Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca 2؉ -ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca 2؉ entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990 -1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.Understanding protein-membrane interaction and the activity of lipophilic drugs and specific lipids like sterols attracts increasing attention and represents a challenge to our current models of protein-solvent interactions and protein dynamics. Sarco/endoplasmic Ca 2ϩ -ATPase (SERCA) 6 offers an attractive model for the analysis of such interactions due to favorable biochemical properties with sensitive assays of function, a large resource of inhibitors, and a wealth of crystal structures related to the functional cycle. In conjunction with plasma membrane Ca 2ϩ -ATPases and Ca 2ϩ channel proteins, SERCA orchestrates spatiotemporal changes in the concentration of Ca 2ϩ inside the cell, providing the background essential for the function of Ca 2ϩ as a second messenger in the regulation of numerous cellular processes (1-3). At the same time, high cytosolic (Ͼ1 M) concentrations of Ca 2ϩ , induced by depletion of the endoplasmic Ca 2ϩ stores and uptake of extracellular Ca 2ϩ by the endoplasmic store-regulated mechanism (4), are involved as essential factors in the induction of apoptotic processes, leading to mitochondrial disruption with release of cytochrome c, activation of intracellular caspases, and cell death (5). As a consequence, interference of cellular function by inhibition of SERCA may lead to apoptotic cell death irrespective of the...

Cytotoxic Phenylpropanoids and an Additional Thapsigargin Analogue Isolated from Thapsia garganica. -Four new phenylpropanoids (I) and a new thapsigargin analogue (II) are isolated from the fruits of the title plant. The former are found to be potent cytotoxins. -(LIU, H.; JENSEN, K. G.; TRAN, L. M.; CHEN, M.; ZHAI, L.; OLSEN, C. E.; SOEHOEL, H.; DENMEADE, S. R.; ISAACS, J. T.; CHRISTENSEN*, S. B.; Phytochemistry 67 (2006) 24, 2651-2658; Dep. Med. Chem., Dan. Univ. Pharm. Sci., DK-2100 Copenhagen, Den.; Eng.) -H. Haber 15-207

Casein kinase ll (CK2) is a serine/threonine kinase and widely distributed in various tissues. CK2 predominantly exists in the form of heterotetramer composed of two catalytic subunits (CK2alpha or CK2alpha prime) and two regulatory subunits (CK2beta). Recently, the CK2alpha inhibition has been revealed to prevent the progression of glomerulonephritis. On the other hand, inhibition of CK2alpha prime in testis affect the spermatogenesis. In order to develop of novel CK2 inhibitor for nephritis, we determined the first structure of CK2alpha prime complexed with a potent inhibitor and compared with the structure of CK2alpha. The crystal structure of a C-terminal deletion mutant of human Ck2alpha prime was solved and refined to 3.2 Å resolution. Two isozymes, CK2alpha and CK2alpha prime, reveal the high similarity of the overall structure. The largest structural difference between CK2alpha prime and human Ck2alpha occurs at the loop connecting the strands beta 4 and beta 5. The corresponding region belongs on one hand to the CK2alpha/ CK2beta interface in the holoenzyme and on the other hand to the catalytic core, which is structurally highly conserved among the eukaryotic protein kinases. This observation is consistent with the growing evidence that CK2alpha prime and CK2alpha may possess the difference of the relation in vivo with CK2beta and the substrate recognition.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.