Immunophilin Ligands Research Articles

Preferential neuroprotective effect of tacrolimus (FK506) on unmyelinated axons following traumatic brain injury

Prior investigations of traumatic axonal injury (TAI), and pharmacological treatments of TAI pathology, have focused exclusively on the role of myelinated axons, with no systematic observations directed towards unmyelinated axon pathophysiology. Recent electrophysiological evidence, however, indicates that unmyelinated axons are more vulnerable than myelinated axons in a rodent model of experimental TAI. Given their susceptibility to TAI, the present study examines whether unmyelinated axons also respond differentially to FK506, an immunophilin ligand with well-established neuroprotective efficacy in the myelinated fiber population. Adult rats received 3.0 mg/kg FK506 intravenously at 30 min prior to midline fluid percussion injury. In brain slice electrophysiological recordings, conducted at 24 h postinjury, compound action potentials (CAPs) were evoked in the corpus callosum, and injury effects quantified separately for CAP waveform components generated by myelinated axons (N 1 wave) and unmyelinated axons (N 2 wave). The amplitudes of both CAP components were suppressed postinjury, although this deficit was 16% greater for the N 2 CAP. While FK506 treatment provided significant neuroprotection for both N 1 and N 2 CAPs, the drug benefit for the N 2 CAP amplitude was 122% greater than that for the N 1 CAPs, and improved postinjury strength-duration and refractoriness properties only in N 2 CAPs. Immunocytochemical observations, of TAI reflected in intra-axonal pooling of amyloid precursor protein, indicated that FK506 reduced the extent of postinjury impairments to axonal transport and subsequent axonal damage. Collectively, these studies further substantiate a distinctive role of unmyelinated axons in TAI, and suggest a highly efficacious neuroprotective strategy to target this axonal population.

778: The Non-Immunosuppressant Immunophilin Ligand FK1706 Enhances the Recovery of Erectile Function Following Bilateral Cavernous Crush Injury in the Rat

778: The Non-Immunosuppressant Immunophilin Ligand FK1706 Enhances the Recovery of Erectile Function Following Bilateral Cavernous Crush Injury in the Rat

FK1706 Enhances the Recovery of Erectile Function Following Bilateral Cavernous Nerve Crush Injury in the Rat

Advances in neurobiology have led to a surge of clinical interest in the development of protective and regenerative neuromodulatory strategies, as surgical therapies for prostate cancer often result in neuronal damage and debilitating loss of sexual function. To investigate the dose-dependent efficacy of FK1706, a nonimmunosuppressant immunophilin ligand, for the recovery of erectile function following bilateral cavernous nerve crush injury in the rat. Recovery of erectile function was assessed by cavernous nerve electrostimulation and reported as maximal increase of intracavernous pressure (ICP) and area under the curve (AUC). Changes in animal weights, percentage completion of treatment course, and survival were compared between groups. METHODS; Thirty-five Sprague-Dawley male rats were randomly divided into five equal groups: seven animals received a sham operation, whereas 28 animals underwent bilateral cavernous nerve crush injury, followed by subcutaneous injection of vehicle alone (1.0 mL/kg), or low (0.1 mg/kg), medium (0.32 mg/kg), or high dose (1.0 mg/kg) FK1706 5 days per week for 8 weeks. Erectile dysfunction did not occur in the sham group (mean maximal ICP increase of 100.8 +/- 6.3 cmH(2)O), whereas nerve injury and vehicle treatment produced a significant reduction in ICP response to 34.4 +/- 12.8 cmH(2)O. The mean ICP increase for high-dose FK106 treatment was 73.9 +/- 6.3 cmH(2)O (P < 0.01 vs. vehicle) compared with 58.3 +/- 7.4 cmH(2)O and 56.9 +/- 8.3 for low and medium doses (P > 0.05). Similar stepwise findings were observed using AUC data. No significant maximal aortic blood pressure or weight differences occurred between groups and all animals completed treatment. High-dose subcutaneous FK1706 therapy promoted recovery of erectile function following bilateral cavernous nerve crush injury in the rat. No significant differences between groups were observed for changes in weight, and the 8-week treatment course was completed for all animals.

Neuroprotective and Antiretroviral Effects of the Immunophilin ligand GPI 1046

HIV infection results in a neurodegenerative disorder for which currently there is no effective therapy available. Currently, available antiretroviral therapy has no impact on the production of early regulatory HIV proteins once the virus is integrated. Of these proteins, Tat was shown to be toxic to neurons. We, thus, used an in vitro neuronal culture system to determine if immunophilin ligands could protect against Tat-induced neurotoxicity. We found that GPI 1046 had potent neuroprotective effects in this model. The compound was able to protect the neurons even though it only partially obliterated Tat-induced oxidative stress in neurons, suggesting that other mechanisms may be important in mediating its neuroprotective effect. Furthermore, GPI 1046 showed inhibition of HIV replication and Tat-mediated long terminal repeat (LTR) activation suggesting that this class of compounds may be worthy of further exploration as a potential treatment for HIV dementia.

