| Part I. Myo-inositol (Ins) and myo-inositol phosphates (InsPs) are widely distributed in plants and animals. The evaluation of Ins and InsPs distribution in cells and plant sources can impact the understanding of their role in nutrition, cellular processes and diseases, and how they may be modulated by diet. We developed an anion-exchange chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the separation and simultaneous quantitation of Ins and different naturally occurring phosphorylated inositol compounds. Chromatographic separation was achieved in 30 min on a commercial anion exchange column (150 x 0.5 mm) using a gradient of 200 mM ammonium carbonate buffer (pH 9.0) and 5% methanol in H2O. Analytes were identified by selective reaction monitoring using a triple quadrupole mass spectrometer in negative ion electrospray mode. Adenosine 5'-monophosphate was used as a general internal standard for quantitation. Using this approach, Ins and InsPs were measured in three different plant samples and in cultured cell, illustrating significant differences in the distribution of inositol compounds in food sample compared to cells and between cell types. Ins and InsPs are cellular second messenger with potential roles in cancer prevention and therapy. It typically is difficult to attribute specific pharmacological activity to a single inositol phosphate because they are rapidly metabolized by phosphatases and kinases. We designed stable analogs of Ins(4,5)P2 and Ins(1,4,5)P3 that maintain the biological activity, but are phosphatase and kinase-resistant LC/MS/MS analysis of the enzyme-catalyzed reaction products indicates the phosphorothioate analog of Ins(1,4,5)P3 is stable towards alkaline phosphatase, and under the same condition, the natural ligand was extensively hydrolyzed. Analogs developed in this study are potential chemical probes for understanding mechanisms of inositol phosphate actions that may be elucidated by eliciting specific and prolonged activation of the Ins(1,4,5)P3 receptor.;Part II. Acylfulvenes (AFs) are a class of antitumor agents with favorable cytotoxic selectivity profiles compared to their natural product precursor, illudin S. Illudin S readily reacts with thiol-containing small molecules such as cysteine, glutathione, and cysteine-containing peptides in slightly acidic pH (pH 6) in aqueous solution; reduced cellular glutathione levels can affect illudin S toxicity. However, AFs are less reactive towards these small thiols under the same conditions. By analogy, these compounds were proposed to have similar reactivity towards thiol-containing proteins, which may be responsible for AFs improved anticancer selectivity. The glutathione system (GSH and glutathione reductase (GR)) and thioredoxin system (thioredoxin reductase (TrxR) and thioredoxin (Trx)) are the major cellular redox-regulating systems that maintain cellular redox homeostasis, offering protection against oxidative stress. Disrupting this system will affect cell viability and lead to apoptosis. Furthermore, overexpression of thioredoxin and thioredoxin reductase in certain tumors is associated with higher proliferation capacities and lower apoptosis rates. The enzymes involved in these systems all have critical cysteine residues at the active site. The reactivities of illudin S and AFs toward these thiol-containing proteins were evaluated.;The inhibition potency of illudin S and AFs is in the same order for GR, TrxR, and Trx, which is opposite what was expected based on their reactivity with small thiols, i.e., HMAF > AF > illudin S, however, both AFs irreversibly inhibit TrxR and Trx by targeting the active site cysteines. For GR, HMAF is an irreversible inhibitor, while AF is a reversible inhibitor. GR and TrxR share great structural and activity similarity, but TrxR has a selenocysteine (Sec) at the active site, which makes TrxR more reactive toward electrophiles. The IC50s for TrxR were in low micromolar range, while that for GR were hundred-fold higher. However, the presence of Sec is not the only reason in dictating the difference reactivity towards these two enzymes, since no covalent interaction occurs between AFs and glutathione peroxidase (Gpx), another redox-regulating enzyme containing Sec at active site. Furthermore, the inhibition of cellular GR and TrxR activity and reduction of Trx cellular protein level upon AFs treament suggest that they are AFs' cellular targets. Quenching of intrinsic fluorescence of GR and Trx suggests extensive conformational changes of by illudin S and AFs, which may facilitate the interaction between AFs and enzymes, but not the same case for illudin S. The differential reactivity of illudin S anda AFs towards these thiol-containing enzymes suggests that compared to small thiol-containing molecules, accessibility and reactivity of the enzyme active sites account for the inhibitory effects of AFs. The disruption of cellular redox systems by AFs may contribute to their improved anticancer selectivity compared to illudin S. The data obtained in this study are valuable in further understanding the role of modulating cellular redox enzymes in the anticancer effects of alkylating agents. |
