A Novel Bongkrekic Acid Analog-Mediated Modulation of the Size of Lipid Droplets: Evidence for the Appearance of Smaller Adipocytes.

Thiazolidinediones (TZDs) are known as peroxisome proliferator-activated receptor γ (PPARγ) activators, and are used in the treatment of diabetes. Although the usefulness of TZDs has been demonstrated, some of their side effects are becoming an obstacle to their clinical applicability; edema is known to be evoked by the "structural characteristics" of TZD, but not by the PPARγ activation. Thus, novel therapeutic modalities (i.e., non-TZD-type PPARγ activators) having different structures to those of TZDs are desired. We previously identified bongkrekic acid (BKA) as a PPARγ activator using the human breast cancer MCF-7 cell line as a model system. In the present study, we newly synthesized BKA analogs and examined the usefulness of BKA and its analogs as PPARγ activators in differentiated adipocyte cells. Among the chemicals investigated, one of the BKA analogs (BKA-#2) strongly stimulated PPARγ and the differentiation of 3T3-L1 cells similar to pioglitazone, a positive control. Furthermore, BKA-#2 reduced the size of lipid droplets in the mature adipocyte cells. The possible modulation mechanism by BKA-#2 is discussed.

Efficient Total Synthesis of Bongkrekic Acid and Apoptosis Inhibitory Activity of Its Analogues.

Bongkrekic acid (BKA), isolated from the bacterium Burkholderia cocovenenans, is an inhibitor of adenine nucleotide translocator, which inhibits apoptosis, and is thus an important tool for the mechanistic investigation of apoptosis. An efficient total synthesis of BKA has been achieved by employing a three-component convergent strategy based on Kocienski-Julia olefination and Suzuki-Miyaura coupling. It is noteworthy that segment B has been prepared as a new doubly functionalized coupling partner, which contributes to shortening of the number of steps. Torquoselective olefination with an ynolate has also been applied for the efficient construction of an unsaturated ester. Furthermore, it is revealed that 1-methyl-2-azaadamantane N-oxyl is an excellent reagent for final oxidation to afford BKA in high yield. Based on the total synthesis, several BKA analogues were prepared for structure-activity relationship studies, which indicated that the carboxylic acid moieties were essential for the apoptosis inhibitory activity of BKA. More easily available BKA analogues with potent apoptosis inhibitory activity were also developed.

Bongkrekic acid analogue, lacking one of the carboxylic groups of its parent compound, shows moderate but pH-insensitive inhibitory effects on the mitochondrial ADP/ATP carrier.

Bongkrekic acid, isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In this study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, performed by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, KH-7, KH-16, and KH-17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of bongkrekic acid for its pH-dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated.

Bongkrekic acid as a selective activator of the peroxisome proliferator-activated receptor γ (PPARγ) isoform.

Bongkrekic acid (BKA), an antibiotic isolated from Pseudomonas cocovenans, is an inhibitory molecule of adenine nucleotide translocase. Since this translocase is a core component of the mitochondrial permeability transition pore (MPTP) formed by apoptotic stimuli, BKA has been used as a tool to abrogate apoptosis. However, the other biochemical properties of BKA have not yet been resolved. Although the definition of a fatty acid is a carboxylic acid (-COOH) with a long hydrocarbon chain (tail), when focused on the chemical structure of BKA, the molecule was revealed to be a branched unsaturated tricarboxylic acid that resembled the structure of polyunsaturated fatty acids (PUFAs). Peroxisome proliferator-activated receptors (PPARs) consist of a subfamily of three isoforms: α, ß, and γ, the ligands of which include PUFAs. Using completely synthesized BKA together with simplified BKA derivatives (purity: > 98%), we herein demonstrated the utility of BKA as a selective activator of the human PPARγ isoform, which may not be associated with the anti-apoptotic nature of BKA. We also discussed the possible usefulness of BKA.

Molecular design, synthesis, and evaluation of novel potent apoptosis inhibitors inspired from bongkrekic acid.

