Patent Application
20140024677
The sequence listing that is contained in the file named “MESCP0063US_ST25.txt”, which is 4 KB (as measured in Microsoft Windows®) and was created on Jun. 26, 2013, is filed herewith by electronic submission and is incorporated by reference herein.
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods and compositions for inducing mitochondrial biogenesis.
2. Description of Related Art
Mitochondrial dysfunction is associated with the etiology of multiple diseases including Alzheimer's disease, Parkinson's disease, diabetes, as well as renal, liver and myocardial injury (Hagen et al., 2002; Baloyannis, 2006; Civitarese and Ravussin, 2008; Seo et al., 2010; Tran et al., 2011). Mitochondrial dysfunction is also a common cause and consequence of ischemia/reperfusion, trauma, and drug/toxicant-induced organ injury. In ischemic/reperfusion and lipopolysaccharide induced acute kidney injury (AKI), the loss of mitochondrial function results in oxidative stress and persistent energy depletion (Funk et al., 2010; Tran et al., 2011). Mitochondrial damage may hinder critical energy-dependent repair mechanisms and lead to irreversible cell injury, limiting restoration of organ function. Thus, the development of therapies to promote mitochondrial biogenesis (MB) has the potential to treat multiple pathologies and restore organ function after injury.
Mitochondria are constantly being renewed through the processes of biogenesis, fusion, fission, and mitophagy (Seo et al., 2010). MB occurs under homeostatic conditions, and can be induced as an adaptive response initiated by cells to meet energetic demands resulting from injury, genetic, metabolic, and dietary events, thereby impacting health and disease (Medeiros, 2008). MB is a complex process requiring the coordination of both nuclear and mitochondrial DNA (mtDNA) that encode for mitochondrial proteins. A primary regulator of MB is the nuclear transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α). PGC-1α interacts with cAMP response element-binding protein and nuclear respiratory factors 1 and 2 to regulate the transcription of multiple genes (Wu et al., 2006). PGC-1α provides a direct link between external physiological stimuli and the regulation of MB, and is inducible both physiologically and pharmacologically.
There are a limited number of chemicals known to induce MB. The Spiegelman group conducted a high-throughput screen that examined the effects of 3,000 compounds in skeletal muscle cells and 82 compounds (2.5%) increased PGC-1α mRNA (Arany et al., 2008). More recently, isoflavones, a 5HT2 agonist (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride; DOI), and a SIRT1 activator (N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide; SRT1720) can MB in renal proximal tubular cells (Rasbach and Schnellmann, 2008; Funk et al., 2010; Rasbach et al., 2010). Furthermore, the inventors determined that stimulation of MB after the initiation of cellular injury accelerates cell repair and regeneration. For example, after t-butylhydroperoxide (TBHP) induced oxidant injury, exposure of primary renal proximal tubule cells (RPTC) to DOI, SRT1720 or over expression of PGC1-α accelerated recovery of mitochondrial and ATP-dependent cellular functions (Rasbach and Schnellmann, 2007; Funk et al., 2010; Rasbach et al., 2010). Therefore, targeted stimulation of MB may be a valuable approach in the development of new therapies for the treatment of injury and disease characterized by mitochondrial impairment (Funk et al., 2010).
Acute kidney injury (AKI) is a clinical disorder characterized by a rapid decrease in kidney function and subsequent retention of nitrogenous waste products, metabolic acids, and increased potassium and phosphate concentrations. AKI is prevalent in approximately 60% of patients during intensive care admission and has a mortality rate of up to 90% in critically ill and aged patients. In addition, AKI is costly to treat and has become a significant financial burden on the healthcare system. Current treatments are limited to mechanical support by dialysis. Historically, the vast majority of research efforts for AKI has focused on pretreatments, and is less clinically relevant as AKI presents as an unpredictable acute onset. Taken together the high mortality rates, financial burden, and limitations in treatment demonstrate a significant clinical need for discovery of novel approaches to therapeutics that promote recovery of renal function following AKI.
The present invention overcomes limitations in the prior art by providing new compounds and methods for inducing mitochondrial biogenesis in a subject, e.g., a patient with a mitochondrial dysfunction or disease. In certain aspects, specific β-2 adrenergic receptor agonists and norepinepherine reuptake inhibitors have been identified that can induce mitochondrial biogenesis. In various embodiments, mitochondrial biogenesis may be induced in vitro or in vivo. Specific compounds have been identified which may be used to treat muscle wasting or acute kidney injury (AKI).
