Strategies for the design of inhibitors of aldose reductase, an enzyme showing pronounced induced-fit adaptations

Aldose reductase is involved in the polyol pathway, catalyzing the reduction of glucose to sorbitol. However, due to pronounced binding site adaptations, the enzyme can operate on a broad palette of structurally diverse substrates ranging from small aliphatic and aromatic aldehydes up to steroid-type ligands. A comparative analysis of the presently accessible crystal structures of aldose reductase complexes reveals four binding-competent protein conformations. Additional relevant conformers are detected through molecular dynamics simulations. They indicate an equilibrium of several conformers which is shifted towards the binding-competent geometries upon ligand binding. Such a manifold system with several alternative binding site conformers requires some tailored concepts in virtual screening. We followed two strategies, both successfully suggesting new micromolar inhibitors. In a first attempt, we concentrated on one preferred conformer and performed a virtual screening, assuming that the binding pocket of aldose reductase adopts only this conformation. In a second approach, we followed a ligand superpositioning method. Ligands were extracted in their bound conformations from three different crystal structures, all accommodating the ligands with different active site conformations. After merging these ligands into one supermolecule, mutual alignments were computed, taking candidate ligands from a screening database. The latter strategy also retrieved several structurally new inhibitors of micromolar potency.     

FK506 sensitizes mammalian cells to high osmolarity by modulating p38 MAP kinase activation

The immunosuppressants tacrolimus (FK506) and cyclosporin A (CsA) have increased the survival rates in organ transplantation. Both drugs inhibit the protein phosphatase calcineurin (CaN) in activated T cells, exhibiting similar side-effects. Diabetes is observed more often in FK506 than CsA therapy, probably due to inhibition of new molecular targets other than CaN. We studied FK506 toxicity in mammalian cells. FK506, but not CsA, regulated p38 activation by osmotic stress, and decreased viability in osmostressed cells. In addition, FK506 treatment strongly increased the phosphorylation of the eukaryotic initiation factor-2a (eIF-2a) subunit. eIF-2a phosphorylation, p38 inhibition and cell lethality were relieved by addition of excess amino acids to the medium, suggesting that amino acid availability mediated FK506 toxicity. Therefore, these FK506-dependent responses could be relevant to the non-therapeutic effects of FK506 therapy.Received 16 October 2003; received after revision 8 January 2004; accepted 14 January 2004     

Ubiquitin-proteasome pathway as a primary defender against TRAIL-mediated cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death as well as expression of proinflammatory genes such as CXCL8 in malignant human astrocytoma cells. However, the molecular mechanisms that determine the fate of cells are not yet understood. The ubiquitin (Ub)-proteasome pathway regulates a wide range of cellular functions through degradation of various regulatory proteins; given this, we hypothesized that this pathway may play a central role in TRAIL-mediated signaling. We demonstrate here that inhibition of the Ub-proteasome pathway enhanced TRAIL-mediated cell death of human astrocytoma CRT-MG cells within hours by blocking degradation of active caspase-8 and -3. Proteasome inhibitors suppressed TRAIL-mediated activation of NF-B; however, inhibition of the NF-B pathway alone was not sufficient to enhance TRAIL-mediated cell death. Collectively, these results suggest that the Ub-proteasome pathway may play an important role as an antiapoptotic surveillance system by eliminating activated caspases as well as mediating NF-B-dependent signals.Received 30 December 2003; received after revision 9 February 2004; accepted 13 February 2004     

Administration of the antitumor drug mitoguazone protects normal thymocytes against spontaneous and etoposide-induced apoptosis

The suggestion has been made that polyamines may be involved in the control of cell death, since exceedingly high or low levels induce apoptosis in different cell systems. For a deeper insight into the relationship between apoptosis and polyamine metabolism, we investigated in vitro the effect on rat thymocytes of mitoguazone (MGBG, which inhibits S-adenosylmethionine decarboxylase, i.e. a key enzyme in the polyamine biosynthetic pathway). Thymocytes were selected as an especially suitable model system, since they undergo spontaneous apoptosis in vivo and can be easily induced to apoptose in vitro by etoposide, used here as an apoptogenic agent. MGBG protected thymocytes from both spontaneous and drug-induced apoptosis, and this protective effect was associated with a decrease in polyamine oxidase activity and total polyamine levels.Received 7 July 2004; received after revision 2 September 2004; accepted 9 September 2004     

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of α-lactalbumin

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human -lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.Received 30 January 2004; received after revision 5 March 2004; accepted 16 March 2004     

Alexander disease: putative mechanisms of an astrocytic encephalopathy

Alexander disease (AXD) is the first primary astrocytic disorder. This encephalopathy is caused by dominant mutations in the glial fibrillary acidic protein (GFAP) gene, encoding the main intermediate filament of astrocyte. Pathologically, this neurodegenerative disease is characterised by dystrophic astrocytes containing intermediate filament aggregates associated with myelin abnormalities.More than 20 GFAP mutations have been reported. Many of them cluster in highly conserved regions between several intermediate filaments. Contrary to other intermediate filament-related diseases, AXD seems to be the consequence of a toxic gain of function induced by aggregates. This is supported by the phenotype of mice overexpressing human GFAP. Nevertheless, GFAP null mice display myelin abnormalities and blood-brain barrier dysfunction that are present in AXD.Given the pivotal role of astrocytes in brain physiology, there are many possibilities for astrocytes to dysfunction and to impair the functions of other cells. Physiopathological hypotheses are discussed in the frame of AXD.Received 11 April 2003; received after revision 22 July 2003; accepted 31 July 2003Both authors contributed equally to this work.     

Gelsolin superfamily proteins: key regulators of cellular functions

Cytoskeletal rearrangement occurs in a variety of cellular processes and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins control actin organization by severing filaments, capping filament ends and nucleating actin assembly [1]. Gelsolin is the founding member of this family, which now contains at least another six members: villin, adseverin, capG, advillin, supervillin and flightless I. In addition to their respective role in actin filament remodeling, these proteins have some specific and apparently non-overlapping particular roles in several cellular processes, including cell motility, control of apoptosis and regulation of phagocytosis (summarized in table 1). Evidence suggests that proteins belonging to the gelsolin superfamily may be involved in other processes, including gene expression regulation. This review will focus on some of the known functions of the gelsolin superfamily proteins, thus providing a basis for reflection on other possible and as yet incompletely understood roles for these proteins.     

Proton motive force mediates a reorientation of the cytosolic domains of the multidrug transporter LmrP

LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.