Synthesis and structure-activity relationship of novel pyridyl ethers for the nicotinic acetylcholine receptor.

The preparation of novel pyridyl ethers as ligands for the nicotinic acetylcholine receptor (nAChR) is described. Variations of the ring size of the azacycle and substitution on the pyridine had dramatic effects on receptor binding affinity with IC50s at the alpha4beta2 nAChR ranging from 22 to >10,000 nM. The most potent molecule was (R)-2-chloro-3-(4-cyanophenyl)-5-((3-pyrrolidinyl)oxy)pyridine 27f with an IC50 of 22 nM.

The solid-phase synthesis of novel 14-membered macrocycles for high throughput screening.

A simple and efficient synthesis of novel 14-membered macrocycles from a resin-bound orthogonally protected lysine residue is described. Reductive alkylation of the lysine alpha-nitrogen introduces the first diversity element. Acylation of the resultant secondary amine with an Fmoc-amino acid introduces the second diversity element providing a resin-bound protected di-peptide precursor. Removal of the Fmoc-group is followed by acylation with a succinic anhydride to introduce the final diversity elements. Removal of the methyltrityl-group from the amino group followed by macrocyclization provides the desired macrocycles, after TFA cleavage, in excellent yield and purity.

Dual agonistic and antagonistic property of nonpeptide angiotensin AT1 ligands: susceptibility to receptor mutations.

Two nonpeptide ligands that differ chemically by only a single methyl group but have agonistic (L-162,782) and antagonistic (L-162,389) properties in vivo were characterized on the cloned angiotensin AT1 receptor. Both compounds bound with high affinity (K(I) = 8 and 28 nM, respectively) to the AT1 receptor expressed transiently in COS-7 cells as determined in radioligand competition assays. L-162,782 acted as a powerful partial agonist, stimulating phosphatidylinositol turnover with a bell-shaped dose-response curve to 64% of the maximal level reached in response to angiotensin II. Surprisingly, L-162,389 also stimulated phosphatidylinositol turnover, albeit only to a small percentage of the angiotensin response. The prototype nonpeptide AT1 agonist L-162,313 gave a response of approximately 50%. The apparent EC50 values for all three compounds in stimulating phosphatidylinositol turnover were similar, approximately 30 nM, corresponding to their binding affinity. Each of the three compounds also acted as angiotensin antagonists, yet in this capacity the compounds differed markedly, with IC50 values ranging from 1.05 x 10(-7) M for L-162,389 to 6.5 x 10(-6) for L-162,782. A series of point mutations in the transmembrane segments (TMs) of the AT1 receptor had only minor effect on the binding affinity of the nonpeptide compounds, with the exception of A104V at the top of TM III, which selectively impaired the binding of L-162,782 and L-162,389. Substitutions in the middle of TM III, VI, or VII, which did not affect the binding affinity of the compounds, impaired or eliminated the agonistic efficacy of the nonpeptides but with only minor or no effect on the angiotensin potency or efficacy. Thus, in the N295D rat AT1 construct, L-162,782, L-162,313, and L-162,389 all antagonized the angiotensin-induced phosphatidylinositol turnover with surprisingly similar IC50 values (90-180 nM), and they all bound with unaltered, high affinity (22-36 nM). However, L-162,313 and L-162,782 could stimulate phosphatidylinositol turnover to only 20% of that of angiotensin. It is concluded that minor chemical modifications of either the compound or the receptor can dramatically alter the agonistic efficacy of biphenyl imidazole compounds on the AT1 receptor without affecting their affinity, as determined in binding assays, and that a number of substitutions in the middle of the TM segments affect the efficacy of nonpeptide agonists as opposed to angiotensin.

Discovery of L-162,313: a nonpeptide that mimics the biological actions of angiotensin II.