An aspect of the present invention relates to a method of inducing mitochondrial biogenesis in a subject, comprising administering to a subject, identified as in need of increased mitochondrial biogenesis, a pharmacologically effective dose of compound, wherein the compound is a beta-2 adrenergic receptor agonist or a norepinepherine reuptake inhibitor. The beta 2 adrenergic receptor agonist may be tomoxetine, nisoxetine, fenoterol, formoterol, or procaterol. In some embodiments, the method may comprise administering to the subject a pharmacologically effective dose of tomoxetine, nisoxetine, fenoterol, formoterol, or procaterol. The compound may be a beta-2 adrenergic receptor agonist. The beta-2 adrenergic receptor agonist may be fenoterol, formoterol, terbutaline, ritodrine, cyclopentylbutanephrine, metaproteranol, procaterol, nisoxetine, or tomoxetine. In some embodiments, the beta-2 adrenergic receptor agonist is not formoterol. In some embodiments, the compound is a norepinepherine reuptake inhibitor. The norepinepherine reuptake inhibitor may be nisoxetine or tomoxetine. The compound may be a compound from Table 1, such as, e.g., CB—3 6431725 or CB—2 5144525. The compound may be administered to the subject orally, intravenously, intramuscularly, intraperitoneally, topically, or via inhalation. The compound may be comprised in a pharmaceutically acceptable formulation.
In some embodiments, the subject is a human. In some embodiments, the subject has been identified as having deficient mitochondrial biogenesis by a muscle biopsy test, a metabolic test, or a genetic test. For example, the blood test may comprise, e.g., evaluation of basic chemistries, a complete blood count, analysis of lactate and/or pyruvate, analysis of amino acids, ammonia levels, creatinine kinase (CPK) levels, plasma acylcarnitine analysis, or analysis of urine organic acids. The muscle biopsy test may comprise evaluation of liver tissue respirometry, muscle pathology, oxidative phosphorylation enzymology, protein chemistry, or COQ10 levels. The genetic testing may evaluate sequencing mitochondrial DNA or nuclear DNA. The subject may have a mitochondrial injury. The mitochondrial injury may result from neurodegeneration, a heart disease, heart attack, a stroke, renal dysfunction, type 2 diabetes, a central nervous system disorder, Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemia/reperfusion, trauma, a drug/toxicant-induced organ injury, a traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), or an acute kidney injury (AKI). The subject has a mitochondrial disease. For example, the mitochondrial disease may be Leber's hereditary optic neuropathy, diabetes mellitus, a mental disorder or disease, Leigh's disease, mitochondrial encephalomyopathy, lactic acidosis, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), “Neuropathy, ataxia, retinitis pigmentosa, and ptosis” (NARP), Wolff-Parkinson-White syndrome, or stroke-like episodes (MELAS), or a muscle wasting disease.
Another aspect of the present invention relates to a method of treating an acute kidney injury in an individual comprising administering to the individual a pharmacologically effective amount of tomoxetine, nisoxetine, fenoterol, formoterol, or procaterol. In some embodiments, a pharmacologically effective amount of tomoxetine, nisoxetine, fenoterol, or procaterol is administered to the subject. The subject may be a human.
Yet another aspect of the present invention relates to a method of treating muscle wasting or a muscle wasting disease in an individual comprising administering to the individual a pharmacologically effective amount of tomoxetine, nisoxetine, fenoterol, or procaterol. In some embodiments, the muscle wasting disease is cachexia (e.g., as a result of cancer, increased age, spinal injury, etc.), muscle atrophy resulting from cancer (cancer cachexia), wasting disease, or muscle atrophy resulting from a spinal cord injury or neurological injury or disease. The cachexia may result from a chronic illness such as type I diabetes, multiple sclerosis, HIV, or cancer. The subject may be a human.
In some embodiments, tomoxetine((3R)-N-methyl-3-(2-methylphenoxy)-3-phenylpropan-1-amine; (R)-N-methyl-3-phenyl-3-(o-tolyloxy)propan-1-amine; also called Atomoxetine) may be administered orally, e.g., to a human subject at a dosage of about 10-100 mg/day to promote mitochondrial biogenesis. In some embodiments, 20-60 mg/day of tomoxetine may be administered orally to a human subject to promote mitochondrial biogenesis or treat a disease as described herein. In some embodiments, a dosage of 0.25-2 mg/kg/day, or 0.5-1.2 mg/kg/day may be administered to an individual to promote mitochondrial biogenesis or treat a disease as described herein. In some embodiments, concentrations of tomoxetine may be used to promote mitochondrial biogenesis that are lower than the dosages of tomoxetine that are used to treat ADHD. For example, less than about 0.25 mg/kg/day tomoxetine, from about 0.05 mg/kg/day to less than about 0.25 mg/kg/day, from about 0.1 mg/kg/day to less than about 0.25 mg/kg/day, about 0.1-0.24 mg/kg/day, about 0.05-0.24 mg/kg/day, about 0.05-0.20 mg/kg/day, about 0.05-0.15 mg/kg/day, or 0.05-0.1 mg/kg/day, or any range derivable therein, may be used to induce mitochondrial biogenesis in a subject, such as a human patient.
