Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides.

Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several ß-alanines that occurred in a repeating ααß motif, causing the compound to adopt a unique right-handed 311 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI50 0.55 ± 0.04 µM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.

TrkB agonist, 7,8-dihydroxyflavone, reduces the clinical and pathological severity of a murine model of multiple sclerosis.

7,8-Dihydroxyflavone (DHF), is a recently described TrkB agonist that readily crosses the blood brain barrier. We treated C57Bl/6 mice with MOG--induced EAE daily with DHF starting on the day of disease induction. Clinical severity of impairment was reduced throughout the course of disease. Pathological examination of brains and spinal cords on day 28 showed that DHF treatment increased the phosphorylation of TrkB and activated downstream signaling pathways including AKT and STAT3 and reduced inflammation, demyelination and axonal loss compared to EAE controls. DHF treatment duplicated the central nervous system effects of brain derived neurotrophic factor in the EAE.

Overexpression of SIRT1 protein in neurons protects against experimental autoimmune encephalomyelitis through activation of multiple SIRT1 targets.

Treatment of experimental autoimmune encephalomyelitis (EAE) with resveratrol, an activator of sirtuin 1 (SIRT1), reduces disease severity. This suggested that activators of SIRT1, a highly conserved NAD-dependent protein deacetylase, might have immune-modulating or neuroprotective therapeutic effects in EAE. Previously, we showed that SIRT1 expression increases in EAE, suggesting that it is an adaptive response. In this study, we investigated the potential function of SIRT1 in regulating EAE using SIRT1-overexpressing mice. The current studies examine potential neuroprotective and immunomodulatory effects of SIRT1 overexpression in chronic EAE induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35-55. SIRT1 suppressed EAE clinical symptoms compared with wild-type EAE mice and prevented or altered the phenotype of inflammation in spinal cords; as a result, demyelination and axonal injury were reduced. Significant neuroprotective effects were observed, with fewer apoptotic cells found in the spinal cords of SIRT1-overexpressing EAE mice associated with increased brain-derived neurotrophic factor and NAD levels. Earlier, we showed that brain-derived neurotrophic factor and NAD play crucial neuroprotective roles in EAE. These results suggest that SIRT1 reduces neuronal loss in this chronic demyelinating disease model and that this is associated with a reduction in inflammation.

Use of engineered bone marrow stem cells to deliver brain derived neurotrophic factor under the control of a tetracycline sensitive response element in experimental allergic encephalomyelitis.

Brain derived neurotrophic factor (BDNF) has neuroprotective properties but its use has been limited by poor penetration of the blood brain barrier. Treatment using bone marrow stem cells (BMSC) or retroviruses as vectors reduces the clinical and pathological severity of experimental allergic encephalomyelitis (EAE). We have refined the BMSC based delivery system by introducing a tetracycline sensitive response element to control BDNF expression. We have now tested that construct in EAE and have shown a reduction in both the clinical and pathological severity of the disease. Further, we looked for changes in sirtuin1 and nicotinamide phosphoribosyltransferase expression that would be consistent with a neuroprotective effect.

C5b-9-activated, K(v)1.3 channels mediate oligodendrocyte cell cycle activation and dedifferentiation.

Voltage-gated potassium (K(v)) channels play an important role in the regulation of growth factor-induced cell proliferation. We have previously shown that cell cycle activation is induced in oligodendrocytes (OLGs) by complement C5b-9, but the role of K(v) channels in these cells had not been investigated. Differentiated OLGs were found to express K(v)1.4 channels, but little K(v)1.3. Exposure of OLGs to C5b-9 modulated K(v)1.3 functional channels and increased protein expression, whereas C5b6 had no effect. Pretreatment with the recombinant scorpion toxin rOsK-1, a highly selective K(v)1.3 inhibitor, blocked the expression of K(v)1.3 induced by C5b-9. rOsK-1 inhibited Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. These data strongly suggest a role for K(v)1.3 in controlling the Akt activation induced by C5b-9. Since Akt plays a major role in C5b-9-induced cell cycle activation, we also investigated the effect of inhibiting K(v)1.3 channels on DNA synthesis. rOsK-1 significantly inhibited the DNA synthesis induced by C5b-9 in OLG, indicating that K(v)1.3 plays an important role in the C5b-9-induced cell cycle. In addition, C5b-9-mediated myelin basic protein and proteolipid protein mRNA decay was completely abrogated by inhibition of K(v)1.3 expression. In the brains of multiple sclerosis patients, C5b-9 co-localized with NG2(+) OLG progenitor cells that expressed K(v)1.3 channels. Taken together, these data suggest that K(v)1.3 channels play an important role in controlling C5b-9-induced cell cycle activation and OLG dedifferentiation, both in vitro and in vivo.

