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Generically engineered cells in drug discovery and pharmacology
CELL ENGINEERING AND EXPRESSION OF IoN CHANNELS M. Taglialatelam, E. Fickera), B. Wible12),and A.M. Brown12) mDept. Neurosciences .- Section of Pharmacology, 2nd School of Medicine, University of Naples "Federico Ir', Via S. Pansini 5, 80121 Naples, Italy; Ph. and Fax 39-81-7463323; ('4Rammelkamp Center for Research, Case Western Univ. 2500 MetroHealth Dr., Cleveland,OH 44109-1998, USA. The specific pattern of ion channels expressedat the membrane level determinescritical cell fimctionslike excitability,volumeregulation,growthand and differentiation.Importanthuman diseases like cystic fibrosis, myotoniaand paramyotoniacongenita,have been related to an alterationof transmembraneion fluxes in specific tissues. The recent introduction of molecular biological techniquestogetherwith the new developmentof modernelectrophysiologyallow structure-function experiments, where the altered behavior of genetically engineered channels provides insights into the possible molecular determinants of specific functions like permeation, gating and pharmacological blockade. K÷ are the most widespread and heterogeneousclass of ion channels. Inwardly rectifying K+ channels (IRKs) serve a dual purpose: 1) to maintainthe resting membrane potential close to the equilibrium potentialfor K÷ ions, and 2) to allow the long duration of the depolarizingplateau phase of the cardiac action potential. We have used a combinedapproach of heterologousexpression in Xenopus oocytes of two cloned members of the IRK family, IRK1 and ROMK1, and mutagenesis of their genes, with the purpose of localizing the structural domains specifyinginward rectification.Tlae results obtained suggest that: 1. IRKI and ROMK1 differ strongly in their affinity for Mg2~,and that the structural determinantsfor the difference are localized in the carboxyl terminus of IRK1; 2. intrinsic gating has been localized to a single negatively-charged aspartate (D) residue in the putative second transmembranedomain of IRKI; 3.physiologicalintracellularconcentrationsof the naturally occurringpolyamines (PAs) spermine and spermidine act as gating molecules in IRK1. These results suggest that, since PAs play a crucial role in cell growth and division and IRK channels are major determinants of potassium fluxes and, as a result, cellular transport and volume, a more direct link between PAs and IRKs may have significance for cell growth.
GENERICALLY ENGINEERED CELLS D I S C O V E R Y AND P H A R M A C O L O G Y
IN
DRUG
A. Maggi, S. Santagati, E. Vegeto, P. Agrati, C. Patrone and Z.Q. Ma Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences University of.Milan, Via Balzaretti 9 20133-Milan, Italy The possibility to express any cloned cDNA in eukaryotic cells provided the pharmacologist with very efficient tools for the dissection of the molecular mechanisms involved in the activation (and inactivation) of specific receptor proteins. Utilizing this technique we have generated a series of human cell lines stably transfected with either the human estrogen or progesterone receptor cDNA. The cell lines generated were of various origin. The goal of the study was the understanding of the establishment of the phenomenon of down-regulation of hormonally-stimulated steroid receptors. Furthermore, we investigated for the basis of a cellular specificity in the downregulation of sex steroid receptors. The final aim of our studies is to originate novel, more specific antagonists for this important class of receptor.
GENETICALLY ENGINEERED CELLS FOR DRUG METABOLISM STUDIES: ADVANTAGES AND LIMITATIONS
J. Doehmer I n s t i t u t ftir Toxikologie und Umwelthygiene, T e c h n i s c h e Universittit Mfinchen, Lazarettstr. 62, 80636 Mtinchen, Germany
W.-D. Schleuning Schering Research Laboratories, M~illerstraBe Berlin, Germany
P H A R M A C O L O G Y OF INTRACELLULAR R E C E P T O R IN ENGINEERING CELLS
178, D-13342
Since the introduction o f methods for gene transfer and heterologous gene expression the roster o f tools available to pharmacologists has greatly expanded. Whereas in previous days receptor binding assays depended on tissue extracts, such tests are now routinely performed with pure receptors produced by genetically engineered cells, and distortions generated by receptor heterogeneity are avoided. Specific combinations :of subunits o f ion channels such as the GABA receptor can be reconstituted in cell culture and individually evaluated by patchclamp electrophysiology. Genetically engineered yeast has proved useful for the assessment o f steroid and other hormone activities. The method o f "phage display" allows the expression o f a receptor or o f parts o f it on the surface of bacterial viruses. By random or site-directed mutagnesis, data on the struc{ure/function relationship o f receptor proteins can be generated with unprecedented speed. If protein domains are fused to a specific yeast transcription factor that requires a protein cofactor "double hybrid" systems are generated which are extremely ueful for the "fishing" o f binding partners for proteins. This method can be employed to find ligands for orphan receptors. It is therefore evident, that molecular biology in drug research and pharmacology reaches far beyond the cloning and expression o f therapeutic proteins, and has become the prime ferment o f innovation in pharmaceutical research.