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Neuroimmunophilin Ligands Protect Cavernous Nerves after Crush Injury in the Rat: New Experimental Paradigms

ObjectivesWe investigated the effects of the orally bioavailable non-immunosuppressive immunophilin ligand GPI 1046 (GPI) on erectile function and cavernous nerve (CN) histology following unilateral or bilateral crush injury (UCI, BCI, respectively) of the CNs. MethodsAdult male Sprague-Dawley rats were administered GPI 15mg/kg intraperitoneally (ip) or 30mg/kg orally (po), FK506 1mg/kg, ip, or vehicle controls for each route of administration just prior to UCI or BCI and daily up to 7 d following injury. At day 1 or 7 of treatment, erectile function induced by CN electrical stimulation was measured, and electron microscopic analysis of the injured CN was performed. ResultsIntraperitoneal administration of GPI to rats with injured CN protected erectile function, in a fashion similar to the prototypic immunophilin ligand FK506, compared with vehicle-treated animals (93%±9% vs. 70%±5% vs. 45%±1%, p<0.01, respectively). Oral administration of GPI elicited the same level of significant protection from CN injury. GPI administered po at 30mg/kg/d, dosing either once daily or four times daily with 7.5mg/kg, provided nearly complete protection of erectile function. In a more severe BCI model, po administration of GPI maintained erectile function at 24h after CN injury. Ultrastructural analysis of injured CNs indicated that GPI administered at the time of CN injury prevents degeneration of about 83% of the unmyelinated axons at 7 d after CN injury. ConclusionsThe orally administered immunophilin ligand GPI neuroprotects CNs and maintains erectile function in rats under various conditions of CN crush injury.

Recovery of sexual function after prostate cancer treatment

The estimated disease-free survival rates are approximately equivalent across standard treatments for localized prostate cancer. We aim to review the efforts being made to reduce posttreatment erectile dysfunction, a major morbidity of these therapies. Potency as an important factor in a patient's decision about choosing a form of therapy has been demonstrated in the literature. For nerve-sparing surgery, though some proponents of laparoscopic radical prostatectomy believe it may confer an advantage over the open surgical techniques, the published data is scarce and has yet to demonstrate a true difference. Enthusiasm has declined for sural nerve grafting because of the associated complexity of the procedure and inconsistent results. Concurrent implantation of a penile prosthesis is an option for certain patients who already have some baseline erectile dysfunction or are not candidates for nerve-sparing surgery. Agents such as phosphodiesterase inhibitors, immunophilin ligands, and recombinant human erythropoietin have demonstrated potential benefits in early reports of both in-vitro and ongoing clinical trials. Currently, no standard treatment or prophylaxis exists for posttreatment erectile dysfunction. Neuro-protective and regenerative therapies, including the immunophilin ligands, hold promise to reduce the morbidity of localized prostate cancer therapy.

Immunophilin-ligands FK506 and CsA inhibit HIF1α expression by a VHL- and ubiquitin-independent mechanism

Hypoxia inducible factor-1alpha (HIF1α) plays a key role in the regulation of genes controlling oxygen supply, glucose metabolism and angiogenesis. Its expression in tumors appears to confer an adaptive advantage to their hypoxic microenvironment. We have evaluated the effect of the immunophilin ligands FK506 and cyclosporin A on HIF1α levels in different tumor cell lines. Our results indicate that both drugs are potent suppressors of HIF1α expression by accelerating the proteasomal degradation of the protein. Unexpectedly, the suppressive effect of these compounds was found to be independent of the presence of von Hippel Lindau factor and the degree of hydroxylation of the HIF1α protein. Moreover, HIF1α degradation induced by these compounds did not required ubiquitination, as it was also induced in E1 ligase-incompetent cells.

Therapeutic potential of natural product signal transduction agents

Modern drug discovery embraces a strategy of targeting cellular signal transduction pathways as a means of finding new therapeutic agents. Historically, natural products derived from microorganisms have played an important role as drug leads and clinical candidates under this paradigm. The future drug potential of natural products as signal transduction agents looks promising, as illustrated by two key examples. First, substantial advances have been made in the development of inhibitors based on immunophilin ligand polyketides, which target the TOR-mediated pathways and can modulate processes including cell proliferation and cell-cycle arrest. Second, the discovery of natural product inhibitors of the ubiquitin-proteasome proteolytic signal transduction pathway represents an emerging field. Given these examples, together with the diversity of as yet undiscovered agents, natural product signal transduction agents offer great potential for future drug discovery efforts.

