Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC-APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Šresolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA's high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate-binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.

The calcimimetic R-568 attenuates subarachnoid hemorrhage-induced vasospasm through PI3K/Akt/eNOS signaling pathway in the rat model.

Cerebral vasospasm (CVS) causes mortality and morbidity in patients after subarachnoid hemorrhage (SAH). The mechanism and adequate treatment of CVS are still elusive. R-568 is a calcimimetic agent known to exert a vasodilating effect. However, there is no report on its vasodilator effect against SAH-induced vasospasm. In the present study, we investigated the therapeutic effect of R-568 on the SAH-induced CVS model in rats. Seventy-two adult male Sprague-Dawley rats were divided into 8 groups: sham surgery; SAH only; SAH + Vehicle, SAH + R-568; SAH + R-568 + Wortmannin (the PI3K inhibitor); SAH + Wortmannin; SAH + R-568 + Calhex-231 (a calcilytic agent); SAH + Calhex-231. SAH was induced by blood (0.3 mL) given by intracisternal injection. R-568 (20 µM) was administered intracisternal immediately prior to experimental SAH. Basilar arteries (BAs) were obtained to evaluate PI3K/Akt/eNOS pathway (immunoblotting) and morphological changes 48 h after SAH. Perimeters of BAs were decreased by 24.1% in the SAH group compared to the control group and the wall thickness was increased by 75.3%. With R-568 treatment, those percentages were 9.6% and 29.6%, respectively, indicating that vasospasm was considerably improved when compared with the SAH group (P < 0.001 in both). While p-PI3K/PI3K and p-Akt/Akt ratio and eNOS protein expression were markedly decreased in the SAH rats, treatment with R-568 resulted in a significant increase in these levels. The beneficial effects of R-568 were partially blocked in the presence of Calhex-231 and completely blocked in the presence of Wortmannin. Herein, we found that treatment with R-568 would attenuate SAH-induced CVS through the PI3K/Akt/eNOS pathway and demonstrate therapeutic promise in CVS treatment following SAH.

Magnetic properties of nanoparticles as a function of their spatial distribution on liposomes and cells.

The aggregation processes of magnetic nanoparticles in biosystems are analysed by comparing the magnetic properties of three systems with different spatial distributions of the nanoparticles. The first one is iron oxide nanoparticles (NPs) of 14 nm synthesized by coprecipitation with two coatings, (3-aminopropyl)trimethoxysilane (APS) and dimercaptosuccinic acid (DMSA). The second one is liposomes with encapsulated nanoparticles, which have different configurations depending on the NP coating (NPs attached to the liposome surface or encapsulated in its aqueous volume). The last system consists of two cell lines (Pan02 and Jurkat) incubated with the NPs. Dynamic magnetic behaviour (AC) was analysed in liquid samples, maintaining their colloidal properties, while quasi-static (DC) magnetic measurements were performed on lyophilised samples. AC measurements provide a direct method for determining the effect of the environment on the magnetization relaxation of nanoparticles. Thus, the imaginary (χ'') component shifts to lower frequencies as the aggregation state increases from free nanoparticles to those attached or embedded into liposomes in cell culture media and more pronounced when internalized by the cells. DC magnetization curves show no degradation of the NPs after interaction with biosystems in the analysed timescale. However, the blocking temperature is shifted to higher temperatures for the nanoparticles in contact with the cells, regardless of the location, the incubation time, the cell line and the nanoparticle coating, supporting AC susceptibility data. These results indicate that the simple fact of being in contact with the cells makes the nanoparticles aggregate in a non-controlled way, which is not the same kind of aggregation caused by the contact with the cell medium nor inside liposomes.

Spermine oxidase promotes bile canalicular lumen formation through acrolein production.

Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H2O2) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

Evaluation of phenylcyclopropylamine compounds by enzymatic assay of lysine-specific demethylase 2 in the presence of NPAC peptide.

Lysine-specific demethylase 2 (LSD2) demethylates mono- and dimethylated Lys-4 of histone H3 (H3K4me1 and H3K4me2). NPAC protein is known to interact with LSD2 and promote its H3K4 demethylase activity. In this study, we established a demethylation assay system that utilizes recombinant LSD2 in the presence of a synthetic NPAC peptide. Several phenylcyclopropylamine (PCPA)-based inhibitors were examined for their LSD2 inhibitory activity in the LSD2 enzymatic assay with the NPAC peptide. The assay results showed that the PCPA derivatives, including NCD41, selectively inhibited LSD1 in preference to LSD2.

