A novel mechanism of modulation of 5-HT₃A receptors by hydrocortisone

Modulation of Cys-loop receptors by steroids is of physiological and therapeutical relevance. Nonetheless, its molecular mechanism has not been elucidated for serotonin (5-HT) type 3 receptors. We deciphered the mechanism of action of hydrocortisone (HC) at 5-HT type 3A receptors. Single-channel currents from the high-conductance form (∼4.7 pA, −70 mV) appear as a series of long opening events forming bursts, which group into long clusters. Although they are very infrequent, subconductance events (∼2.4 pA) are detected within clusters. HC produces a significant concentration-dependent reduction in open and burst durations, demonstrating open-channel block. In addition, it increases the appearance of subconductance levels in a concentration- and slightly voltage-dependent manner. The amplitude of the subconductance level does not change with HC concentration and its open duration is briefer than that of full amplitude events, indicating lower open-channel stability. Dual effects are distinguished from macroscopic responses: HC reduces amplitude by acting from either open or closed states, and it increases decay rates from the open state. Thus, HC acts as a negative modulator of 5-HT type 3A receptors by different mechanisms: It acts as an open-channel blocker and it favors opening to a preexisting subconductance level. The latter constitutes a novel, to our knowledge, mechanism of channel modulation, which might be applicable to other steroids and channels.     

Mechanism of action of snake venom α-neurotoxins on nicotinic acetylcholine receptors of rabbit sympathetic ganglion neurons

Currents evoked by iontophoretic applications of acetylcholine and postsynaptic currents evoked by single stimulation of the cervical sympathetic nerve were recorded in neurons of the isolated rabbit superior cervical ganglion with membrane voltage clamped and muscarinic acetylcholine receptors blocked by atropine (10^?6 M). The α-neurotoxins from snake venom (α-bungarotoxin and α-cobratoxin) in a concentration of 10^?6 M caused an increase in amplitude (potentiation) of the acetylcholine current, inhibition of that current, or initial potentiation followed by inhibition, in different neurons. Spectral analysis of the fluctuations of this current showed that α-neurotoxins affect neither the current through a single channel nor the duration of the open state of long-living channels (evidently extrasynaptic), but they approximately double the duration of the open state of long-living channels. This last effect in all probability causes potentiation of the acetylcholine current. The α-neurotoxins also depressed the amplitude of the postsynaptic current evoked by sympathetic nerve stimulation (on average by 44%) and lengthened its decline (on average by 24%). It is postulated that α-neurotoxins may both block and modify activity of the receptor-channel complex in the neurons tested, lengthening the duration of its open state. This latter mechanism of action of α-neurotoxins is exhibited only in long-living channels, evidence that the phamacological properties of the two populations of channels connected with nicotinic acetylcholine receptors are not identical.     

Structure–activity relationships of N-substituted 4-(trifluoromethoxy)benzamidines with affinity for GluN2B-containing NMDA receptors

GluN2B subtype-selective NMDA antagonists represent promising therapeutic targets for the symptomatic treatment of multiple CNS pathologies. A series of N-benzyl substituted benzamidines were synthesised and the benzyl ring was further replaced with various polycyclic moieties. Compounds were evaluated for activity at GluN2B containing NMDA receptors where analogues 9, 12, 16 and 18 were the most potent of the series, replacement of the benzyl ring with polycycles resulted in a complete loss of activity.     

Structure–activity relationships of non-opioid [des-Arg7]-dynorphin A analogues for bradykinin receptors

In our earlier studies, bradykinin receptors (BRs) were identified as a potential target for the neuroexcitatory effects of dynorphin A (Dyn A) in the central nervous system (CNS), and [des-Arg⁷]-Dyn A-(4–11) (6) was discovered as a lead ligand to modulate Dyn A-(2–13) induced neuroexcitatory effects in the CNS as an antagonist. In an effort to gain insights into key structural features of the Dyn A for the BRs, we pursued further structure–activity relationships (SAR) study on the [des-Arg⁷]-Dyn A analogs and confirmed that all of the [des-Arg⁷]-Dyn A analogues showed good binding affinities at the BRs.     