Nisoxetine ((RS)-3-(2-methoxyphenoxy)-N-methyl-3-phenylpropan-1-amine) may be administered to a subject, such as a human subject to promote mitochondrial biogenesis. For example, a dose of about 0.05-2 mg/kg, or about 0.1 mg/kg, may be used to promote mitochondrial biogenesis or treat a disease as described herein.
In some embodiments, fenoterol ((RR,SS)-5-(1-hydroxy-2-{[2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)benzene-1,3-diol) may be administered to a subject, such as a human subject, to promote mitochondrial biogenesis or treat a disease as described herein. In some embodiments, fenoterol is delivered to a subject via aerosol delivery or orally. In some embodiments, 0.007-0.035 mg/kg may be administered to a human subject about every 6 hours via inhalation. 0.028-0.14 mg/kg/day may be administered to an individual via an aerosol delivery. A dosage of about 300-600 mcg/day may be administered to an individual. In some embodiments, about 100-200 mcg may be administered to a subject orally about every 8 hours. In some embodiments, about 0.01 mg/kg/day may be administered to a subject.
In some embodiments, procaterol ((±)-(1R,2S)-rel-8-Hydroxy-5-[1-hydroxy-2-(isopropylamino)butyl]-quinolin-2(1H)-one) may be administered to a subject, such as a human subject, to promote mitochondrial biogenesis or treat a disease as described herein. In some embodiments, 0.25-125 mcg/day, or 0.5-100 mcg/day of procaterol may be administered to an individual orally. For pediatric patients, a dose of about 0.625-2.5 ug/kg may be used. Procaterol may be administered, in various embodiments, orally or via inhalation. In some embodiments, the amount of procaterol administered to a subject to promote mitochondrial biogenesis may be lower than the dose that would be used clinically to treat asthma. For example, in some embodiments, 0.007-0.035 mg/kg may be administered to a human subject about every 6 hours via inhalation. 0.028-0.14 mg/kg/day may be administered to an individual via an aerosol delivery. A dosage of about 300-600 mcg/day may be administered to an individual. In some embodiments, about 100-200 mcg may be administered to a subject orally about every 8 hours. In some embodiments, about 0.01 mg/kg/day may be administered to a subject.
Formoterol (rac-(R,R)—N-[2-hydroxy-5-[1-hydroxy-2-[1-(4-methoxyphenyl)propan-2-ylamino]ethyl]phenyl]formamide) may be administered to a subject, such as a human subject, to promote mitochondrial biogenesis or treat a disease as described herein. The formoterol may be administered orally or via inhalation. In some embodiments, about 12-24 mcg/day may be administered to an individual, e.g., as an inhalation powder. In some embodiments, about 20-40 μg/2 mL of formoterol may be administered via aerosol once or twice per day. In some embodiments, the amount of formotorol administered to a subject to promote mitochondrial biogenesis may be lower than the dose that would be used clinically to treat asthma or chronic obstructive pulmonary disease. For example, in some embodiments, 0.007-0.035 mg/kg may be administered to a human subject about every 6 hours via inhalation. 0.028-0.14 mg/kg/day may be administered to an individual via an aerosol delivery. A dosage of about 300-600 mcg/day may be administered to an individual. In some embodiments, about 100-200 mcg may be administered to a subject orally about every 8 hours. In some embodiments, about 0.01 mg/kg/day may be administered to a subject.
The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.
The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
The use of the term “or” in the claims is used to mean “and/or ” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
As described herein, specific compounds have been identified as being particularly effective for promoting mitochondrial biogenesis. The stimulation of mitochondrial biogenesis (MB) via cell surface G-protein coupled receptors is a promising strategy for cell repair and regeneration. As shown in the below examples, the specificity and quantitative structure rationale of a panel of β2-AR agonists was evaluated for possible effects on MB. β2-AR agonists elicit three distinct phenotypes: full MB, partial MB, and non-MB. Full MB compounds represent two chemical scaffolds but three distinct chemical clusters. Clusters were subjected to pharmacophore and 2D-QSAR modeling and identified unique features conserved amongst full MB compounds and distinct features unique to MB. A novel cluster was further investigated via a combined virtual screening and experimental validation approach leading to the identification of five novel MB agents. Using informatics, the three pharmacophore models were coalesced into a consensus pharmacophore, elucidating the spatial and chemical features required to stimulate MB. Via superposition of the consensus model within the β2-AR x-ray structure theonists binding modes were used to evaluate possible MB-specific interactions. Additional studies were conducted that have shown these compounds to be particularly effective in treating diseases including muscle wasting diseases and acute kidney disease (AKI).
