David M. Armistead

A nonimmunosuppressant FKBP-12 ligand increases nerve regeneration

The immunosuppressant drugs FK506 and cyclosporin A inhibit T-cell proliferation via a common mechanism: calcineurin inhibition following binding to their respective binding proteins, the peptidyl prolyl isomerases FKBP-12 and cyclophilin A. In contrast, FK506, but not cyclosporin A, accelerates nerve regeneration. In the present study, we show that the potent FKBP-12 inhibitor V-10,367, which lacks the structural components of FK506 required for calcineurin inhibition, increases neurite outgrowth in SH-SY5Y neuroblastoma cells and speeds nerve regeneration in the rat sciatic nerve crush model. In SH-SY5Y cells, V-10,367 increased the lengths of neurite processes in a concentration-dependent (between 1 and 10 nM) fashion over time (up to 168 h). Daily subcutaneous injections of V-10,367 accelerated the onset of clinical signs of functional recovery in the hind feet compared to vehicle-treated control animals. Interdigit distances (between the first and fifth digits) measured on foot prints obtained during walking showed an increase in toe spread in V-10,367-treated rats compared to vehicle-treated controls. Electron microscopy demonstrated larger regenerating axons distal to the crush site in the sciatic nerve from V-10,367-treated rats. Quantitation of axonal areas in the soleus nerve revealed a shift to larger axonal calibers in V-10,367-treated rats (400 or 200 mg/kg/day); mean axonal areas were increased by 52 and 59%, respectively, compared to vehicle-treated controls. FKBP-12 ligands lacking calcineurin inhibitory activity represent a new class of potential drugs for the treatment of human peripheral nerve disorders.

Cellular and biochemical characterization of VX-710 as a chemosensitizer ; reversal of P-glycoprotein-mediated multidrug resistance in vitro

VX-710 or (S)-N[2-Oxo-2-(3,4,5-trimethoxyphenyl)acetyl]-piperidine-2-carboxylic acid 1,7-bis(3-pyridyl)-4-heptyl ester, a novel non-macrocyclic ligand of the FK506-binding protein FKBP12, was evaluated for its ability to reverse P-glycoprotein-mediated multidrug resistance in vitro. VX-710 at 0.5-5 microM restored sensitivity of a variety of multidrug resistant cells to the cytotoxic action of doxorubicin, vincristine, etoposide or paclitaxel, including drug-selected human myeloma and epithelial carcinoma cells, and human MDR1 cDNA-transfected mouse leukemia and fibroblast cells. Uptake experiments showed that VX-710 at 0.5-2.5 microM fully restored intracellular accumulation of [14C]doxorubicin in multidrug resistant cells, suggesting that VX-710 inhibits the drug efflux activity of P-glycoprotein. VX-710 effectively inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine or [125I]iodoaryl azidoprazosin with EC50 values of 0.75 and 0.55 microM. Moreover, P-glycoprotein was specifically labeled by a tritiated photoaffinity analog of VX-710 and unlabeled VX-710 inhibited analog binding with an EC50 of 0.75 microM. VX-710 also stimulated the vanadate-inhibitable P-glycoprotein ATPase activity 2- to 3-fold in a concentration-dependent manner with an apparent k(a) of 0.1 microM. These data indicate that a direct, high-affinity interaction of VX-710 with P-glycoprotein prevents efflux of cytotoxic drugs by the MDR1 gene product in multidrug resistant tumor cells.

Non-FK506-binding protein-12 neuroimmunophilin ligands increase neurite elongation and accelerate nerve regeneration.

Neurotrophic activity of neuroimmunophilin ligands (FK506 and its nonimmunosuppressant derivatives) has been assumed to be mediated by the FK506‐binding protein‐12 (FKBP‐12). We recently showed that activity is retained in hippocampal neurons from FKBP‐12 knockout mice, indicating that binding to FKBP‐12 is not necessary. Here we show that three nonimmunosuppressant FK506 derivatives (V‐13,450, V‐13,629, and V‐13,670) that do not bind FKBP‐12 (>12.5 mM affinity) are equipotent to FKBP‐12 ligands (FK506, V‐10,367, and V‐13,449) for increasing neurite elongation in SH‐SY5Y cells. One non‐FKBP‐12 ligand (V‐13,670) is also shown to accelerate functional recovery and nerve regeneration in the rat sciatic nerve crush model. Surprisingly, it exhibited an unusual dose‐response effect upon oral administration, showing a novel bimodal dose‐response for behavioral functional recovery and myelination, but not for axonal size, suggesting both Schwann cell and neuronal targets. Orally active non‐FKBP‐12 neuroimmunophilin ligands may be useful for the treatment of human neurological disorders without any potential side effects resulting from FKBP‐12 binding.

Chapter 22. Immunophilins and Immunosuppressive Drug Action

Publisher Summary This chapter discusses the progress in understanding the biochemical and biophysical basis for the action of the immunophilins and immunosuppresive drugs. Numerous studies have examined the cellular and molecular mechanism for cyclic peptide cyclosporin A (CsA), macrolide antibiotic (FK-506), and rapamycin mediated immunosuppression. CsA is currently the principle agent used for immunosuppressive therapy in solid organ transplantation and results of ongoing clinical trials with FK-506 suggest a therapeutic index that may surpass CsA. Despite their dissimilar structures, both CsA and FK-506 inhibit Ca +2 dependent signal transduction events essential for the transcription of early T cell activation genes, including IL-2, IL-3,lL-4, and GM-CSF. Initial investigations by Handschumacher and colleagues identified cyclophilin (CyP-A) as a specific cytosolic receptor for CsA. Subsequent studies identified a common receptor for FK-506 and rapamycin, FK-506 binding protein, FKBP12, specifying the 11,820 M r drug receptor protein. Other immunophilin family members have been identified from a wide variety of species, including Saccharomyces , Neurospofa, Drosophile , E. coli , and others. FKBPl2 and FKBP13 have been characterized from several species and more than 20 cyclophilins have been described to date, with the pace of immunophilin protein discovery, exceeding the definition of their physiologic cellular functions. Early investigations examined the direct role of immunophilins in immunosuppression to address if PPlase inhibition was the sole requisite property for drug action. Several studies examined the correlation between immunophilin affinity, immunosuppressive activity, or nephrotoxicity. Consensus states that immunophilin binding is “necessary, but not sufficient” for immunosuppression. The solution conformation of unlabeled CsA and MeAla6-CsA complexed to fully deuterated CyP has been discussed in the chapter and the results bear striking resemblance to the analogous investigations of inactive FK-506 analogs. As has been previously determined, the binding core of CsA is buried in a hydrophobic cleft of CyP with roughly 55% of the ligand left exposed to solvent.