New insight into active muscarinic receptors with the novel radioagonist [³H]iperoxo.

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M2 receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [³H]iperoxo - unlike the common radioagonists [³H]acetylcholine and [³H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [³H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M2 and M4 receptor in the picomolar range. Probing ternary complex formation on the M2 receptor, [³H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M2, the preferred Gi protein acted as a positive allosteric modulator of [³H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM2 cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [³H]iperoxo. Taken together, the muscarinic superagonist [³H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.

Investigation of the molecular mechanism of the α7 nicotinic acetylcholine receptor positive allosteric modulator PNU-120596 provides evidence for two distinct desensitized states.

Although α7 nicotinic acetylcholine receptors are considered potentially important therapeutic targets, the development of selective agonists has been stymied by the α7 receptor's intrinsically low probability of opening (P(open)) and the concern that an agonist-based therapeutic approach would disrupt endogenous cholinergic function. Development of α7 positive allosteric modulators (PAMs) holds promise of avoiding both issues. N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596) is one of the most effective α7 PAMs, with a mechanism associated, at least in part, with the destabilization of desensitized states. We studied the mechanism of PNU-120596 potentiation of α7 receptors expressed in Xenopus laevis oocytes and outside-out patches from BOSC 23 cells. We identify two forms of α7 desensitization: one is destabilized by PNU-120596 (D(s)), and the other is induced by strong episodes of activation and is stable in the presence of the PAM (D(i)). Our characterization of prolonged bursts of single-channel currents that occur with PNU-120596 provide a remarkable contrast to the behavior of the channels in the absence of the PAM. Individual channels that avoid the D(i) state show a 100,000-fold increase in P(open) compared with receptors in the nonpotentiated state. In the presence of PNU-120596, balance between D(s) and D(i) is dynamically regulated by both agonist and PAM binding, with maximal ion channel activity at intermediate levels of binding to both classes of sites. In the presence of high agonist concentrations, competitive antagonists may have the effect of shifting the balance in favor of D(s) and increasing ion channel currents.

Synergistic action of the novel HSP90 inhibitor NVP-AUY922 with histone deacetylase inhibitors, melphalan, or doxorubicin in multiple myeloma.

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP-AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM-2, RPMI-8226, U-266, LP-1, MM1.S, and primary myeloma cells were exposed to NVP-AUY922 and one of the combination partners histone deacetylase inhibitor NVP-LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP-AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP-AUY922 with SAHA, NVP-LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI-8226 cell line and relative resistance of some primary myeloma cells against NVP-AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP-AUY922 with melphalan, doxorubicin, NVP-LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.