β-AR receptor agonists are approved therapeutic agents for the management of asthma and other diseases. The β2-AR has been successfully targeted for drug discovery using a ligand-based approach, resulting in the creation of multiple receptor-specific drugs (Wishart et al., 2006). Recently, Kolb et al. (2009), used the X-ray structure of the β2-AR to conduct a structure-based screen of nearly one million commercially available molecules. Utilizing the β2-AR crystallized in complex with the inverse agonist carazozol, the researchers identified 25 novel compounds that fit the receptor, and six of the compounds were confirmed as inverse agonists. However the therapeutic potential of β2-AR agonists as inducers of mitochondrial biogenesis (MB) has not fully been explored (Ortega and Peters, 2010).
In the below examples the inventors use a phenotypic approach recently developed by Beeson et al. (2010), to examine the potential of β2-AR agonism to induce MB in both the kidney and the heart. The Seahorse Biosciences extracellular flux analyzer was used to evaluate the effects of β2-AR agonists on maximal mitochondrial respiration in primary cultures of renal proximal tubules and cardiomyocytes, two cells highly dependent on aerobic metabolism. In vivo and ligand-based in silico studies were then employed to integrate the effects of β2-AR agonists on mitochondrial function and gene expression, with detailed analysis of chemical structure. These data were used to develop a discrete pharmacophore model capable of predicting novel compounds with MB properties.
While recent studies show that inducing mitochondrial biogenesis (MB) stimulates cell repair and regeneration, only a limited number of chemicals are known to induce MB. As shown in the examples below, to examine the impact of the β-adrenergic signaling pathway on mitochondrial biogenesis, primary renal proximal tubule cells (RPTC) and adult feline cardiomyocytes (AFC) were exposed for 24 hours to multiple β-adrenoceptor (β-AR) agonists: isoproterenol (non-selective β-AR agonist), BRL 37344 (selective β3-AR agonist), and formoterol (selective β2-AR agonist). The Seahorse Biosciences Extracellular Flux (XF) analyzer was used to quantify FCCP-uncoupled oxygen consumption rate (OCR), a marker of maximal electron transport chain activity. Isoproterinol and BRL 37244 did not significantly alter mitochondrial respiration at any of the concentrations examined. Formoterol exposure resulted in significant increases in both FCCP-uncoupled OCR and mitochondrial DNA (mtDNA) copy number. The effect of formoterol on OCR in RPTC was inhibited by the β-AR antagonist propranolol. Mice exposed to formoterol for 24 or 72 h exhibited increases in kidney and heart mtDNA copy number, PGC-1α, and multiple genes involved in the mitochondrial electron transport chain (ATP6, ND1, ND6, and NDUFB8). Cheminformatic modeling, virtual chemical library screening, and experimental validation identified two chemically similar compounds from the Sigma Library of Pharmacologically Active Compounds (tomoxetine and nisoxetine), and two from the ChemBridge DIVERSet™ that caused significant increases in mitochondrial respiratory capacity. These data provide compelling evidence for the use of β2-AR ligands for therapeutic MB.
Abbreviations Used: AFC, adult feline cardiomyocytes; ATP6, ATP synthase F0 subunit 6 of transmembrane F-type ATP synthase; β-AR, β-adrenoceptor; DOI, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; i.p., intraperitoneal injection; LOPAC, Library of Pharmacologically Active Compounds; MB, mitochondrial biogenesis; mtDNA, mitochondrial DNA; ND1, NADH dehydrogenase subunit 1; ND6, NADH dehydrogenase subunit 6; NDUFB8, NADH dehydrogenase[ubiquinone] 1 beta subcomplex subunit 8; OCR, oxygen consumption rate; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-α; RPTC, renal proximal tubule cells; SMILES, Simplified Molecular Input Line Entry Specification; SRT1720, N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide; TBHP, t-butylhydroperoxide; TC, tanimoto coefficient; XF-96, Seahorse Biosciences extracellular flux analyzer.
The mitochondrion is an intricate organelle, with components derived from both the nuclear and mitochondrial genomes, whose activity must be carefully coupled to cellular metabolism and signaling (Wagner et al., 2008). Mitochondrial health is essential for cell and organ function. Multiple diseases are characterized by mitochondrial impairment including diabetes, Alzheimer's disease, Parkinson's disease and diet-induced obesity. Ischemia/reperfusion injury of the kidney, heart and liver all show deleterious consequences on mitochondrial function. Therefore, the development of novel pharmaceuticals that induce MB may have broad usage in diverse diseases. Despite the potential for treating disorders characterized by mitochondrial impairment, very few therapies target the mitochondria to promote its function (Funk et al., 2010). Likewise, there are no current databases matching mitochondrial biogenic activity to chemical pharmacophores or specific chemotypes.