Patents related to therapeutic activation of K(ATP) and K(2P) potassium channels for neuroprotection: ischemic/hypoxic/anoxic injury and general anesthetics.

BACKGROUND: Mechanisms of neuroprotection encompass energy deficits in brain arising from insufficient oxygen and glucose levels following respiratory failure; ischemia or stroke, which produce metabolic stresses that lead to unconsciousness and seizures; and the effects of general anesthetics. Foremost among those K(+) channels viewed as important for neuroprotection are ATP-sensitive (K(ATP)) channels, which belong to the family of inwardly rectifying K(+) channels (K(ir)) and contain a sulfonylurea subunit (SUR1 or SUR2) combined with either K(ir)6.1 (KCNJ8) or K(ir)6.2 (KCNJ11) channel pore-forming alpha-subunits, and various members of the tandem two-pore or background (K(2P)) K(+) channel family, including K(2P)1.1 (KCNK1 or TWIK1), K(2P)2.1 (KCNK2 or TREK/TREK1), K(2P)3.1 (KCNK3 or TASK), K(2P)4.1 (KCNK4 or TRAAK), and K(2P)10.1 (KCNK10 or TREK2). OBJECTIVES: This review covers patents and patent applications related to inventions of therapeutics, compound screening methods and diagnostics, including K(ATP) channel openers and blockers, as well as K(ATP) and K(2P) nucleic/amino acid sequences and proteins, vectors, transformed cells and transgenic animals. Although the focus of this patent review is on brain and neuroprotection, patents covering inventions of K(ATP) channel openers for cardioprotection, diabetes mellitus and obesity, where relevant, are addressed. RESULTS/CONCLUSIONS: Overall, an important emerging therapeutic mechanism underlying neuroprotection is activation/opening of K(ATP) and K(2P) channels. To this end substantial progress has been made in identifying and patenting agents that target K(ATP) channels. However, current K(2P) channels patents encompass compound screening and diagnostics methodologies, reflecting an earlier 'discovery' stage (target identification/validation) than K(ATP) in the drug development pipeline; this reveals a wide-open field for the discovery and development of K(2P)-targeting compounds.

The human WW45 protein enhances MST1-mediated apoptosis in vivo.

Mammalian sterile 20-like kinase 1 (MST1) is a serine/threonine protein kinase that is activated in response to a variety of apoptotic stimuli and causes apoptosis when over-expressed in mammalian cells. The physiological regulation and cellular targets of MST1 are not well understood. Using a yeast two-hybrid system, we identified human WW45 (hWW45, also called hSav1) as an MST1-binding protein. The association between the two proteins was confirmed by immunofluorescence and co-immunoprecipitation, and hWW45 was present in both the cytoplasm and nucleus. When hWW45 alone was over-expressed, it weakly induced apoptosis. However, hWW45 augmented MST1-induced apoptosis when the two were co-expressed. Conversely, RNA interference-mediated depletion of endogenous hWW45 suppressed MST1-induced apoptosis. These results indicate that hWW45 is required to enhance MST1-mediated apoptosis in vivo and thus is a critical player in an MST1-driven cell death signaling pathway.

Dendritic cells are abundant in non-lesional gray matter in multiple sclerosis.

We have analyzed the localization of dendritic cells (DCs) in non-lesional gray matter (NLGM) in comparison to non-lesional white matter (NLWM) and acute or chronic active multiple sclerosis (MS) lesions. Immunohistochemistry was performed on cryostat sections for DCs markers (CD209, CD205, CD83) and other markers for inflammatory cells (CD68, CD8, CD4, CD3, CCR7, CCR5). We found cells expressing CD209 and containing myelin basic protein in both perivascular and parenchymal areas of NLGM. Our findings showing the expression of CD209(+) cells in NLGM parenchymal areas are surprising relative to the previous literature which reported the presence of CD209(+) DCs only in MS plaque perivascular areas. Although less numerous than CD209(+) cells, NLGM cells expressing mature DCs marker CD205 were consistently detected in perivascular cuffs of most lesions. In double labeling experiments, some but not all of the CD209(+) cells also expressed CD68 and CCR5. We also found CD209(+) cells in close contact with CD3(+) lymphocytes suggesting that DCs might contribute to the local activation of pathogenic T cells in the NLGM. Since injury to the NLGM is one of the key factors associated with disability accumulation, targeting DCs may represent a possible new therapeutic approach in MS to prevent disease progression.

Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells.

Apoptosis results in cell death within 10 min after initiation by Bcl-2 family proteins and mitochondria; however, cells enter the apoptotic pathway at different elapsed times after being triggered. Intrinsic factors related to chemical or physical cell damage can initiate apoptosis at a specific cell cycle phase; it is not clear whether cells insulted via an extrinsic pathway also die at a specific cell cycle phase, or how apoptosis is related to cell cycle progression in cells. To illustrate the kinetic changes of apoptosis during cell cycle progression, we examined both intrinsically and extrinsically induced apoptosis in MOLT-4 and Jurkat lymphocytic leukemia cells and in cultured peripheral blood lymphocytes (PBLs) using a recently modified annexin V and propidium iodide method, which detects cell cycle-specific apoptosis. Apoptosis predominantly occurred at a specific cell cycle phase. Leukemia cells were sensitive to induction by both intrinsic (X-rays, UV light, camptothecin, arsenic trioxide, and the traditional Chinese medicine Jinke, which is an extract of Auricularia auricula) and extrinsic factors (via Fas and TNF receptor pathways). The phase at which leukemia cells entered apoptosis depended on the nature of the insult (X-ray or UV, G1-phase; camptothecin, S-phase; arsenic, G1/S phases; Jinke, G1/S phases; and TNF or Fas ligand, G1/S phases), whereas PBLs did not exhibit such insult-dependent differences. PHA-stimulated PBLs entered apoptosis, and additional cells were recruited following additional insults. Unstimulated PBLs remained unresponsive to apoptosis, and proliferating cells became insensitive to the insults after the cell cycle checkpoint was abolished by caffeine. Confluent or starving PBLs were also unresponsive to apoptotic triggers. Thus, apoptotic cell death is a cell cycle event with most, if not all, apoptosis being initiated during a particular cell cycle phase, and changes in the cell cycle result in changes in the apoptotic pattern and schedule. The coordination of apoptosis and proliferation in cells offers a mechanism for the integration of both cell cycle and apoptotic signals.

Evaluation of combination gene therapy with PTEN and antisense hTERT for malignant glioma in vitro and xenografts.

Telomerase activation is a critical event in cell immortalization, and an increase in human telomerase reverse transcriptase (hTERT) expression is the key step in activating telomerase. The phosphatase and tensin homolog (PTEN) gene encodes a double-specific phosphatase that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death. Here, we evaluated a combined PTEN and antisense hTERT gene therapy for experimental glioma in vitro and in vivo. We demonstrated that infection with antisense-hTERT and wild-type-PTEN adenoviruses significantly inhibited human U251 glioma cell proliferation in vitro and glioma growth in a xenograft mouse model. The efficacy of therapy was obviously higher in the tumor xenografts infected with both PTEN and antisense hTERT than in the gliomas infected with either agent alone at the same total viral dose. Consistent with these results, we showed that telomerase activity and hTERT protein levels were markedly reduced in the glioma cells following adenovirus infection. In contrast, the levels of PTEN protein expression were dramatically increased in these cells. Our data indicate that combination treatment with antisense hTERT and wild-type PTEN effectively suppresses the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach in glioma gene therapy that warrants further investigation.

Potassium channel blockers and openers as CNS neurologic therapeutic agents.

