Analytical
Approach
Fluorescent Molecular The tyranny of large numbers of drug compounds to screen is the raison d'être for a fluorescent assay system.
T
he discovery of biologically active molecules relies on testing many compounds and mixtures, such as natural extracts, and on identifying biological systems that provide a measure of activity. For millennia, humans discovered active compounds by ingestion. This dangerous practice has since given way to biochemical and cell-based methods for primary screening of drug candidates. During this century, both the medical conditions thought to be amenable to drug treatment and the number of compounds available for testing have grown explosively. Today, large pharmaceutical companies possess in excess of 100,000 discrete compounds in their repositories and have more than a dozen clinical targets in development at any given time. In the future, drug companies will have access to thousands of potential drug targets because of the massive effort presently being undertaken in genomics biotechnology companies and the imminent availability of the genetic of the entire human genome {1 2) With the advent of combinatorial chemistry and its cousin automated parallel synthesis compound inventories of manv Pharmaceutical companip*; will continnp to crrow
Gregor Z l o k a r n i k Aurora Biosciences
bounds Although testing large numbers of compounds against large numbers of targets improves the chances for successful drug discoveries, it poses two major challenges for compound screening. How can assays be generated faster for the many emerging new drug targets and how can active compounds be efficiently detected in large and continuously growing compound libraries? No one can currently test all the compounds in a 100,000-member compound library against hundreds of targets This Report focuses on a recently developed fluorescent assay system and its use in ac-
322 A Analytical Chemistry News & Features, May 1, 1999
celerating screen development and drug candidate screening. Agonist or agony?
Membrane receptors are the largest class of validated drug targets. Of these, G-proteincoupled receptors (GPCRs) are targeted by up to 60% of modern drugs (3). An assay ii needed to search for compounds that can antagonize (block) or agonize (activate) a receptor. Traditionally, compounds that bind to the ligand-binding domain of a membrane receptor are discovered by screening for displacement of a labeled ligand. Often the label is a radioactive atom incorporated into the ligand (4), and ligand displacement from an immobilized tor is quantified by the change in radioactivity associated with either soluble or insoluble fractions In addition to a labeled ligand this assay also requires an isolated receptor Obtaining sufficient quantities of purified receptors for screening challenge that or generating a source of the receptor protein It also entails an elaborate purification procedure that enriches the desired protein and retains its ligand-binding characteristics. Sometimes, a truncated soluble form of the receptor can be engineered that retains the binding properties of the native receptor (5). The truncated receptor lacks the transmembrane segments and cytoplasmic tail, making it soluble in the absence of lipids and thus facilitates purification. This approach is limited to receptors that have a large extracellular domain, contain the ligand-binding site, and can be stably expressed. It is unsuitable for receptors that are assembled from multiple membranespanning proteins and receptors with ligand-binding domains located close to or in the membrane interface Also a binding assay cannot detect compounds that modulate receptor activity by binding to a site
Sensor for Drug Discovery distinctfromthe docking site for the labeled ligand. Something is sticking
An alternative approach to finding molecules that bind to the receptor is based on receptor signaling pathways. Many of these pathways lead to the modulation of gene transcription events via expression control elements (promoters), which are responsive to signals from the receptor. In the case of GPCRs, receptor activation initially leads to the activation of G-proteins followed by a change in the concentration of intracellular messengers (such as the free cytosolic calcium, cyclic nucleotide monophosphates, insitol phosphates and plasma membrane-resident lipids) or by a change in membrane potential (6-9) Changes in intracellular messenger levels have been used to detect receptor activation and screen for agonists and antagonists (10). For instance, changes in the concentration of intracellular free calcium levels are conveniently measured with fluorescent calcium indicators (11,12). Changes in membrane potential can also be detected optically (13,14). Most of these signals are transient, however, requiring a kinetic measurement to detect the change. A consequence of these signaling events is the activation or inactivation of transcription factors which regulate the expression of genes A stable readout for receptor-ligand interactions can be obtained by measuring receptor-regulated gene expression which is tightly controlled bv the activation status of the tor Introducing foreign genes
