Stimulation of σ1-receptor restores abnormal mitochondrial Ca mobilization and ATP production following cardiac hypertrophy

Background

We previously reported that the σ₁-receptor (σ₁R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ₁R stimulation with the selective σ₁R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ₁R localized in the sarcoplasmic reticulum (SR).

Methods

We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ₁R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ₁R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).

Results

σ₁R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca^2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca^2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.

Conclusions

σ₁R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca^2 + mobilization and ATP production via σ₁R stimulation.

General significance

Our observations suggest that σ₁R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.     

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc‐SPPS while synthesizing the N‐terminal Xaa‐Asp‐Yaa motif. This termination was caused by the formation of piperazine‐2,5‐diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine‐2,5‐dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six‐membered ring is formed whereas the theoretically conceivable seven‐membered 1,4‐diazepine‐2,5‐dione is not found. We propose a mechanism where nucleophilic attack of the N‐terminal amino function takes place at the α‐carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

p-Methylthioamphetamine and 1-(m-chlorophenyl)piperazine,two non-neurotoxic 5-HT releasers in vivo,differ from neurotoxic amphetamine derivatives in their mode of action at 5-HT nerve endings in vitro

The mechanism underlying the serotoninergic neurotoxicity of some amphetamine derivatives, such as p-chloroamphetamine (pCA) and 3,4-methylenedioxymethamphetamine (MDMA), is still debated. Their main acute effect, serotonin (5-HT) release from nerve endings, involves their interaction with 5-HT transporters (SERTs), as substrates. Although this interaction is required for the neurotoxic effects, 5-HT release alone may not be sufficient to induce long-term 5-HT deficits. Some non-neurotoxic compounds, including p-methylthioamphetamine (MTA) and 1-(m-chlorophenyl)piperazine (mCPP), have 5-HT releasing properties in vivo and in brain slices comparable to that of neurotoxic amphetamine derivatives. We measured 5-HT release in superfused rat brain synaptosomes preloaded with [3H]5-HT, a model that distinguishes a releasing effect from reuptake inhibition. MTA and mCPP induced much lower release than pCA and MDMA. The striking difference between our findings in synaptosomes and those obtained in vivo or in brain slices is probably related to a different compartmentalisation of 5-HT in the different experimental models. Studies in synaptosomes, where the vesicular storage of 5-HT is predominant, could therefore bring to light differences between neurotoxic and non-neurotoxic 5-HT releasing agents which cannot be appreciated in other experimental models and might be useful to identify the mechanisms responsible for the neurotoxicity induced by amphetamine derivatives.     

Base-induced side reactions in Fmoc-solid phase peptide synthesis:Minimization by use of piperazine as Nα-deprotectionreagent

Base-induced aspartimide (cyclic imide) and subsequentbase adduct formation in the Fmoc-solid phasesynthesis of sensitive sequences are serious sidereactions that are difficult to both anticipate andcontrol. The effect of extended treatment ofpiperazine as N-Fmoc deprotection reagenton two sensitive peptide sequences was examined. Forcomparison, other bases were also investigated,including piperidine, 1-hydroxypiperidine,tetrabutylammonium fluoride, and1,8-diazabicyclo[5.4.0]undec-7-ene. The results showedthat all bases induced varying degrees of bothaspartimide and, in some cases, base adduct formation,although piperazine caused the least side reaction.Use of N-(2-hydroxy-4-methoxybenzyl) peptidebackbone amide protection was confirmed to confercomplete protection against side reaction. In theabsence of such protection, for all bases, the use of1-hydroxybenzotriazole as additive had some, but notcomplete, beneficial effect in further reducing sidereaction. Best results were obtained with piperazinecontaining 0.1M 1-hydroxybenzotriazole indicating thatthis reagent merits serious consideration forN-deprotection in the Fmoc-solid phasesynthesis of base-sensitive sequences. A furtheradvantage of this reagent is that it causes littleracemisation of resin-bound C-terminal cysteine, anoccasionally serious base-mediated problem in Fmoc-solidphase assembly.     

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.     

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.     

Structural Basis for the SUMO2 Isoform Specificity of SENP7

SUMO proteases or deSUMOylases regulate the lifetime of SUMO-conjugated targets in the cell by cleaving off the isopetidic bond between the substrate and the SUMO modifier, thus reversing the conjugation activity of the SUMO E3 ligases. In humans the deSUMOylating activity is mainly conducted by the SENP/ULP protease family, which is constituted of six members sharing a homologous catalytic globular domain. SENP6 and SENP7 are the most divergent members of the family and they show a unique SUMO2/3 isoform preference and a particular activity for dismantling polySUMO2 chains. Here, we present the crystal structure of the catalytic domain of human SENP7 bound to SUMO2, revealing structural key elements for the SUMO2 isoform specificity of SENP7. In particular, we describe the specific contacts between SUMO2 and a unique insertion in SENP7 (named Loop1) that is responsible for the SUMO2 isoform specificity. All the other interface contacts between SENP7 and SUMO2, including the SUMO2 C-terminal tail interaction, are conserved among members of the SENP/ULP family. Our data give insight into an evolutionary adaptation to restrict the deSUMOylating activity in SENP6 and SENP7 for the SUMO2/3 isoforms.     

Novel 1-(2-pyrimidin-2-yl)piperazine derivatives as selective monoamine oxidase (MAO)-A inhibitors

In the present study, a new series of 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-substituted piperazine-1-carbodithioate derivatives (2a-n) were synthesized and screened for their monoamine oxidase A and B inhibitory activity. The structures of compounds were elucidated using spectroscopic methods and some physicochemical properties of new compounds were predicted using Molinspiration and MolSoft programs. Compounds 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-(4-nitrophenyl)piperazine-1-carbodithioate (2j) and 2-[4-(pyrimidin-2-yl)piperazin-1-yl]-2-oxoethyl 4-benzhydrylpiperazine-1-carbodithioate (2m) exhibited selective MAO-A inhibitory activity with IC_50?=_?23.10, 24.14?µM, respectively. Some of the biological results were found in accordance with the obtained in silico data based on Lipinski’s fule of five.     

Base-induced side reactions in Fmoc-solid phase peptide synthesis:Minimization by use of piperazine as Nα-deprotectionreagent

Summary Base-induced aspartimide (cyclic imide) and subsequent base adduct formation in the Fmoc-solid phase synthesis of sensitive sequences are serious side reactions that are difficult to both anticipate and control. The effect of extended treatment of piperazine as N^α-Fmoc deprotection reagent on two sensitive peptide sequences was examined. For comparison, other bases were also investigated, including piperidine, 1-hydroxypiperidine, tetrabutylammonium fluoride, and 1,8-diazabicyclo[5.4.0]undec-7-ene. The results showed that all bases induced varying degrees of both aspartimide and, in some cases, base adduct formation, although piperazine caused the least side reaction. Use ofN-(2-hydroxy-4-methoxybenzyl) peptide backbone amide protection was confirmed to confer complete protection against side reaction. In the absence of such protection, for all bases, the use of 1-hydroxybenzotriazole as additive had some, but not complete, beneficial effect in further reducing side reaction. Best results were obtained with piperazine containing 0.1M 1-hydroxybenzotriazole indicating that this reagent merits serious consideration for N^α-deprotection in the Fmoc-solid phase synthesis of base-sensitive sequences. A further advantage of this reagent is that it causes little racemisation of resin-bound C-terminal cysteine, an occasionally serious base-mediated problem in Fmoc-solid phase assembly. A preliminary account of this work was presented at the 25th European Peptide Symposium, Budapest, Hungary, 1998.