Enantioselective synthesis and selective functionalization of 4-aminotetrahydroquinolines as novel GLP-1 secretagogues.

Polysubstituted tetrahydroquinolines were obtained in moderate to high yields (28% to 92%) and enantiomeric ratios (er 89:11 to 99:1) by a three-component Povarov reaction using a chiral phosphoric acid catalyst. Significantly, post-Povarov functional group interconversions allowed a rapid access to a library of 36 enantioenriched 4-aminotetrahydroquinoline derivatives featuring five points of diversity. Selected analogs were assayed for their ability to function as glucagon-like peptide-1 (GLP-1) secretagogues.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

A Topical Mitochondria-Targeted Redox-Cycling Nitroxide Mitigates Oxidative Stress-Induced Skin Damage.

Skin is the largest human organ, and it provides a first line of defense that includes physical, chemical, and immune mechanisms to combat environmental stress. Radiation is a prevalent environmental stressor. Radiation-induced skin damage ranges from photoaging and cutaneous carcinogenesis caused by UV exposure, to treatment-limiting radiation dermatitis associated with radiotherapy, to cutaneous radiation syndrome, a frequently fatal consequence of exposures from nuclear accidents. The major mechanism of skin injury common to these exposures is radiation-induced oxidative stress. Efforts to prevent or mitigate radiation damage have included development of antioxidants capable of reducing reactive oxygen species. Mitochondria are particularly susceptible to oxidative stress, and mitochondrial-dependent apoptosis plays a major role in radiation-induced tissue damage. We reasoned that targeting a redox cycling nitroxide to mitochondria could prevent reactive oxygen species accumulation, limiting downstream oxidative damage and preserving mitochondrial function. Here we show that in both mouse and human skin, topical application of a mitochondrially targeted antioxidant prevents and mitigates radiation-induced skin damage characterized by clinical dermatitis, loss of barrier function, inflammation, and fibrosis. Further, damage mitigation is associated with reduced apoptosis, preservation of the skin's antioxidant capacity, and reduction of irreversible DNA and protein oxidation associated with oxidative stress.

Small Molecule Antagonists of the Nuclear Androgen Receptor for the Treatment of Castration-Resistant Prostate Cancer.

After a high-throughput screening campaign identified thioether 1 as an antagonist of the nuclear androgen receptor, a zone model was developed for structure-activity relationship (SAR) purposes and analogues were synthesized and evaluated in a cell-based luciferase assay. A novel thioether isostere, cyclopropane (1S,2R)-27, showed the desired increased potency and structural properties (stereospecific SAR response, absence of a readily oxidized sulfur atom, low molecular weight, reduced number of flexible bonds and polar surface area, and drug-likeness score) in the prostate-specific antigen luciferase assay in C4-2-PSA-rl cells to qualify as a new lead structure for prostate cancer drug development.

Deciphering of mitochondrial cardiolipin oxidative signaling in cerebral ischemia-reperfusion.

It is believed that biosynthesis of lipid mediators in the central nervous system after cerebral ischemia-reperfusion starts with phospholipid hydrolysis by calcium-dependent phospholipases and is followed by oxygenation of released fatty acids (FAs). Here, we report an alternative pathway whereby cereberal ischemia-reperfusion triggered oxygenation of a mitochondria-specific phospholipid, cardiolipin (CL), is followed by its hydrolysis to yield monolyso-CLs and oxygenated derivatives of fatty (linoleic) acids. We used a model of global cerebral ischemia-reperfusion characterized by 9 minutes of asphyxia leading to asystole followed by cardiopulmonary resuscitation in postnatal day 17 rats. Global ischemia and cardiopulmonary resuscitation resulted in: (1) selective oxidation and hydrolysis of CLs, (2) accumulation of lyso-CLs and oxygenated free FAs, (3) activation of caspase 3/7 in the brain, and (4) motor and cognitive dysfunction. On the basis of these findings, we used a mitochondria targeted nitroxide electron scavenger, which prevented CL oxidation and subsequent hydrolysis, attenuated caspase activation, and improved neurocognitive outcome when administered after cardiac arrest. These data show that calcium-independent CL oxidation and subsequent hydrolysis represent a previously unidentified pathogenic mechanism of brain injury incurred by ischemia-reperfusion and a clinically relevant therapeutic target.