A fluorometric method for determination of catalytic activity of CYP51b1 (sterol 14α-demethylase) with coumarin derivatives

A method for direct registration of CYP51b1 (sterol-14α-demethylase) activity with coumarin derivatives as substrate has been proposed. Determination of catalytic activity of this enzyme with 7-aminocoumarin-4-acetic acid (ACAC) is based on registration of the increase of fluorescence (λ_excitation = 360 nm and λ_emission = 435 nm) at 30°C. In the model system for assay of CYP51b1 activity BMR (a flavin domain of the bacterial cytochrome P450BM-3) may serve as the electron donor. The developed method is simple, highly sensitive and accurate; it can be used for screening of sterol 14α-demethylase inhibitors.     

Identification of a PPARδ agonist with partial agonistic activity on PPARγ

The discovery and optimization of a series of potent PPARδ full agonists with partial agonistic activity against PPARγ is described.     

Antagonizing α7 nicotinic receptors with methyllycaconitine (MLA) potentiates receptor activity and memory acquisition

α7 nicotinic acetylcholine receptors (α7nAChRs) have been targeted to improve cognition in different neurological and psychiatric disorders. Nevertheless, no α7nAChR activating ligand has been clinically approved. Here, we investigated the effects of antagonizing α7nAChRs using the selective antagonist methyllycaconitine (MLA) on receptor activity in vitro and cognitive functioning in vivo. Picomolar concentrations of MLA significantly potentiated receptor responses in electrophysiological experiments mimicking the in vivo situation. Furthermore, microdialysis studies showed that MLA administration substantially increased hippocampal glutamate efflux which is related to memory processes. Accordingly, pre-tetanus administration of low MLA concentrations produced longer lasting potentiation (long-term potentiation, LTP) in studies examining hippocampal plasticity. Moreover, low doses of MLA improved acquisition, but not consolidation memory processes in rats. While the focus to enhance cognition by modulating α7nAChRs lies on agonists and positive modulators, antagonists at low doses should provide a novel approach to improve cognition in neurological and psychiatric disorders.     

Cholesterol and Clioquinol modulation of Aβ(1-42) interaction with phospholipid bilayers and metals

The β-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-β peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, Aβ(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state ³¹P and ²H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in β-sheet, were observed using specifically ¹³C-labelled Aβ(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely ¹⁵N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric β-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.     

Structure–activity relationships of bisphenol A analogs at estrogen receptors (ERs): Discovery of an ERα-selective antagonist

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we synthesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonistic/antagonistic activities at ERs and ERα/ERβ subtype selectivity. Among the compounds examined, 18 was found to be a potent ERα-antagonist with high selectivity over ERβ and androgen receptor under our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic conformation of ERα. ERα-selective antagonists, such as 18, are candidate agents for treatment of breast cancer.     

Does infusion of ANG II increase muscle sympathetic nerve activity in patients with primary aldosteronism?

Patients with primary aldosteronism (PA) were shown to have suppressed muscle sympathetic nerve activity (MSNA) in our previous study. Although baroreflex inhibition probably accounts in part for this reduced MSNA in PA, we hypothesized that the lowered activity of the renin-angiotensin system in PA may also contribute to the suppressed SNA. We recorded MSNA in 9 PA and 16 age-matched normotensive controls (NC). In PA, the resting mean blood pressure (MBP) and serum sodium concentrations were increased, and MSNA was reduced. We examined the effects of infusion of a high physiological dose of ANG II (5.0 ng.kg(-1).min(-1)) on MSNA in 6 of 9 PA and 9 of 16 NC. Infusion of ANG II caused a greater pressor response in PA than NC, but, in spite of the greater increase in pressure, MSNA increased in PA, whereas it decreased in NC. Simultaneous infusion of nitroprusside and ANG II, to maintain central venous pressure at the baseline level and reduce the elevation in MBP induced by ANG II, caused significantly greater increases in MSNA in PA than in NC. Baroreflex sensitivity of heart rate, estimated during phenylephrine infusions, was reduced in PA, but baroreflex sensitivity of MSNA was unchanged in PA compared with NC. All the abnormalities in PA were eliminated following unilateral adrenalectomy. In conclusion, the suppressed SNA in PA depends in part on the low level of ANG II in these patients.     

Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp²³-Lys²⁸ salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly³³, a residue involved in Met³⁵ oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.     

Structure–activity relationship (SAR) of parthenin analogues with pro-apoptotic activity: Development of novel anti-cancer leads

Analogues of parthenin were synthesized by substitutions at different reaction centres to establish a structure–activity relationship (SAR). Some of the molecules have displayed significant cytotoxicity in human cervical carcinoma (HeLa) and human myeloid leukemia (HL-60) cells. A few of the compounds also induced apoptosis in HL-60 cells measured in terms of sub-Go/G1 DNA fraction. Also one of the lead molecules has been shown to be the inhibitor of both telomerase and topoisomerase-II.     

