Biomedical subjects
Publications and source records attributed to E J Corey.
[formula: see text] Highly enantioselective (up to 130:1) epoxidation of a variety of alpha,beta-enones to form alpha,beta-epoxy ketones is described along with a rational analysis of the mechanistic basis for this strong absolute stereochemical control by the chiral catalyst 2.
[reaction: see text] Enantiomerically pure chiral 5-silylated 2-cyclohexenones are easily prepared in high yield using as a key step kinetic resolution with a commercially available lipase. Fully active enzyme can be recovered very efficiently for reuse. The synthetic steps are outlined in Schemes 1 and 3. Enantiomerically pure 2-cyclohexenones such as 1 and 2 are versatile intermediates for the synthesis of a multitude of chiral targets by means of a variety of diastereoselective reactions such as those illustrated in Scheme 2.
[structure: see text] The antiviral agent (+/-)-virantmycin has been synthesized from two simple building blocks (2 and 3) in eight steps, as outlined in Scheme 2.
[formula: see text] A short synthesis of saframycin A is described which begins with a readily available intermediate previously utilized for the total synthesis of ecteinascidin 743. A key step in this synthesis is the use of 1-fluoro-3,5-dichloropyridinium triflate to oxidize a phenolic ring to a 1,4-benzoquinone unit while simultaneously cleaving a methoxymethyl ether of a different phenolic ring to the corresponding phenol (4-->5). The common intermediate (2) for the synthesis of saframycin A (1) and ecteinascidin 743 also allowed the synthesis of two hybrids of these structures (6 and 7). Whole cell bioassays for antitumor activity using lung, colon, melanoma, and prostate-derived tumor cell lines allowed a clear correlation of structure with biological activity in this series.
[formula: see text] A novel catalytic enantioselective Strecker synthesis of chiral alpha-amino nitriles and alpha-amino acids is described and analyzed with regard to the possible mechanistic basis for stereoselectivity. Key features of the enantioselective process include (1) the use of the chiral bicyclic guanidine 1 as catalyst and (2) the use of the N-benzhydryl substituent on the imine substrate.
A series of totally synthetic molecules that are structurally related to the marine natural product ecteinascidin 743 (Et 743) has been prepared and evaluated as antitumor agents. The most active of these, phthalascidin, is very similar to Et 743 with regard to in vitro potency and mode of action across a variety of cell types. The antiproliferative activity of phthalascidin (IC50 = 0.1-1 nM) is greater than that of the agents Taxol, camptothecin, adriamycin, mitomycin C, cisplatin, bleomycin, and etoposide by 1-3 orders of magnitude, and the mechanism of action is clearly different from these currently used drugs. Phthalascidin and Et 743 induce DNA-protein cross-linking and, although they seem to interact with topoisomerase (topo) I (but not topo II), topo I may not be the primary protein target of these agents. Phthalascidin and Et 743 show undiminished potency in camptothecin- and etoposide-resistant cells. Phthalascidin is more readily synthesized and more stable than Et 743, which is currently undergoing clinical trials. The relationship of chemical structure and antitumor activity for this class of molecules has been clarified by this study.
LXRalpha and -beta are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRalpha-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.
Proteases play an important role in the pathogenic mechanisms and differentiation events of protozoan parasites; the proteasome/ubiquitin system is essential for maintaining the differentiation state of many cell types. A single input of the specific inhibitor of proteasomes, lactacystin, prevented encystation of the protozoan parasite Entameoba invadens, whereas a cysteine protease inhibitor, E64, only delayed encystation. The ameba target of lactacystin was purified and it displayed the features typical of eukaryotic 20S proteasome complexes. In addition, transcripts encoding ubiquitin were detectable in trophozoites stage cells, disappeared immediately following transfer of amoebae to encystation induction medium, and reappeared at the same time during encystation as other encystation-specific transcripts. These results demonstrate that proteasome function is required during the conversion of the disease-causing trophozoite into the infectious cyst stage of Entamoeba parasites, and that ubiquitin transcript levels undergo an unusual decrease during the early stages of this differentiation process.
