Antifungal activities of antineoplastic agents: Saccharomyces cerevisiae as a model system to study drug action

Abstract

Recent evolutionary studies reveal that microorganisms including yeasts and fungi are more closely related to mammals than was previously appreciated. Possibly as a consequence, many natural-product toxins that have antimicrobial activity are also toxic to mammalian cells. While this makes it difficult to discover antifungal agents without toxic side effects, it also has enabled detailed studies of drug action in simple genetic model systems. We review here studies on the antifungal actions of antineoplasmic agents. Topics covered include the mechanisms of action of inhibitors of topoisomerases I and II; the immunosuppressants rapamycin, cyclosporin A, and FK506; the phosphatidylinositol 3-kinase inhibitor wortmannin; the angiogenesis inhibitors fumagillin and ovalicin; the HSP90 inhibitor geldanamycin; and agents that inhibit sphingolipid metabolism. In general, these natural products inhibit target proteins conserved from microorganisms to humans. These studies highlight the potential of microorganisms as screening tools to elucidate the mechanisms of action of novel pharmacological agents with unique effects against specific mammalian cell types, including neoplastic cells. In addition, this analysis suggests that antineoplastic agents and derivatives might find novel indications in the treatment of fungal infections, for which few agents are presently available, toxicity remains a serious concern, and drug resistance is emerging.

FIG. 1

Model of the human topoisomerase I-camptothecin interaction. A binding model of the three-dimensional structure of camptothecin (shown in green and labelled CPT) bound to the DNA-topoisomerase I complex is shown. Arg-364 and Asp-533 on the enzyme are hydrogen bonded to functional groups on the camptothecin E ring. A prominent feature of the model is that to accommodate camptothecin, the guanosine in the +1 position on the scissile DNA strand (labelled +1 Gua) is proposed to be flipped out of the DNA double helix. This could provide additional interactions, allowing the guanine ring to stack above the five-member ring system of the camptothecin molecule. This model will probably prove valuable in understanding the activity of camptothecin analogs and in developing novel analogs that specifically target human or fungal topoisomerase I enzymes. This figure is based on the X-ray structure of the DNA-topoisomerase I complex solved by Redinbo et al. (279) and was kindly provided by Matthew Redinbo and Wim Hol. It is important to note that this represents a binding model for the enzyme-drug-DNA complex and is not an experimentally determined binding mode for camptothecin association with the enzyme-DNA complex.

FIG. 2

Structures of rapamycin, wortmannin, CsA, and FK506. The molecular structures of several natural products that inhibit signal transduction cascades are depicted. CsA is a cyclic peptide, rapamycin and FK506 are structurally related macrolide antibiotics, and wortmannin is related to the steroids.

FIG. 3

Structures of the FKBP12-rapamycin-mTOR complex. (A) Structure of the ternary complex between the FKBP12-rapamycin binding domain on the FRAP/mTOR molecule (blue), rapamycin (green), and FKBP12 (red). (B) Detailed view of the FRB domain of FRAP/mTOR (blue) bound to rapamycin (green). Rapamycin binds into a hydrophobic pocket on the surface of TOR composed of the highly conserved residues Phe2108, Tyr2105, Trp2101, Phe2039, and Tyr2038 (side chains all in gold). These residues are conserved from yeast to humans and provide critical interactions with the inhibitor. This figure is based on the X-ray crystal structure of the FRB-rapamycin-FKBP12 complex solved by Choi et al. (54) and was kindly provided by Jon Clardy.

FIG. 3

Structures of the FKBP12-rapamycin-mTOR complex. (A) Structure of the ternary complex between the FKBP12-rapamycin binding domain on the FRAP/mTOR molecule (blue), rapamycin (green), and FKBP12 (red). (B) Detailed view of the FRB domain of FRAP/mTOR (blue) bound to rapamycin (green). Rapamycin binds into a hydrophobic pocket on the surface of TOR composed of the highly conserved residues Phe2108, Tyr2105, Trp2101, Phe2039, and Tyr2038 (side chains all in gold). These residues are conserved from yeast to humans and provide critical interactions with the inhibitor. This figure is based on the X-ray crystal structure of the FRB-rapamycin-FKBP12 complex solved by Choi et al. (54) and was kindly provided by Jon Clardy.