Mitochondria-targeted ROS scavenger improves post-ischemic recovery of cardiac function and attenuates mitochondrial abnormalities in aged rats.

Mitochondria-generated reactive oxygen species (ROS) play a crucial role in the pathogenesis of aging and age-associated diseases. In this study, we evaluated the effects of XJB-5-131 (XJB), a mitochondria-targeted ROS and electron scavenger, on cardiac resistance to ischemia-reperfusion (IR)-induced oxidative stress in aged rats. Male adult (5-month old, n=17) and aged (29-month old, n=19) Fischer Brown Norway (F344/BN) rats were randomly assigned to the following groups: adult (A), adult+XJB (AX), aged (O), and aged+XJB (OX). XJB was administered 3 times per week (3mg/kg body weight, IP) for four weeks. At the end of the treatment period, cardiac function was continuously monitored in excised hearts using the Langendorff technique for 30 min, followed by 20 min of global ischemia, and 60-min reperfusion. XJB improved post-ischemic recovery of aged hearts, as evidenced by greater left ventricular developed-pressures and rate-pressure products than the untreated, aged-matched group. The state 3 respiration rates at complexes I, II and IV of mitochondria isolated from XJB-treated aged hearts were 57% (P<0.05), 25% (P<0.05) and 28% (P<0.05), respectively, higher than controls. Ca(2+)-induced swelling, an indicator of permeability transition pore opening, was reduced in the mitochondria of XJB-treated aged rats. In addition, XJB significantly attenuated the H2O2-induced depolarization of the mitochondrial inner membrane as well as the total and mitochondrial ROS levels in cultured cardiomyocytes. This study underlines the importance of mitochondrial ROS in aging-induced cardiac dysfunction and suggests that targeting mitochondrial ROS may be an effective therapeutic approach to protect the aged heart against IR injury.

An uncharged oxetanyl sulfoxide as a covalent modifier for improving aqueous solubility.

Low aqueous solubility is a common challenge in drug discovery and development and can lead to inconclusive biological assay results. Attaching small, polar groups that do not interfere with the bioactivity of the pharmacophore often improves solubility, but there is a dearth of viable neutral moieties available for this purpose. We have modified several poorly soluble drugs or drug candidates with the oxetanyl sulfoxide moiety of the DMSO analog MMS-350 and noted in most cases a moderate to large improvement of aqueous solubility. Furthermore, the membrane permeability of a test sample was enhanced compared to the parent compound.

Can Radiosensitivity Associated with Defects in DNA Repair be Overcome by Mitochondrial-Targeted Antioxidant Radioprotectors.

Radiation oncologists have observed variation in normal tissue responses between patients in many instances with no apparent explanation. The association of clinical tissue radiosensitivity with specific genetic repair defects (Wegner's syndrome, Ataxia telangiectasia, Bloom's syndrome, and Fanconi anemia) has been well established, but there are unexplained differences between patients in the general population with respect to the intensity and rapidity of appearance of normal tissue toxicity including radiation dermatitis, oral cavity mucositis, esophagitis, as well as differences in response of normal tissues to standard analgesic or other palliative measures. Strategies for the use of clinical radioprotectors have included modalities designed to either prevent and/or palliate the consequences of radiosensitivity. Most prominently, modification of total dose, fraction size, or total time of treatment delivery has been necessary in many patients, but such modifications may reduce the likelihood of local control and/or radiocurability. As a model system in which to study potential radioprotection by mitochondrial-targeted antioxidant small molecules, we have studied cell lines and tissues from Fanconi anemia (Fancd2(-/-)) mice of two background strains (C57BL/6NHsd and FVB/N). Both were shown to be radiosensitive with respect to clonogenic survival curves of bone marrow stromal cells in culture and severity of oral cavity mucositis during single fraction or fractionated radiotherapy. Oral administration of the antioxidant GS-nitroxide, JP4-039, provided significant radioprotection, and also ameliorated distant bone marrow suppression (abscopal effect of irradiation) in Fancd2 (-/-) mice. These data suggest that radiation protection by targeting the mitochondria may be of therapeutic benefit even in the setting of defects in the DNA repair process for irradiation-induced DNA double strand breaks.

