Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

Activation and allosteric modulation of a muscarinic acetylcholine receptor.

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the ß2 adrenergic receptor (ß2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the ß2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

Muscarinic receptors as model targets and antitargets for structure-based ligand discovery.

G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse ß-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

Structure-Based Design and Discovery of New M2 Receptor Agonists.

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Protein kinase C delta activation and translocation to the nucleus are required for fatty acid-induced apoptosis of insulin-secreting cells.

Insulin resistance as well as pancreatic beta-cell failure can be induced by elevated free fatty acid (FFA) levels. We studied the mechanisms of FFA-induced apoptosis in rat and human beta-cells. Chronic treatment with high physiological levels of saturated fatty acids (palmitate and stearate), but not with monounsaturated (palmitoleate and oleate) or polyunsaturated fatty acids (linoleate), triggers apoptosis in approximately 20% of cultured RIN1046-38 cells. Apoptosis restricted to saturated FFAs was also observed in primary cultured human beta-cells, suggesting that this mechanism is potentially relevant in vivo in humans. To further analyze FFA-induced signaling pathways leading to apoptosis, we used RIN1046-38 cells. Apoptosis was accompanied by a rapid (within 15 min) nuclear translocation of protein kinase C (PKC)-delta and subsequent lamin B1 disassembly. This translocation was impaired by the phospholipase C inhibitor U-73122, which also substantially reduced apoptosis. Furthermore, lamin B1 disassembly and apoptosis were decreased by cell transfection with a dominant-negative mutant form of PKC-delta. These data suggest that nuclear translocation and kinase activity of PKC-delta are both necessary for saturated fatty acid-induced apoptosis.

Involvement of protein kinase Cdelta and extracellular signal-regulated kinase-2 in the suppression of microglial inducible nitric oxide synthase expression by N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast).

Excess nitric oxide (NO) in the brain released by microglial cells contributes to neuronal damage in various pathologies of the central nervous system (CNS) including neurodegenerative diseases and multiple sclerosis. N-[3,4-Dimethoxycinnamoyl]-anthranilic acid (tranilast, TNL) is an anti-allergic compound which suppresses the activation of monocytes. We show that inducible nitric oxide synthase (iNOS) mRNA and protein expression and the release of NO from N9 microglial cells stimulated with the bacterial endotoxin lipopolysaccharide (LPS) are inhibited when the cells are exposed to TNL. TNL fails to modulate LPS-stimulated nuclear factor-kappaB (NF-kappaB) reporter gene activity and phosphorylation of inhibitory kappaB (IkappaB), indicating that NF-kappaB is not involved in the TNL-mediated suppression of LPS-induced iNOS expression. Moreover, TNL inhibits LPS-induced phosphorylation of extracellular signal-regulated kinase 2 (ERK-2). Finally, TNL abolishes translocation of protein kinase Cdelta (PKCdelta) to the nucleus and suppresses the phosphorylation of the PKCdelta substrate, myristoylated alanin-rich C kinase substrate (MARCKS). We conclude that the anti-allergic compound TNL suppresses microglial iNOS induction by LPS via inhibition of a signalling pathway involving PKCdelta and ERK-2.

[Apoptosis induced by free fatty acids]. / Fettsäureinduzierte beta-Zell-Apoptose.

BACKGROUND AND AIMS: Elevated free fatty acid (FFA) levels are believed to be one of the major contributing factors in the pathogenesis of type 2 diabetes. FFAs enhance peripheral insulin resistance, promote beta-cell dysfunction, and trigger beta-cell death. The purpose of this study was to investigate whether the lipoapoptotic effect of FFAs is determined by the degree of saturation and the chain length, and which pathways might be involved. MATERIAL AND METHODS: After incubation of RIN1046-38 rat insulinoma cells and primary human islet cells with different FFAs, apoptosis was assessed by flow cytometry (sub-G1-DNA formation) and TUNEL assay, respectively. Intracellular distribution of protein kinase C-delta (PKC-delta) was determined by confocal laser microscopy. RESULTS: In RIN1046-38 cells, the sub-G1 fraction increased 40-fold over basal (0.5% to approximately 20%) after treatment with the saturated FFAs palmitate and stearate (1 mM, 24 h), while the monounsaturated FFAs palmitoleate and oleate as well as the polyunsaturated FFA linoleate had no proapoptotic effect. Unsaturated FFAs even more prevented palmitate- and stearate-induced apoptosis completely. Furthermore, these results could be confirmed in primary beta-cells of human islets. Investigating potential underlying signaling pathways, it was found that only the saturated FFAs were able to induce nuclear translocation of PKC-delta. CONCLUSIONS: FFAs exert different effects on beta-cell survival. Saturated FFAs turned out to be pro-apoptotic, whereas unsaturated FFAs protected from saturated FFA-induced apoptosis. Furthermore, our data suggest an involvement of PKC-delta in apoptosis induced by saturated FFAs.

Different role of saturated and unsaturated fatty acids in beta-cell apoptosis.

It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human beta-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in beta-cells. RIN1046-38 cells and isolated human beta-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24-72 h with 1mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human beta-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.