FK506 binding proteins as targets in anticancer therapy

FK506 binding proteins (FKBPs) are the intracellular ligands of FK506 and rapamycin, two natural compounds with powerful and clinically efficient immunosuppressive activity. In recent decades, a relevant role for immunosuppressants as anticancer agents has emerged. Especially, rapamycin and its derivatives are used, with successful results, across a variety of tumors. Of note, rapamycin and FK506 bind to FKBP12, and the resulting complexes interfere with distinct intracellular signaling pathways driven, respectively, by the mammalian target of rapamycin and calcineurin phosphatase. These pathways are related to T-cell activation and growth. Hyperactivation of the mammalian target of rapamycin (mTOR), particularly in cancers that have lost the tumor suppressor gene PTEN, plays an important pathogenetic role in tumor transformation and growth. The signaling pathway involving calcineurin and nuclear factors of activated T-lymphocytes is also involved in the pathogenesis of different cancer types and in tumor metastasis, providing a rationale for use of FK506 in anticancer therapy. Recent studies have focused on FKBPs in apoptosis regulation: Targeting of FKBP12 promotes apoptosis in chronic lymphocytic leukemia, FKBP38 knockdown sensitizes hepatoma cells to apoptosis, and FKBP51 silencing overcomes resistance to apoptosis in acute lymphoblastic leukemia, prostate cancer, melanoma, and glioma. Interestingly, derivatives of FK506 that have the same FKBP12-binding properties as FK506 but lack functional immunosuppressant activity, exert the same apoptotic effect as FK506 in chronic lymphocytic leukemia.These findings suggest that a direct FKBP inhibition represents a further mechanism of immunosuppressants.' anticancer activity. In this review, we focus on the role of FKBP members in apoptosis control and summarize the data on the antitumor effect of selective targeting of FKBP.     

The immunophilins

Summary The structurally unrelated drugs cyclosporin A (CsA) and FK-506 are powerful immunosuppressants, used prophylactically following transplant surgery to prevent organ rejection. Although neither drug was originally identified by a mechanism-based screen, these extraordinarily effective immunosuppressants have become important tools for understanding T-lymphocyte activation. Both drugs bind to receptors that have been given the generic name immunophilins to describe immunosuppressant binding proteins. The immunophilins are divided into two distinct families, based upon their binding specificity for drug and upon their amino acid sequence similarity. The CsA binding proteins are termed cyclophilins (CyPs) and the FK-506 binding proteins have been given the acronym FKBP. The FKBPs also bind rapamycin, an immunosuppressant structurally related to FK-506 [1], but which has a biochemical mechanism of action distinct from that of CsA and FK-506. CsA and FK-506 are inactive alone and are more aptly described as co-drugs that must first bind to their cognate immunophilin to form the active immunosuppressive species. Surprisingly, since the structures of the drugs are different and the two immunophilin families are unrelated phylogenetically, both drug-immunophilin complexes converge upon protein phosphatase 2B, calcineurin (CaN), a key regulatory component of the T-cell activation pathway. Immunosuppression results from inhibition of CaN phosphatase activity. Both classes of immunophilins are themselves enzymes which catalyze the cis-to-trans isomerization of Xaa-Pro amide bonds (peptidyl-prolyl isomerase or PPI) in polypeptides and proteins. The enzymology of the proline isomerization reaction has been studied intensively, yet the precise mechanism remains to be firmly established. Although binding of CsA or FK-506 to their respective immunophilins inhibits the PPI activity, inhibition of PPI activity is irrelevant to the mechanism of immunosuppression because PPI activity is not required for T-lymphocyte activation. Within each family, the different cyclophilin and FKBP members bind to their cognate drug with varying degrees of affinity. Furthermore, not all members of each family are relevant to the immunosuppressive action of CsA or FK-506. This is because not all CyP-CsA complexes, nor all FKBP-FK-506 complexes, are equally capable of binding to CaN. Despite our understanding of how the immunophilins function during immunosuppressive therapy, it is unlikely that these proteins evolved to bind immunosuppressive molecules derived from microorganisms. Recent studies concerning the endogenous functions of the immunophilins have indicated that they bind to other proteins and that they have roles in protein folding, translocation and regulation [2].     

