No abstract

The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are "atypical", having steep concentration-effect curves and not interacting competitively with "typical" allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M 2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4Ј-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1Ј-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg 2ϩ , and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M 2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M 2 /M 5 -receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M 2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the "common" allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M 2 -receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer.A rapidly increasing number of G protein-coupled receptors have been discovered to be sensitive to allosteric modulation (Christopoulos and Kenakin, 2002). Potentially favorable features for a clinical application of such modulation include the enhancement of the binding of endogenous ligands (but also exogenous agonists and antagonists), absolute subtype selectivity of action, and self-limiting effects on receptor function (see Christopoulos and Kenakin, 2002 for review).Over the past decade, allosteric interactions at muscarinic acetylcholine receptors have been intensively studied (e.g., Ellis, 1997;Mohr et al., 2003, Birdsall andLazareno, 2005). All five muscarinic receptor subtypes are sensitive to alloste-

Bis(2-N,N-dimethylamino-indenyJ) zirconium dichloride, (2-(CH 3)2 N-C 9 H 6)2 ZrC1 2 • and dimethylsilyl-bridged bis(2-N,N-dimethylamino-indenyl) zirconium dichloride. (CH3)2Si(2-(CH3)2N-C9Hs)2ZrCI2' were prepared by reaction of the corresponding ligand lithium salts with ZrCl" in toluene. Diffractometric structure determinations reveal C 2-symmetrlc complex geometries for both complexes. An increased electron density at the Zr center of the dimethylamino-substituted complexes is indicated by reduction potentials which are 0.3-0.4 V morr negative than those of their unsubstituted analogs. When activated with methyl aluminoxane in toluene solution. (CH3)2Si(2-(CH3)2N-C9Hs)2ZrCI2 catalyzes the polymerization of propene to polymers with a microstructure comparable with that of polymers produced with other Mel Si-bridged bis(indenyJ)ZrCl 2 complexes. but with a substantially increased fraction of i-propyl end groups derived from alkyl exchange between Zr-polymer and AI-Me species.

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.

ansa-Metallocene derivatives xxv *. Synthesis, crystal structure and reactions of a tetramethylethano-bridged vanadocene dichloride, (CH3)4C2(CsH4)zVClz