Pharmacologic profiling of phosphoinositide 3-kinase inhibitors as mitigators of ionizing radiation-induced cell death.

Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death.

Pharmacological mitigation of tissue damage during brain microdialysis.

Microdialysis sampling in the brain is employed frequently in the chemical analysis of neurological function and disease, but implanting the probes, which are substantially larger than the size and spacing of brain cells and blood vessels, is injurious and triggers ischemia, gliosis, and cell death at the sampling site. The nature of the interface between the brain and the microdialysis probe is critical to the use of microdialysis as a neurochemical analysis technique. The objective of the work reported here was to investigate the potential of two compounds, dexamethasone, a glucocorticoid anti-inflammatory agent, and XJB-5-131, a mitochondrially targeted reactive oxygen species scavenger, to mitigate the penetration injury. Measurements were performed in the rat brain striatum, which is densely innervated by axons that release dopamine, an electroactive neurotransmitter. We used voltammetry to measure electrically evoked dopamine release next to microdialysis probes during the retrodialysis of dexamethasone or XJB-5-131. After the in vivo measurements, the brain tissue containing the microdialysis probe tracks was examined by fluorescence microscopy using markers for ischemia, neuronal nuclei, macrophages, and dopamine axons and terminals. Dexamethasone and XJB-5-131 each diminished the loss of evoked dopamine activity, diminished ischemia, diminished the loss of neuronal nuclei, diminished the appearance of extravasated macrophages, and diminished the loss of dopamine axons and terminals next to the probes. Our findings confirm the ability of dexamethasone and XJB-5-131 to mitigate, but not eliminate, the effects of the penetration injury caused by implanting microdialysis probes into brain tissue.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells.

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria.

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

Bridged tetrahydroisoquinolines as selective NADPH oxidase 2 (Nox2) inhibitors.

(1SR,4RS)-3,3-Dimethyl-1,2,3,4-tetrahydro-1,4-(epiminomethano)naphthalenes were synthesized in 2-3 steps from commercially available materials and assessed for specificity and effectiveness across a range of Nox isoforms. The N-pentyl and N-methylenethiophene substituted analogs 11g and 11h emerged as selective Nox2 inhibitors with cellular IC50 values of 20 and 32 µM, respectively.

Targeting of XJB-5-131 to mitochondria suppresses oxidative DNA damage and motor decline in a mouse model of Huntington's disease.

Oxidative damage and mitochondrial dysfunction are implicated in aging and age-related neurodegenerative diseases, including Huntington's disease (HD). Many naturally occurring antioxidants have been tested for their ability to correct for deleterious effects of reactive oxygen species, but often they lack specificity, are tissue variable, and have marginal efficacy in human clinical trials. To increase specificity and efficacy, we have designed a synthetic antioxidant, XJB-5-131, to target mitochondria. We demonstrate in a mouse model of HD that XJB-5-131 has remarkably beneficial effects. XJB-5-131 reduces oxidative damage to mitochondrial DNA, maintains mitochondrial DNA copy number, suppresses motor decline and weight loss, enhances neuronal survival, and improves mitochondrial function. The findings poise XJB-5-131 as a promising therapeutic compound.

Allylic Amines as Key Building Blocks in the Synthesis of (E)-Alkene Peptide Isosteres.

Nucleophilic imine additions with vinyl organometallics have developed into efficient, high yielding, and robust methodologies to generate structurally diverse allylic amines. We have used the hydrozirconation-transmetalation-imine addition protocol in the synthesis of allylic amine intermediates for peptide bond isosteres, phosphatase inhibitors, and mitochondria-targeted peptide mimetics. The gramicidin S-derived XJB-5-131 and JP4-039 and their analogs have been prepared on up to 160 g scale for preclinical studies. These (E)-alkene peptide isosteres adopt type II' ß-turn secondary structures and display impressive biological properties, including selective reactions with reactive oxygen species (ROS) and prevention of apoptosis.