FK1706, a novel non-immunosuppressive immunophilin ligand, modifies the course of painful diabetic neuropathy.

FK1706, a derivative of FK506, is a non-immunosuppressive immunophilin ligand with significant neurotrophic activity mediated via FKBP-52 and the RAS/RAF/MAPK signaling pathway. Here, we tested the effect of FK1706 on painful diabetic neuropathy in rat model of diabetes induced by streptozotocin (STZ). FK1706 ameliorated mechanical allodynia in this model at doses over 0.32 mg/kg, p.o., even if treatment was initiated after neuropathy was established, and did not affect plasma glucose levels. Furthermore, this improvement continued at least 4 weeks after the last administration. In morphological analysis, FK1706 treatment also restored intraepidermal nerve fiber density in footpad skin to almost normal levels. Gabapentin also improved mechanical allodynia in the same model, but efficacy disappeared the day after administration stopped. Allodynia responses were potentiated by co-administration of both compounds. Thus, FK1706 ameliorated painful diabetic neuropathy via a different mechanism from gabapentin and improved morphological outcomes, indicating that FK1706 improves painful diabetic neuropathy by modifying the underlying disease pathology.

FK1706, a novel non-immunosuppressant neurophilin ligand, ameliorates motor dysfunction following spinal cord injury through its neuroregenerative action.

Injured spinal cord axons fail to regenerate in part due to a lack of trophic support. While various methods for replacing neurotrophins have been pursued, clinical uses of these methods face significant barriers. FK1706, a non-immunosuppressant neurophilin ligand, potentiates nerve growth factor signaling, suggesting therapeutic potential for functional deficits following spinal cord injury. Here, we demonstrate that FK1706 significantly improves behavioral outcomes in animal models of spinal cord hemisection and contusion injuries in rats. Furthermore, we show that FK1706 is effective even if administration is delayed until 1 week after injury, suggesting that FK1706 has a reasonable therapeutic time-window. Morphological analysis of injured axons in the dorsal corticospinal tract showed an increase in the radius and perimeter of stained axons, which were reduced by FK1706 treatment, suggesting that axonal swelling and retraction balls observed in injured spinal cord were improved by the neurotrophic effect of FK1706. Taken together, FK1706 improves both behavioral motor function and the underlying morphological changes, suggesting that FK1706 may have therapeutic potential in meeting the significant unmet needs in spinal cord injury.

Advances in small molecules promoting neurotrophic function.

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons and have been implicated in the pathophysiology of numerous disease states. Although the therapeutic potential of neurotrophins has generated much excitement over the past decade, inconvenient pharmacokinetics and adverse side-effect profiles have limited the clinical usefulness of neurotrophic factors themselves. Compounds that mimic neurotrophin signaling and overcome the pharmacokinetic and side-effect barriers may have greater therapeutic potential. Here, we review the progress to date of clinical trials with direct neurotrophin modulators and describe alternative strategies to target (modulate) neurotrophin production and/or their signal transduction pathways. Particular emphasis is placed on small molecules that are able to modulate neurotrophin function in diseases of the nervous system. These alternative strategies show promise in preclinical studies, with some advancing into clinical development. Moreover, the recognition that clinically effective therapeutics, such as antidepressants and immunophilin ligands, can modulate neurotrophin function suggests that the concept of small molecule therapeutics that promote neurotrophic function may still be viable.

A simple, flexible, nonfluorescent system for the automated screening of neurite outgrowth.