Everolimus and sirolimus antagonize tacrolimus based calcineurin inhibition via competition for FK-binding protein 12

The calcineurin inhibitors cyclosporin A and tacrolimus and the inhibitors of the mTOR, sirolimus and everolimus bind immunophilins that are required for their immunosuppressive action. In contrast to cyclosporin A, tacrolimus and the mTOR inhibitors (MTIs) share common immunophilins, the FK506-binding proteins (FKBPs). We investigated the immunosuppressive interactions of MTIs on tacrolimus based immune suppression, since insights in immunological drug-drug interactions can be very relevant for optimization of immunosuppressive regimens in allograft transplantation medicine.Isolated peripheral blood mononuclear cells from healthy volunteers were incubated with combinations of MTIs and calcineurin inhibitors and when monitored for calcineurin activity and IL-2 excretion after mitogen stimulation, tacrolimus IC₅₀ concentrations shifted to higher concentrations in the presence of MTIs. This antagonism was absent for cyclosporin A, reproducible for 10 healthy volunteers (p < 0.001) and stronger for sirolimus than for everolimus. When cell lysate was treated with and without MTI, tacrolimus and FKBP12, FKBP12 could increase calcineurin inhibition by tacrolimus and reverse the MTI antagonism for both MTIs. These results demonstrate that FKBP12 can be rate limiting for calcineurin inhibition at high tacrolimus concentrations and that the antagonism of sirolimus and everolimus on tacrolimus based immune suppression is mediated via saturation of FKBP12.     

FK506 induces chondrogenic differentiation of clonal mouse embryonic carcinoma cells, ATDC5

FK506 (Tacrolimus) and cyclosporin A exert their immunosuppressive effects via a common mechanism, calcineurin inhibition, after binding to intracellular proteins termed immunophilins: FK506-binding protein (FKBP) and cyclophilin. In this study, FK506 was found to induce chondrogenic differentiation of ATDC5 cells (clonal mouse embryonal carcinoma cells) in a concentration-dependent manner (0.1-1000 ng/ml). Immunohistochemical staining showed that ATDC5 cells induced to differentiate by FK506 produced proteoglycan and type II collagen, main components of the extracellular matrix of cartilage. Rapamycin, an immunosuppressant that binds to FKBP, antagonized the effect of FK506. Cyclosporin A did not induce chondrogenesis at concentrations up to 1000 ng/ml. Taken together, these results suggest that FK506 induces chondrogenic differentiation of ATDC5 cells via a calcineurin-independent mechanism, after binding to FKBP.     

Structural studies on FK-506, cyclosporin A and their immunophilin complexes

Summary The immunosuppressants FK-506 and cyclosporin A (CsA), along with their macromolecular receptors FKBP12 and cyclophilin A (CyPA), have become important targets for structure-based drug design. In the last few years X-ray diffraction and NMR spectroscopy have combined to provide high-resolution structures of FK-506, CyA, FKBP12, CyPA, FKBP12-FK-506, CyPA-CyA, and other complexes. This review summarizes these structural studies and some of their implications. Because the immunosuppressant-immunophilin complex forms a composite binding surface that interacts with yet another protein, structure-based drug design in this area is unusually challenging.     

New eremophilane sesquiterpenoid compounds, eremoxylarins A and B directly inhibit calcineurin in a manner independent of immunophilin

In the course of our screening program for a new Ca2+-signal transduction inhibitor using the hypersensitive mutant strain of Saccharomyces cerevisiae (zds1Delta erg3Delta pdr1Delta pdr3Delta), new eremophilane sesquiterpenoid compounds eremoxylarins A and B were found to restore the growth inhibition caused by the hyperactivated Ca2+-signal. These compounds showed lethal activity against the mpk1Delta strain, specifically, compared to the cnb1Delta strain, and ion-sensitive activity against the wild-type strain in the presence of LiCl, indicating that their molecular target might be the calcineurin pathway. They inhibited calcineurin directly without immunophilins at IC50=2.7 and 1.4 microM with competitive inhibition in vitro. The eremophilane sesquiterpenoid structure in eremoxylarins could be a good leading compound for immunosuppressants and anti-allergy drugs.     

FKBP51 regulation of AKT/protein kinase B phosphorylation

FK506 binding protein 51 (FKBP51, also called FKBP5) belongs to a family of immunophilins, FK506 binding proteins (FKBPs). FKBP family members are targets for drugs such as rapamycin. Although FKBP51 shares characteristics with other FKBPs, it also has unique features, especially the role in its regulation of important signaling pathways such as the AKT kinase/protein kinase B pathway. In this review, we will focus on the function of FKBP51 as a scaffolding protein in the regulation of AKT activation and, in turn, its role in tumorigenesis and response to chemotherapy.     

32-Indolyl ether derivatives of ascomycin: three-dimensional structures of complexes with FK506-binding protein.

