Structure-activity relationship analysis of the selective inhibition of transglutaminase 2 by dihydroisoxazoles - PubMed (original) (raw)

Structure-activity relationship analysis of the selective inhibition of transglutaminase 2 by dihydroisoxazoles

R Edward Watts et al. J Med Chem. 2006.

Abstract

Human transglutaminase 2 (TG2) is believed to play an important role in the pathogenesis of various human disorders including celiac sprue, certain neurological diseases, and some types of cancer. Selective inhibition of TG2 should therefore enable further investigation of its role in physiology and disease and may lead to effective clinical treatment. Recently we showed that certain 3-halo-4-,5-dihydroisoxazole containing compounds are selective inhibitors of human TG2 with promising pharmacological activities. Here, we present definitive evidence that this class of compounds targets the active site of human TG2. Structure-activity relationship studies have provided insights into the structural prerequisites for selectivity and have led to the discovery of an inhibitor with about 50-fold higher activity than a prototypical dihydroisoxazole inhibitor with good in vivo activity. A method for preparing enantiomerically enriched analogues was also developed. Our studies show that the 5-(S)-dihydroisoxazole is a markedly better inhibitor of human TG2 than its 5-(R) stereoisomer.

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Figures

Figure 1

Figure 1

Dihydroisoxazole inhibitors covalently bind the active site cysteine of transglutaminase 2. Human TG2 was incubated with or without inhibitor 1 before being digested with trypsin and analyzed by mass spectrometry. LC-MS chromatogram of uninhibited TG2 (A) and inhibited TG2 (B) filtered to only display peptide peaks corresponding to the predicted weight of the peptide fragment (YGQ_C_WVFAAVACTVLR) containing the active site cysteine (1787 a.m.u. (+1 peak) + 894 amu (+2 peak)) or inhibited active site cysteine (1092 amu (+2 peak)). The fragment containing the active site cysteine is the smaller peak eluting at 23.9 minutes. Note that there is a log difference in the scale of the 1092 plots. MS/MS spectrum (C) of the 1092 peak for the inhibited TG2 sample with 15 “y” and “b” peaks identified, proving the identity of the peptide fragment.

Scheme 1

Scheme 1

General synthesis of inhibitors in this study. a) LiBH4, THF b) p-nitro-phenylchloroformate, _N_-methylmorpholine, CH2Cl2 c) 1. amino acid methyl ester, 11, DMF 2. MeOH, THF, H2O, LiOH d) EDCI, HOBT, DMF, 12.

Scheme 2

Scheme 2

Chiral resolution of dihydroisoxazoles. a) 15% v/v acetone/0.1M pH7 phosphate buffer, Amano Lipase PS b) MeOH, 20% aq. K2CO3 c) DIAD, PPh3, tetrachlorophthalimide, THF d) ethylenediamine, ACN, THF, EtOH.

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