Interferon inducing activity of (A)n·(U)n complexes of varying chain length

Evidence is presented that the interferon-inducing activity of (A)_n·(U)_n in primary rabbit kidney cells with respect to the chain length of the constituting (A)_n and (U)_n strands is governed by the following criteria: (1) the activity increases with the length of the uninterrupted double-stranded segment in the complex whereby both chains are equally important and the number of such segments for complex molecule is without effect, (2) at a constant total concentration of constituting nucleotides, the activity increases with the number of double-stranded molecular entities available to the cell, and (3) complexes with the (U)_n strand considerably overlapping the (A)_n strand are inactive due to the formation of triple-stranded structures.     

Interaction of thymic peptide thymosinα 1 with vasoactive intestinal peptide (VIP) receptors

Thymic peptide thymosin ₁ (10^–9 to 3 x 1010^–7 M) is shown to inhibit the specific binding of [¹²⁵I]VIP to rat blood mononuclear cells and liver plasma membranes. Thymosin ₁ was 160 and 6250 times less potent that VIP at inhibiting [¹²⁵I]VIP binding to blood mononuclear cells and liver plasma membranes, respectively. Thymosin ₁ (10^–10 to 1010^–7 M) was weak in stimulating adenylate cyclase activity. Its efficacy is about 25 % and 27 % that of native VIP in blood mononuclear cells and liver plasma membranes, respectively. Thymosin ₁ may behave as a partial VIP agonist in rat.Abbreviations GRF growth hormone releasing factor - PHI porcine intestinal peptide having N-terminal histidine and C-terminal isoleucine amide - GIP gastric inhibitory polypeptide - VIP vasoactive intestinal peptide     

Synthesis and structure–activity relationships of cassiarin A as potential antimalarials with vasorelaxant activity

Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothelium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria. Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthesized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom without any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibition of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials with different mode of actions.     

Effects of neuropeptide Y (NPY) at the sympathetic neuroeffector junction. Can pre- and postjunctional receptors be distinguished?

Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic postganglionic neurons, NPY coexists with noradrenaline. NPY and its structural relative peptide YY (PYY) appear to exert three principally different effects at the sympathetic neuroeffector junction. Firstly, NPY has a direct postjunctional effect; this effect is manifested as a vasoconstriction when studied on the guinea pig iliac vein. Secondly, NPY has an indirect postjunctional effect in that it potentiates the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as vasoconstrictors. Thirdly, NPY acts prejunctionally in that it suppresses the release of noradrenaline from sympathetic nerve terminals; this was studied in the rat vas deferens. The aim of the investigation was to examine whether the three effects of NPY were mediated by the same type of receptor. For this purpose, we examined the effects of a series of NPY-related peptides, namely NPY, PYY, desamido-NPY, and five C-terminal fragments (NPY 19-36, NPY 24-36, PYY 13-36, PYY 24-36 and PYY 27-36). NPY and PYY were active in all three assay systems. The C-terminal amide appears to be crucial for maintaining the biological activity, since desamido-NPY was inactive in the three test systems. Interestingly, PYY 13-36 was almost as active as NPY and PYY in suppressing the electrically evoked contractions of the vas deferens; PYY 13-36 was inactive in the two other test systems. None of the shorter fragments had any biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

What sets the long-term level of sympathetic nerve activity: is there a role for arterial baroreceptors?

Much of our knowledge of the influence of the sympathetic nervous system on the control of blood pressure is built on experimental approaches that focus very much on time scales <24 h. Although direct recordings of sympathetic nerve activity (SNA) over short time scales provide important information, it is difficult to place their relevance over the longer term where the development of chronic changes in blood pressure are likely to be a mixture of hormonal, renal, and neural influences. Recently new experimental approaches are now revealing a possible role for arterial baroreceptors in the chronic regulation of SNA. These studies reveal that chronic increases in blood pressure are associated with chronic changes in SNA that may be due to nonresetting of the blood pressure-SNA baroreflex relationship. This review discusses the implications of such information, highlighting new technologies for long-term recording of SNA that appear to hold much promise for revealing the role of SNA to the kidney for the long-term control of blood pressure.