To obtain a convenient readout for the expression of a gene, molecular biology techniques in which genes of interest (under
the control of gene regulatory elements of choice, or promoters) are inserted into the genome of mammalian cells can be used. With these so-called "transfections", exogenous genes (also called transgenes) can be introduced into cells under the transcriptional control of a receptor-regulated promoter. Cell lines generated from these transfected cells express the transgene under receptor control. The binding of an agonist to a receptor leads to the induction (or repression) of the transcription of the transgene depending on whether the regulatory element is activational or inhibitory. An assay for the presence of then the activation status of the receptor and hence the gene introduced is ap-
propriately called a "reporter gene" (Figure 1). An advantage of reporter gene assays over binding assays is that neither a labeled ligand nor a purified receptor preparation is needed. Spying on genetic activity
A recent review described using reporter assays for determining analyte concentrations (15). In that appllcation, the signal obtainedfromthe reporter reflected the concentration of the analyte. The authors engineered bacteria to express (produce) luciferase, a light-producing enzyme, in amounts that were dependent on the concentration of antimonite present in the sample.
Figure 1 . Reporter conveys receptor activation. Binding of a ligand (agonist) to the receptor initiates a cascade of intracellular events accompanied by a change in intracellular messenger concentrations. Consequently, transcription factors (tf) activate and translocate to the nucleus where they bind to their target promoter sequences. When the transcription factor constructively interacts with the promoter, the reporter gene is transcribed, and the corresponding messenger RNA (mRNA) is generated. The RNA message is translated into the reporter enzyme protein. The reporter enzyme catalyzes the conversion of substrates into conveniently detectable products. Analytical Chemistry News & Features, May 1, 1999 323 A
Analytical
Approach
In drag discovery, reporter genes are configured to convey information about the properties of compounds as opposed to their concentrations. The properties in question include affinity for a specific re ceptor and whether binding to the recep tor leads to a change in receptor signal ing. In this case, reporter signals allow the determination of binding constants and the assessment of whether com pounds are agonists or antagonists of the receptor. In either application, reporter genes are often chosen to encode enzymes whose presence is easily monitored by measuring the conversion of a chromogenic, fluorogenic, luminogenic or radioactive substrate (16,17). Ideally, the reporter gene en codes a protein that is not endogenously expressed in cells of the organism under study. Therefore, many reporter genes cur rently in use for studying gene expression in mammalian cells are of bacterial or in vertebrate origin. Historically, one of the first reporter genes introduced into a mammalian cell encoded bacterial chloramphenicol acetyl transferase. Acetyl transferase levels in a cell population can be measured by moni toring the conversion of radioactively la beled chloramphenicol to the acetyl deriva tive by TLC, and the result can be visual ized by exposure tofilm.The activity of glucosidase enzymes, such as bacterial B-galactosidase and glucuronidase, can be measured using fluorogenic sugar deriva tives of fluorescent dyes. In whole-tissue preparations p-galactosidase activity is indi cated by the enzyme-catalyzed of an indigo precipitate The location of the orecipitate indicates the location of the en7vme in the tissue Bacterial andfireflyluciferases are detected by their catalvsis of a lip,ht-p,eneratin£T reapj-ion ir, which a Iuciferin ATP and oxveen
directly detected by measuring its inherent fluorescence (18). And then there w a s light (or fluorescence) Reporter enzymes, such as luciferases or secreted alkaline phosphatase, are most sensitively detected by enzyme-catalyzed light emission. This measurement re quires an efficient method of collecting light for detection by the photon counter. To capture a maximum amount of the emitted light, microtiter plates made from white reflective plastic are used to reflect the light toward a photomultiplier tube located above the plate. Alternatively, the
