RESUMO
Calpains are intracellular calcium-activated cysteine proteases whose unregulated proteolysis following the loss of calcium homeostasis can lead to acute degeneration during ischemic episodes and trauma, as well as Alzheimer's disease and cataract formation. The determination of the crystal structure of the proteolytic core of mu-calpain (muI-II) in a calcium-bound active conformation has made structure-guided design of active site inhibitors feasible. We present here high-resolution crystal structures of rat muI-II complexed with two reversible calpain-specific inhibitors employing cyclic hemiacetal (SNJ-1715) and alpha-ketoamide (SNJ-1945) chemistries that reveal new details about the interactions of inhibitors with this enzyme. The SNJ-1715 complex confirms that the free aldehyde is the reactive species of the cornea-permeable cyclic hemiacetal. The alpha-ketoamide warhead of SNJ-1945 binds with the hydroxyl group of the tetrahedral adduct pointing toward the catalytic histidine rather than the oxyanion hole. The muI-II-SNJ-1945 complex shows residue Glu261 displaced from the S1' site by the inhibitor, resulting in an extended "open" conformation of the domain II gating loop and an unobstructed S1' site. This conformation offers an additional template for structure-based drug design extending to the primed subsites. An important role for the highly conserved Glu261 is proposed.
Assuntos
Calpaína/antagonistas & inibidores , Cristalografia por Raios X , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calpaína/química , Calpaína/genética , Calpaína/metabolismo , Domínio Catalítico , Sequência Conservada , Cisteína/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Leupeptinas/química , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Soluções/química , Água/químicaRESUMO
The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.
Assuntos
Calpaína/química , Glicoproteínas/química , Leucina/análogos & derivados , Leucina/química , Leupeptinas/química , Sequência de Aminoácidos , Animais , Calpaína/antagonistas & inibidores , Domínio Catalítico , Catepsina K , Catepsinas/química , Catepsinas/metabolismo , Cristalização , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Glicoproteínas/metabolismo , Leucina/metabolismo , Leupeptinas/metabolismo , Dados de Sequência Molecular , Papaína/química , Papaína/metabolismo , Estrutura Terciária de Proteína , RatosRESUMO
This paper reports the use of DryLab, a computer simulation software package, to assist in the development and optimization of a reversed-phase high-performance liquid chromatographic (HPLC) method for the separation of a model drug candidate and its degradation products. Prior to the optimization process, columns with various bonded phases are evaluated for their chromatographic performance using the sample of interest. Simultaneous optimization of two separation variables and the use of resolution maps to predict the optimal conditions are illustrated. Options to optimize column conditions (column length and flow-rate) to further reduce run time are briefly discussed. The accuracy of DryLab-predicted retention times and resolution is compared with experimental values. The DryLab software used in this study provided satisfactory predictions for the selected model, with average errors of less than 3.5 and 11.8% for retention time and resolution, respectively.