L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n- butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]- imadazo[4,5-b]pyridine) is a nonpeptide that mimics the biological actions of angiotensin II (ANG II). The intravenous administration of L-162,313 increased blood pressure in the rat. The maximum increase in mean arterial pressure (MAP) was not different from the maximum response to ANG II in the same preparation. However, the duration of the pressor response after L-162,313 greatly exceeded that of ANG II. Pretreatment with ANG II receptor antagonists, L-158,809 (AT1 selective) or saralasin, blocked the L-162,313-induced increase in MAP. Enalaprilat, an angiotensin-converting enzyme inhibitor, failed to block the MAP response to L-162,313. In vitro, L-162,313-activated phosphoinositide turnover in rat aortic smooth muscle cell cultures was also blocked by L-158,809 and losartan (DuP-753). Therefore, L-162,313 is the first reported nonpeptide ANG II receptor agonist.

Non-peptide angiotensin agonist. Functional and molecular interaction with the AT1 receptor.

Non-peptide ligands for peptide receptors for the G-protein-coupled type are generally antagonists, except in the opiate system. Recently, it was observed that a subset of biphenylimidazole derivatives surprisingly possessed angiotensin-like activity in vivo. In COS-7 cells transfected with the rat AT1 receptor a prototype of these compounds, L-162,313 stimulated phosphoinositide hydrolysis with an EC50 of 33 +/- 11 nM. The maximal response to the compound was 50% of that of angiotensin II in COS-7 cells but only 3% in stably transfected Chinese hamster ovary cells. The agonistic effect of L-162,313 was blocked by the AT1-specific antagonist L-158,809 and was not observed in untransfected cells. In Chinese hamster ovary cells, L-162,313 also acted as an insurmountable antagonist of the angiotensin stimulated phosphoinositide hydrolysis. In contrast to previously tested non-peptide ligands, L-162,313 bound with reasonably high affinity to the Xenopus laevis AT1 receptor. In the human receptor, the binding of L-162,313 was found to be unaffected by point mutations in transmembrane segments III and VII, which impaired the binding of biphenylimidazole antagonists. Substitutions in the extracellular domains of the human and rat receptor, which impaired the binding of angiotensin II, did not affect the binding of L-162,313. It is concluded that a subset of biphenylimidazole compounds can act as high affinity partial agonists on the AT1 receptor. These compounds have molecular interactions with the receptor which appear to differ both from that of the structurally similar non-peptide antagonists and from that of their functional counterpart, the peptide agonist.

[Immunologic changes in sickle-cell anemia]. / Alteraciones inmunológicas en la anemia drepanocítica.

In order to assess the status of their immunologic system, a study was carried out in 38 adults with sickle-cell anaemia. Fifty healthy blood donors were used as control group. Significant decrease of serum albumin (p less than 0.02) and increase of alpha-globulins (p less than 0.01) and gamma-globulins (p less than 0.001) were present in the patients. They showed also significantly decreased percentage of spontaneous rosette-forming lymphocytes (p less than 0.01) and of lymphocytes responding to anti-CD3 monoclonal antibody (p less than 0.05) with respect to the control group. Such relative T-cell decrease in peripheral blood seemingly took place by means of decreasing CD4-positive subpopulations, whose percentage was significantly lower (p less than 0.001) in the patients than in the control subjects. Functional studies showed a significant decrease (p less than 0.001) of the activity of natural cytotoxic cells. None of the patients had antibodies against human immunodeficiency viruses type 1 and type 2, and 60% of them were positive to cytomegalovirus test. No statistical correlation was found between the immunological findings and the presence of antibodies against such virus, neither such alterations correlated with the number of blood units received by the patients.

Density separation of spleen cells increases fusion frequency and yield of Ig-producing hybridomas.

The efficiency of hybridoma formation and growth after cell fusion can be much improved by fractionation of the mouse splenocytes. A simple procedure is described in which splenocytes with a specific gravity of more than 1.065 g/cm3 are selected by centrifugation on a Percoll gradient. The resulting cell suspension is largely depleted of macrophages and fibroblasts while the cell viability is improved. In fusion experiments performed with these cells, overgrowth of hybridomas by macrophages, fibroblasts and P-cells is avoided. The fusion efficiency and the frequency of immunoglobulin-secreting hybridomas is increased compared with fusions carried out with unfractionated spleen cells.