S-allyl-mercapto-captopril: a novel compound in the treatment of Cohen-Rosenthal diabetic hypertensive rats.

S-allyl-mercapto-captopril (CPSSA) is a conjugate of captopril with allicin, an active principle in garlic with multiple beneficial actions on metabolic syndrome abnormalities, including weight preservation, observed by the authors in fructose-induced hypertensive hyperinsulinemic rats and in Koletsky rats. The aim of the study was to examine blood pressure (BP) and glucose levels in the Cohen-Rosenthal Diabetic Hypertensive (CRDH) model as well as to follow their weight preservation. CRDH rats (n=14) were fed the sugar-rich copper-free diet essential for the development of diabetes mellitus. Two months later BP and blood glucose levels were measured. CPSSA was diluted in drinking water and administered at a final dose of 53.5 mg/kg/d (n=8). Control rats (n=6) received no drug (vehicle group). In contrast to control group, CPSSA prevented progressive weight gain, without a detectable effect on food and water intake. CPSSA was effective in attenuating systolic and diastolic BP (P<0.01) as well as significantly reducing glucose levels (P<0.01). Control rats continued to gain weight, whereas the groups fed CPSSA did not. CPSSA was shown to have additional beneficial effects on improving BP and glucose level, as well as preserving weight gain. The authors conclude that the combined molecule CPSSA integrates the antihypertensive feature of both allicin and captopril, making it a potential antidiabetic and cardiovascular protective agent.

Therapy of murine pulmonary aspergillosis with antibody-alliinase conjugates and alliin.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.

Thiol-disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum).

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5'-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S--S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored --SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two --SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free --SH groups. This allowed the oriented conjugation of alliinase, via the --SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.

Allicin inhibits cell polarization, migration and division via its direct effect on microtubules.

Allicin (diallyl thiosulfinate) is a major biologically active component of garlic that is known to inhibit cell proliferation and induce apoptosis. The effects of allicin are attributed to its ability to react with thiol groups. However, the mechanism underlying the cytostatic activity of allicin, as well as the identity of the relevant subcellular targets, are not known. In the present study, we found that the effects of allicin on cell polarization, migration, and mitosis are similar to the effects of microtubule-depolymerizing drugs such as nocodazole. Moreover, treatment of cultured fibroblasts with micromolar doses of allicin results in microtubule depolymerization in cells within minutes of its application, without disrupting the actin cytoskeleton or inducing direct cytotoxic effects. Furthermore, allicin blocks the polymerization of pure tubulin in vitro in a concentration-dependent manner, suggesting that it acts directly on tubulin dimers. Sulfhydryl (SH)-reducing reagents such as 2-mercaptoethanol and dithiothreitol abolish the effect of allicin on microtubule polymerization. Thus, allicin is a potent microtubule-disrupting reagent interfering with tubulin polymerization by reaction with tubulin SH groups.

Two structures of alliinase from Alliium sativum L.: apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the PLP cofactor.

Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent alpha, beta-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L-cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to "short chain complex vacuole type" oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the epsilon-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.

Apoptotic killing of B-chronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-alliinase conjugate.

Allicin, a highly active component from freshly crushed garlic, is produced upon the reaction of the small molecular weight molecule alliin, with the enzyme alliinase (EC 4.4.1.4). Because allicin was shown to be toxic to various mammalian cells in vitro, we devised a novel approach for the therapy of B-cell malignancies based on site-directed generation of allicin. Alliinase was conjugated to the monoclonal antibody rituximab, which recognizes the CD20 antigen, and the resulting conjugate was targeted to CD20+ B chronic lymphocytic leukemia (B-CLL) and other B-cell lymphomas. Upon addition of alliin, allicin was formed in situ, killing the CD20+ tumor B cells via apoptosis. Following a 72-hour treatment, an 85% and 96% reduction was observed in the number of viable B-CLL and EBV-transformed B cells, respectively. Using the human/mouse radiation chimera for the evaluation of allicin targeting in a preclinical animal model, we showed a significant reduction in the number of recovered B-CLL, mantle cell lymphoma, or EBV-transformed B cells. We conclude that our system offers a new powerful and less toxic therapy for B-CLL and other B-cell malignancies. Furthermore, combining alliinase with the appropriate monoclonal antibody may extend the application of this approach to other conditions in which the elimination of a specific cell population is desired.

Efficacy of allicin, the reactive molecule of garlic, in inhibiting Aspergillus spp. in vitro, and in a murine model of disseminated aspergillosis.

OBJECTIVES: The evaluation of allicin, the biologically active compound responsible for the antimicrobial activities of freshly crushed garlic cloves, in inhibiting Aspergillus spp. in vitro and in a murine model of disseminated aspergillosis. METHODS: Pure allicin was prepared by reacting synthetic alliin with a stabilized preparation of the garlic enzyme alliinase. We tested the in vitro efficacy of pure allicin against 31 clinical isolates of Aspergillus spp. using a microdilution broth method and following the NCCLS guidelines (document M-38P). Subsequently, the in vivo efficacy of allicin was tested in immunocompetent mice infected intravenously (iv) with Aspergillus fumigatus conidia. Allicin (5 mg/kg body weight) was administered iv once daily for 5 days post-infection or orally (po) (9 mg/kg body weight) for 5 days pre-infection and 10 days post-infection. No ill effects were observed in allicin-treated uninfected mice. RESULTS: The in vitro MICs and MFCs of allicin were between 8 and 32 mg/L, indicating that allicin in its pure form may be an effective fungicide in vitro. Time-kill studies indicate that allicin exerts its fungicidal activity within 2-12 h of administration in vitro. Allicin treatment significantly prolonged survival of infected mice (P < 0.01) from mean survival time (MST) = 7.7 days in untreated mice to MST = 21.3 and 13.9 days for allicin iv and po treated mice, respectively. Allicin iv treatment led to a significant (P < 0.001) 10-fold reduction in fungal burden in A. fumigatus infected mice as evaluated by quantitative fungal cultures of kidney tissue samples. CONCLUSIONS: These favourable results, despite the short half-life of this compound in vivo, support further studies of controlled sustained release or more prolonged administration of allicin as a treatment for aspergillosis.

A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.

Alliin lyase (alliinase) from garlic (Allium sativum): crystallization and preliminary X-ray characterization.

The enzyme alliinase has been isolated from garlic bulbs and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 70.191, b = 127.006, c = 108.085 A, beta = 93.384 degrees. They diffract to 2.2 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function suggests that the crystals contain two dimeric molecules per asymmetric unit.