Annalisa De Palma

Identification of the yeast ACR1 gene product as a succinate‐fumarate transporter essential for growth on ethanol or acetate

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over‐produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source.

New N-(phenoxydecyl)phthalimide derivatives displaying potent inhibition activity towards α-glucosidase

Several members of a new family of non-sugar-type alpha-glucosidase inhibitors, bearing a phthalimide moiety connected to a variously substituted phenoxy ring by an alkyl chain, were synthesized and their activities were investigated. The efficacy of the inhibition activity appeared to be governed by the chain length of the substrate. Substrates possessing 10 carbons afforded the highest levels of activity, which were one to two orders of magnitude more potent than the known inhibitor 1-deoxynojirimycin (dNM). Furthermore, structure-activity relationship studies indicated a critical role of electron-withdrawing substituents at the phenoxy group for the activity. Derivatives bearing a chlorine atom along with a strong electron-withdrawing group, such as a nitro group, were the most potent of the series.

Synthesis and Toxicopharmacological Evaluation of m-Hydroxymexiletine, the First Metabolite of Mexiletine More Potent Than the Parent Compound on Voltage-Gated Sodium Channels

The first synthesis of m-hydroxymexiletine (MHM) has been accomplished. MHM displayed hNav1.5 sodium channel blocking activity, and tests indicate it to be ∼2-fold more potent than the parent mexiletine and to have more favorable toxicological properties than mexiletine. Thus, MHM and possible related prodrugs might be studied as agents for the treatment of arrhythmias, neuropathic pain, and myotonias in substitution of mexiletine (metabolite switch), which has turned out to be tainted with common toxicity.

Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria.

The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrates.

The mitochondrial carnitine/acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155.

BACKGROUND The carnitine/acylcarnitine carrier (CAC or CACT) mediates transport of acylcarnitines into mitochondria for the β-oxidation. CAC possesses Cys residues which respond to redox changes undergoing to SH/disulfide interconversion. METHODS The effect of H2S has been investigated on the [(3)H]carnitine/carnitine antiport catalyzed by recombinant or native CAC reconstituted in proteoliposomes. Site-directed mutagenesis was employed for identifying Cys reacting with H2S. RESULTS H2S led to transport inhibition, which was dependent on concentration, pH and time of incubation. Best inhibition with IC50 of 0.70 μM was observed at physiological pH after 30-60 min incubation. At longer times of incubation, inhibition was reversed. After oxidation of the carrier by O2, transport activity was rescued by H2S indicating that the inhibition/activation depends on the initial redox state of the protein. The observed effects were more efficient on the native rat liver transporter than on the recombinant protein. Only the protein containing both C136 and C155 responded to the reagent as the WT. While reduced responses were observed in the mutants containing C136 or C155. Multi-alignment of known mitochondrial carriers, highlighted that only the CAC possesses both Cys residues. This correlates well with the absence of effects of H2S on carriers which does not contain the Cys couple. CONCLUSIONS Altogether, these data demonstrate that H2S regulates the CAC by inhibiting or activating transport on the basis of the redox state of the protein. GENERAL SIGNIFICANCE CAC represents a specific target of H2S among mitochondrial carriers in agreement with the presence of a reactive Cys couple.

Nitric oxide inhibits the mitochondrial carnitine/acylcarnitine carrier through reversible S -nitrosylation of cysteine 136

S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71μM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.

Discovery of Potent Dual Binding Site Acetylcholinesterase Inhibitors via Homo- and Heterodimerization of Coumarin-based Moieties

Acetylcholinesterase (AChE) inhibitors still comprise the majority of the marketed drugs for Alzheimers disease (AD). The structural arrangement of the enzyme, which features a narrow gorge that separates the catalytic and peripheral anionic subsites (CAS and PAS, respectively), inspired the development of bivalent ligands that are able to bind and block the catalytic activity of the CAS as well as the role of the PAS in beta amyloid (Aβ) fibrillogenesis. With the aim of discovering novel AChE dual binders with improved drug‐likeness, homo‐ and heterodimers containing 2H‐chromen‐2‐one building blocks were developed. By exploring diverse linkages of neutral and protonatable amino moieties through aliphatic spacers of different length, a nanomolar bivalent AChE inhibitor was identified (3‐[2‐({4‐[(dimethylamino)methyl]‐2‐oxo‐2H‐chromen‐7‐yl}oxy)ethoxy]‐6,7‐dimethoxy‐2H‐chromen‐2‐one (6 d), IC50=59 nm) from originally weakly active fragments. To assess the potential against AD, the disease‐related biological properties of 6 d were investigated. It performed mixed‐type AChE enzyme kinetics (inhibition constant Ki=68 nm) and inhibited Aβ self‐aggregation. Moreover, it displayed an outstanding ability to protect SH‐SY5Y cells from Aβ1‐42 damage.

