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Discovery of potent and specific dihydroisoxazole inhibitors of human transglutaminase 2.

Klöck C, Herrera Z, Albertelli M, Khosla C - J. Med. Chem. (2014)

Bottom Line: Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown.Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity.Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.

View Article: PubMed Central - PubMed

Affiliation: Departments of †Chemistry, ‡Chemical Engineering and §Comparative Medicine, Stanford University , MC 5080, Stanford California 94305, United States.

ABSTRACT
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.

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Plasma concentrationsof TG inhibitors after oral dosing. Followingoral administration of sets of TG inhibitors to mice at 50 mg/kg,blood was sampled at 5, 20, and 60 min time points. The concentrationof each compound in plasma was quantified, and the resulting profilesplotted as time versus concentration curves, as depicted. Whereassome inhibitors, such as CK996 and CK999, have negligible systemicavailability, others, such as ERW1041E and ZH147A, reach peak concentrationsof 2–3 μg/mL.
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fig4: Plasma concentrationsof TG inhibitors after oral dosing. Followingoral administration of sets of TG inhibitors to mice at 50 mg/kg,blood was sampled at 5, 20, and 60 min time points. The concentrationof each compound in plasma was quantified, and the resulting profilesplotted as time versus concentration curves, as depicted. Whereassome inhibitors, such as CK996 and CK999, have negligible systemicavailability, others, such as ERW1041E and ZH147A, reach peak concentrationsof 2–3 μg/mL.

Mentions: All compounds were rapidly clearedfrom circulation (Figure 4). Relative to theirplasma concentrations at 20min, the equivalent values at 60 min were at least 4-fold lower, verifyingthat their half-lives were shorter than 20 min. No compound appearedto possess a considerably longer half-life than the others; as such,this parameter did not provide any basis for the selection of a specificlead compound. Knowing that this class of compounds achieves highpeak concentrations in plasma after intraperitoneal administration(ca. 20–40 μg/mL, data not shown), we sought to establishif any compound could achieve at least 10% of this value range afteroral dosing. Compound 4b (aka ZH147A), harboring the4-cis fluoro moiety, was most promising in this regard,with plasma concentrations of 2.9 ± 1.5 and 2.1 ± 1.1 μg/mLat 5 and 20 min, respectively. The unsubstituted parent ERW1041E (1) also had reasonable oral bioavailability, achieving plasmaconcentrations of 2.1 ± 1.1 and 1.2 ± 0.6 μg/mL at5 and 20 min, respectively. The plasma levels of the more potent derivativeswith a 4-aryl moiety (7a/e) (aka CK805/CK937)were 3–4-fold lower than that of 4b, whereas the4-arylamido derivatives (9d/e) were muchless bioavailable, suggesting that these compounds are either inherentlyimpermeable or that they undergo more rapid presystemic metabolism(possibly even in the intestinal lumen).


Discovery of potent and specific dihydroisoxazole inhibitors of human transglutaminase 2.

Klöck C, Herrera Z, Albertelli M, Khosla C - J. Med. Chem. (2014)

Plasma concentrationsof TG inhibitors after oral dosing. Followingoral administration of sets of TG inhibitors to mice at 50 mg/kg,blood was sampled at 5, 20, and 60 min time points. The concentrationof each compound in plasma was quantified, and the resulting profilesplotted as time versus concentration curves, as depicted. Whereassome inhibitors, such as CK996 and CK999, have negligible systemicavailability, others, such as ERW1041E and ZH147A, reach peak concentrationsof 2–3 μg/mL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234452&req=5

fig4: Plasma concentrationsof TG inhibitors after oral dosing. Followingoral administration of sets of TG inhibitors to mice at 50 mg/kg,blood was sampled at 5, 20, and 60 min time points. The concentrationof each compound in plasma was quantified, and the resulting profilesplotted as time versus concentration curves, as depicted. Whereassome inhibitors, such as CK996 and CK999, have negligible systemicavailability, others, such as ERW1041E and ZH147A, reach peak concentrationsof 2–3 μg/mL.
Mentions: All compounds were rapidly clearedfrom circulation (Figure 4). Relative to theirplasma concentrations at 20min, the equivalent values at 60 min were at least 4-fold lower, verifyingthat their half-lives were shorter than 20 min. No compound appearedto possess a considerably longer half-life than the others; as such,this parameter did not provide any basis for the selection of a specificlead compound. Knowing that this class of compounds achieves highpeak concentrations in plasma after intraperitoneal administration(ca. 20–40 μg/mL, data not shown), we sought to establishif any compound could achieve at least 10% of this value range afteroral dosing. Compound 4b (aka ZH147A), harboring the4-cis fluoro moiety, was most promising in this regard,with plasma concentrations of 2.9 ± 1.5 and 2.1 ± 1.1 μg/mLat 5 and 20 min, respectively. The unsubstituted parent ERW1041E (1) also had reasonable oral bioavailability, achieving plasmaconcentrations of 2.1 ± 1.1 and 1.2 ± 0.6 μg/mL at5 and 20 min, respectively. The plasma levels of the more potent derivativeswith a 4-aryl moiety (7a/e) (aka CK805/CK937)were 3–4-fold lower than that of 4b, whereas the4-arylamido derivatives (9d/e) were muchless bioavailable, suggesting that these compounds are either inherentlyimpermeable or that they undergo more rapid presystemic metabolism(possibly even in the intestinal lumen).

Bottom Line: Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown.Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity.Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.

View Article: PubMed Central - PubMed

Affiliation: Departments of †Chemistry, ‡Chemical Engineering and §Comparative Medicine, Stanford University , MC 5080, Stanford California 94305, United States.

ABSTRACT
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.

Show MeSH
Related in: MedlinePlus