Fluoresceinated FKBP12 ligands for a high-throughput fluorescence polarization assay

Several fluoresceinated FKBP12 ligands have been prepared for a high-throughput fluorescence polarization assay. K(i)s for FKBP12 rotamase inhibition by these ligands range from 1.3 microM to 32 nM, and their design is based on X-ray crystal structures of FKBP12 complexed with known immunophilin ligands.     

A yeast sensor of ligand binding.

We describe a biosensor that reports the binding of small-molecule ligands to proteins as changes in growth of temperature-sensitive yeast. The yeast strains lack dihydrofolate reductase (DHFR) and are complemented by mouse DHFR containing a ligand-binding domain inserted in a flexible loop. Yeast strains expressing two ligand-binding domain fusions, FKBP12-DHFR and estrogen receptor-alpha (ERalpha)-DHFR, show increased growth in the presence of their corresponding ligands. We used this sensor to identify mutations in residues of ERalpha important for ligand binding, as well as mutations generally affecting protein activity or expression. We also tested the sensor against a chemical array to identify ligands that bind to FKBP12 or ERalpha. The ERalpha sensor was able to discriminate among estrogen analogs, showing different degrees of growth for the analogs that correlated with their relative binding affinities (RBAs). This growth assay provides a simple and inexpensive method to select novel ligands and ligand-binding domains.     

Synthesis, molecular modeling and biological evaluation of aza-proline and aza-pipecolic derivatives as FKBP12 ligands and their in vivo neuroprotective effects

Nonimmunosuppressant ligands, exemplified by GPI 1046 (1), for the peptidyl-prolyl isomerase FKBP12 have been found to unexpectedly possess powerful neuroprotective and neuroregenerative effects in vitro and in vivo. We have extensively explored the therapeutic utility of FKBP12 ligands based on analogues of proline and pipecolic acid. As part of our ongoing program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands containing aza-proline and aza-pipecolic acid analogues. Details of the synthetic studies, together with biological activity will be presented.     

An FKBP12 binding assay based upon biotinylated FKBP12.

A binding assay was developed for measuring the affinity of FKBP12 ligands. A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase. The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with [3H]FK506 for FKBP12 sites on the plate. The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values. The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules.     

A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

The FRB domain of mTOR: NMR solution structure and inhibitor design

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.     

Design and structure-based study of new potential FKBP12 inhibitors

Based on the structure of FKBP12 complexed with FK506 or rapamycin, with computer-aided design, two neurotrophic ligands, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester and (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-phenylalanine benzyl ester, were designed and synthesized. Fluorescence experiments were used to detect the binding affinity between FKBP12 and these two ligands. Complex structures of FKBP12 with these two ligands were obtained by x-ray crystallography. In comparing FKBP12-rapamycin complex and FKBP12-FK506 complex as well as FKBP12-GPI-1046 solution structure with these new complexes, significant volume and surface area effects and obvious contact changes were detected which are expected to cause their different binding energies-showing these two novel ligands will become more effective neuron regeneration drugs than GPI-1046, which is currently undergoing phase II clinical trail as a neurotrophic drug. Analysis of volume and surface area effects also gives a new clue for structure-based drug design.     

Synthesis and evaluation of chiral bicyclic proline FKBP12 ligands

As part of our ongoing program to explore novel structural classes of FKBP12 ligands, we herein wish to report a new class of FKBP12 ligands containing chiral bicyclic proline analogues. Details of the synthetic routes, together with preliminary biological activity, will be presented.     

Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.     

FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate beta-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate beta-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.     

High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.     

Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening

Although the idea of homogeneous electrochemical immunoassay using antibody and an electroactive modified antigen as a probe looks to be very useful for high-throughput drug screening, there have been few reports. One reason for this is the difficulty experienced making an electroactive probe, because the introduction of electroactive compounds to antigens often interferes with the antigen-antibody interaction. To apply a homogeneous electrochemical assay to drug screening, we have designed new probes referring to the information of immobilization on beads which could identify the drug receptor. FK506 (also called Tacrolimus), immunosuppressive agent is modified with ferrocene derivatives as an electron mediator between glucose oxidase and an electrode, at a non-obstructing part. One of the probes still indicated the electrochemical activity as a mediator and had the specific binding capability for FKBP12 (FK506 binding protein). The current decrease in response to the additional FKBP12, detected with constant voltage amperometry using the probe, was observed within 5 min. Then, free FK506 as a leader drug, rapamycin and cyclosporine A as unknown drugs were used as a model for drug screening. Since the order of response currents at the same concentration of each drug reflected their binding constants, it was shown that binding capacity of an unknown drug candidate could be estimated by comparison of response currents between the leader drug and the unknown drug candidate. Thus, this glucose oxidase assisted homogeneous electrochemical drug-receptor binding assay has been proved to be a useful tool for drug screening.     

A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc

Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity.     

Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex.

The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12.FK-506.calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807-815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506-binding protein. Our characterization of the FKBP12.FK-506.CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12.FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12.FK-506, FKBP51.FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.     

Analysis of type 1 ryanodine receptor-12 kDa FK506-binding protein interaction.

Although dissociation of the 12 kDa FK506 binding protein (FKBP12)-type 1 ryanodine receptor (RyR1) complex by macrolide immunosuppressants is well documented, effects of many solutes and drugs have not been quantitated. In the current study, the influence of these on binding between solubilised RyR1 and an FKBP12-glutathione-S-transferase fusion protein was analysed using a novel assay. Association between these two proteins is stable, and is not greatly altered by changes in temperature, pH, cations, and endogenous solutes over physiological ranges. Ascomycin, an FK506 analogue, was identified for the first time as a drug which can disrupt the FKBP12-RyR1 complex.     

The full electron structure of the FKBP12/FK506 complex

We present a study of FKBP12/FK506 using an electron structure calculation. These calculations employ a novel technique called eCADD on the protein’s full electron structure along with its hydrophobic pocket and the frontier-orbital-perturbation theory. We first obtain the energy bands and orbital coefficients of protein FKBP12. On this basis, we found that the activity atoms and activity residues of FKBP12 were in good agreement with X-ray crystallography experiments. The results indicate that the interactions occur only between the LUMOs of FKBP12 and the HOMO of FK506, not between the HOMOs of FKBP12 and the LUMO of FK506. In other words, the activity sites of protein FKBP12 are located on its LUMOs, not HOMOs. The electron structures of FKBP12/FK506 give us a clearer understanding of their interaction mechanism and will help us design new ligands of FKBP12.     

Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor

The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis.