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1.  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.
PMCID: PMC4007306  PMID: 23711747
alternative substrates; enzyme kinetics; AdoMet synthetase; pathogenic bacteria; quorum sensing
2.  Largazole, a class I histone deacetylase inhibitor, enhances TNF-α to induce ICAM-1 and VCAM-1 expression in Rheumatoid Arthritis Synovial Fibroblasts 
In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1-5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (1-5 μM) inhibited the constitutive expression of HDAC1 (0-30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ~220% with a concomitant decrease in HDAC5 [30-58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α+LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA.
PMCID: PMC3766723  PMID: 23632129
3.  Largazole and Analogues with Modified Metal-Binding Motifs Targeting Histone Deacetylases: Synthesis and Biological Evaluation 
Journal of medicinal chemistry  2011;54(21):7453-7463.
The histone deacetylase inhibitor, largazole 1 was synthesized by a convergent approach which involved several efficient and high yielding single pot multistep protocols. Initial attempts using t-butyl as thiol protecting group proved problematic and synthesis was accomplished by switching to trityl protecting group. This synthetic protocol provides a convenient approach to many new largazole analogues. Three side chain analogues with multiple heteroatoms for chelation with Zn2+ were synthesized and their biological activities were evaluated. They were less potent than largazole 1 in growth inhibition of HCT116 colon carcinoma cell line and in inducing increases in global H3 acetylation. Largazole 1 and the three side chain analogues had no effect on HDAC6 as indicated by the lack of increased acetylation of α-tubulin.
PMCID: PMC3208063  PMID: 21936551

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