174: Immunophilin Ligand, GPI-1046, Attenuates Neuronal Cell Death Following Radiation

Nonimmunosuppressive immunophilin ligands act by binding to immunophilin receptor proteins such as FKBP-12 resulting in a decrease in the morbidity associated with neuronal crush injury. It is not known whether radiation-induced neuronal injury also results in alteration of FKBP-12 levels or whether the resulting injury can be modulated by immunophilin ligand binding to FKBP-12. To study this, we investigated the relationship between FKBP-12 expression patterns and radiation exposure as well as performed clonogenic survival assays to determine the protective effects of GPI-1046, an immunophilin ligand, in both an in vitro mammalian neuronal model as well as in human prostate cancer cell lines.

3‐Normeridamycin: A Potent Non‐Immunosuppressive Immunophilin Ligand is Neuroprotective in Dopaminergic Neurons.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

A multiplexed proteomics approach to differentiate neurite outgrowth patterns

We report here a method for proteomics pattern discovery by utilizing a self-organizing map approach to analyze data obtained from a novel multiplex iTRAQ™ proteomics method. Through the application of this technique, we were able to delineate the early molecular events preceding dorsal root ganglia neurite outgrowth induced by either nerve growth factor (NGF) or an immunophilin ligand, JNJ460. Following pattern analysis we discovered that each neurotrophic agent promoted mostly distinct increases in protein expression with few overlapping patterns. In the NGF-treated group, proteins possessing “biosynthesis function” ( p < 0.002) and “ribosome localization” ( p < 0.0003) were increased, while proteins promoting “organogenesis” ( p < 0.004) and related “signal transduction” ( p < 0.008) functions were notably increased in the JNJ460-treated group. This study suggests that the properties of neurite outgrowth triggered by NGF and JNJ460 can be distinguished at the proteome level. Multiplexed proteomics analysis, along with pattern discovery bioinformatics tools, has the capability to differentiate subtle neuroproteomics patterns.

The immunophilin ligand GPI1046 protects neurons from the lethal effects of the HIV‐1 proteins gp120 and Tat by modulating endoplasmic reticulum calcium load

The dysfunction and death of neuronal cells is thought to underlie the cognitive manifestations of human immunodeficiency virus (HIV)-associated neurological disorders. Although HIV-infected patients are living longer owing to the effectiveness of anti-retroviral therapies, the number of patients developing neurological disorders is on the rise. Thus, there is an escalating need for effective therapies to preserve cognitive function in HIV-infected patients. Using HIV-protein-induced neurotoxicity as a model system, we tested the effectiveness of a non-immunosuppressive immunophilin ligand to attenuate gp120 and Tat-induced modification of neuronal function. The immunophilin ligand GPI1046 attenuated endoplasmic reticulum (ER) calcium release induced by gp120 and Tat and protected neurons from the lethal effect of these neurotoxic HIV proteins. Both inositol 1,4,5 trisphosphate (IP(3)) and ryanodine-sensitive ER calcium release was attenuated by pre-incubation with GPI1046. Using the sarco/endoplasmic reticulum calcium pump inhibitor thapsigargin to release ER calcium, we determined that GPI1046 reduced the total ER calcium load. These findings suggest that non-immunosuppressive immunophilin ligands may be useful neuroprotective drugs in HIV dementia.

The aggregation of alpha‐synuclein is stimulated by FK506 Binding Proteins as shown by Fluorescence Correlation Spectroscopy

The aggregation of alpha-synuclein (α-SYN) plays a key role in Parkinson's disease (PD). We have used Fluorescence Correlation Spectroscopy (FCS) to study α-SYN aggregation in vitro and show that we can observe oligomeric intermediates. We also discovered that aggregation is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The α-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of α-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and α-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.

3-Normeridamycin: A Potent Non-Immunosuppressive Immunophilin Ligand is Neuroprotective in Dopaminergic Neurons

3-Normeridamycin (1), isolated from fermentation extracts of the soil actinomycete Streptomyces sp. LL-C31037, demonstrated potent neuroprotective activity. When challenged with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), known to induce parkinsonism, 1 restored functional dopamine uptake in a concentration-dependent manner, with an EC50 of 110 nM in dopaminergic neurons. The structure of 1 was determined via spectroscopic methods, and the immunosuppressive and immunophilin binding properties of the compound were also measured.

The aggregation of alpha‐synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.