Synthesis, antiarrhythmic activity, and toxicological evaluation of mexiletine analogues

Four mexiletine analogues have been tested for their antiarrhythmic, inotropic, and chronotropic effects on isolated guinea pig heart tissues and to assess calcium antagonist activity, in comparison with the parent compound mexiletine. All analogues showed from moderate to high antiarrhythmic activity. In particular, three of them (1b,c,e) were more active and potent than the reference drug, while exhibiting only modest or no negative inotropic and chronotropic effects and vasorelaxant activity, thus showing high selectivity of action. All compounds showed no cytotoxicity and 1b,c,d did not impair motor coordination. All in, these new analogues exhibit an interesting cardiovascular profile and deserve further investigation.

Mannich base approach to 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone: A water-soluble prodrug for a multitarget inhibition of cholinesterases, beta-amyloid fibrillization and oligomer-induced cytotoxicity

&NA; Targeting protein aggregation for the therapy of neurodegenerative diseases remains elusive for medicinal chemists, despite a number of small molecules known to interfere in amyloidogenesis, particularly of amyloid beta (A&bgr;) protein. Starting from previous findings in the antiaggregating activity of a class of indolin‐2‐ones inhibiting A&bgr; fibrillization, 5‐methoxyisatin 3‐(4‐isopropylphenyl)hydrazone 1 was identified as a multitarget inhibitor of A&bgr; aggregation and cholinesterases with IC50s in the low &mgr;M range. With the aim of increasing aqueous solubility, a Mannich‐base functionalization led to the synthesis of N‐methylpiperazine derivative 2. At acidic pH, an outstanding solubility increase of 2 over the parent compound 1 was proved through a turbidimetric method. HPLC analysis revealed an improved stability of the Mannich base 2 at pH 2 along with a rapid release of 1 in human serum as well as an outstanding hydrolytic stability of the parent hydrazone. Coincubation of A&bgr;1–42 with 2 resulted in the accumulation of low MW oligomers, as detected with PICUP assay. Cell assays on SH‐SY5Y cells revealed that 2 exerts strong cytoprotective effects in both cell viability and radical quenching assays, mainly related to its active metabolite 1. These findings show that 2 drives the formation of non‐toxic, off‐pathway A&bgr; oligomers unable to trigger the amyloid cascade and toxicity. Graphical abstract Figure. No caption available.

Nitro-substituted tetrahydroindolizines and homologs: Design, kinetics, and mechanism of α-glucosidase inhibition

A series of 1-[(methylsulfonyl)methyl]-2-nitro-5,6,7,8-tetrahydroindolizines and homologs were designed, prepared, and evaluated as non-sugar-type α-glucosidase inhibitors. The inhibitory activity appeared to be related to cyclo homologation with the best congeners being tetrahydroindolizines. The introduction of a methoxycarbonyl group as an additional hydrogen bond acceptor into the exocyclic methylene group was beneficial affording the most potent congener 3e (half maximal inhibitory concentration, IC50=8.0±0.1μM) which displayed 25-fold higher inhibitory activity than 1-deoxynojirimycin (2, IC50=203±9μM)-the reference compound. Kinetic analysis indicated that compound 3e is a mixed inhibitor with preference for the free enzyme over the α-glucosidase-substrate complex (Ki,free=3.6μM; Ki,bound=7.6μM). Molecular docking experiments were in agreement with kinetic results indicating reliable interactions with both the catalytic cleft and other sites. Circular dichroism spectroscopy studies suggested that the inhibition exerted by 3e may involve changes in the secondary structure of the enzyme. Considering the relatively low molecular weight of 3e together with its high fraction of sp3 hybridized carbon atoms, this nitro-substituted tetrahydroindolizine may be considered as a good starting point towards new leads in the area of α-glucosidase inhibitors.