tacrolimus and geldanamycin

Neurodegenerative diseases, whether acute or chronic, are a tremendous medical problem in the modern world. Therapies are rare and only applied after a vast amount of neurons are lost. Many efforts have been made to develop new strategies to treat these disorders, but so far, there has been no breakthrough. A characteristic shared by some experimental neuroprotective substances is the induction of the heat shock response, in particular the expression of the heat shock proteins Hsp70 and Hsp27. These Hsps protect cells from cell death induced by various noxious stimuli and inhibit various cellular death pathways. Gene therapy, transgenic mice and drugs inducing Hsps in the brain decrease the infarction area after ischemia and protect neurons and nonneuronal cells of the brain. Furthermore, recent data hint toward a protective role of Hsps in chronic neurological diseases. The induction of Hsps as a possible treatment for stroke, Alzheimer's disease and Huntington's disease is discussed.

Topics: Alzheimer Disease; Animals; Apoptosis; Benzoquinones; Heat-Shock Proteins; Humans; Huntington Disease; Lactams, Macrocyclic; Neuroprotective Agents; Quinones; Stroke; Tacrolimus

In summary, FKBP-12 does not mediate the neurite outgrowth-promoting properties of neuroimmunophilin ligands (e.g., FK506). Instead, the neurotrophic properties of neuroimmunophilin ligands (FK506) and steroid hormones are mediated by disruption of steroid-receptor complexes. It remains unclear which component mediates neurite outgrowth, although the most likely candidates are FKBP-52, hsp-90, and p23 [42]. Regardless of the underlying mechanism involved, the FKBP-52 antibody data reveal that it should be possible to design, based on the structure of FK506, non-FKBP-12-binding (nonimmunosuppressant) compounds selective for FKBP-52 and test these new libraries for their ability to augment nerve regeneration. It may also be possible to exploit the structure of geldanamycin to develop a new class of hsp-90-binding compounds for use in nerve regeneration.

Topics: Animals; Antibodies; Benzoquinones; Humans; Immunophilins; Immunosuppressive Agents; Lactams, Macrocyclic; Mice; Mice, Knockout; Molybdenum; Nerve Regeneration; Neurites; Quinones; Steroids; Tacrolimus; Tacrolimus Binding Proteins

Many bacteria and fungi produce natural products that are toxic to other microorganisms and have a variety of physiological effects in animals. Recent studies have revealed that, in several cases, the targets of these agents are components of conserved signal-transduction cascades. This article looks at the mechanisms of action of five natural products--the immunosuppressants cyclosporin A, FK506 and rapamycin, and the antiproliferative agents wortmannin and geldanamycin. These mechanisms reveal the importance of signal-transduction cascades as targets for therapeutic intervention and the enormous utility of studies of natural-product action in simple model genetic systems.

Topics: Androstadienes; Animals; Apoptosis; Benzoquinones; Calcineurin; Cell Cycle; Cyclosporine; Enzyme Inhibitors; Fungi; HSP90 Heat-Shock Proteins; Humans; Immunosuppressive Agents; Lactams, Macrocyclic; Protein Biosynthesis; Quinones; Signal Transduction; Sirolimus; Tacrolimus; Wortmannin

The multicomponent heat-shock protein (hsp) 90-based chaperone system is an ubiquitous protein-folding system in the cytoplasm of eukaryotes. Several signal transduction systems utilize an interaction with hsp90 as an essential component of the signaling pathway. The steroid and dioxin receptors are bound to hsp90 through their hormone-binding domains, and several of them must be bound to hsp90 in order to have a ligand-binding site. The binding of ligands to these receptors promotes their dissociation from hsp90, an event that is the first step in their signaling pathways. Several protein kinases, including the Src and Raf components of the MAP kinase system, are also bound to hsp90. Genetic studies in yeast have demonstrated that hsp90 is required for normal signaling via steroid and dioxin receptors and for the activity of Src in vivo. The hsp90-based chaperone system has been reconstituted from purified components, permitting detailed analysis of the molecular basis of the chaperone's role in signal transduction.

Topics: Benzoquinones; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Molecular Chaperones; Protein Binding; Quinones; Receptors, Aryl Hydrocarbon; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Signal Transduction; Tacrolimus

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.

