Nicole A. Horenstein

Activation and desensitization of nicotinic alpha7-type acetylcholine receptors by benzylidene anabaseines and nicotine.

Nicotinic receptor activation is inextricably linked to desensitization. This duality affects our ability to develop useful therapeutics targeting nicotinic acetylcholine receptor (nAChR). Nicotine and some α7-selective experimental partial agonists produce a transient activation of α7 receptors followed by a period of prolonged residual inhibition or desensitization (RID). The object of the present study was to determine whether RID was primarily due to prolonged desensitization or due to channel block. To make this determination, we used agents that varied significantly in their production of RID and two α7-selective positive allosteric modulators (PAMs): 5-hydroxyindole (5HI), a type 1 PAM that does not prevent desensitization; and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a type 2 PAM that reactivates desensitized receptors. The RID-producing compounds nicotine and 3-(2,4-dimethoxybenzylidene)anabaseine (diMeOBA) could obscure the potentiating effects of 5HI. However, through the use of nicotine, diMeOBA, and the RID-negative compound 3-(2,4-dihydroxybenzylidene)anabaseine (diOHBA) in combination with PNU-120596, we confirmed that diMeOBA produces short-lived channel block of α7 but that RID is because of the induction of a desensitized state that is stable in the absence of PNU-120596 and activated in the presence of PNU-120596. In contrast, diOHBA produced channel block but only readily reversible desensitization, whereas nicotine produced desensitization that could be converted into activation by PNU-120596 but no demonstrable channel block. Steady-state currents through receptors that would otherwise be desensitized could also be produced by the application of PNU-120596 in the presence of a physiologically relevant concentration of choline (60 μM), which may be significant for the therapeutic development of type 2 PAMs.

A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors.

Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.

Molecular dissection of tropisetron, an α7 nicotinic acetylcholine receptor-selective partial agonist

The alpha7 nicotinic acetylcholine receptor (nAChR)-selective partial agonist tropisetron is a conjugate of an indole and a tropane group. We tested compounds structurally related to either the indole or tropane domains of tropisetron on oocytes expressing human alpha7. alpha4beta2, or alpha3beta4 nAChR or rat 5HT(3A) receptors. The simple compounds tropane and tropinone had alpha7-selective agonist activity comparable to that of tropisetron. Tropinone was more efficacious than tropisetron but 100-fold less potent. Some tropane compounds had antagonist activity on alpha3beta4 nAChR but no effect on alpha4beta2 nAChR. Some tropanes also affected the responses of 5HT3 receptors to serotonin. Tropisetron was more potent at inhibiting alpha3beta4 receptors (IC(50)=1.8+/-0.6) than was tropane or tropinone, suggesting that the presence of the indole group has a large impact on the potency of tropisetron, both as an alpha7 agonist and as an alpha3beta4 antagonist. The further reduced structures of dimethyl piperidinium and 1-methylpyrrolidine also had agonist activity on alpha7 receptors, suggesting that the minimal activating pharmacophore of these compounds, as with tetramethylammonium, may simply be the charged nitrogen, while additional structure elements impact subtype selectivity, potency, and efficacy. It has previously been reported that 5-hydroxyindole (5HI) can potentiate alpha7 receptor responses to acetylcholine (ACh). However, the site where 5HI binds to the receptor is not known. We tested the hypothesis that the tropisetron binding site might overlap the 5HI site and thereby produce a block of 5HI potentiation. Our results indicate that the indole portion of tropisetron is not likely to be binding to the same site where 5HI binds to potentiate alpha7 receptor responses since 5HI can greatly potentiate responses of tropisetron, tropinone, and other partial agonists such as 4OH-GTS-21.

Multiple Pharmacophores for the Selective Activation of Nicotinic α7-Type Acetylcholine Receptors

The activation of heteromeric and homomeric nicotinic acetylcholine receptors was studied in Xenopus laevis oocytes to identify key structures of putative agonist molecules associated with the selective activation of homomeric α7 receptors. We observed that selectivity between α7 and α4β2 was more readily obtained than selectivity between α7 and α3β4. Based on structural comparisons of previously characterized selective and nonselective agonists, we hypothesize at least three chemical motifs exist that, when present in molecules containing an appropriate cationic center, could be associated with the selective activation of α7 receptors. We identify the three distinct structural motifs based on prototypical drugs as the choline motif, the tropane motif, and the benzylidene motif. The choline motif involves the location of an oxygen-containing polar group such as a hydroxyl or carbonyl separated by two carbons from the charged nitrogen. The tropane motif provides α7-selectivity based on the addition of multiple small hydrophobic groups positioned away from the cationic center in specific orientations. We show that this motif can convert the nonselective agonists quinuclidine and ethyltrimethyl-ammonium to the α7-selective analogs methyl-quinuclidine and diethyldimethyl-ammonium, respectively. We have shown previously that the benzylidene group of 3-2,4, dimethoxy-benzylidene anabaseine (GTS-21) converts anabaseine into an α7-selective agonist. The benzylidene motif was also applied to quinuclidine to generate another distinct family of α7-selective agonists. Our results provide insight for the further development of nicotinic therapeutics and will be useful to direct future experiments with protein structure-based modeling and site-directed mutagenesis.

The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.

Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors.

Homomeric α7 and heteromeric α4β2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in α7 receptors for which the α7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in α4 receptors co-expressed with β2 resulted in α4β2 receptors for which ACh potency was relatively unchanged, although the efficacy of the α7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in α7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both α7 and α4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the α4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.

Mechanisms for nucleophilic aliphatic substitution at glycosides

Abstract Much of carbohydrate chemistry and biochemistry is centered on bond forming and bond breaking reactions at the anomeric carbon of glycosides. No single mechanism adequately covers the scope of these reactions, because differences in sugar substituents, stereochemistry, leaving groups, nucleophiles, and catalysts can influence the mechanistic pathway taken. The influence of solvent is only now beginning to become apparent in greater detail. Several methods exist to probe the mechanisms of these reactions; they include a variety of kinetic studies, including isotope effects, and computational methods. It has been found that typical reactions will uniquely utilize a mechanism somewhere within a continuum between A N D N and D N +A N mechanisms. With knowledge of the factors that determine the mechanism, synthetic method development will be furthered and a deeper understanding of biological catalysis is likely to be gained.

The Minimal Pharmacophore for Silent Agonism of the α7 Nicotinic Acetylcholine Receptor

The minimum pharmacophore for activation of the human α7 nicotinic acetylcholine receptor (nAChR) is the tetramethylammonium cation. Previous work demonstrated that larger quaternary ammonium compounds, such as diethyldimethylammonium or 1-methyl quinuclidine, were α7-selective partial agonists, but additional increase in the size of the ammonium cation or the quinuclidine N-alkyl group by a single carbon to an N-ethyl group led to a loss of efficacy for ion channel activation. We report that although such compounds are ineffective at inducing the normal channel open state, they nonetheless regulate the induction of specific conformational states normally considered downstream of channel activation. We synthesized several panels of quaternary ammonium nAChR ligands that systematically varied the size of the substituents bonded to the central positively charged nitrogen atom. In these molecular series, we found a correlation between the molecular volume of the ligand and/or charge density, and the receptor’s preferred distribution among conformational states including the closed state, the active state, a nonconducting state that could be converted to an activated state by a positive allosteric modulator (PAM), and a PAM-insensitive nonconducting state. We hypothesize that the changes of molecular volume of an agonist’s cationic core subtly impact interactions at the subunit interface constituting the orthosteric binding site in such a way as to regulate the probability of conversions among the conformational states. We define a new minimal pharmacophore for the class of compounds we have termed “silent agonists,” which are able to induce allosteric modulator-dependent activation but not the normal activated state.

Cysteine accessibility analysis of the human alpha7 nicotinic acetylcholine receptor ligand-binding domain identifies L119 as a gatekeeper.

A large number of structurally diverse ligands have been produced to selectively target α7 nicotinic acetylcholine receptors (nAChRs). We applied the method of scanning cysteine accessibility mutations (SCAM) to the ligand-binding domain of the α7 nAChR to identify subdomains of particular importance to the binding and subsequent activation by select agonists. We evaluated the activity of four structurally distinct α7 agonists on wild-type human α7 and 44 targeted mutants expressed in Xenopus oocytes. Responses were measured prior and subsequent to the application of the sulfhydryl reagent methanethiosulfonate ethylammonium (MTSEA). One mutant (C116S) served as a Cys-null control, and the additional mutants were made in the C116S background. In many cases, the insertion of free cysteines into the agonist-binding site had a negative effect on function, with 12 of 44 mutants showing no detectable responses to ACh, and with only 19 of the 44 mutants showing sufficiently large responses to permit further study. Several of the cysteine mutations, including W55C, showed selectively reduced responses to the largest agonist tested, 2-methoxy,4-hydroxy-benzylidene anabaseine. Interestingly, although homology models suggest that most of the introduced cysteine mutations should have had good solvent accessibility, application of MTSEA had no effect or produced only modest changes in the agonist response profile of most mutants. Consistent with previous studies implicating W55 to play important roles in agonist activation, MTSEA treatment further decreased the functional responses of W55C to all the test agonists. While the cysteine mutation at L119 itself had relatively little effect on receptor function, treatment of L119C receptors with MTSEA or alternative cationic sulfhydryl reagents profoundly decreased activation by all agonists tested, suggesting a general block of gating. The homologous mutation in heteromeric nAChRs produced similar results, provided that the mutation was placed in the beta subunit complementary surface of the ligand-binding domain. Structural models locate the L119 residue directly across the subunit interface from the C-loop of the primary face of the binding domain. Our data suggest that a covalent modification of L119C by MTSEA or other cationic reagents might block the binding of even small agonists such as TMA through electrostatic interactions. Reaction of L119C with small non-polar reagents increases activation by small agonists but can block the access of large ligands such as benzylidene anabaseines to the ligand-binding domain.

Tethered Agonist Analogs as Site-Specific Probes for Domains of the Human α7 Nicotinic Acetylcholine Receptor that Differentially Regulate Activation and Desensitization

Homomeric α7 nicotinic acetylcholine receptors represent an important and complex pharmaceutical target. They can be activated by structurally diverse agonists and are highly likely to enter and remain in desensitized states at rates determined by the structures of the agonists. To identify structural elements regulating this function, we introduced reactive cysteines into the α7 ligand-binding domain allowing us to bind sulfhydryl-reactive (SH) agonist analogs or control reagents onto specific positions in the ligand binding domain. We identified four α7 mutants (S36C, L38C, W55C, and L119C) in which the tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor. However, after selective reaction with SH agonist analogs, the type II allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl-3-isoxazolyl)-urea (PNU-120596) could reactivate L119C and W55C mutants and receptors with a reduced or modified C-loop. Modified S36C and L38C mutants were insensitive to reactivation by PNU-120596, whether they were reacted with agonist analogs or alternative SH reagents. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in a similar but nonidentical position underneath the C-loop. The orientation assumed by the ligand tethered to 119C was approximately 3-fold more sensitive to PNU-120596 than the alternative pose at 55C. Our results support the hypothesis that a single ligand can bind within the receptor in different ways and, depending on the specific binding pose, may variously promote activation or desensitization, or, alternatively, function as a competitive antagonist. This insight may provide a new approach for drug development.