FIG. 4

Rapamycin mechanism of action. Rapamycin inhibits a conserved signaling cascade that drives cell proliferation in response to interleukin-2 (IL-2) and other cytokines. The target of rapamycin kinase (TOR) functions to regulate the activities of the translational regulators PHAS-I and p70 S6 kinase. Signaling via the Tor cascade provides a proliferative signal that can in part prevent apoptosis. As a consequence, rapamycin promotes apoptosis under certain conditions.

FIG. 5

Structure of the FKBP12-FK506-calcineurin complex. The ternary complex between CnAB, FK506, and FKBP12 is depicted. Calcineurin is a heterodimer, composed of the CnA catalytic subunit (blue) and the CnB regulatory subunit (green). A molecule of phosphate bound in the calcineurin active site is shown in yellow, and the N-terminal myristoyl group on CnB is shown in purple. FKBP12 (red) bound to FK506 (grey) binds in a hydrophobic groove composed of an extended α-helical arm of CnA and regions of CnB. Note that the FKBP12-FK506 inhibitor complex does not bind in the active site of calcineurin but probably inhibits by occluding the docking of large protein substrates to the phosphatase. Modified from reference with permission of the publisher.

FIG. 6

Structures of angiogenesis inhibitors. The structures of the natural-product angiogenesis inhibitors fumagillin and ovalicin and of the derivative TNP-470, currently in clinical trials, are depicted.

FIG. 7

Structure of the fumagillin-MetAP2 complex. (A) Schematic view of fumagillin bound to the active site of MetAP2. (B) Structure of the human MetAP2 enzyme (blue and red) and the bound molecule of fumagillin (gold). Notably, the active-site residue His231 is covalently linked to a reactive ring epoxide on fumagillin. This figure is based on the structure of the aminopeptidase-fumagillin complex solved by Liu et al. (200) and was generously provided by Maria C. Nonato and Jon Clardy.

FIG. 7

Structure of the fumagillin-MetAP2 complex. (A) Schematic view of fumagillin bound to the active site of MetAP2. (B) Structure of the human MetAP2 enzyme (blue and red) and the bound molecule of fumagillin (gold). Notably, the active-site residue His231 is covalently linked to a reactive ring epoxide on fumagillin. This figure is based on the structure of the aminopeptidase-fumagillin complex solved by Liu et al. (200) and was generously provided by Maria C. Nonato and Jon Clardy.

FIG. 8

The HSP90 chaperone complex is targeted by geldanamycin. (A) Schematic view of geldanamycin bound in the ATPase active site of HSP90. (B) Stereoview of the geldanamycin-HSP90 active-site complex, with HSP90 shown in blue, side chains shown in purple, and geldanamycin shown in white and yellow. Interactions between geldanamycin and HSP90 are indicated by green dotted lines. (C) Space-filling model of the complex between geldanamycin, which snugly docks into the ATPase binding site on the surface of HSP90 (blue). Modified from reference with permission of the publisher and kindly provided by Nikola Pavletich.

FIG. 8

The HSP90 chaperone complex is targeted by geldanamycin. (A) Schematic view of geldanamycin bound in the ATPase active site of HSP90. (B) Stereoview of the geldanamycin-HSP90 active-site complex, with HSP90 shown in blue, side chains shown in purple, and geldanamycin shown in white and yellow. Interactions between geldanamycin and HSP90 are indicated by green dotted lines. (C) Space-filling model of the complex between geldanamycin, which snugly docks into the ATPase binding site on the surface of HSP90 (blue). Modified from reference with permission of the publisher and kindly provided by Nikola Pavletich.

FIG. 9

Sphingolipid metabolic cascades are conserved between yeast and humans. Differences in the de novo synthesis of sphingolipids between human and fungi are shown. Inhibitors of specific enzymes are shown in italics.

FIG. 10

Inhibitors of sphingolipid biosynthesis. The structures of three inhibitors of IPC synthase, aureobasidin A (a cyclic peptide), khafrefungin, and rustmicin, are shown.

FIG. 11

Schematic view of the yeast three-hybrid assay for drug target discovery. The two-hybrid system for detecting protein-protein interactions was modified to develop a genetic method to identify drug targets. The GAL4 DNA binding domain was fused to a steroid receptor ligand binding domain. The GAL4 activation domain was fused to a library. The third hybrid is a conjugate drug in which dexamethosone (A) is conjugated to a second ligand (B). This hybrid drug is added to cells, and reporter gene expression is monitored to identify clones from the library that encode proteins that interact with the ligand of interest.