32-Indole ether derivatives of tacrolimus and ascomycin retain the potent immunosuppressive activity of their parent compounds but display reduced toxicity. In addition, their complexes with the 12-kDa FK506-binding protein (FKBP) form more stable complexes with the protein phosphatase calcineurin, the molecular target of these drugs. We have solved the three-dimensional structures of the FKBP complexes with two 32-indolyl derivatives of ascomycin. The structures of the protein and the macrolide are remarkably similar to those seen in the complexes with tacrolimus and ascomycin. The indole groups project away from the body of the complex, and multiple conformations are observed for the linkage to these groups as well as for a nearby peptide suggesting apparent flexibility in these parts of the structure. Comparison of these structures with that of the ternary complex of calcineurin, FKBP, and tacrolimus suggests that the indole groups interact with a binding site comprising elements of both the calcineurin alpha- and beta-chains and that this interaction is responsible for the increased stability of these complexes.     

Exploration of pipecolate sulfonamides as binders of the FK506-binding proteins 51 and 52

FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid hormone receptors. Single nucleotide polymorphisms in the gene encoding FKBP51 have been associated with a variety of psychiatric disorders. Rapamycin and FK506 are two macrocyclic natural products, which tightly bind to most FKBP family members, including FKBP51 and FKBP52. A bioisosteric replacement of the α-ketoamide moiety of rapamycin and FK506 with a sulfonamide was envisaged with the retention of the conserved hydrogen bonds. A focused solid support-based synthesis protocol was developed, which led to ligands with submicromolar affinity for FKBP51 and FKBP52. The molecular binding mode for one sulfonamide analogue was confirmed by X-ray crystallography.     

The immunophilin-ligands FK506 and V-10,367 mediate neuroprotection by the heat shock response

(1) The macrolid FK506 is widely used in transplantation to suppress allograft rejection. FK506 and its derivatives are powerful neuroprotective molecules, but the underlying mechanisms remain to be resolved. We have previously shown that the FK506 mediated neuroprotection against oxygen radicals is independent of the inhibition of calcineurin but depends on de novo protein synthesis. (2) Here, we have shown that FK506 mediates protection against H(2)O(2), UV-light or thapsigargin in neuronal cell lines, but not in non-neuronal cells such as R3T3 fibroblasts. We compared in detail the effect of FK506 on apoptotic features in PC12 cells after H(2)O(2) with V-10,367 which binds to FKBPs but does not inhibit calcineurin. Both molecules exert the same neuroprotective effect after H(2)O(2) stimulation. FK506, but not V-10,367, inhibited the cytochrome c release out of the mitochondria and the caspase 3 activation, while both molecules inhibited the cleavage of Poly-(ADP-ribose)-polymerase (Parp) and prevented the expression of p53. (3) FK506 and V-10,367 rapidly induced the expression of Hsp70 and Hsp27, but not Hsp90. Their neuroprotective actions could be completely blocked by quercetin, a functional inhibitor of the heat shock proteins. (4) We conclude that immunophilin-ligands such as FK506 and V-10,367 exert their neuroprotection independent of calcineurin through the induction of the heat shock response. The identification of the underlying signal transduction from application of immunophilin ligands to the expression of heat shock proteins represents a novel target cascade for neuroprotection.     

FK506 and its analogs - therapeutic potential for neurological disorders

Immunophilin ligands such as FK506 and Cyclosporin A, used in immunosuppression, are well-characterized drugs. In the past, they had been the center of attention as a putative therapeutic strategy for neuroregeneration and neuroprotection. In contrast to Cyclosporin A, FK506 readily crosses the brain-blood-barrier and, thus together with its derivatives, may represent a novel approach to the treatment of neurological disorders. FK506 exerts profound neuroprotective and neuroregenerative effects in vivo and in vitro. The mechanism underlying neuroregeneration is fairly well understood. It is independent of the inhibition of calcineurin, which is responsible for the immunosuppression, but operates via the binding of FKBP52 and the heat shock protein (Hsp) 90. In contrast, the underlying pathways of neuroprotection are far less understood. Protection is apparently independent of calcineurin, as shown by non-calcineurin inhibiting derivatives, such as V-10,367 and GPI-1046, but the intracellular actions remain to be defined. FK506 has been shown to interfere with the apoptotic pathway of neuronal cells, including inhibiting JNK activity, cytochrome c release, caspase 3 activation, and CD95 ligand expression. These effects are in part mediated by the inhibition of calcineurin and may not contribute to protection. Our recent studies suggest that the protective properties of FK506 and its non-calcineurin inhibiting derivatives are realized by a fast induction of heat shock proteins. The induction of the heat shock response by immunophilin ligands might prove to be an interesting target for neuroregeneration and neuroprotection.     