Potassium (K+) channels are the most heterogeneous and widely distributed class of ion channels. K(+) channels are dynamic pore-forming transmembrane proteins known to play important roles in all cell types underlying both normal and pathophysiological functions. Essential for such diverse physiological processes as nerve impulse propagation, muscle contraction, cellular activation and the secretion of biologically active molecules, various K(+) channels are recognized as potential therapeutic targets in the treatment of multiple sclerosis, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, brain tumors, brain/spinal cord ischemia, pain and schizophrenia, migraine, as well as cardiac arrhythmias, pulmonary hypertension, diabetes, cervical cancer, urological diseases and sepsis. In addition to their importance as therapeutic targets, certain K(+) channels are gaining attention for their beneficial roles in anesthesia, neuroprotection and cardioprotection. The K(+) channel gene families (subdividing into multiple subfamilies) include voltage-gated (K(v): K(v)1-K(v)12 or KCNA-KCND, KCNF-KCNH, KCNQ, KCNS), calcium-activated (K(Ca): K(Ca)1-K(Ca)5 or KCNM-KCNN), inwardly rectifying (K(ir): K(ir)1-K(ir)7 or KCNJ) and background/leak or tandem 2-pore (K(2P): K(2P)1-K(2P)7, K(2P)9-K(2P)10, K(2P)12-K(2P)13, K(2P)15-K(2P)18 or KCNK) K(+) channels. Worldwide, the pharmaceutical industry is actively developing better strategies for targeting ion channels, in general, and K(+) channels, in particular, already generating over $6 billion in sales per annum from drugs designed to block or modulate ion channel function. This review provides an overview of recent patents on emerging K(+) channel blockers and activators (openers) with potential for development as new and improved nervous system therapeutic agents.

Voltage-gated potassium channels in multiple sclerosis: Overview and new implications for treatment of central nervous system inflammation and degeneration.

Inflammatory tissue damage and the presence of reactive immunocompetent T lymphocytes, macrophages, microglia, and dendritic cells (DCs) are characteristic features in the human chronic inflammatory demyelinating disease, multiple sclerosis (MS). Together, these cells orchestrate the inflammation and immunopathogenesis underlying the MS autoimmune disease processes and all up-regulate the same voltage-gated potassium (K(v)) channel, K(v)1.3, when fully activated. Only microglia, which mediate central nervous system (CNS) inflammatory processes (possibly playing a dual role of CNS protection and mediation of neuroinflammation/ neurodegeneration), and DC, which are pivotal to the induction of T cell responses, express the distinct K(v)1.5 prior to K(v)1.3 up-regulation. Although the precise functional roles of first K(v)1.5 and then K(v)1.3 channels are unclear, their differential expression is likely a common mechanism used by both microglia and DC, revealing K(v)1.5 (in addition to K(v)1.3) as a potentially important target for the development of new immunomodulatory therapies in MS.

Potassium channels Kv1.3 and Kv1.5 are expressed on blood-derived dendritic cells in the central nervous system.

OBJECTIVE: Potassium (K(+)) channels on immune cells have gained attention recently as promising targets of therapy for immune-mediated neurological diseases such as multiple sclerosis (MS). We examined K(+) channels on dendritic cells (DCs), which infiltrate the brain in MS and may impact disease course. METHODS: We identified K(+) channels on blood-derived DCs by whole-cell patch-clamp analysis, confirmed by immunofluorescent staining. We also stained K(+) channels in brain sections from MS patients and control subjects. To test functionality, we blocked K(v)1.3 and K(v)1.5 in stimulated DCs with pharmacological blockers or with an inducible dominant-negative K(v)1.x adenovirus construct and analyzed changes in costimulatory molecule upregulation. RESULTS: Electrophysiological analysis of DCs showed an inward-rectifying K(+) current early after stimulation, replaced by a mix of voltage-gated K(v)1.3- and K(v)1.5-like channels at later stages of maturation. K(v)1.3 and K(v)1.5 were also highly expressed on DCs infiltrating MS brain tissue. Of note, we found that CD83, CD80, CD86, CD40, and interleukin-12 upregulation were significantly impaired on K(v)1.3 and K(v)1.5 blockade. INTERPRETATION: These data support a functional role of K(v)1.5 and K(v)1.3 on activated human DCs and further define the mechanisms by which K(+) channel blockade may act to suppress immune-mediated neurological diseases.

