Protein kinase C (PKC), a key enzyme family involved in cellular signal transduction, is the main target of the potent phorbol ester-type tumor promoters. In order to elucidate at the molecular level the role of PKC in tumor promotion and signal transduction, the first and the second cysteine-rich domains (CRD's) of rat brain PKCγ and mouse skin PKCη (γ-CRD1, γ-CRD2, η-CRD1, and η-CRD2) have been prepared by solid phase synthesis. These peptides except for η-CRD1 properly folded upon zinc treatment to produce PKC regulatory domain surrogates that bind [3H]phorbol 12, 13-dibutyrate (PDBu) with high affinity in the presence of phosphatidylserine. The binding affinity of η-CRD2 was especially high (Kd =0.91 nM), comparable to that reported for native PKCη (Kd = 0.87 nM). The similar binding behavior of these peptides and native PKC suggests that these peptides serve as useful molecular probes for elucidation of the structural requirements for the recognition of phorbol ester-type tumor promoters. We have recently used these peptides for binding assay of new conformationally restricted analogues of teleocidins and for affinity labeling by photolabile phorbol esters.
A number of artificially modified substrates for prenyltransferases were designed, including Z-3- methyl-3-pentenyl diphosphate and its E-isomer, , which could be converted into the R- and S enantiomers of 4-methyl-E, E- farnesol, respectively. Chiral synthesis and configurational determination of faranal and 4-methyl juvenile hormone I were achieved by exploiting the stereospecificity of farnesyl diphosphate synthase (FPS) reaction. Molecular cloning and overexpression of the gene for thermostable FPS made it possible to produce sufficient amounts of crystals of the enzyme. Site-directed mutagenesis studies were conducted extensively with FPS, providing detailed information about essential amino acid residues involved in catalysis and substrate binding. Based on these results coupled with those from studies with artificial substrates, a hypothetical mechanism is proposed for the FPS reaction.
In order to understand roles of amino acid residues around heme vicinity in the oxygen activation by cytochrome P-450 and peroxidases, we have employed two strategies. One was the usage of synthetic heme models and the other was mutation of key residues in hemoproteins. Through the model studies, we have characterized all conceivable intermediates postulated in enzymic systems. In addition, push-pull effect on the oxygen activation was experimentally demonstrated. On the other hand, mutant myoglobins having cysteine as the proximal ligand were prepared to understand roles of thiolate ligand in the oxygen activation by cytochrome P-450. Further, hydrogen bonding between Asn and distal His in peroxidases has been shown to be important to control both the oxygen activation and the substrate oxidation. On the basis of these enzymic and model studies, we have synthesized artificial peroxide-dependent heme enzymes by site-directed double point mutation of sperm whale myoglobin. The resulting Mb mutants exhibit remarkable oxidation activities with high enantioselectivities in sulfoxi-dation and epoxidation
Just after the discovery of PGI2, we started to find chemically and metabolically stable PGI2 derivatives with longer duration of action. Extensive studies led us to a new class of stable PGI2 analogue, 5, 6, 7-trinor-4, 8-inter-m-phenylenePGI2 that has a phenol moiety instead of enol-ether linkage in PGI2. In order to accomplish synthesis of the m-phenylenePGI2, novel synthetic methods were developed, for instance, ortho-selective metal-halogen exchange reaction of bromoanisoles by means of Grignard reagent, copper-catalyzed SN2' cyclization to prepare dihydrocyclopenta [b] benzofuran, and regioselective and stereo-selective elongation of ω-side chain by Prins reaction. Further efforts were devoted to synthesize derivatives of m-phenylenePGI2 with enhanced pharmacological activity and less adverse reaction. Finally, we attained to Beraprost sodium which is the first launched drug as an orally active PGI2. This paper will focus on the study of total syntheses of m-phenylenePGI2.
Oncogene product functions are mainly distUrbance of cellular signal transduction and modification of specific gene expression. Several oncogenes are known to be related to human neoplastic diseases. If these onocogenes play important roles in the progression of cancer, it should be possible to develop new antitumor drugs inhibiting oncogene product activities. We have isolated various signal transduction inhibitors such as tyrosine kinase inhibitors, Ras function inhibitors, and phosphatidylinositol turnover inhibitors from microorganisms and plants. In the present review, we describe isolation, synthesis and biological activities of tyrosine phosphatase and Ras function inhibitors which were recently isolated. Intracellular tyrosine phosphorylation should be regulated by both tyrosine kinases and tyrosine phosphatases. Therefore, we screened microbial culture filtrates for tyrosine phosphatase inhibitors and isolated a novel nitrosamine dephostatin from Streptomyces. We also established a practical synthetic route for dephostatin. Since dephostatin is not stable enough, we synthesized several structurally related dephostatin analogs. Among them a regioisomer of dephostatin showed tyrosine phosphatase inhibitory activity equivalent to that of dephostatin, and also had increased stability. We isolated conophylline and the novel aglaiastatin from tropical plant extracts as Ras function inhibitors. Conophyline and aglaiastatin induced normal phenotypes in K-ras-expressing cells. Since farnesl protein transferase (FPTase) is necessary for the Ras activity, we also screened microbial culture filtrates for FPTase inhibitors. As a result, we isolated 4 novel compounds, valinoctin A and B, and saquayamaycin E and F. The absolute configuration of valinoctin A was determined by a synthetic method together with crystallographic analysis.
Aplyronine A (1), isolated as a very minute constituent of the sea hare Aplysia kurodai, is a new potent antitumor macrolide that interacts with actin, the protein in cytoskeleton. The absolute stereostructure of aplyronine A (1) was determined by the spectroscopic analysis and the enantioselective synthesis of the fragments derived from 1. The enantioselective synthesis of aplyronine A (1) was achieved by a convergent approach. Three segments 28, 29, and 30 were prepared using the Evans aldol reaction and the Sharpless epoxidation as key steps. The segment 28 was combined with two segments 31 and 32 successively to afford segment 33, while the Julia coupling reaction between segments 29 and 30 gave segment 34. Julia olefination between segments 33 and 34 and the subsequent four-carbon homologation reaction led to seco acid 35, which was converted into aplyronine A (1) by Yamaguchi lactonization followed by the introduction of two amino acids.