Cell-penetrating magnetic nanoparticles for highly efficient delivery and intracellular imaging of siRNA.

RNA interference is one of the most promising technologies for cancer therapeutics, while the development of a safe and effective small interfering RNA (siRNA) delivery system is still challenging. Here, amphipol polymer and protamine peptide were employed to modify magnetic nanoparticles to form cell-penetrating magnetic nanoparticles (CPMNs). The unique CPMN could efficiently deliver the eGFP siRNA intracellularly and silence the eGFP expression in cancer cells, which was verified by fluorescent imaging of cancer cells. Compared with lipofectamine and polyethyleneimine (PEI), CPMNs showed superior silencing efficiency and biocompatibility with minimum siRNA concentration as 5 nm in serum-containing medium. CPMN was proved to be an efficient siRNA delivery system, which will have great potential in applications as a universal transmembrane carrier for intracellular gene delivery and simultaneous MRI imaging.

Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing.

An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.

Site-specific protein propargylation using tissue transglutaminase.

Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines. Herein we report the first FRET-based activity assay for the direct detection of the ligation (transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and 2-azidoethyl amine were validated independently as TG2 substrates with K(M) values of 44 ± 4 µM, and 0.99 ± 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled through a dipolar cycloaddition reaction with an azido-fluorescein conjugate. This application demonstrates the strong potential of using TG2 for site-specific protein modification through a combination of enzymatic and bioorthogonal chemistry.

Carboxylate and aromatic active-site residues are determinants of high-affinity binding of ω-aminoaldehydes to plant aminoaldehyde dehydrogenases.

The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.

The effects of nitric oxide-oxidase and putative glutathione-peroxidase activities of ceruloplasmin on the viability of cardiomyocytes exposed to hydrogen peroxide.

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia-reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H(2)O(2)), the former activity by consuming H(2)O(2) and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO(+) species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H(2)O(2). In contrast to human CP, bovine CP consumed H(2)O(2) in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H(2)O(2) could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H(2)O(2)-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CP's putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue.

A phase I clinical trial of tiopronin, a putative neuroprotective agent, in aneurysmal subarachnoid hemorrhage.

BACKGROUND: The neurotoxic aldehyde 3-aminopropanal (3-AP) contributes to brain injury following cerebral ischemia. Tiopronin (N-2-mercaptopropionyl-glycine[N-2-MPG]) is a US Food and Drug Administration (FDA)-approved drug for the treatment of cystinuria and a putative neuroprotective agent that has been shown to bind and neutralize 3-AP and reduce infarct volumes. OBJECTIVE: The objective of this trial was to establish the safety of tiopronin administration in patients with aneurysmal subarachnoid hemorrhage (aSAH) in preparation for further trials of its efficacy as a neuroprotective agent in this disease process. METHODS: This Phase I dose-escalation trial enrolled three-patient cohorts using a conventional "3+3" study design. Tiopronin dose began at 1 g/d until aSAH Day 14. Each subsequent cohort received a dose of tiopronin based on predetermined guidelines. A maximum dose of 3 g/d was selected, because this is the maximum FDA-approved dose for long-term cystinuria treatment. Subjects were monitored for known side effects of tiopronin. RESULTS: Nine patients were enrolled, the minimum number required based on the study design. None of these patients experienced serious side effects attributable to tiopronin, and no adverse events were noted that could not be attributed to the pathophysiology of aSAH. CONCLUSION: The administration of 3 g/d of tiopronin following aSAH for up to 14 days appears to be safe and without the side effects associated with long-term use. Plans for a randomized, placebo-controlled Phase II trial of tiopronin for neuroprotection following aSAH are underway.

Interactions of attention-deficit/hyperactivity disorder therapeutic agents with the efflux transporter P-glycoprotein.

