Amarjit Luniwal

Bilirubin Binding to PPARα Inhibits Lipid Accumulation

Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin.

Alternative substrates selective for S-adenosylmethionine synthetases from pathogenic bacteria

S-adenosyl-l-methionine (AdoMet) synthetase catalyzes the production of AdoMet, the major biological methyl donor and source of methylene, amino, ribosyl, and aminopropyl groups in the metabolism of all known organism. In addition to these essential functions, AdoMet can also serve as the precursor for two different families of quorum sensing molecules that trigger virulence in Gram-negative human pathogenic bacteria. The enzyme responsible for AdoMet biosynthesis has been cloned, expressed and purified from several of these infectious bacteria. AdoMet synthetase (MAT) from Neisseria meningitidis shows similar kinetic parameters to the previously characterized Escherichia coli enzyme, while the Pseudomonas aeruginosa enzyme has a decreased catalytic efficiency for its MgATP substrate. In contrast, the more distantly related MAT from Campylobacter jejuni has an altered quaternary structure and possesses a higher catalytic turnover than the more closely related family members. Methionine analogs have been examined to delineate the substrate specificity of these enzyme forms, and several alternative substrates have been identified with the potential to block quorum sensing while still serving as precursors for essential methyl donation and radical generation reactions.

Total syntheses of racemic and natural glycinol.

Total syntheses of racemic and (-)-glycinol (1) are described. A Wittig reaction produced the isoflav-3-ene from which a Sharpless dihydroxylation introduced either the racemic or enantiomeric 6a-hydroxy group. A 5.5% overall yield of racemic material was obtained after 12 steps. A method was devised for a one-pot switch of protecting groups masking a sensitive resorcinolic para-functionality, and conditions were optimized to prompt spontaneous closure of the pterocarpanolic dihydrofuran upon subsequent exposure of its ortho-functionality. These improvements eliminated two steps and increased the overall yield to 9.8% during production of the natural enantiomer.

Molecular docking and enzymatic evaluation to identify selective inhibitors of aspartate semialdehyde dehydrogenase

Microbes that have gained resistance against antibiotics pose a major emerging threat to human health. New targets must be identified that will guide the development of new classes of antibiotics. The selective inhibition of key microbial enzymes that are responsible for the biosynthesis of essential metabolites can be an effective way to counter this growing threat. Aspartate semialdehyde dehydrogenases (ASADHs) produce an early branch point metabolite in a microbial biosynthetic pathway for essential amino acids and for quorum sensing molecules. In this study, molecular modeling and docking studies were performed to achieve two key objectives that are important for the identification of new selective inhibitors of ASADH. First, virtual screening of a small library of compounds was used to identify new core structures that could serve as potential inhibitors of the ASADHs. Compounds have been identified from diverse chemical classes that are predicted to bind to ASADH with high affinity. Next, molecular docking studies were used to prioritize analogs within each class for synthesis and testing against representative bacterial forms of ASADH from Streptococcus pneumoniae and Vibrio cholerae. These studies have led to new micromolar inhibitors of ASADH, demonstrating the utility of this molecular modeling and docking approach for the identification of new classes of potential enzyme inhibitors.

Early Stage Efficacy and Toxicology Screening for Antibiotics and Enzyme Inhibitors

The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Toward this end, we have established a microplate-based, dual-assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage development. The bacterial assay uses nonpathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with selected agents from the ASADH inhibitor fragment library show some compounds with antibiotic activity, but as expected, most of these early agents display higher than desired mammalian cell toxicity.

Practical synthesis of a chromene analog for use as a retinoic acid receptor alpha antagonist lead compound.

Retinoic acid receptor alpha (RARα) selective compounds may guide the design of drugs that can be used in conjunction with hormonal adjuvant therapy in the treatment of breast cancer. Herein we report a modified synthesis of a known RARα antagonist, 2-fluoro-4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid and a synthesis of its unknown, desfluoro analog, 4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid. The modified route allows for facile reaction workups, increased yields, lower cost and incorporates a green alternative step. Structure-activity relationship studies determined through functional cell-based assays, demonstrated antagonism to RARα for both compounds. Molecular modeling within the RARα binding pocket was used to compare binding interactions of the desfluoro analog to a known RAR antagonist.

Syntheses of 2,3-Diarylated 2H-Benzo[e][1,2]Thiazine 1,1-Dioxides and their 3,4-Dihydro Derivatives, and Assessment of their Inhibitory Activity Against MCF-7 Breast Cancer Cells

A practical synthesis of 2,3-diarylated 2H-benzo[e][1,2]thiazine 1,1-dioxides and their 3,4-dihydro derivatives was developed. ortho-Methyl lithiation of N-aryl-o-toluenesulfonamide followed by reaction with aryl aldehydes gave carbinol sulfonamides, which were either converted directly, or first oxidized to their ketones and converted, to 2,3-diarylated six-membered benzosultams via a TMSCl-NaI-MeCN mediated cyclization. A library of benzosultams was synthesized and evaluated for inhibitory activity against MCF-7 cells. Compound 3 in the 3,4-dihydro (saturated) series and compound 8 in the unsaturated series exhibited the highest potencies with growth inhibition (GI50) values of 0.8 and 18.0 μM, respectively. Molecular modeling studies suggest that these compounds can associate with the colchicine binding site on microtubules. However, experimental assessments of that and other mechanistic possibilities are still ongoing.