Very few studies have explored MB as a therapeutic target and those studies that have focused on skeletal muscle and assay systems using surrogate markers of MB (e.g. PGC-1α mRNA). Surrogate markers may suffer from false-positives and negatives, and the use of cell lines that are highly glycolytic with limited aerobic capacity may not be sensitive to MB. The use of primary cultures of primary RPTC grown under improved conditions resolves the cell line issue and the use of a phenotypic respirometric assay minimizes the limitations of surrogate markers. As shown below, formoterol, a potent and selective agonist for the β2-AR, stimulates MB in both RPTC and AFC within 24 h of a 30 nM exposure (
Researchers have showed that exercise-induced increases in PGC-1α mRNA in skeletal muscle, is blocked by the β2-AR inhibitor ICI 118,551 and β-AR inhibitor propranolol (Miura et al., 2007; Sutherland et al., 2009). Additionally PGC-1α in skeletal muscle 24 h increased after injection of formoterol (100 μg/kg) (Pearen et al., 2009). Here, the same dose of formoterol induced MB in the kidney and heart of male mice as indicated by increased mRNA expression of multiple mitochondrial proteins that are both nuclear and mitochondrially-encoded (NDUFB8, AT6, ND1, ND6) (
Currently there are no known pharmacophores associated with MB. The chemical structure of formoterol was utilized to search the Sigma LOPAC of 1280 compounds to find current pharmacological compounds that were similar in structure to formoterol and therefore may induce MB. 29 compounds were identified and were tested for their ability to increase the FCCP-OCR in RPTC after a 24 h 5 μM exposure. Tomoxetine and nisoxetine were identified as potent inducers (30-300 nM) of MB. Nisoxetine is reported to be a potent and selective inhibitor of noradrenaline uptake with little or no affinity for a range of other similar neurotransmitter receptors (Wong and Bymaster, 1976; Cheetham et al., 1996; Mochizucki, 2004). Tomoxetine is also a potent and selective noradrenaline re-uptake inhibitor, and is clinically used as an antidepressant (Zerbe et al., 1985). Currently there are no data suggesting a role for either nisoxetine or tomoxetine in inducing MB. Interestingly these compounds were aligned with formoterol and a preliminary pharmacophore was developed with six features common between all three compounds (
In addition to finding pharmacologically active compounds with MB activity, the formoterol-based pharmacophore was utilized to probe diverse chemical space. The pharmacophore designed from formoterol, nisoxetine and tomoxetine was used to screen the 50,000 small molecule ChemBridge DIVERSet™. The in silico screen identified only 16 compounds that matched all six chemical features that defined the pharmacophore. When tested utilizing RPTC, three of the compounds (19% accuracy in prediction, enrichment factor of 6.9) increased FCCP-OCR 24 h after a 5 μM exposure. A concentration response curve for the three compounds revealed CB2 and CB3 increased MB at 30 nM while CB1 had no effect at concentrations below 5 μM. The positive compounds were aligned, resulting in the development of a refined pharmacophore with six features present within 100% of the compounds (
There is an abundance of evidence showing that exercise induces MB in skeletal muscle, and recent data suggests that β-AR agonism is required for exercise-mediated alterations in mitochondrial function (Miura et al., 2007; Higashida et al., 2008; Sutherland et al., 2009; Little et al., 2011). However, the potential use for β2-AR agonists for the treatment of mitochondrial dysfunction and injury has not yet been explored. Although evidence suggests that chronic exposure to formoterol does decrease oxidative capacity in the heart, acute exposures to β2-AR agonists do have the potential to activate MB after insult with limited detrimental effects (Ortega and Peters, 2010; Léger et al., 2011). Agonists for the β2-AR are promising therapeutics for the treatment of mitochondrially-related organ dysfunction found in diseases including diabetes, as well as to promote recovery after injury, including acute kidney injury. The inventors have identified a role of formoterol in the induction of MB in both the kidney and the heart. Additionally the chemical structure of formoterol was utilized to identify four new mitochondrial biogenic compounds. Future studies will focus on the potential of formoterol exposure to promote recovery of kidney and heart mitochondrial function in both disease and injury models.
Table 1 below shows Formoterol pharmacophore extraction of the 1280 compound Sigma Library of Pharmacologically Active Compounds (LOPAC). Chemical fingerprints defined within the MOE software package were used to cluster compounds based on molecular similarity as measured from the Tanimoto Coefficient (TC). Analysis of the data utilizing a TC of 60% identified 23 compounds out of 1280 (or 1.8%) compounds from LOPAC that matched formoterol based on chemical similarity. RPTC were treated with the 23 identified LOPAC compounds (5 μM) for 24 hr and examined for changes in FCCP-OCR. This assay identified two compounds, nisoxetine and tomoxetine, that increase FCCP-OCR 15% above vehicle control. Fold change in FCCP-OCR relative to DMSO controls appears in the bottom left of each compound. CAS numbers appear to the bottom right.
