Synthesis and pharmacological properties of new potent direct activators of heterotrimeric G proteins are described. Compounds were synthesized from protected amino acids with alkylamines using coupling reagents (CDI, DCC, and EDC). Alkyl-substituted amino acid amides and their corresponding di- and triamines were subjected to structure-activity analysis. All compounds activated membrane-bound HL-60 GTPases in a pertussis toxin-sensitive fashion. This suggests a specific effect of compounds on the carboxy terminus of a defined subclass of heterotrimeric G proteins, i.e., members of the G alpha i subfamily. Elongation of the alkyl chain and increasing the number of amino groups enhanced the potency of compounds on HL-60 membrane-bound GTPase. N-(2,5-Diaminopentyl)dodecylamine (21) was selected to study its mode of action employing purified pertussis toxin-sensitive G proteins. It stimulated G alpha subunits by inducing the release of bound GDP. In contrast to receptors G beta gamma complexes were not required for 21-mediated activation of G alpha. Moderate isoform selectivity of its action was observed within a group of highly homologous members of the Gi subfamily with G alpha o1 being activated at lowest concentrations, whereas higher concentrations were necessary for the stimulation of G alpha i1 or transducin. We conclude that these compounds represent important tools for studying G protein-dependent cellular functions.

We have previously shown that alkyl-substituted amino acid derivatives directly activate G i/o proteins. N-Dodecyl-N ␣ ,N ⑀ -(bis-l-lysinyl)-l-lysine amide (FUB132) is a new representative of this class of compounds with increased efficacy. Here, we characterized the molecular mechanism of action of this class of compounds. FUB132 and its predecessor FUB86 were selective receptomimetics for G i/o because they stimulated the guanine nucleotide exchange reaction of purified G i/o as documented by an increased rate of GDP release, GTP␥S binding, and GTP hydrolysis. In contrast to the receptomimetic peptide mastoparan, stimulation of G proteins by lipoamines required the presence of neither G␤␥-dimers nor lipids. On the contrary, G␤␥-dimers suppressed the stimulatory effect of FUB132. The stimulation of G i/o by lipoamines and by mastoparan was not additive. A peptide derived from the C terminus of G␣ o3 , but not a corresponding G␣ q -derived peptide, quenched the FUB132-induced activation of G␣ o . In membranes prepared from human embryonic kidney 293 cells that stably expressed the G i/ocoupled human A 1 -adenosine receptor, lipoamines impeded high-affinity agonist binding. In contrast, antagonist binding was not affected. We conclude that alkyl-substituted amines target a site, most likely at the C terminus of G␣ i/o -subunits, that is also contacted by receptors. However, because G␤␥-dimers blunt rather than enhance their efficacy, their mechanism of action differs fundamentally from that of a receptor. Thus, despite their receptomimetic effect in vitro, alkyl-substituted amines and related polyamines are poor direct G protein activators in vivo. In the presence of G␤␥, they rather antagonize G protein-coupled receptor signaling.Heterotrimeric G proteins play a pivotal role in the communication of a given cell with the environment. They are stimulated by cell surface receptors, members of the superfamily of heptahelical receptors [G protein-coupled receptors (GPCR)] that catalyze the exchange of G␣-bound GDP for GTP (Hamm, 1998;Freissmuth et al., 1999). Consequently, the GTP-bound G protein dissociates into two signaling entities: the G␣-subunit and the G␤␥-subunit complex. With the notable exception of G␤ 5 CA

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