Potassium channel blockers in multiple sclerosis: neuronal Kv channels and effects of symptomatic treatment.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by demyelination, with a relative sparing of axons. In MS patients, many neurologic signs and symptoms have been attributed to the underlying conduction deficits. The idea that neurologic function might be improved if conduction could be restored in CNS demyelinated axons led to the testing of potassium (K(+)) channel blockers as a symptomatic treatment. To date, only 2 broad-spectrum K(+) channel blockers, 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP), have been tested in MS patients. Although both 4-AP and 3,4-DAP produce clear neurologic benefits, their use has been limited by toxicity. Here we review the current status of basic science and clinical research related to the therapeutic targeting of voltage-gated K(+) channels (K(v)) in MS. By bringing together 3 distinct but interrelated disciplines, we aim to provide perspective on a vast body of work highlighting the lengthy and ongoing process entailed in translating fundamental K(v) channel knowledge into new clinical treatments for patients with MS and other demyelinating diseases. Covered are (1) K(v) channel nomenclature, structure, function, and pharmacology; (2) classic and current experimental morphology and neurophysiology studies of demyelination and conduction deficits; and (3) a comprehensive overview of clinical trials utilizing 4-AP and 3,4-DAP in MS patients.

The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain.

Multiple Sclerosis (MS) is characterized by central nervous system perivenular and parenchymal mononuclear cell infiltrates consisting of activated T cells and macrophages. We recently demonstrated that elevated expression of the voltage-gated potassium channel, Kv1.3, is a functional marker of activated effector memory T (T(EM)) cells in experimental allergic encephalomyelitis and in myelin-specific T cells derived from the peripheral blood of patients with MS. Herein, we show that Kv1.3 is highly expressed in postmortem MS brain inflammatory infiltrates. The expression pattern revealed not only Kv1.3(+) T cells in the perivenular infiltrate but also high expression in the parenchyma of demyelinated MS lesions and both normal appearing gray and white matter. These cells were uniformly chemokine receptor 7 negative (CCR7(-)), consistent with an effector memory phenotype. Using double-labeling immunohistochemistry and confocal microscopy, we demonstrated colocalization of Kv1.3 with CD3, CD4, CD8, and some CD68 cells. The expression patterns mirrored in vitro experiments showing polarization of Kv1.3 to the immunological synapse. Kv1.3 was expressed in low to moderate levels on CCR7(+) central memory T cells from cerebrospinal fluid, but, when these cells were stimulated in vitro, they rapidly became Kv1.3(high)/CCR7(-) T(EM), suggesting that a subset of cerebrospinal fluid cells existed in a primed state ready to become T(EM). These studies provide further rationale for the use of specific Kv1.3 antagonists in MS.

Determinants of 4-aminopyridine sensitivity in a human brain kv1.4 k(+) channel: phenylalanine substitutions in leucine heptad repeat region stabilize channel closed state.

The biophysical and pharmacological effects of individual phenylalanine-for-leucine (Phe-for-Leu) substitutions in the leucine heptad repeat region located at the cytosolic surface of the channel pore, on whole-cell K(+) currents, were studied in cloned and mutated human brain Kvl.4 K(+) channels (hKvl.4) transiently transfected into HeLa cells. Although L2 and L5 are not considered part of the 4-aminopyridine (4-AP) binding site, unlike the L4 heptad leucine, Phe substitutions at L2 (L464) or L5 (L485) increase 4-AP sensitivity by 400-fold, as seen previously in the L4F mutant channel. Greater depolarizing shifts manifest in the voltage dependence of activation and inactivation in L2F (20 mV) and L5F (30 mV) than in L4F (10 mV) relative to hKv1.4. L1F (L457) and L3F (L471) increase 4-AP sensitivity by 8- and 150-fold, respectively, and produce depolarizing shifts in activation of approximately 5 mV without affecting inactivation. The apparent free energy differences of 4-AP binding in each mutant suggest enhanced drug-channel interactions (L2F > or = L4F > or = L5F > L3F > L1F). Deactivation kinetics are accelerated in L2F (11-fold), L5F (8-fold), L1F (5-fold), and L3F (2-fold), at -50 mV. All Phe-for-heptad-Leu substitutions produce gating changes suggesting variable stabilization of the channel closed state conformation, with L1F, L2F, and L5F exhibiting the strongest correlations between altered gating and increased 4-AP sensitivity. If 4-AP blocks the open channel by promoting closure of the activation gate (recent Armstrong-Loboda model), then changes in the leucine heptad repeat that stabilize the channel closed state may contribute to increased 4-AP sensitivity by amplifying the mechanism of 4-AP block.