Table 2 below shows the 6-point formoterol pharmacophore model was used to search the ChemBridge DIVERSet™ for similar compounds. This library contains 50,080 unique drug-like compounds that cover pharmacophore diversity. These compounds were used to create a 1,420,467 entry conformer library. An in silico search identified 16 compounds (or 0.03%) containing all six chemical features with a RMSD<1 Å to the pharmacophore model for all features. RPTC were exposed to the 16 identified ChemBridge compounds (5 μM) for 24 hr and examined for changes in FCCP-OCR. This assay identified three novel compounds (CB1-3) that increase FCCP-OCR 15% above the vehicle control. Fold change in FCCP-OCR relative to DMSO controls appears in the bottom left of each compound. CAS numbers appear to the bottom right.
Table 3 below shows Formoterol, nisoxetine, tomoxetine, and (CB 2) ChemBridge #5144525. The original pharmacophore was built using formoterol, nisoxetine and tomoxetine. CB 1-3 were found using the original pharmacophore from a search of the ChemBridge DIVERSet™ 50,000 small molecule conformational library. ChemBridge compounds 2 and 3 were confirmed biogenic; however, CB 2 was the only compound to add features to the pharmacophore model.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Chemistry
All solvents and reagents, unless otherwise stated, were supplied by Sigma-Aldrich Chemical Co. Ltd. and were used as supplied.
(±)-(R*,R*)-[4-[2-[[2-(3-Chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium hydrate (BRL 37344), (R*,R*)—N-[2-Hydroxy-5-[1-hydroxy-2-[[2-(4-methoxyphenyl)-1-methylethyl]amino]ethyl]phenyl]formamide fumarate (formoterol furmarate dihydrate), and (R)-3,4-Dihydroxy-α-(isopropylaminomethyl)benzyl alcohol hydrochloride (isoproterenol hydrochloride) were purchased from Sigma-Aldrich Chemical Co. Ltd. and were confirmed to be 98% pure by HPLC. The Library of Pharmacologically Active Compounds (LOPAC) was purchased from Sigma-Aldrich Chemical Co. Ltd. and the compounds were confirmed to be 95% pure by HPLC. The ChemBridge DIVERSet™ 50,000 compound library was purchased from ChemBridge Corporation and the compounds were confirmed to be 95% pure by HPLC.
Biological Evaluation of Compounds
Isolation of proximal tubules. Female New Zealand white rabbits (1.5-2.0 kg) were purchased from Myrtle's Rabbitry (Thompson Station, Tenn., USA). Rabbit renal tubules were isolated using the iron oxide perfusion method as described in Nowak et al. (1995). The resulting proximal tubules were plated on 100-mm tissue culture-grade plastic Petri dishes constantly swirled on an orbital shaker at 80 rpm. The culture medium was a 50:50 mixture of Dulbecco's modified Eagle's essential medium and Ham's F12 (lacking glucose, phenol red, and sodium pyruvate; Gibco BRL) supplemented with 5 μg/ml human transferrin, 5 ng/ml selenium, 50 nM hydrocortisone, and 10 nM bovine insulin. After three days the de-differentiated cells were trypsinized and re-plated onto XF-96 polystyrene cell culture microplates at a concentration of 18,000 cells/well and maintained in a 37° C. incubator for two days prior to experimentation (Beeson et al., 2010).
Isolation of primary adult feline cardiomyocytes. Adult feline cardiomyocytes were isolated to 95% purity as published previously (Mann et al., 1989). In the present study, glass-bottom dishes (MatTek) coated with laminin were used for culturing cardiocytes in Piper's medium, which was prepared in M199 cell culture medium (Invitrogen) containing the following additional ingredients: 2% bovine serum albumin, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 0.25 mM L-ascorbate, 10 μM cytosine arabinoside, 200 units/ml penicillin and 200 mg/ml streptomycin (Invitrogen). The freshly isolated adult cardiomyocytes were plated into XF-9696 Polystyrene Cell Culture Microplates and were maintained in a 37° C. incubator for two days prior to experimentation.
Respirometry assay. The OCR measurements were performed using a Seahorse Bioscience XF-96 instrument according to the protocol outlined in Beeson et al. (2010). Each experimental plate was treated with vehicle controls (DMSO<0.5%), a positive control (SRT1720, 10 μM), blank controls, and the appropriate concentration of the compound of interest. The XF-96 protocol consists of five measurements of basal OCR (1 measurement/1.5 min), injection of FCCP (0.5 μM), and three measurements of uncoupled OCR (1 measurement/1.5 min). The consumption rates were calculated from the continuous average slope of the O2 partitioning among plastic, atmosphere, and cellular uptake (Gerencser et al., 2009). Quality control evaluations considered the basal and uncoupled rates of the vehicle control, positive control, and variances between duplicate treatment wells. Based on preliminary studies the positive threshold value was >1.15 for the mean ratio of (chemical treatment FCCP-OCR/vehicle control FCCP-OCR). This threshold is ≧1 S.D. above the historic mean for the vehicle control.
Dosing of C57BL/6 mice. Male C57BL/6 mice (NCI) were between six and eight weeks old. The mice were housed in groups of three in a temperature-controlled room, under a 12/12 h light/dark cycle. Mice were randomly assigned to either saline control or formoterol treated groups (n=3-6 mice/group). Treated mice received daily i.p. injections of formoterol for up to 72 h (100 μg/kg/day) and control mice received an equivalent volume of sterile saline.
Real time RT-PCR. Total RNA was isolated from renal cortex and heart using TRIzol reagent (Invitrogen). cDNA was synthesized from 2 μg of RNA template using SuperScript II Reverse Transcriptase kit (Invitrogen). PCR products were amplified from 5 μl of cDNA template in a 25 μl reaction containing 12.5 μl of 2× SYBR Premix (Stratagene) and 400 nM of each primer (Integrated DNA Technologies) (Supp. Table 1). The average fold induction was calculated by comparing the CT (threshold cycle) of the target gene to that of tublin (the reference gene). The gene expression of the reference gene remained consistent throughout each treatment. The CT of each of the technical replicates was averaged, and that average was used in the following formulas:
C
T(target gene)−CT(β-actin)={CT
ΔCT(treatment)−ΔCT(control)=ΔΔCT
2(−ΔΔCT)=fold change
Mitochondrial DNA Content. Real-time PCR was used to determine relative quantities of mtDNA content in both RPTC and mouse kidney and heart tissues. Genomic DNA was extracted using the DNeasy Blood and Tissue kit (QIAGEN, Valencia, Calif.). PCR products were amplified from 25 ng of cellular DNA in a 25 μl reaction containing 12.5 μl of 2× SYBR Premix and 400 nM of each primer.
For estimation of mtDNA in RPTC the ND6 gene was amplified. The nuclear-encoded tubulin gene was used for normalization (Supp. Table 1). For estimation of mtDNA in mice the control region (D-loop) of mouse mtDNA was amplified. The nuclear-encoded apoB gene was used for normalization (Supp. Table 1) (Fuke et al., 2011).
Computational Procedures
Molecular modeling of potential mitochondrial biogenic compounds. Modeling, simulations and visualizations were performed using MOE Version 2010.10 (Chemical Computing Group Inc.). Computational procedures were performed using a Dell XPS T3400 with an Intel Core2 Duo processor E8500@3.16 GHz with 3GB RAM using a Windows XP OS. The Sigma LOPAC and the ChemBridge DiverSet™ were searched using the Tanimoto score with MACCS structural keys (166 keys) fingerprinting (Maccs II; Molecular Design Ltd.). Using tables of the compounds represented as Simplified Molecular Input Line Entry Specification (SMILES) strings were imported into MOE as an mdb database. Molecules were rigidly aligned manually, and then subjected to MOE flexible body refinement (configuration limit 100, alpha 1, gradient test 0.01, RMSD tolerance 0.5, maximum steps 500). The ChemBridge DIVERSet™ conformers were a stochastic force field based library generated using MOE conformation import with no import filters. Total number of possible conformations per molecule was 50 and used default settings including a strain limit of 4 kcal/mol, RMSD test for structural diversity of 0.15, trans conformations were enforced, and an energy minimization gradient test of 0.01 kcal/mol. Consensus pharmacophores were calculated using a distance parameter (tolerance) maintained at the default value of 1.2 angstroms and a threshold of 100%. Pharmacophore feature projections including aromatic rings (PiN), H-bond acceptors (Don2), and H-bond acceptors (Acc2) were not included in the analysis.
Statistical Analysis. Data are presented as means±s.e.m. and were tested for normality. Data that was confirmed to have a normal distribution were subjected to one-way analysis of variance (ANOVA). The respiration data failed a normality test, therefore a Kruskal-Wallis one-way ANOVA on ranks was conducted. Multiple means were compared utilizing Dunn's post hoc test and were considered statistically different when P<0.05. RPTCs and AFCs isolated from a single animal represented an individual experiment (N=1) and were repeated until an N≧four was obtained. Rodent studies were repeated until an N≧three was obtained.