Lactacystin (1) and the related beta-lactone omuralide (2) are remarkably selective and potent irreversible inhibitors of the 20 S proteasome, a large polymolecular protein machine which is responsible for the degradation of ubiquitin-labeled proteins. Because of this fact 1 and 2 have emerged as important tools in biochemistry and cell biology. The challenge of synthesis has been accepted by several research groups with the result that 1 and 2 and their analogs can now be synthesized by a variety of synthetic approaches. This review summarizes the synthetic processes which have been developed to date for the production of such compounds. The study of biological activity of analogs of 1 and 2 has clearly defined the structural features which are responsible for the potency of 1 and 2, as described in the closing section of this account. It is concluded that 1 and 2 are nearly optimal for the irreversible inactivation of the 20 S proteasome.
Proteasomes degrade most of the proteins inside eukaryotic cells, including transcription factors and regulators of cell cycle progression. Here we show that nanomolar concentrations of lactacystin, a specific irreversible inhibitor of the 20S proteasome, inhibit development of the exoerythrocytic and erythrocytic stages of the malaria parasite. Although lactacystin-treated Plasmodium berghei sporozoites are still invasive, their development into exoerythrocytic forms (EEF) is inhibited in vitro and in vivo. Erythrocytic schizogony of P. falciparum in vitro is also profoundly inhibited when drug treatment of the synchronized parasites is prior, but not subsequent, to the initiation of DNA synthesis, suggesting that the inhibitory effect of lactacystin is cell cycle specific. Lactacystin reduces P. berghei parasitemia in rats, but the therapeutic index is very low. Along with other studies showing that lactacystin inhibits stage-specific transformation in Trypanosoma and Entamoeba spp., these findings highlight the potential of proteasome inhibitors as drugs for the treatment of diseases caused by protozoan parasites.
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A prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan.
A Schizosaccharomyces pombe cDNA encoding lanosterol synthase was cloned by complementing a Saccharomyces cerevisiae lanosterol synthase mutant. The predicted 83-kDa protein is 54-58% identical to other lanosterol synthases. The previously known lanosterol synthases contain 229 conserved residues, which should encompass the catalytically essential amino acids. This number is decreased dramatically by including the Sc. pombe lanosterol synthase in the analysis; 42 residues are no longer conserved and therefore are catalytically nonessential. We have begun mutagenic studies to identify catalytic residues from the remaining conserved residues. Mutant Sa. cerevisiae lanosterol synthase genes were generated in which phenylalanine was specifically substituted for conserved tryptophan residues. All of the resultant mutant enzymes retained the ability to complement the Sc. cerevisiae lanosterol synthase mutant, suggesting that these conserved tryptophan residues are not catalytically essential.
Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] catalyzes the cyclization of (S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus. We report herein the cloning and characterization of the human gene (OSC) encoding lanosterol synthase, a predicted 83 kDa protein of 732 amino acids. The deduced amino acid sequence is 36-40% identical to known yeast and plant homologues and 83% identical to Rattus norvegicus lanosterol synthase. The new gene was shown to encode lanosterol synthase. The yeast lanosterol synthase deficient mutant SMY8 was complemented by the human gene, and a cell-free homogenate of SMY8 expressing the human gene was shown to convert 2,3-oxidosqualene to lanosterol.
Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma cell line. Tritium-labeled lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) were inhibited by lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited, and the ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component of an amino-terminal-threonine protease activity of the proteasome.
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Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacystin altered only in the N-acetylcysteine moiety are active, while structural or stereochemical modifications of the gamma-lactam ring or the hydroxyisobutyl group lead to partial or complete loss of activity. The inactive compounds do not antagonize the effects of lactacystin in either neurite outgrowth or cell cycle progression assays. The response to lactacystin involves induction of a predominantly bipolar morphology that is maximal 16-32 h after treatment and is distinct from the response to several other treatments that result in morphological differentiation. Neurite outgrowth in response to lactacystin appears to be dependent upon microtubule assembly, actin polymerization, and de novo protein synthesis. The observed structure-activity relationships suggest that lactacystin and its related beta-lactone may act via acylation of one or more relevant target molecule(s) in the cell.