decrease the incidence of failure in the Phase I clinical trial in human volunteers.
 
HTS Metabolism Studies
In order to increase the speed of metabolism studies or to decrease the animal utilization in the metabolism studies, in vitro techniques were developed. Isolated human or animal liver microsomes are incubated along with the drug of interest and at periodical interval the aliquots are subjected for LC-MS or LC-NMR to elucidate the metabolites. Sometimes the major metabolites are isolated and subjected to primary in vitro screening to elucidate whether they are active metabolites or not. Liver microsomes are prepared by the homogenization of a liver, followed by centrifugation of the homogenate at 10000 g to obtain a supernatant fraction known as S10 fraction. The S10 fraction at 100,000 g pellets out smooth endoplasmic reticulum where the enzymes responsible for Phase I oxidation, including the CYP450 monooxygenases reside. However, most of the Phase II enzymes are cytosolic which are absent from the liver microsomes. Apart from microsomes, isolated hepatocytes (or hepatocytes-derived microsomes), cDNA-expressed enzymes and liver slices are also used to study the metabolic stability of drugs. The screening of metabolic stability with liver microsomes or human hepatocytes can be performed in 96-well plates to enhance throughput.2011
 
HTS-cytochrome (CYP) Inhibition and Induction Studies
During the drug development process, pharmacokinetic drug-drug interaction is also an undesirable factor. For example, ketoconazole, a potent inhibitor of CYP3A4, causes drug-drug interaction with drugs that are substrates for the same enzyme. Cryopreserved hepatocytes retain both Phase I and II drug metabolizing enzyme activities and, therefore, are used extensively for drug interaction studies. To get more information about the newly developed compound's metabolic interaction in specific cytochrome, liver microsome incubation studies are carried out using specific inhibitors. For this study phenacetin (for CYP1A2), coumarin (for CYP2A6), tolbutamide (for CYP2C9), s-mephenytoin (for CYP2C19), dextromethorphan (for CYP2D6), chlorzoxazone (for CYP2E1) and testosterone (for CYP3A4) are used. For HTS enzyme induction studies, the mechanism of CYP3A4 induction has been defined. Compounds, which are capable of inducing CYP3A4, also induce pregnane-X-receptor (PXR), that bind to the response element in the CYP3A4 gene called the pregnane-X-receptor response element (PXRE). An HTS method has been developed using a genetically engineered cell line that expresses a PXRE-luciferase reporter gene. Using this method, an induction of CYP3A4 by the xenobiotic-mediated binding of PXR to PXRE leads to the activation of luciferase synthesis, which can be quantified using a chemiluminescent substrate (luciferin).
 
HTS Hepatotoxicity Studies
The isolated hepatocytes are also used to study the possible xenobiotic induced hepatotoxicity in vitro. After the incubation of hepatocytes along with the drug candidate, the hepatocytes are subjected for measurement of ATP content in microtiter plates using chemiluminescence with the help of a luciferin-luciferase assay. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay is performed to quantify mitochondrial toxicity. LDH measurement, neutral red uptake test, thymidine uptake test and estimation of glutathione give more information on the possible hepatotoxicity of the xenobiotics.
 
High Speed Drug Synthesis-combinatorial Chemistry
In order to cater to the increased requirements of compounds for drug screening, conventional time-consuming drug synthesis has given place for the development of modern synthetic combinatorial chemistry methods. Normally, drug synthesis is a long process, which involves heating at higher temperature for hours, laborious extraction procedures, and removal of solvents, confirming the structure, removal of reactants and other impurities. The first combinatorial chemistry methods were presented by Geysen and coworkers21 for peptide synthesis. Later, this has been utilized for classical heterocyclic and other small molecule organic compounds.22 Advances in microwave technology quickened the reactions in a more efficient way.
 
High Speed Natural Product Isolation Techniques
Natural products (NP) are the most successful sources of leads to the evolutionary development of biologically active metabolic byproducts. Out of 520 new drugs developed and approved between 1983–1994, 39% of them were either NP or their derivatives.23 However, the long-pending problems with the NP are the isolation and identification of the active ingredients as well as ensuring their pharmacological properties. Seasonal, regional, geographical, 12taxonomical variations and processing methods make traditional activity guided NP development very long. Many pharmaceutical giants stopped using whole natural product extract for biological activities. Due to the development of successful two-dimensional high performance isolation units like SEPBOX (SEPIAtec, GmbH, Germany) for multiparallel HPLC, it is now possible to load up to 5 gm of NP extract and to isolate all compounds in 70–80% purity within 24 hr and in amounts sufficient and in microtiter formats amenable for direct HTS.
This SEPBOX system works by using gradient elution and polarity based trapping in solid phase extraction (SPE) trap columns. After the successful entrapment of compounds, they are eluted to obtain pure compounds. Coupling the SEPBOX to photodiode array (PDA) detector (enhanced UV detector), and evaporative light-scattering detector (ESD) in series enables the identification and quantitation of the significant compounds. NMR or mass spectroscopy is further used for the complete identification and structure elucidation. NP database containing about 10000 different structurally characterized compounds is being commercialized [Chapman and Hall Dictionary of Natural Products (DNP), Ver. 7:2, 8:2 on CD-ROM and Antibase, Ver 3.0] using a combination of mass spectroscopy and 1 and 2 D NMR.
A recent study conducted by Aventis Pharma AG (Vitry sur Seine, France) and AnalytiCon Discovery (Berlin, Germany) was jointly conducted to test the feasibility of high-throughput profiling, isolation and structure-elucidation technology for NP. A project named Megabolite was initiated to get minimum 5 mg samples of 4000 pure compounds from microbes and plants. They have used the above (SEPBOX) technology for the isolation of the active compounds. The plants were selected from the families of Leguminosae, Euphorbiaceae, Umbelliferae, Solanaceae, Borganiaceae, Compositeae, Labiatae, Apocynaceae, Rubiaceae, Meliaceae and Araliaceae. A total of 2242 compounds from microorganisms and 1758 compounds from plants were isolated in the course of the project. About 2400 pure compounds isolated from NP were tested against biological targets using HTS assays (scintillation proximity assay and homogeneous time-resolved fluorescence). The compounds isolated from NP gave a higher confirmation rate as compared to the Rhone-Poulenc Rorer synthetic compound collection (>50000 single compounds) and combinatorial library (>50000 single compounds). Therefore, the development of advanced HPLC techniques can go beyond the synthetic process in making drugs having higher success rate in screens.24
 
CONCLUSION
Human Genome project has been nearing completion and the first blue print was released on 25th June 2000 and the third map was released in 2001 thereby, throwing light on the hidden biological targets. They need to be evaluated for their involvement in various cellular functions and their utilization in various altered physiological conditions. It has been reported that human genome revealed the availability of 750 new GPCRs, 100 ligand-gated ion channels, 60 nuclear receptors, 50 cytokines and 20 reuptake/transport proteins. They are all yet to be evaluated for their function.
High Throughput Screening is a remarkable achievement in the drug discovery process to speed up preclinical drug discovery process. This automation in the process is further supported by the excellent software packages.13
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