The objective of this study was to assess the potential interactions of the drug transporter P-glycoprotein with attention-deficit/hyperactivity disorder (ADHD) therapeutic agents atomoxetine--and the individual isomers of methylphenidate, amphetamine, and modafinil utilizing established in vitro assay. An initial ATPase assay indicated that both d- and l-methylphenidate have weak affinity for P-glycoprotein. The intracellular accumulation of P-glycoprotein substrates doxorubicin and rhodamine123 in the P-glycoprotein overexpressing cell line LLC-PK1/MDR1 was determined to evaluate potential inhibitory effects on P-glycoprotein. The results demonstrated that all compounds, except both modafinil isomers, significantly increased doxorubicin and rhodamine123 accumulation in LLC-PK1/MDR1 cells at higher concentrations. To investigate the P-glycoprotein substrate properties, the intracellular concentrations of the tested compounds in LLC-PK1/MDR1 and P-glycoprotein negative LLC-PK1 cells were measured in the presence and absence of the P-glycoprotein inhibitor PSC833. The results indicate that the accumulation of d-methylphenidate in LLC-PK1 cells was 32.0% higher than in LLC-PK1/MDR1 cells. Additionally, coadministration of PSC833 leads to 52.9% and 45.6% increases in d-modafinil and l-modafinil accumulation, respectively, in LLC-PK1/MDR1 cells. Further studies demonstrated that l-modafinil transport across LLC-PK1/MDR1 cell monolayers in the basolateral-to-apical (B-A) direction was significantly higher than in the apical-to-basolateral (A-B) direction. PSC833 treatment significantly decreased the transport of l-modafinil in B-A direction. In conclusion, our results suggest that all tested agents with the exception of modafinil isomers are relatively weak P-glycoprotein inhibitors. Furthermore, P-glycoprotein may play a minor role in the transport of d-methylphenidate, d-modafinil, and l-modafinil.

A structural insight into the inhibition of human and Leishmania donovani ornithine decarboxylases by 1-amino-oxy-3-aminopropane.

The critical role of polyamines in key processes such as cell growth, differentiation and macromolecular synthesis makes the enzymes involved in their synthesis potential targets in the treatment of certain types of cancer and parasitic diseases. Here we present a study on the inhibition of human and Leishmania donovani ODC (ornithine decarboxylase), the first committed enzyme in the polyamine biosynthesis pathway, by APA (1-amino-oxy-3-aminopropane). The present study shows APA to be a potent inhibitor of both human and L. donovani ODC with a K(i) value of around 1.0 nM. We also show that L. donovani ODC binds the substrate, the co-enzyme pyridoxal 5'-phosphate and the irreversible inhibitor alpha-difluoromethylornithine (a curative agent of West African sleeping sickness) with less affinity than human ODC. We have also determined the three-dimensional structure of human ODC in complex with APA, which revealed the mode of the inhibitor binding to the enzyme. In contrast with earlier reports, the structure showed no indication of oxime formation between APA and PLP (pyridoxal 5'-phosphate). Homology modelling suggests a similar mode of binding of APA to L. donovani ODC. A comparison of the ODC-APA-PLP structure with earlier ODC structures also shows that the protease-sensitive loop (residues 158-168) undergoes a large conformational change and covers the active site of the protein. The understanding of the structural mode of APA binding may constitute the basis for the development of more specific inhibitors of L. donovani ODC.

Mucin-Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people suffering from cystic fibrosis (CF). In CF airways, P. aeruginosa forms surface-associated communities called biofilms. Compared with free-swimming cultures, biofilms resist clearance by the host immune system and display increased resistance to antimicrobial agents. In this study we developed a technique to coat surfaces with molecules that are abundant in CF airways in order to investigate their impact on P. aeruginosa biofilm development. We found that P. aeruginosa biofilm development proceeds differently on surfaces coated with the glycoprotein mucin compared with biofilm development on glass and surfaces coated with actin or DNA. Biofilms formed on mucin-coated surfaces developed large cellular aggregates and had increased tolerance to the antibiotic tobramycin compared with biofilms grown on glass. Analysis of selected mutant backgrounds in conjunction with time-lapse microscopy revealed that surface-associated motility was blocked on the mucin surface. Furthermore, our data suggest that a specific adhesin-mucin interaction immobilizes the bacterium on the surface. Together, these experiments suggest that mucin, which may serve as an attachment surface in CF airways, impacts P. aeruginosa biofilm development and function.

Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA(C)) is a ligand-gated ion channel with pharmacological properties distinct from the GABA(A) receptor. To date, only three binding domains in the recombinant rho1 GABA(C) receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the rho1 GABA(C) receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA(C) receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a beta-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the rho1 GABA(C) receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA(C) receptor binding pocket was proposed and discussed in comparison with the GABA(A) receptor binding pocket.

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus Sclerotinia sclerotiorum.

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40-50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.

New propylamine oligopyrrole carboxamides linked to a heterocyclic or anthraquinone system: synthesis, DNA binding, topoisomerase I inhibition and cytotoxicity.

Continuing our studies on combilexines, compounds consisting of a DNA intercalator linked to a minor groove ligand, new results are presented. The synthesis of a series of new propylamine oligopyrrole carboxamides closely related to netropsin and distamycin A, linked to a heterocyclic or anthraquinone system is reported. The cytotoxic activity in vitro, the DNA binding characteristics and the inhibition of the topoisomerase I of the compounds were studied in order to explain the biological mechanism of action of these new potential combilexines. Some of the synthesised compounds showed cytotoxic activity against human tumour cell lines, as well as DNA binding and topoisomerase I inhibiting properties.

3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin.

Cytotoxic polyamine-derived amino aldehydes, formed during cerebral ischaemia, damage adjacent tissue (the so-called 'penumbra') not subject to the initial ischaemic insult. One such product is 3-aminopropanal (3-AP), a potent cytotoxin that accumulates in ischaemic brain, although the precise mechanisms responsible for its formation are still unclear. More relevant to the present investigations, the mechanisms by which such a small aldehydic compound might be cytotoxic are also not known, but we hypothesized that 3-AP, having the structure of a weak lysosomotropic base, might concentrate within lysosomes, making these organelles a probable focus of initial toxicity. Indeed, 3-AP leads to lysosomal rupture of D384 glioma cells, a process which clearly precedes caspase activation and apoptotic cell death. Immunohistochemistry reveals that 3-AP concentrates in the lysosomal compartment and prevention of this accumulation by the lysosomotropic base ammonia, NH(3), protects against 3-AP cytotoxicity by increasing lysosomal pH. A thiol compound, N-(2-mercaptopropionyl)glycine, reacts with and neutralizes 3-AP and significantly inhibits cytoxocity. Both amino and aldehyde functions of 3-AP are necessary for toxicity: the amino group confers lysosomotropism and the aldehyde is important for additional, presently unknown, reactions. We conclude that 3-AP exerts its toxic effects by accumulating intralysosomally, causing rupture of these organelles and releasing lysosomal enzymes which initiate caspase activation and apoptosis (or necrosis if the lysosomal rupture is extensive). These results may have implications for the development of new therapeutics designed to lessen secondary damage arising from focal cerebral ischaemia.

Neuroprotection in cerebral ischemia by neutralization of 3-aminopropanal.

Cerebral ischemia stimulates increased activity of polyamine oxidase, a ubiquitous enzyme that catabolizes polyamines to produce 3-aminopropanal. 3-Aminopropanal is a reactive aldehyde that mediates progressive neuronal necrosis and glial apoptosis. Here we report that increased levels of 3-aminopropanal-modified protein levels in humans after aneurysmal subarachnoid hemorrhage correlate with the degree of cerebral injury as measured by admission Hunt/Hess grade. In vitro screening of clinically approved drugs reveals that N-2-mercaptopropionyl glycine (N-2-MPG), an agent clinically approved for prevention of renal stones in patients with cysteinuria, significantly inhibits the cytotoxicity of 3-aminopropanal. N-2-MPG reacts with 3-aminopropanal to yield a nontoxic thioacetal adduct, as confirmed by electrospray ionization mass spectroscopy. Administration of N-2-MPG in clinically relevant doses to rats significantly reduces cerebral 3-aminopropanal-modified protein immunoreactivity and infarct volume in a standardized model of middle cerebral artery occlusion, even when the agent is administered after the onset of ischemia. These results implicate 3-aminopropanal as a therapeutic target for cerebral ischemia.