"β-Lactamase is an attractive reporter becauseitis absent in mammalian cells." sample can be viewed through a clearbottom vessel from below with a high nu merical aperture objective, which effi ciently collects a wide cone of emitted light that is detected with a cooled CCD camera, allowing spatial analysis of light emission (19). (A commercial vendor of luciferase reagents currently advertises detection levels of 0.1 fM firefly lucif erase, using a 96-well plate luminometer for light detection [20].) In luminescence assays, signal intensity is proportional to readout time increase in sensitivity comes at the cost of decreased sample throughput Reporter assays in which the readout is based on the conversion of a fluoro genic substrate to a fluorescent product by the reporter enzyme need an excita tion light source and detectors for emit ted light. Fluorescence assays tend to be somewhat less sensitive than lumines cence assays because of background fluo rescence in the sample, but these assays compensate, in that, the enzymatic reac tion does not need to be monitored con tinuously. If the fluorescent product is
are rnnQiimprl trt eive'an oxyluciferin AMP and nvrronhosnhate Secreted alkaline phospharase ran be detected via its activation of a dioxetane derivative, which then spontaneously decomposes, emitting light in the process. Reporter molecules that are not enzymes include human growth hormone, which is detected colonmetncally by an enzymelinked immunosorbent assay, and the jelly fish green fluorescent protein that can be 324 A Analytical Chemistry News & Features, May 1, 1999
allowed to accumulate, the amount of product generated is an integrated mea sure of reporter activity over time. Irrespective of the amount of product generated, its fluorescence can be mea sured in a fraction of a second. This inte grating feature means that sample volumes can be as small as 1 uL wiihout having to increase sample readout times. Microtiter plates for fluorescence analysis are made of black plastic to avoid blinding the detector with reflected excitation light during analy ses in top-read mode. Alternatively, plates can be manufactured with clear bottoms that allow for exciting the fluorescent dye and collecting its fluorescence from below. That nasty bacterial enzyme B-Lactamase is the bacterial enzyme that catalyzes the hydrolysis of B-lactam anti biotics, such as penicillins and cephalospo rins, making bacteria resistant to these antibiotics. Despite its infamous role in infectious disease, B-lactamase has re cently also been developed as a reporter. Its enzymatic activity can be detected with fluorogenic as well as chromogenic substrates (21,22). B-Lactamase cleaves the B--actam ring in penicillin and cepha losporin antibiotics. This enzyme is an attractive reporter because it is absent in mammalian cells. Also, its substrates are more hydrophobic than the products it creates, allowing for the delivery of substrates into living cells by diffusion and cellular retention of the products because of their increased polar ity. The lack of these properties in the sub strates of glycosidases, phosphatases, and sulphatases requires that the substrate be delivered to cells under harsh loading con ditions with the concomitant difficulty of retaining the more hydrophobic product inside the cells. Therefore, assays using these often performed on cell lysates FRETting in a β- lactamase substrate In the fluorogenic B--actamase substrate, CCF2, two fluorescent dyes are linked via a cephalosporin linker. The dyes are a matched pair in which the emission spec trum of the shorter wavelength dye significandy overlaps the excitation spectrum of
the longer wavelength dye. The short sepa ration of the dyes by the cephalosporin linker ensures that the electronic states of both dyes can interact. Resonance energy is transferred from the excited state of the short wavelength dye (donor) to the ground state of its longer wavelength partner (accep tor). The donor fluorophore is a derivative of 7-hydroxycoumarin-3-carboxamide, which excites at 409 nm in visible violet light, which is distinct from endogenous, cellbased fluorescent substances that excite in the UV; and is a good antenna for the excit ing light (molar extinction coefficient of - 40 000 cm_1M"1) In the intact substrate excitation of the hydroxycoumarin fluoro phore with violet light leads to nonradiative fluorescence resonance energy transfer (FRET) with preen fluorescence emission (maximiim at S20 nm) fmm the enerfrv-
accepting fluorescein derivative The two fluoroDhores are in close proximity t o PPPVI
other C
