Phenolic Lipids Derived from Cashew Nut Shell Liquid to Treat Metabolic Diseases.

Metabolic diseases are increasing at staggering rates globally. The peroxisome proliferator-activated receptors (PPARα/γ/δ) are fatty acid sensors that help mitigate imbalances between energy uptake and utilization. Herein, we report compounds derived from phenolic lipids present in cashew nut shell liquid (CNSL), an abundant waste byproduct, in an effort to create effective, accessible, and sustainable drugs. Derivatives of anacardic acid and cardanol were tested for PPAR activity in HEK293 cell co-transfection assays, primary hepatocytes, and 3T3-L1 adipocytes. In vivo studies using PPAR-expressing zebrafish embryos identified CNSL derivatives with varying tissue-specific activities. LDT409 (23) is an analogue of cardanol with partial agonist activity for PPARα and PPARγ. Pharmacokinetic profiling showed that 23 is orally bioavailable with a half-life of 4 h in mice. CNSL derivatives represent a sustainable source of selective PPAR modulators with balanced intermediate affinities (EC50 ∼ 100 nM to 10 µM) that provide distinct and favorable gene activation profiles for the treatment of diabetes and obesity.

A Heck-Based Strategy To Generate Anacardic Acids and Related Phenolic Lipids for Isoform-Specific Bioactivity Profiling.

A synthetic strategy for phenolic lipids such as anacardic acid and ginkgolic acid derivatives using an efficient and selective redox-relay Heck reaction followed by a stereoselective olefination is reported. This approach controls both the alkene position and stereochemistry, allowing the synthesis of natural and unnatural unsaturated lipids as single isomers. By this strategy, the activities of different anacardic acid and ginkgolic acid derivatives have been examined in a matrix metalloproteinase inhibition assay.

Enzyme kinetics and inhibition of histone acetyltransferase KAT8.

Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT.

Discovery of protein disulfide isomerase P5 inhibitors that reduce the secretion of MICA from cancer cells.

In order to regulate the activity of P5, which is a member of the protein disulfide isomerase family, we screened a chemical compound library for P5-specific inhibitors, and identified two candidate compounds (anacardic acid and NSC74859). Interestingly, anacardic acid inhibited the reductase activity of P5, but did not inhibit the activity of protein disulfide isomerase (PDI), thiol-disulfide oxidoreductase ERp57, or thioredoxin. NSC74859 inhibited all these enzymes. When we examined the effects of these compounds on the secretion of soluble major histocompatibility complex class-I-related gene A (MICA) from cancer cells, anacardic acid was found to decrease secretion. In addition, anacardic acid was found to reduce the concentration of glutathione up-regulated by the anticancer drug 17-demethoxygeldanamycin in cancer cells. These results suggest that anacardic acid can both inhibit P5 reductase activity and decrease the secretion of soluble MICA from cancer cells. It might be a novel and potent anticancer treatment by targeting P5 on the surface of cancer cells.

Discovery of a novel activator of 5-lipoxygenase from an anacardic acid derived compound collection.

Lipoxygenases (LOXs) and cyclooxygenases (COXs) metabolize poly-unsaturated fatty acids into inflammatory signaling molecules. Modulation of the activity of these enzymes may provide new approaches for therapy of inflammatory diseases. In this study, we screened novel anacardic acid derivatives as modulators of human 5-LOX and COX-2 activity. Interestingly, a novel salicylate derivative 23a was identified as a surprisingly potent activator of human 5-LOX. This compound showed both non-competitive activation towards the human 5-LOX activator adenosine triphosphate (ATP) and non-essential mixed type activation against the substrate linoleic acid, while having no effect on the conversion of the substrate arachidonic acid. The kinetic analysis demonstrated a non-essential activation of the linoleic acid conversion with a KA of 8.65 µM, αKA of 0.38µM and a ß value of 1.76. It is also of interest that a comparable derivative 23d showed a mixed type inhibition for linoleic acid conversion. These observations indicate the presence of an allosteric binding site in human 5-LOX distinct from the ATP binding site. The activatory and inhibitory behavior of 23a and 23d on the conversion of linoleic compared to arachidonic acid are rationalized by docking studies, which suggest that the activator 23a stabilizes linoleic acid binding, whereas the larger inhibitor 23d blocks the enzyme active site.

Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

Chagas' disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of the essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl- and 6-n-dodecylsalicilic acids, have IC(50) values of 28 and 55 microM, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor.

Design and evaluation of anacardic acid derivatives as anticavity agents.

On the basis of antibacterial anacardic acids, 6-pentadecenylsalicylic acids, isolated from the cashew apple, Anacardium occidentale L. (Anacardiaceae), a series of 6-alk(en)ylsalicylic acids were synthesized and tested for their antibacterial activity against Streptococcus mutans ATCC 25175. Among them, 6-(4',8'-dimethylnonyl)salicylic acid was found to exhibit the most potent antibacterial activity against this cariogenic bacterium with the minimum inhibition concentration (MIC) of 0.78 microg/ml.

Characterization of novel inhibitors of histone acetyltransferases.

Modification of proteins by histone acetyltransferases (HAT) or histone deacetylases plays an important role in the control of gene expression, and its dysregulation has been linked to malignant transformation and other diseases. Although histone deacetylase inhibitors have been extensively studied and several are currently in clinical trials, there is little information available on inhibitors of HATs (HATi). Starting from the natural product lead HATi anacardic acid, a series of 28 analogues was synthesized and investigated for HAT-inhibitory properties and effects on cancer cell growth. The compounds inhibited up to 95% HAT activity in vitro, and there was a clear correlation between their inhibitory potency and cytotoxicity toward a broad panel of cancer cells. Interestingly, all tested compounds were relatively nontoxic to nonmalignant human cell lines. Western blot analysis of MCF7 breast carcinoma cells treated with HATi showed significant reduction in acetylation levels of histone H4. To directly show effect of the new compounds on HAT activity in vivo, MCF7 cells were cotransfected with the p21 promoter fused to firefly luciferase and a full-length p300 acetyltransferase, and luciferase activity was determined following treatment with HATi. Significant inhibition of p300 activity was detected after treatment with all tested compounds except one. Effects of the new HATi on protein acetylation and HAT activity in vivo make them a suitable tool for discovery of molecular targets of HATs and, potentially, for development of new anticancer therapeutics.

Molecular design of anti-MRSA agents based on the anacardic acid scaffold.

A series of anacardic acid analogues possessing different side chains viz. phenolic, branched, and alicyclic were synthesized and their antibacterial activity tested against methicillin-resistant Staphylococcus aureus (MRSA). The maximum activity against this bacterium occurred with the branched side-chain analogue, 6-(4',8'-dimethylnonyl)salicylic acid, and the alicyclic side-chain analogue, 6-cyclododecylmethyl salicylic acid, with the minimum inhibitory concentration (MIC) of 0.39 microg/mL, respectively. This activity was superior to that of the most potent antibacterial anacardic acid isolated from the cashew Anacardium occidentale (Anacardiaceae), apple and nut, that is, the 6-[8'(Z),11'(Z),14'-pentadecatrienyl]salicylic acid.

Synthesis of isonicotinoylhydrazones from anacardic acid and their in vitro activity against Mycobacterium smegmatis.

Isonicotinoylhydrazones were synthesized from a natural product anacardic acid, a major constituent of cashew nut shell liquid. The unsaturated side chain in anacardic acid and its 5-nitro derivative were converted into C(8')-aldehydes by oxidative cleavage. C(8')-aldehydes are then coupled with isoniazid (an anti-TB drug) to obtain N-isonicotinoyl-N'-8-[(2'-carbohydroxy-3'-hydroxy) phenyl] octanal hydrazone (5) and N-isonicotinoyl-N'-8-[(2'-carbohydroxy-3'-hydroxy-6-nitro) phenyl] octanal hydrazone (6). These isonicotinoylhydrazones of anacardic aldehydes showed potent antimycobacterial activity against Mycobacterium smegmatis mc(2)155. The synergistic studies of 5 and 6 with isoniazid showed more inhibitory activities than isoniazid alone. Compounds 5 and 6 also showed activity against Mycobacterium tuberculosis H(37)Rv.