Biologicals and Immunologicals Overview: Recent developments in macrolide immunosuppressants

Rapamycin and FK506 are structurally related macrolide antibiotic compounds which exhibit potent immunosuppressive properties in higher eukaryotes. It is now well established that these powerful immunosuppressive macrolides, as well as the structurally unrelated undecapeptide Cyclosporin A, all mediate their immunosuppressive effects via inhibition of the specific signal transduction pathways that induce T-lymphocyte activation. Despite the structural similarity between rapamycin and FK506, and that both FK506 and rapamycin bind to FK506 binding proteins through a common transition state mimic structure, the FK506 binding protein 12-rapamycin complex does not bind to calcineurin. FK506 acts at an early stage of the distinct intracellular signal transduction pathway, inhibiting T-cell receptor-mediated transition from a resting state, Go, to the G₁ phase of the cell cycle (activation). Thus, the FK506-FKBP12 and cyclosporin A-cyclophilin complexes act similarly to each other, inhibiting a family of Ca²⁺-dependent signalling pathways by forming a termolecular complex with calcineurin, a Ca²⁺, calmodulin-dependent protein phosphatase target. In contrast to FK506, rapamycin inhibits cytokine-driven T-lymphocyte proliferation rather than activation of cells induced by antigen-T-cell receptor interaction. Rapamycin selectively interacts with a family of Ca²⁺-dependent and Ca²⁺-independent signalling pathways, and as a consequence induces its effects at a later stage of the intracellular signalling pathway, arresting the transition from the G₁ phase to the S phase of the cell cycle (progression). Recent studies have indicated that rapamycin mediates its immunosuppressive activity by selectively inhibiting a specific protein phosphorylation p70 S6 serine/threonine protein kinase cascade. This overview describes recent developments in both the current understanding of the molecular basis of the mechanism of action by which these macrolide immunosuppressive agents exert their activity and the influence on the development of several new diagnostic screening assays. The activity of an extensive number of novel semi-synthetic and naturally produced macrolide compounds in vitro and in vivo tests are evaluated, as well as a broad spectrum of new therapeutic applications.     

Evaluation of synthetic FK506 analogues as ligands for the FK506-binding proteins 51 and 52

The FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid hormone receptors. Both proteins have been implicated in prostate cancer. Furthermore, single nucleotide polymorphisms in the gene encoding FKBP51 have been associated with a variety of psychiatric disorders. Rapamycin and FK506 are two macrocyclic natural products that bind to these proteins indiscriminately but with nanomolar affinity. We here report the cocrystal structure of FKBP51 with a simplified α-ketoamide analogue derived from FK506 and the first structure-activity relationship analysis for FKBP51 and FKBP52 based on this compound. In particular, the tert-pentyl group of this ligand was systematically replaced by a cyclohexyl ring system, which more closely resembles the pyranose ring in the high-affinity ligands rapamycin and FK506. The interaction with FKBPs was found to be surprisingly tolerant to the stereochemistry of the attached cyclohexyl substituents. The molecular basis for this tolerance was elucidated by X-ray cocrystallography.     

Rapamycin and FK506 induce long-term potentiation by pairing stimulation via an intracellular Ca(2+) signaling mechanism in rat hippocampal CA1 neurons

Immunophilin-CsA and -FK506 complexes bind to calcineurin (CaN) and inhibit its phosphatase activity leading to enhancement of neuronal activities. However, inhibition of CaN activity is not the mediator of modulatory activity for IP3 and ryanodine receptors and does not mediate the neurotrophic actions of FK506. FK506 binding protein (FKBP)-12 also binds rapamycin, another immunosuppressant which does not affect CaN activity. Using whole-cell patch clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded and we analyzed the effect of immunosuppressants on the synaptic potentiation induced by pairing weak presynaptic stimulation with postsynaptic depolarization in CA1 neurons of rat hippocampal slices. We found that postsynaptic application of rapamycin or FK506, at low concentrations, but not cyclosporin A, in conjunction with weak pairing stimulation, induced NMDA-dependent long-term potentiation (LTP). The rapamycin-induced LTP was blocked by chelating intracellular Ca(2+) or by inhibiting the intracellular Ca(2+) release. Thus, Ca(2+) release from intracellular Ca(2+) stores is required for the induction of LTP by weak pairing stimulation in the presence of rapamycin or FK506 at postsynaptic sites. We propose that postsynaptic FKBP-12 regulates synaptic transmission by stabilizing the postsynaptic Ca(2+) signaling mechanism in rat hippocampal CA1 neurons.