Gsa-selective G protein antagonists

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the Ga subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for Gsa. Two compounds have been identified: NF449 (4,4*,4(,4*(-[carbonyl-bis[imino-5,1,3benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3disulfonate) and NF503 (4,4*-[carbonylbis[imino-3,1-phenylene(2,5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (i) suppress the association rate of guanosine 5*-[g-thio]triphosphate ([35S]GTP[gS]) binding to Gsa-s but not to Gia-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc2 membranes (deficient in endogenous Gsa) by exogenously added Gsa-s, and (iii) block the coupling of b-adrenergic receptors to Gs with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A1-adenosine receptor with its cognate G proteins (Gi/Go) at concentrations that are >30fold higher than those required for uncoupling of b-adrenergic receptor/Gs tandems; similarly, the angiotensin II type-1 receptor (a prototypical Gq-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for Gsa-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition. In current pharmacotherapy the input into G proteinregulated signaling is manipulated by targeting the receptor with appropriate agonists and antagonists. Several arguments, however, suggest that elements of the receptor-activated, downstream signaling cascade, in particular G proteins, may be considered as drug targets per se. (i) The molecular diversity of G protein a, b, and g subunits is large, and the number of distinct abg oligomers that can be produced by combinatorial association of subunit is excessive (1). (ii) The interaction of a given receptor with the cellular complement of G proteins may be governed by both exquisite specificity and promiscuity; in the first case, only one defined oligomer supports the ability of a receptor to regulate an effector in an intact cell (for review see refs. 2 and 3). (iii) On the other hand, many receptors couple to multiple G proteins (2, 3); this is exemplified by the thyrotropin receptor (4), which can activate essentially all G protein a subunits expressed in the thyroid (i.e., members of all subfamilies of Ga other than the transducins). Thus, cellular stimulation by a receptor often results in the concerted activation of several distinct G proteins such that multiple effector pathways are recruited to produce the biological response. In theory, it may be desirable to block signaling of a receptor via one type of G protein but not via the other G proteins (resulting in ‘‘biased inhibition of receptor/G protein tandem formation’’), a goal that cannot be achieved by receptor antagonists but that may be achieved by compounds that bind selectively to individual G proteins. In addition, several human diseases arise from activation of G protein a subunits by point mutations (for a brief overview, see ref. 5); appropriate G protein antagonists are desirable under these circumstances. Suramin has been shown previously to act directly on G protein a subunits (6) and to block their activation by receptors (7–9). In the present work, we have searched for compounds that inhibit Gsa directly. Two compounds of remarkable selectivity were identified that suppress the coupling of b-adrenergic receptors to Gs, whereas they affect a prototypical Gi/Goand Gq-coupled receptors (A1-adenosine and angiotensin II receptor, respectively) to a much lesser extent. MATERIALS AND METHODS Materials and Chemical Synthesis. Suramin, chloroglycouril, DTT, Hepes, angiotensin II, and saralasin were purchased from Sigma; [125I], [125I]CYP (iodocyanopindolol), [35S]GTP[gS], and [a-32P]ATP were from NEN. The synthesis of NF503 [C41H28N8O9S2Na2; disodium-4.49-[carbonylbis[imino-3.1phenylene-(2.5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate)] has been described (10); NF445 [C45H36N6O17S4Na4; tetrasodium-4.49. 40.490-[carbonylbis[imino-2.1. 4-benzenetriylbis(carbonyliminomethylene)]]tetrakis-(benzenesulfonate)] and NF449 [C41H24N6O298Na8; octasodium-4.49. 40.490-[carbonyl-bis [imino-5.1. 3-benzenetriylbis-(carbonylimino)]]tetrakis-(benzene1.3-disulfonate)] were synthesized using methods described in detail for other suramin analogues (11). For NF445, 3-(aminomethyl)benzenesulfonic acid (50 mmol in 50 ml of H2O) was treated at pH 8.5 and room temperature with 2-nitroterephtalic acid dichloride (30 mmol dissolved in 100 ml of toluene). The precipitate formed during the reaction was filtered, recrystallized from H2O, and hydrogenated in aqueous solution (1%) using Pd/C as catalyst. After filtration, the concentrated filtrate (10% original volume) was reacted with a large excess of phosgene (20% in toluene) at room temperature and pH 4. The aqueous phase of the reaction mixture was evaporated to dryness. The residue was recrystallized from H2O/CH3OH; overall yield was 75% (yellowish powder). NF449 was synthesized in a similar way starting from aminobenezene-2.4-disulfonic acid and 5-nitroisophtalic acid dichloride; overall yield was 25% (yellowish powder). All reaction steps were followed up by TLC. The purity of the final products was . 95% as determined by HPLC (12). Saralasin and angiotensin II were iodinated by incubating the peptides (7 nmol) and carrier-free Na125I (1 mCi) in 40 ml of 0.1 M NaPi, pH 7.4, in a tube precoated with 20 mg of chloroglycouril for 10 min on ice. The reaction products were resolved by HPLC (Merck Lichrospher 100 RP-C18; 4.6 3 250 mm; 5 mm) using a 35-ml linear gradient of 15 to 40% acetonitrile in 0.1% trifluoroacetic acid. Saralasin, monoiodinated saralasin, and diThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y95346-6$2.00y0 PNAS is available online at http:yywww.pnas. org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: [125I]HPIA, N6-(3-iodo-4-hydroxyphenylisopropyl)adenosine; [35S]GTP[gS], guanosine 59-[g-thio]triphosphate. †To whom reprint requests should be addressed. e-mail: michael. freissmuth@univie.ac. at.