m18m
The key e n z y m e s in b i o t r a n s f o r m a t i o n are t h e c y t o c h r o m e s P450 (CYP). The i d e n t i f i c a t i o n of t h e metabolically competent CYP isoform, enzyme kinetics, a n d metabolite profile are an integral part in drug design a n d development. A multitude of CYP may be expressed depending on gender, age, daytime, diet, and tissue. This explains to a great extent a large variation in individual CYP expression. A system is needed for standardised CYP content. The classical approach was purificatio n and isolation of a single CYP and reconstitution for enzyme activity. Beside technical problems, CYP may be difficult to obtain in sufficient quality and quantity. This is certainly the case w i t h CYP of h u m a n origin. Cloning CYP genes for heterologuous expression in cultivated ceils is the m o s t advanced technology for gaining access to individual and functionally active CYP. In this way, many CYP have b e e n e x p r e s s e d in b a c t e r i a l , y e a s t , insect, a n d mammalian cells, Each of these expressions systems is unique in terms of their cell biology and physiology in general, and their CYP expression s t a t u s in particular. For this reason, expression s y s t e m s m a y have a d v a n t a g e s and limitations, depending on the problem to be solved. This refers also to the more classical approach by using primary hepatocytes. There is no s u c h thing like a n ultimate in vitro system. A critical view on the performance of t h e s e s y s t e m s is an essential prerequisite in order to make the most appropiate choice for a particular expression system. Only t h e n t h e s e s y s t e m s may serve as a n analytical tool to dissect the complex In vivo situation in order to s t u d y a n d to u n d e r s t a n d details. Especially with the avaflibility of h u m a n genes, it is possible to perform studies of h u m a n relevance already at the preclinical stage. This includes studies on efficacy of drugs, metabolism studies, drug interaction, and toxicity testing.
GENERICALLY ENGINEERED CELLS D I S C O V E R Y AND P H A R M A C O L O G Y
IN
DRUG
A. Maggi, S. Santagati, E. Vegeto, P. Agrati, C. Patrone and Z.Q. Ma Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences University of.Milan, Via Balzaretti 9 20133-Milan, Italy The possibility to express any cloned cDNA in eukaryotic cells provided the pharmacologist with very efficient tools for the dissection of the molecular mechanisms involved in the activation (and inactivation) of specific receptor proteins. Utilizing this technique we have generated a series of human cell lines stably transfected with either the human estrogen or progesterone receptor cDNA. The cell lines generated were of various origin. The goal of the study was the understanding of the establishment of the phenomenon of down-regulation of hormonally-stimulated steroid receptors. Furthermore, we investigated for the basis of a cellular specificity in the downregulation of sex steroid receptors. The final aim of our studies is to originate novel, more specific antagonists for this important class of receptor.
GENETICALLY ENGINEERED CELLS FOR DRUG METABOLISM STUDIES: ADVANTAGES AND LIMITATIONS
J. Doehmer I n s t i t u t ftir Toxikologie und Umwelthygiene, T e c h n i s c h e Universittit Mfinchen, Lazarettstr. 62, 80636 Mtinchen, Germany
W.-D. Schleuning Schering Research Laboratories, M~illerstraBe Berlin, Germany
P H A R M A C O L O G Y OF INTRACELLULAR R E C E P T O R IN ENGINEERING CELLS
178, D-13342
Since the introduction o f methods for gene transfer and heterologous gene expression the roster o f tools available to pharmacologists has greatly expanded. Whereas in previous days receptor binding assays depended on tissue extracts, such tests are now routinely performed with pure receptors produced by genetically engineered cells, and distortions generated by receptor heterogeneity are avoided. Specific combinations :of subunits o f ion channels such as the GABA receptor can be reconstituted in cell culture and individually evaluated by patchclamp electrophysiology. Genetically engineered yeast has proved useful for the assessment o f steroid and other hormone activities. The method o f "phage display" allows the expression o f a receptor or o f parts o f it on the surface of bacterial viruses. By random or site-directed mutagnesis, data on the struc{ure/function relationship o f receptor proteins can be generated with unprecedented speed. If protein domains are fused to a specific yeast transcription factor that requires a protein cofactor "double hybrid" systems are generated which are extremely ueful for the "fishing" o f binding partners for proteins. This method can be employed to find ligands for orphan receptors. It is therefore evident, that molecular biology in drug research and pharmacology reaches far beyond the cloning and expression o f therapeutic proteins, and has become the prime ferment o f innovation in pharmaceutical research.
m18m
The key e n z y m e s in b i o t r a n s f o r m a t i o n are t h e c y t o c h r o m e s P450 (CYP). The i d e n t i f i c a t i o n of t h e metabolically competent CYP isoform, enzyme kinetics, a n d metabolite profile are an integral part in drug design a n d development. A multitude of CYP may be expressed depending on gender, age, daytime, diet, and tissue. This explains to a great extent a large variation in individual CYP expression. A system is needed for standardised CYP content. The classical approach was purificatio n and isolation of a single CYP and reconstitution for enzyme activity. Beside technical problems, CYP may be difficult to obtain in sufficient quality and quantity. This is certainly the case w i t h CYP of h u m a n origin. Cloning CYP genes for heterologuous expression in cultivated ceils is the m o s t advanced technology for gaining access to individual and functionally active CYP. In this way, many CYP have b e e n e x p r e s s e d in b a c t e r i a l , y e a s t , insect, a n d mammalian cells, Each of these expressions systems is unique in terms of their cell biology and physiology in general, and their CYP expression s t a t u s in particular. For this reason, expression s y s t e m s m a y have a d v a n t a g e s and limitations, depending on the problem to be solved. This refers also to the more classical approach by using primary hepatocytes. There is no s u c h thing like a n ultimate in vitro system. A critical view on the performance of t h e s e s y s t e m s is an essential prerequisite in order to make the most appropiate choice for a particular expression system. Only t h e n t h e s e s y s t e m s may serve as a n analytical tool to dissect the complex In vivo situation in order to s t u d y a n d to u n d e r s t a n d details. Especially with the avaflibility of h u m a n genes, it is possible to perform studies of h u m a n relevance already at the preclinical stage. This includes studies on efficacy of drugs, metabolism studies, drug interaction, and toxicity testing.