Biomedical Engineering Reference
In-Depth Information
The nuclear receptors consist of a ligand-binding domain, a DNA binding domain, and a trans-
activation domain. Upon activation, two receptors dimerize, as homo- or heterodimers, and bind to
specii c recognition sites on the DNA. Coactivators will then associate with the dimeric receptor
and initiate transcription of the target gene(s). Each receptor recognizes specii c DNA sequences,
also known as the hormone response elements, which are located upstream of the genes that are
regulated. 3D high-resolution structures of both ligand- and DNA-binding domains have been deter-
mined. In drug research, the main focus has been on the structures of the ligand-binding domains,
which, for several receptors, have been determined in the absence and in the presence of ligands.
12.3 RECEPTORPHARMACOLOGY
12.3.1 R ECOMBINANT VERSUS IN S ITU A SSAYS
The previous decade has had a profound impact on how receptor pharmacology is performed. As
mentioned in the introduction, receptor cloning was initiated in the mid-1980s and today the major-
ity of receptors have been cloned. Thus, it is now possible to determine the effect of ligands on indi-
vidual receptor subtypes expressed in recombinant systems rather than on a mixture of receptors in,
e.g., an organ. This is very useful given that receptor selectivity is a major goal in terms of decreas-
ing side effects of drugs and the development of useful pharmacological tools that can be used to elu-
cidate the physiological function of individual receptor subtypes. Furthermore, recombinant assays
allow one to assay cloned human receptors, which would otherwise not have been possible. Most
receptors are more than 95% identical between humans and rodents, but due to the small differences
in primary amino acid sequence there have been cases of drugs developed for rats rather than for
humans, because the compounds were active on the rat receptor but not on the human receptor.
It should be noted that the use of organ and whole animal pharmacology is still required. As
previously noted, the cellular effects of receptor activation depend on the intracellular contents of
the proteins involved in, e.g., the signaling cascades. These effects can only be determined when the
receptor is situated in its natural environment rather than in a recombinant system. In most situations,
both recombinant and in situ assays are thus used to fully evaluate the pharmacological proi le of new
ligands. Furthermore, once a compound with the desired selectivity proi le has been identii ed in the
recombinant assays, it is important to coni rm that this compound has the predicted physiological
effects in, e.g., primary nonrecombinant cell lines, isolated organs and/or whole animals.
12.3.2 B INDING VERSUS F UNCTIONAL A SSAYS
Binding assays were used as the method of choice for primary pharmacological evaluation, mainly
due to the ease of these assays compared to functional assays that generally required more steps than
binding assays. However, several factors have changed this perception: (1) biotechnological func-
tional assays have evolved profoundly and have decreased the number of assay steps and increased
the throughput, (2) functional assay equipment has been automated, (3) ligand binding requires a
high-afi nity ligand, which for many targets identii ed in genome projects simply does not exist,
(4) binding assays are unable to discriminate between agonists and antagonists, and (5) binding
assays will only identify compounds binding to the same site as the radioactively labeled tracer.
The Fluorometric Imaging Plate Reader (FLIPR ) illustrates this development toward functional
assays. Cells transfected with a receptor coupled to increase in intracellular calcium levels (e.g., a
G αq coupled GPCR or a Ca 2+ permeable ligand-gated ion channel) are loaded with the dye Fluo-3,
which in itself is not l uorescent. However, as shown in Figure 12.10, the dye becomes l uores-
cent when exposed to Ca 2+ in the cell in a concentration-dependent manner. In this manner, ligand
concentration-response curves can be generated on the FLIPR very fast as it automatically reads
all wells of a 96-, 384-, or 1534-well tissue culture plate. Many other functional assays along these
lines have been developed in recent years.
Search WWH ::




Custom Search