Measurement of neurite outgrowth is a common assay of neurotrophic activity. However, currently available techniques for measuring neurite outgrowth are either time or resource intensive. The authors established a system in which chronic treatment of a subcloned SH-SY5Y cell line with aphidicolin and various concentrations of nerve growth factor (NGF) induced discernable alterations in proliferation and differentiation. Cells were fixed, labeled with a nonfluorescent dye, and evaluated both manually and with an automated analysis system. NGF increased multiple parameters of differentiation, including neurite length, the proportion of cells extending neurites, and branching, as well as promoting cellular survival/proliferation. Interestingly, although NGF treatment increased the total number of branches, it actually decreased the proportion of branches per neurite length. The authors observed no differences in results obtained using the manual and automated systems, but the automated system was orders of magnitude faster. To demonstrate the flexibility of the system, the authors also show that they could measure changes in differentiation induced by a small-molecule Rho kinase inhibitor, as well as by retinoic acid cotreatment with brain-derived neurotrophic factor. In addition to this flexibility, this system does not require specialized equipment or fluorescent antibodies for analysis and therefore provides a less resource-intensive alternative to fluorescence-based systems.

FK1706, a novel non-immunosuppressive immunophilin: neurotrophic activity and mechanism of action.

Immunophilin ligands are neuroregenerative agents, characterized by binding to FK506 binding proteins (FKBPs), which stimulate recovery of neurons in a variety of injury paradigms. Here we report the discovery of a novel, non-immunosuppressive immunophilin ligand, FK1706. FK1706, a derivative of FK506, showed similarly high affinity for two FKBP subtypes, FKBP-12 and FKBP-52, but inhibited T-cell proliferation and interleukin-2 cytokine production with much lower potency and efficacy than FK506. FK1706 (0.1 to 10 nM) significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in SH-SY5Y cells, as did FK506. This neurite potentiation could be blocked by an anti-FKBP-52 antibody, as well as by specific pharmacological inhibitors of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), and the Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) signaling pathway. FK1706 also potentiated NGF-induced MAPK activation, with a similar dose-dependency to that necessary for potentiating neurite outgrowth. Taken together, these data suggest that FK1706 is a non-immunosuppressive immunophilin ligand with significant neurotrophic effects, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway, and therefore that FK1706 may have therapeutic potential in a variety of neurological disorders.

RNA editing of the human serotonin 5-HT2C receptor disrupts transactivation of the small G-protein RhoA.

The human serotonin 5-HT2C receptor undergoes adenosineto-inosine RNA editing at five positions, generating multiple receptor isoforms with altered G-protein coupling properties. In the current study, we demonstrate that RNA editing regulates the pattern of intracellular signaling. The non-edited human 5-HT2C receptor isoform INI activates phospholipase D via the G13 heterotrimer G-protein. We present evidence that transactivation of the small G-protein RhoA is required for phospholipase D activation. In contrast, neither transactivation of RhoA nor phospholipase D activation was detected in cells expressing the fully edited VGV isoform. The ability to activate phospholipase C is also reduced in VGV-expressing cells, but not to the extent found for the phospholipase D signal. We conclude that RNA editing represents a novel mechanism for regulating 5-HT2C receptor signaling to pathways linked to actin cytoskeletal organization and regulated exocytosis.

FK506 potentiates NGF-induced neurite outgrowth via the Ras/Raf/MAP kinase pathway.

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems. Neurophilin ligands, including FK506, potentiate NGF-induced neurite outgrowth in several experimental models, although the mechanism of this potentiation is unclear. Therefore, we tested which signaling pathways were involved in FK506-potentiated neurite outgrowth in SH-SY5Y neuroblastoma cells using specific pharmacological inhibitors of various signaling molecules. Inhibitors of Ras (lovastatin), Raf (GW5074), or MAP kinase (PD98059 and U0126) blocked FK506 activity, as did inhibitors of phospholipase C (U73122) and phosphatidylinositol 3' kinase (LY294002). Protein kinase C inhibitors (Go6983 and Ro31-8220) slightly but significantly inhibited neurite outgrowth, whereas inhibitors of p38 MAPK (SB203580) or c-Jun N-terminal kinase (SP600125) had no effect. These data suggest that FK506 potentiates neurite outgrowth through the Ras/Raf/MAP kinase signaling pathway downstream of phospholipase C and phosphatidylinositol 3' kinase.