A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is currently considered the gold standard for peanut allergy diagnosis. However, this procedure that requires the hospitalization of patients, mostly children, in specialized centers for oral exposure to allergens may cause severe reactions requiring emergency measures. Thus, a simpler and safer diagnosis procedure is needed. The aim of this study was to evaluate the diagnostic performance of a new set of in vitro blood tests for peanut allergy. METHODS: The levels of IgE directed towards peanut extract and recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 7, and Ara h 8 were measured in 3 groups of patients enrolled at 2 independent centers: patients with proven peanut allergy (n=166); pollen-sensitized subjects without peanut allergy (n=61), and control subjects without allergic disease (n=10). RESULTS: Seventy-nine percent of the pollen-sensitized patients showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/l. Use of a threshold of 0.23 kU/l for recombinant Ara h 2 increased specificity (96%) at the cost of sensitivity (93%). CONCLUSION: A simple blood test can be used to diagnose peanut allergy with a high level of precision. However, DBPCFC will remain useful for the few cases where immunological and clinical observations yield conflicting results.

Novel peptidyl alpha-keto amide inhibitors of calpains and other cysteine proteases.

A series of new dipeptidyl alpha-keto amides of the general structure R1-L-Leu-D,L-AA-CONH-R2 were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. They combine 10 different N-protecting groups (R1), 3 amino acids residues in P1 (AA), and 44 distinct substituents on the alpha-keto amide nitrogen (R2). In general, calpain II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors displaying dissociation constants (Ki) in the 10-100 nM range. Calpain I was also effectively inhibited, but very low Ki values were observed with a smaller number of inhibitors than with calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best inhibitors for calpain II were Z-Leu-Abu-CONH-CH2-CHOH-C6H5 (Ki = 15 nM), Z-Leu-Abu-CONH-CH2-2-pyridyl (Ki = 17 nM), and Z-Leu-Abu-CONH-CH2-C6H3(3,5(OMe)2) (Ki = 22 nM). The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH2-2-pyridyl (Ki = 19 nM). The peptide alpha-keto amide Z-Leu-Abu-CONH-(CH2)2-3-indolyl was the best inhibitor for cathepsin B (Ki = 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such inhibitors may be useful in elucidating the physiological and pathological events involving these proteases and may become possible therapeutic agents.

Novel pyridinium derivatives as inhibitors for acetylcholinesterase.

The carbamate 1-(methyl-3-(N,N-dimethylcarbamoyloxy)-2-pyridylmethylene)-4 -(4-phenyl) diazinecarboxamide chloride (MHP 133) is the parent for a new class of pyridinium salts which inhibit acetylcholinesterase (AChE) in vitro as well as in vivo. Fourteen new derivatives of MHP 133 have been synthesized with the intention of improving their hydrophobicity while maintaining their propensity to inhibit acetylcholinesterase. Upon prolonged incubation with AChE, the pyridinium salts exhibit progressive time-dependent inhibition according to first order kinetics with kobs/[I] values ranging from 3 to 345 M-1s-1. The enzyme did not regain any activity after prolonged incubation with the inhibitors (1 day). The partition coefficients for each inhibitor were evaluated in octanol/water in order to determine their hydrophobic character as hydrophobicity is a key prerequisite for crossing the blood brain barrier.

New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted m-aminobenzoic acid residue.

In order to obtain selective suicide substrates of trypsin-like proteases including plasminogen activators, plasmin, and thrombin, a series of cyclopeptides cyclo[Arg or Lys-aB(CH2X)-Gly4], in which a substituted o- or m-aminobenzoyl group constitutes a latent electrophile, have been prepared. Treatment of the corresponding phenyl ethers cyclo[P1-aB(CH2OC6H5)-Gly4] with HBr/HOAc or R1R2S/TFA gives the bromides (X = Br) or the sulfonium salts (X = +SR1R2 with R1 = R2 = Me or R1 = Me and R2 = C6H5), respectively. These water-soluble cyclopeptides behave as time-dependent inhibitors of bovine trypsin and human urokinase (u-PA) but have no effect on tissue plasminogen activator (t-PA) and no or poor effect on plasmin and thrombin. The compounds containing a m-aminobenzoic acid residue are more efficient inactivators than their anthranilic analogues. The kinetic criteria expected for a suicide inhibition are met. A mechanism of inhibition involving the formation of a quinonimmonium methide intermediate is proposed. The activity of the inhibitors is very sensitive to the nature of the X benzylic substituent. An increased efficiency for the inactivation of human urokinase is observed with the sulfonium salts. The selectivity of the inactivation of u-PA compared to t-PA could be of therapeutical significance in controlling cell proliferation and invasion.

Evaluation of the inhibitory activity on serine and aspartic proteases of 4-amino-4H-1,2,4-triazole and 5-aminothiazole derivatives structurally related to beta-lactam antibiotics.

Twenty new derivatives of 4-amino-4H-1,2,4-triazole and 5-aminothiazole have been examined for their inhibitory potential towards serine and aspartic proteases. Upon prolonged incubation with enzyme, the phenylacetylaminothiazolium salts exhibit progressive, time-dependent inhibition of chymotrypsin according to a first-order process. The formation of a tetrahedral transition state-like complex by attack of the active-site serine at the C2-position of the pseudobase form of the thiazolium may be responsible for the observed effect. Triazolium salts appeared to be simple competitive inhibitors of this enzyme, effective in the mM range concentration. Poor inhibitions of trypsin and pepsin were also obtained in the triazolium series. In spite of their structural analogy with beta-lactams, the selected derivatives failed to inhibit human leucocyte elastase.

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.

Acyloxybenzyl halides, inhibitors of elastases.

3-Benzyl-6-chloromethyl-3,4-dihydrocoumarin inhibits human leucocyte elastase (HLE) and porcine pancreatic elastase (PPE) through a mechanism-based process characterized by the following apparent enzyme-inhibitor dissociation constants, Ki, and limiting inactivation rate constants k2: 200 microM (HLE), 69 microM (PPE) and 5.10(-2) s-1 (HLE), 17.7.10(-2) s-1 (PPE) at pH 8.0, 37 degrees C. Bis(4-acyloxyphenyl)methane derivatives with a benzylic halogen as potential leaving group have also been synthesized and studied. They transiently inactivate PPE and HLE through the formation of an acyl-enzyme.