Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China.

A monoclonal antibody against tetrodotoxin (TTX) was produced from the hybridoma cell line T6D9, which was established by the fusion of Sp2/0 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with the TTX-keyhole limpet hemocyanin (KLH) conjugate. This monoclonal antibody belongs to the IgG1 subclass; the affinity constant of the antibody is 2.4 × 10(-8) mol l(-1). The relative cross-reactivity of the antibody with TTX was 100%, but with saxitoxin, KLH and bovine serum albumin (BSA) it was less than 1%, respectively. The titre of the antibody in ascites was 6.4 × 10(6); the reference working concentration was 1:1.2 × 10(5). By using this monoclonal antibody, a competitive indirect enzyme-linked immunoabsorbant assay (ELISA) for the analysis of TTX was developed. The linear portion of the dose-response curve of TTX concentration was in range 5-500 ng ml(-1). The limit of detection was 5 ng ml(-1) according 10% inhibition with TTX to anti-TTX monoclonal antibody. The concentration of TTX inhibiting 50% of antibody binding was about 50 ng ml(-1). The recoveries from TTX spiked samples were 79.5-109.5%. In addition, the toxicity of some wild puffer fish specimens captured from south-east China and the Yangzi River in Jiangsu province was determined. The results indicate that the toxicity and toxin tissue distribution vary in different species of wild puffer fish.

Exploiting subsite S1 of trypsin-like serine proteases for selectivity: potent and selective inhibitors of urokinase-type plasminogen activator.

A nonselective inhibitor of trypsin-like serine proteases, 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine (1) (Verner, E.; Katz, B. A.; Spencer, J.; Allen, D.; Hataye, J.; Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.; Luong, C.; Martelli, A.; Radika. K.; Rai, R.; She, M.; Shrader, W.; Sprengeler, P. A.; Trapp, S.; Wang, J.; Young, W. B.; Mackman, R. L. J. Med. Chem. 2001, 44, 2753-2771) has been optimized through minor structural changes on the S1 binding group to afford remarkably selective and potent inhibitors of urokinase-type plasminogen activator (uPA). The trypsin-like serine proteases(1) that comprise drug targets can be broadly categorized into two subfamilies, those with Ser190 and those with Ala190. A single-atom modification, for example, replacement of hydrogen for chlorine at the 6-position of the 5-amidinoindole P1 group on 1, generated up to 6700-fold selectivity toward the Ser190 enzymes and against the Ala190 enzymes. The larger chlorine atom displaces a water molecule (H(2)O1(S1)) that binds near residue 190 in all the complexes of 1, and related inhibitors, in uPA, thrombin, and trypsin. The water molecule, H(2)O1(S1), in both the Ser190 or Ala190 enzymes, hydrogen bonds with the amidine N1 nitrogen of the inhibitor. When it is displaced, a reduction in affinity toward the Ala190 enzymes is observed due to the amidine N1 nitrogen of the bound inhibitor being deprived of a key hydrogen-bonding partner. In the Ser190 enzymes the affinity is maintained since the serine hydroxyl oxygen O gamma(Ser190) compensates for the displaced water molecule. High-resolution crystallography provided evidence for the displacement of the water molecule and validated the design rationale. In summation, a novel and powerful method for engineering selectivity toward Ser190 proteases and against Ala190 proteases without substantially increasing molecular weight is described.

Development of serine protease inhibitors displaying a multicentered short (<2.3 A) hydrogen bond binding mode: inhibitors of urokinase-type plasminogen activator and factor Xa.

Novel scaffolds that bind to serine proteases through a unique network of short hydrogen bonds to the catalytic Ser195 have been developed. The resulting potent serine protease inhibitors were designed from lead molecule 2-(2-hydroxyphenyl)1H-benzoimidazole-5-carboxamidine, 6b, which is known to display several modes of binding. For instance, 6b can recruit zinc and bind in a manner similar to that reported by bis(5-amidino-2-benzimidazolyl)methane (BABIM) (Nature 1998, 391, 608-612).(1) Alternatively, 6b can bind in the absence of zinc through a multicentered network of short (<2.3 A) hydrogen bonds. The lead structure was optimized in the zinc-independent binding mode toward a panel of six human serine proteases to yield optimized inhibitors such as 2-(3-bromo-2-hydroxy-5-methylphenyl)-1H-indole-5-carboxamidine, 22a, and 2-(2-hydroxybiphenyl-3-yl)-1H-indole-5-carboxamidine, 22f. Structure-activity relationships determined that, apart from the amidine function, an indole or benzimidazole and an ortho substituted phenol group were also essential components for optimal potency. The affinities (K(i)) of 22a and 22f, for example, bearing these groups ranged from 8 to 600 nM toward a panel of six human serine proteases. High-resolution crystal structures revealed that the binding mode of these molecules in several of the enzymes was identical to that of 6b and involved short (<2.3 A) hydrogen bonds among the inhibitor hydroxyl oxygen, Ser195, and a water molecule trapped in the oxyanion hole. In summation, novel and potent trypsin-like serine protease inhibitors possessing a unique mode of binding have been discovered.

Glutathione-S-transferase activates novel alkylating agents.

Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.

Isozyme-specific glutathione-S-transferase inhibitors: design and synthesis.

Glutathione-S-transferase (GST) isozyme-selective inhibitors were designed by an empirically guided strategy. In the first phase, literature data were used to select C-terminal modifications which generated maximum variation in the catalytic efficiency (Vmax/Km) for glutathione (GSH) analogs used as substrates with different rat GSTs. Also, on the basis of literature data, the sulfhydryl group was functionalized with a selection of alkyl and aryl groups to maximize potential isozyme specificity. Affinity chromatography sorbents were prepared from these which showed isozyme selectivity for both rat tissue and recombinant human GST isozymes. Some of these compounds also showed selective inhibition of GST activity in catalysis of the reaction of 1-chloro-2,4-dinitrobenzene with GSH. In the second phase, electronic effects were explored through synthesis of an isostructural series of S-benzyl GSH ligands with different substituents on the aromatic ring. GST isozyme specificity for these ligands, measured by binding to derivatized sorbents, varied substantially, with hydrophobic substituents favoring the human GST M1a isozyme and electronegative moieties favoring GST P1. In the third phase, information obtained from testing both series of compounds was combined and used to prepare GSH analogs with chemical features responsible for isozyme specificity at both the C-terminus and the sulfur. This approach gave two new compounds which showed improved potency while still maintaining selectivity in the inhibition of GSTs. A detailed discussion of the logic used in the selection of functional groups for maximum potency and selectivity is included.