Bongkrekic acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Since inhibition of ANT is connected to the inhibition of cytochrome c release from mitochondria, which then results in the suppression of apoptosis, it has been used as a tool for the mechanistic investigation of apoptosis. BKA consists of a long carbon chain with two asymmetric centers, a nonconjugated olefin, two conjugated dienes, three methyl groups, a methoxyl group, and three carboxylic acids. This complicated chemical structure has caused difficulties in synthesis, supply, and biochemical mechanistic investigations. In this study, we designed and synthesized more simple tricarboxylic acids that were inspired by the molecular structure of BKA. Their cytotoxicity and apoptosis-preventing activity in HeLa cells and the effect on the mitochondrial inner membrane potential (ΔΨm) in HL-60 cells were then evaluated. All tested tricarboxylic acid derivatives including BKA showed little toxicity against HeLa cells. BKA and two of the synthesized derivatives significantly suppressed staurosporine (STS)-induced reductions in cell viability. Furthermore, STS-induced ΔΨm collapse was significantly restored by pretreatment with BKA and a tricarboxylic acid derivative. Other derivatives, in which one of three carboxylic acids was esterified, exhibited potent toxicity, especially a derivative bearing a carbon chain of the same length as that of BKA. In conclusion, we have developed a new lead compound as an apoptosis inhibitor bearing three carboxylic acids connected with the proper length of a long carbon chain.

Total synthesis of iso- and bongkrekic acids: natural antibiotics displaying potent antiapoptotic properties.

For over five decades, owing to their antiapoptotic activities, bongkrekic and isobongkrekic acids have generated interest from the scientific community. Here, we disclose full details of our investigation into the synthesis of isobongkrekic acid, which culminated in its first preparation and features various palladium-catalysed cross-couplings and Takai olefination reactions. Access to bongkrekic acid is also reported by this route. These syntheses involve the preparation and use of new general building blocks which could find wider applications.

Total synthesis of the anti-apoptotic agents iso- and bongkrekic acids.

The first convergent total synthesis of isobongkrekic acid is reported involving three different stereospecific palladium cross-couplings for the formation of the diene units. Access to bongkrekic acid by this route is also demonstrated. These syntheses involve the formation of several potentially general building blocks.

Isobongkrekic acid, a new inhibitor of mitochondrial ADP-ATP transport: radioactive labeling and chemical and biological properties.

An isomer of bongkrekic acid, designated as isobongkrekic acid, has been isolated from ethereal extracts of Pseudomonas cocovenenans grown on defatted coconut. Isobongkrekic acid was also obtained by alkaline treatment of bongkrekic acid. Isobongkrekic acid possesses the same ultraviolet spectrum and the same molecular weight as bongkrekic acid; it has a similar infrared spectrum but not the same nuclear magnetic resonance (NMR) spectrum. The differences in NMR data were interpreted to mean that isobongkrekic acid differs from bongkrekic acid by the configuration of the dicarboxylic end; whereas the two carboxylic groups of the dicarboxylic end have the trans configuration in bongkrekic acid, they have the cis configuration in isobongkrekic acid. Differences between bongkrekic and isobongkrekic acids are lost after catalytic hydrogenation of the molecules. Isobongkrekic acid, like bongkrekic acid, is an uncompetitive inhibitor of ADP transport in mitochondria, provided the mitochondria are preincubated in the presence of the inhibitor and a minute concentration of ADP. The inhibitory and binding efficiency of isobongkrekic acid is considerably increased below pH 7. The number of high affinity sites for [3H] isobongkrekic acid is 0.13 to 0.20 nmol/mg protein in rat liver mitochondria and about 1 nmol/mg protein in rat heart mitochondria, i.e., similar to the number of high affinity sites for [3H] bongkrekic acid. Isobongkrekic and bongkrekic acids compete for the same site, but the affinity of isobongkrekic acid for mitochondria is one-half to one-fourth that of bongkrekic acid.