RESUMEN
Protein Kinase RNA-activated (PKR) inhibition is thought to be relevant for immunology due to the potential to reduce macrophage and dendritic cell responses to bacteria and its signaling downstream of TNFα. PKR is also associated with neuroscience indications such as Alzheimer's disease due to its activation by the double stranded DNA (dsDNA) virus HSV1, a virus suggested to be important in the development of AD. Studies exploring the mechanistic role of PKR with existing tool molecules such as the tricyclic oxindole C16 are clouded by the poor selectivity profile of this ATP-competitive, Type I kinase inhibitor. Type II kinase leads such as the benzothiophene or pyrazolopyrimidine scaffolds from literature are equally poor in their selectivity profiles. As such, it became necessary to identify more potent and selective chemical matter to better understand PKR biology. A dual approach was taken. The first step of the strategy included virtual screening of the AbbVie compound collection. A combination of pharmacophore-based and GPU shape-based screening was pursued to identify selective chemical matter from promiscuous leads. The second step of the strategy followed traditional compound design. This step initiated from a literature lead with PKR cross reactivity. Combined, the two parallel efforts led to identification of more selective leads for investigation of PKR biology.
Asunto(s)
Inhibidores de Proteínas Quinasas , Humanos , Enfermedad de Alzheimer/metabolismo , Macrófagos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Descubrimiento de Drogas/métodosRESUMEN
Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.
Asunto(s)
Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Tirosina/metabolismo , Animales , Secuencia de Bases , Catálisis , Cartilla de ADN , Epóxido Hidrolasas/genética , Humanos , Cinética , Ratones , MutaciónRESUMEN
The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme-inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure-based mechanism is proposed for leukotoxin epoxide hydrolysis.
Asunto(s)
Inhibidores Enzimáticos/farmacocinética , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Tirosina , Urea/análogos & derivados , Urea/farmacocinética , Secuencia de Aminoácidos , Toxinas Bacterianas/química , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Epóxido Hidrolasas/antagonistas & inhibidores , Exotoxinas/farmacocinética , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Conformación Proteica , Urea/química , Urea/farmacologíaRESUMEN
The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3) has been determined at 2.8-A resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the alpha/beta hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.