Topics: Aneuploidy; Antifungal Agents; Azoles; Benzoquinones; Calcineurin; Calcineurin Inhibitors; Candida albicans; DNA-Binding Proteins; Drug Combinations; Drug Resistance, Fungal; Ergosterol; Evolution, Molecular; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tacrolimus; Transcription Factors

Due to the limited number of antifungals and the emergence of resistance, new therapies against invasive aspergillosis are needed. We show that calcineurin inhibitors are active in vitro against both azole- and echinocandin-resistant Aspergillus fumigatus strains. The heat shock protein 90 (Hsp90) inhibitor geldanamycin had modest activity when used alone, but its combination with caspofungin or tacrolimus (FK506) resulted in fungicidal activity against azole-resistant strains. Targeting the Hsp90-calcineurin axis is a promising alternative strategy against azole-resistant A. fumigatus strains.

Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Azoles; Benzoquinones; Calcineurin Inhibitors; Caspofungin; Drug Resistance, Multiple, Fungal; Drug Therapy, Combination; Echinocandins; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Lipopeptides; Microbial Sensitivity Tests; Tacrolimus

Heat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance of Candida albicans to azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogen Aspergillus fumigatus. We investigated the role of Hsp90 in A. fumigatus by genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain of hsp90 suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factors brlA, wetA, and abaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.

Topics: Aspergillus fumigatus; Benzoquinones; beta-Glucans; Caspofungin; Cell Wall; Cytosol; Echinocandins; Fungal Proteins; Gene Deletion; Gene Expression Regulation, Fungal; Green Fluorescent Proteins; HSP90 Heat-Shock Proteins; Hyphae; Lactams, Macrocyclic; Lipopeptides; Microbial Viability; Promoter Regions, Genetic; Spores, Fungal; Stress, Physiological; Tacrolimus; Transcription Factors; Transcription, Genetic

FK-506 accelerates nerve regeneration and improves functional recovery in vivo; its immunosuppressive properties, however, limit its clinical utility. Geldanamycin (GA), a non-immunosuppressive agent, shares a common binding target (heat shock protein 90) with FK-506 and may accelerate nerve regeneration through a similar mechanism. GA has been shown to augment neurite outgrowth in vitro but has not been tested in vivo. The current study investigated the effect of GA on the rate of axonal regeneration and functional recovery following peripheral nerve injury. In the first experiment, Thy1-GFP transgenic rats underwent serial transmuscular imaging to quantify the rate of axonal regeneration following saphenous nerve crush injury. In subsequent experiments, Lewis rats underwent tibial nerve crush or transection-and-repair injuries and were assessed for functional recovery by walking track analysis. All animals were randomized to receive daily administration of FK-506 (2mg/kg), GA (0.2mg/kg), or a control vehicle (dimethyl sulfoxide, 1 mL/kg) starting 3 days prior to injury. Both GA and FK-506 significantly increased the rate of axonal regeneration following crush injury in Thy1-GFP rats. In Lewis rats undergoing tibial nerve crush injury, earlier functional recovery occurred at day 5 and day 6 in animals treated with FK-506 and GA respectively, vs. day 13 for controls. Over a truncated 21-day timeframe, Lewis rats undergoing tibial nerve transection-and-repair injury and treated with FK-506 regained function at day 16, whereas those treated with GA or the control vehicle did not regain normal function. GA-treated animals, however, did exhibit significant functional improvement vs. controls. The current study demonstrated that GA accelerates axonal regeneration and enhances functional recovery in vivo. Its ability to increase the rate at which peripheral axons regenerate is comparable to that of FK-506. GA, however, did not match the performance of FK-506 in injury models where Wallerian degeneration (WD) is ongoing in the distal stump. This provides evidence that FK-506 accelerates axonal regeneration through two parallel mechanisms: the first being its well-established effect on neurons; the second is likely a newly described, as-yet poorly defined mechanism that affects WD. Finally, given the decrease in observed toxicity with GA administration, it might be a suitable non-immunosuppressive alternative to FK-506 for accelerating peripheral nerve regenerati

Topics: Analysis of Variance; Animals; Benzoquinones; Cysteine Proteinase Inhibitors; Drug Synergism; Green Fluorescent Proteins; Immunosuppressive Agents; Lactams, Macrocyclic; Locomotion; Muscle Strength; Nerve Regeneration; Peripheral Nerve Injuries; Rats; Rats, Transgenic; Recovery of Function; Tacrolimus; Time Factors

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; HSP90 Heat-Shock Proteins; Hybrid Cells; Lactams, Macrocyclic; Mice; Neurites; Neurons; Protein Binding; Rats; Tacrolimus; Tacrolimus Binding Proteins

