A novel immunophilin ligand: distinct branching effects on dopaminergic neurons in culture and neurotrophic actions after oral administration in an animal model of Parkinson's disease.

Protection or regeneration of the dopaminergic (DA) system would be of significant therapeutic value for Parkinson's disease. Immunophilin ligands, such as FK506, can produce neurotrophic effects in vitro and in vivo, but their immunosuppressive effects make them unsuitable for neurological application. This study demonstrates that a novel, nonimmunosuppressive immunophilin ligand (V-10,367) increased the number of neurites extended by tyrosine hydroxylase positive (TH+) DA neurons in embryonic day 14 primary DA neuronal cultures. In contrast, the immunosuppressive immunophilin ligand FK506 increased the length of TH+ neurites. After oral administration in MPTP-treated mice, V-10,367 completely protected against MPTP-induced loss of striatal TH+ axonal density, while FK506 did not. These experiments demonstrate that nonimmunosuppressive immunophilin ligands specifically increase neurite branching in primary DA neuronal culture and possess neurotrophic actions in vivo with potential application to neurodegenerative disease.

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.

Design, synthesis and structure of non-macrocyclic inhibitors of FKBP12, the major binding protein for the immunosuppressant FK506.

We have synthesized a series of non-macrocyclic ligands to FKBP12 that are comparable in binding potency and peptidyl prolyl isomerase (PPIase) inhibition to FK506 itself. We have also solved the structure of one of these ligands in complex with FKBP12, and have compared that structure to the FK506-FKBP12 complex. Consistent with the observed inhibitory equipotency of these compounds, we observe a strong similarity in the conformation of the two ligands in the region of the protein that mediates PPIase activity. Our compounds, however, are not immunosuppressive. In the FKBP12-FK506 complex, a significant portion of the FK506 ligand, its 'effector domain', projects beyond the envelope of the binding protein in a manner that is suggestive of a potential interaction with a second protein, the calcium-dependent phosphatase, calcineurin, whose inhibition by the FKBP 12-FK506 complex interrupts the T-cell activation events leading to immunosuppression. In contrast, our compounds bind within the surface envelope of FKBP12, and induce significant changes in the structure of the FKBP12 protein which may also affect calcineurin binding indirectly.