Administration of the immunophilin ligand FK506 differentially attenuates neurofilament compaction and impaired axonal transport in injured axons following diffuse traumatic brain injury

Traumatic axonal injury (TAI) following traumatic brain injury (TBI) remains a clinical problem for which no effective treatment exists. TAI was thought to involve intraaxonal changes that universally led to impaired axonal transport (IAT), disconnection and axonal bulb formation. However, recent, immunocytochemical studies employing antibodies to amyloid precursor protein (APP), a marker of IAT and antibodies to neurofilament compaction (NFC), RM014, demonstrated that NFC typically occurs independent of IAT, indicating the existence of different populations of damaged axons. FK506 administration has been shown to attenuate IAT. However, in light of the above, the ability of FK506 to attenuate axonal damage demonstrating NFC requires evaluation. The current study explored the potential of FK506 to attenuate both populations of damaged axons. Rats were administered FK506 (3 mg/kg) or vehicle 30 min preinjury. Three hours post-TBI, tissue was prepared for the visualization of TAI using antibodies targeting IAT (APP) or NFC (RMO14) or a combined labeling strategy. Confirming previous reports, FK506 treatment reduced the number of axons demonstrating IAT in the CSpT, from 411 ± 54.70 to 91.00 ± 33.87 ( P ≤ 0.05) and in the ML from 78.62 ± 16.87 to 41.00 ± 5.80 ( P ≤ 0.05). FK506 treatment failed to reduce the number of axons demonstrating NFC in either the CSpT or ML. FK506's failure to attenuate NFC suggests that additional therapeutic agents may be necessary to blunt the full burden of TAI. Because FK506 targets IAT, calcineurin appears to be a major target for neuroprotection in damaged axons demonstrating IAT.

POLY(ADENOSINE DIPHOSPHATE-RIBOSE) POLYMERASE INHIBITION PRESERVES ERECTILE FUNCTION IN RATS AFTER CAVERNOUS NERVE INJURY

We investigated the involvement of poly(adenosine diphosphate-ribose) (PAR) polymerase (PARP) activation in the development of erectile dysfunction and the therapeutic benefit of the potent PARP inhibitor INO-1001 (Inotek Pharmaceuticals Corp., Beverly, Massachusetts) in a bilateral cavernous nerve crush injury (BCNCI) model in rats. Sprague-Dawley rats were divided into 3 groups, namely sham treated, BCNCI plus vehicle and BCNCI plus the PARP inhibitor INO-1001. One week after surgical intervention all groups underwent in vivo cavernous nerve stimulation. PAR activation, nitrotyrosine and inducible nitric oxide synthase were evaluated by immunohistochemistry and serum levels of INO-1001 were measured by high performance liquid chromatography. Penile tissues were analyzed for levels of malondialdehyde and myeloperoxidase. Data sets were statistically compared in all groups. Neurogenic mediated erectile responses were evaluated. Mean intracavernous pressure (ICP), the ICP-to-blood-pressure ratio and total ICP were significantly decreased in BCNCI plus vehicle rats. These values were not statistically different between the sham and PARP inhibitor treated groups. There was a marked decrease in PAR staining in the treatment group. There was a substantial increase in malondialdehyde tissue levels but not myeloperoxidase in response to BCNCI, which was unchanged with PARP inhibitor treatment. There was a marked increase in tyrosine nitration in the treatment group. Up-regulation of nitric oxide synthase and increased tyrosine nitration were not observed in the penile tissues of the treatment group. These data demonstrate that BCNCI in a rat model causes increased PARP activation, resulting in severe erectile dysfunction. Treatment with the PARP inhibitor INO-1001 decreases the degree of nitrosative stress, prevents PARP activation and provides significant cavernous neuroprotection, which in turn preserves erectile function.

Neuroprotection in the PNS: Erythropoietin and Immunophilin Ligands

Many illnesses that affect the peripheral nervous system (PNS) lead to distal axonal degeneration rather than loss of neuronal cell bodies. Strategies aimed at promoting survival of injured neurons (i.e., preventing cell death) may not be applicable to many PNS illnesses. We have developed in vitro and in vivo animal models to study mechanisms of acquired peripheral neuropathies and used these models to evaluate the therapeutic potential of novel compounds. In recent years, erythropoietin (EPO) has been recognized as a novel neuroprotectant in the central nervous system. In the PNS, we recently showed that Schwann cell-derived EPO acts as an endogenous neuroprotectant and that it is most effective in preventing distal axonal degeneration seen in models of peripheral neuropathy. Similarly, we showed that immunophilin ligands are also neuroprotective in the PNS and prevent axonal degeneration seen in models of peripheral neuropathies. Both EPO and non-immunosuppressive immunophilin ligands are in early clinical development for the treatment of acquired peripheral neuropathies.

Neuroprotection in the PNS: Erythropoietin and Immunophilin Ligands

Neuroprotection in the PNS: Erythropoietin and Immunophilin Ligands