Topics: Antifungal Agents; Aspergillus; Benzoquinones; Biological Evolution; Calcineurin; Calcineurin Inhibitors; Candida albicans; Cyclosporine; Drug Resistance, Fungal; Drug Therapy, Combination; Ergosterol; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mutation; Mycoses; Phenotype; Quinones; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tacrolimus

Programmed cell death 4 (Pdcd4), originally identified as an inhibitor of murine cellular transformation, inhibits protein synthesis by directly interacting with eukaryotic initiation factor 4A (eIF4A) of the translation initiation complex. The relevance of Pdcd4 to a broad range of human cancers derived from multiple tissue sites is unknown. Protein expression patterns from the National Cancer Institute drug-screening panel of 60 human cancer cells (NCI60) were analyzed by Western blot methods and revealed frequent reduction of Pdcd4 protein levels in renal-, lung-, and glia-derived tumors. Greater than mean Pdcd4 protein levels correlated with the antitumor activity of geldanamycin and tamoxifen. Stable expression of antisense PDCD4 significantly reduced the sensitivity of MCF-7 breast cancer cells to geldanamycin and to tamoxifen. Sensitivity to geldanamycin significantly increased in UO-31 renal cancer cells expressing sense PDCD4 cDNA. Increased geldanamycin sensitivity was accompanied by enhanced cell cycle arrest and apoptosis. One primary mode of inactivation of Pdcd4 in human cancers appears to involve down-regulated expression, and this down-regulation causes a decreased sensitivity to geldanamycin cytotoxicity. Thus, up-regulating Pdcd4 expression may be promising for geldanamycin-based combination therapy.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Benzoquinones; Cell Division; Cell Line, Tumor; Drug Screening Assays, Antitumor; G2 Phase; Humans; Kidney Neoplasms; Lactams, Macrocyclic; Mitosis; Neoplasms; Prognosis; Quinones; Retinoblastoma Protein; RNA-Binding Proteins; RNA, Messenger; Tacrolimus; Tamoxifen

The neurotrophic property of the immunosuppressant drug FK506 (tacrolimus) is believed to depend on the 12-kDa FK506-binding protein (FKBP-12). Here, we show that FK506 maintains its neurotrophic activity in primary hippocampal cell cultures from FKBP-12 knockout mice. In human neuroblastoma SH-SY5Y cells, the neurotrophic action of FK506 (10 pM to 10 nM) is completely prevented by the addition of a monoclonal antibody (50-100 nM) to the immunophilin FKBP-52 (also known as FKBP-59 or heat shock protein 56), a component of mature steroid receptor complexes. By itself, the FKBP-52 antibody is also neurotrophic. The neurotrophic activity of dexamethasone (50 nM) is potentiated by FK506, whereas that of beta-estradiol (50 nM) is not altered, suggesting a common mechanisms of action. Geldanamycin (which disrupts mature steroid receptor complexes) is also neurotrophic (0.1-10 nM), whereas it reduces the neurotrophic activity of FK506 and steroid hormones (dexamethasone and beta-estradiol). Conversely, 20 mM molybdate (which prevents the disruption of mature steroid receptor complexes) decreases the neurotrophic activity of FK506, FKBP-52 antibody, dexamethasone, and beta-estradiol. In rats, FK506 (10 mg/kg s.c.) augments the regenerative response of regenerating motor and sensory neurons to nerve injury as shown by its ability to increase the axotomy-induced induction of c-jun expression. A model is proposed to account for the neurotrophic action of both neuroimmunophilin ligands (FK506) and steroid hormones. Components of steroid receptor complexes represent novel targets for the rational design of new neurotrophic drugs.

Topics: Animals; Antibodies; Benzoquinones; Cells, Cultured; Cysteine Proteinase Inhibitors; Dexamethasone; Embryo, Mammalian; Estradiol; Hippocampus; Humans; Immunophilins; Lactams, Macrocyclic; Mice; Mice, Knockout; Molybdenum; Motor Neurons; Nerve Growth Factors; Nerve Regeneration; Neurites; Neuroblastoma; Neurons; Neurons, Afferent; Neuroprotective Agents; Proto-Oncogene Proteins c-jun; Quinones; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Binding Sites; DNA-Binding Proteins; HSP90 Heat-Shock Proteins; Humans; Immunophilins; Immunosorbent Techniques; Lactams, Macrocyclic; Macromolecular Substances; Molecular Weight; Plant Proteins; Protein Binding; Quinones; Recombinant Proteins; Tacrolimus; Tacrolimus Binding Proteins; Triticum