0165-6147/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0165-6147(99)01337-1 TiPS – June 1999 (Vol. 20) 237 M. Freissmuth, Professor of Pharmacology, M. Waldhoer, Research Associate, E. Bofill-Cardona, Research Associate, and C. Nanoff, Associate Professor of Pharmacology, Institute of Pharmacology, University of Vienna, Austria. R E V I E W G protein antagonists Michael Freissmuth, Maria Waldhoer, Elisa Bofill-Cardona and Christian Nanoff Heterotrimeric G proteins couple membrane- bound heptahelical receptors to their cellular effector systems (ion channels or enzymes generating a second messenger). In current pharmacotherapy, the input to G protein- regulated signalling is typically manipulated by targeting the receptor with appropriate agonists or antagonists and, to a lesser extent, by altering second messenger levels, most notably by inhibiting phosphodiesterases that hydrolyse cyclic nucleotides. When stimulated, G proteins undergo a cycle of activation and deactivation in which the -subunits and the -dimers sequentially expose binding sites for their reaction partners (receptors, guanine nucleotides and effectors, as well as regulatory proteins). These domains can be blocked by inhibitors and this produces effects that cannot be achieved by receptor antagonists. Here, the structural and mechanistic information on G protein antagonists is summarized and an outline of the arguments supporting the hypothesis that G proteins per se are also potential drug targets is provided. Activation of a heptahelical receptor by an agonist (a neuro- transmitter, hormone, autacoid, photon, odourant or a drug) generates, in principle, at least two types of signals, namely those propagated by the GTP-liganded G protein -subunits and those transmitted by free G protein - subunit complexes. There is a large molecular diversity within the subunits of heterotrimeric G protein subunits. At the beginning of this decade, the number of known mammalian a-subunits grew rapidly to comprise more than 20 individual proteins 1 . Since the discovery of the gustatory G protein gustducin 2 , no additional mam- malian -subunit has been identified and it seems un- likely that this family will proliferate any further. How- ever, alternative splicing generates XL-G s , extra-large versions of the protein that carry large amino-terminal extensions (~30 kDa); the physiological role of these pro- teins is poorly understood 3 . All -subunits share bio- chemical and structural properties and these are sum- marized in Table 1. They can be assigned to the following structurally and functionally related groups 1,4 : (1)  s -group: the members of this group stimulate the isoforms of adenylate cyclase. (2)  i/o/t -group: this can be divided into  i/o/z and  t/g subunits. The  i/o/z subunits inhibit some isoforms of adenylate cyclase; they also inhibit and stimulate neu- ronal Ca 2+ and K + channels, respectively (an effect that is due to the release of free -dimers). The  t/g subunits are transducins and gustducin, which stimulate the reti- nal cGMP-phosphodiesterases and presumably a related gustatory effector, respectively. (3)  q -group: these subunits activate the -isoenzymes of phospholipase C and non-receptor tyrosine kinases of the btk-family. (4)  12/13 -group: regulate low-molecular-weight G pro- teins of the rho-family (which affect the cytoskeleton) and the Na + –H + -exchanger (however, a direct interaction has not been proven in a cell-free assay). G protein - and -subunits are tightly associated and form stable dimers that cannot be separated unless the pro- teins are denatured. Hence, from a functional point of view they are considered as a single entity, the -dimer. There are five G protein -subunits and at least 12 -subunits 5 ; their general biochemical properties are summarized in Table 2. Some - and -combinations fail to form functional dimers 5 ; nevertheless, the number of -dimers and, most importantly, of oligomeric G proteins that can be produced by combinatorial association is presumably very large 1,5,6 . The implications of this diversity are not fully understood; however, in an intact cell, G protein oligomers of a given subunit composition ( x  y  z ) subserve functions for which other, closely related, oligomers cannot readily substitute. This conclusion is based on experiments in which cells were depleted of individual G protein subunits by injection of antisense oligonucleotides; under these conditions, indi- vidual receptors display a stringent requirement for dis- tinct G protein oligomers to regulate the same effector 6 . Signalling mechanism and potential target sites for drug action The cycle of G protein activation can be broken down into a four-step reaction 1 (Fig. 1): (1) The basal state (Fig. 1a), in which the G protein is an -heterotrimer with GDP bound to the -subunit. In the absence of activation by a receptor, the rate of GDP release (k off  0.1 min -1 ) is much lower than the rate of GTP hydrolysis (k cat  3 min -1 ); this kinetic feature clamps the system in the ‘off’ position. (2) Receptor-mediated GDP-release (Fig. 1b): The agonist-liganded, activated receptor interacts with the cognate G protein(s) and dramatically accelerates the rate of GDP release from the -subunit. In the absence of added GTP (or of its hydrolysis-resistant analogues), agonist (H), receptor (R) and G protein (G) form a ternary com- plex (HRG), in which the agonist is bound with con- siderably higher affinity than if bound to the receptor alone. This high-affinity state is typically seen in binding experiments that use membrane preparations. However, in intact cells, GTP concentrations are high and GTP binds instantaneously to the empty guanine-nucleotide- binding pocket.