INTERACTION OF HYPERICIN WITH BIOLOGICALLY IMPORTANT MOLECULES:  HYPERICIN/GST COMPLEX

GSTs are of special significance because they are a family of enzymes that catalyze the conjugation of reduced glutathione (GSH) to a variety of electrophiles and thereby detoxify carcinogens, pesticides, and reactive products generated under oxidative stress. The conjugation of GSH with a foreign compound generally results in the formation of a nontoxic product that can be readily eliminated . In addition to their catalytic function, GSTs also serve as nonenzymatic binding proteins, known as ligandins, that interact with various lipophilic compounds that include steroid and thyroid hormones.  Finally, reactive oxygen species can trigger apoptosis, programmed cell death.  The production of GSTs, by helping to protect the cell from oxidative damage caused by reactive oxygen species, assists in obviating the induction of apoptosis.

    We are working on characterizing the photophysics of hypericin and two of its analogs, methoxy hypericin and gymnochrome B, in complex with GST A1-1 and GST P1-1 (Figures 1). The role of GST in this study is of extreme importance. Although the hydrophobic nature of hypericin causes it to associate indiscriminately with membranes, proteins, or DNA , GST is the first protein that we know of where there is both a tight binding with hypericin and where different isoforms of the protein influence the ability of hypericin to produce oxidative damage. (We have previously demonstrated tight binding to HSA, human serum albumin.)

    We also wish to determine the mechanism of action of hypericin and its analogs in complex with the GSTs. In particular, does binding to GST only influence the singlet oxygen yield of hypericin? How do the triplet and singlet yields of hypericin vary with binding to the two GSTs? Are photoinduced protons produced in the two GSTs? If so, from what excited-states do the protons originate? Is photoinduced acidification correlated with oxidative damage, and under what conditions?

    Glutathione S-transferases (GSTs) are a family of detoxification enzymes that are known to bind nonsubstrate hydrophobic anions such as hemes and porphyrins (Figure 1). It has recently been demonstrated that two forms of GST bind hypericin very tightly: A1-1 with K_D = 70 nM; P1-1 with K_D = 50 nM (Atkins and coworkers, unpublished results). Binding of hypericin to GSTs inhibits their catalytic activity. An intriguing observation is that the photophysical properties of hypericin can be altered upon binding to GSTs. In particular, when hypericin binds to A1-1, light-induced oxidative damage is produced, as evidenced by the mass spectrometry data obtained after 30 minutes of optical irradiation. On the other hand, no such oxidative damage is observed for P1-1.

Figure 1. Two forms of glutathione S-transferases (GSTs): left, A1-1; right, P1-1. These GSTs exhibit different behavior when hypericin is bound to them in the presence of light.

    Hypericin  is a widely spread, naturally occurring perylene quinone pigment that is of great interest because of its broad spectrum of light-induced biological activities . Hypericin-like molecules have been identified in both the plant and the animal kingdoms . Hypericins are most commonly found in the more than three hundred species of the genus Hypericum of the plant family Guttiferae, where they are believed to function as ingestion deterrents against plants and animals. Hypericin-like molecules are responsible for the photosensory signals in the protozoa Stentor coeruleus and Blepharisma, where the photoresponse is believed to be a result of proton ejection or possibly an electron transfer reaction. Hypericin displays virucidal activity against several types of viruses, including the human immunodeficiency virus (HIV) , as well as antiproliferative and cytotoxic effects on tumor cells . Hypericin is also a potent antidepressant , exhibits light-dependent inhibition of protein kinase C (PKC), which has been suggested to be a locus of its antiviral activity , and is reported to possess numerous other types of biological behavior . Hypericin, like other anticancer drugs, also induces apoptosis .