β2-Adrenergic Agonist Induces MB In Vitro
Kidney proximal tubules require aerobic metabolism to maintain high levels of ATP for transport processes. The primary cultures of RPTC utilized in this study were grown under improved culture conditions with optimized glucose-free media supplemented with 6 mM sodium lactate, and increased oxygen supply (Nowak and Schnellmann, 1995; Nowak and Schnellmann, 1996). RPTC grown under these conditions remain polarized, maintain their differentiated functions, and exhibit respiration and gluconeogenesis rates comparable to in vivo renal proximal tubule cells. The primary culture of adult feline cardiomyocytes (AFC) utilized in the study also maintain differentiated function and exhibit mitochondrial respiration similar to that observed in vivo (Mitcheson et al., 1998).
Primary cultures of RPTC and AFC have been optimized for use with the Seahorse Biosciences 96-well extracellular flux analyzer (XF-96), a multi-well plate-based assay platform that addresses the need for higher throughput cellular respirometric measurements (Ferrick et al., 2008). The XF-96 instrument uses fluorescent detectors to measure oxygen consumption rates (OCR), and can be used to identify compounds that alter mitochondrial respiration (Beeson et al., 2010). Injection of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) uncouples the mitochondrial membrane potential from the production of ATP increasing the OCR. It was determined that this maximum respiratory capacity could be used as a measure of MB (Beeson et al., 2010).
The FCCP-OCR increased in RPTC after a 24 hr exposure to formoterol (30 nM) relative to the vehicle control (
To further document that the increased OCR values were the result of MB, mitochondrial DNA (mtDNA) copy number was assessed. Relative mitochondrial DNA copy number was determined using quantitative real-time PCR and the ratio of a mitochondrial-encoded gene (NADH dehydrogenase subunit 6, ND6) to a nuclear-encoded gene (tubulin). Formoterol (30 nM) treated cells exhibited a significant 2.5-fold increase in mitochondrial copy number (
β2-Adrenergic Agonist Induces MB In Vivo
To determine whether β2-AR agonism in vivo produces MB, male C57BL/6 mice were dosed intraperitonealy (i.p.) with 100 μg/kg formoterol every day for 1 or 3 days. Kidney and heart tissues were collected from the animals and MB was determined by assessing mitochondrial DNA copy number and the mRNA levels of multiple mitochondrial proteins.
Twenty-four hr after formoterol exposure, mRNA levels of multiple genes involved in mitochondrial regulation and function increased in kidneys. PGC-1α was induced 2.5-fold and mitochondrially-encoded ATP synthase F0 subunit 6 of transmembrane F-type ATP synthase (ATP6), ND1, and ND6 were induced 2.5-,4- and 2.5-fold, respectively (
The hearts of mice exposed to 100 μg/kg formoterol for 24 hr exhibited a marked induction of PGC-1α, ATP6, ND1, ND6, and NDUFB8 (3-, 1.5-, 2-, 2- and 2-fold, respectively) (
Formoterol-Based Pharmacophore Identifies Novel Mitochondrial Biogenics
Using formoterol as a basis for chemical similarity and pharmacophore analysis, the inventors explored chemical space that would otherwise be impractical to explore given current limitations in biological assay techniques. Pharmacophores are defined as a collection of steric and electronic features required for molecular interactions with a specific biological target structure, resulting in the activation or deactivation of its biological response (Horvath, 2011). Pharmacophore models have been developed to identify biologically active chemicals responsible for therapeutic activity (Ebalunode et al., 2011). The resulting structures can be used to predict potential pharmacology based on the assumption that compounds containing the same pharmacophore are likely to cause similar effects by targeting the same active site (Horvath, 2011). Currently, there are no known pharmacophores associated with MB. Formoterol was utilized in a ligand-based approach to identify novel compounds with a similar chemical structure and determine if they induced MB.
Formoterol was used to conduct a similarity search of the 1280 compound Sigma Library of Pharmacologically Active Compounds (LOPAC). Similarity is measured using the Tanimoto Coefficient (TC). TC is a similarity metric of one-dimensional chemical descriptors that identifies the presence of molecular elements, and facilitates rapid initial comparisons (Willett, 2006). A TC similarity search of LOPAC found 23 compounds 60% similar to formoterol. RPTC were treated with the 23 identified LOPAC compounds (5 μM) for 24 hr and examined for changes in FCCP-uncoupled OCR. This assay identified two compounds, nisoxetine and tomoxetine (also known as atomoxetine), that increase FCCP-uncoupled OCR above vehicle control (Table 1). A subsequent concentration response evaluation determined that nisoxetine and tomoxetine increased FCCP-uncoupled OCR in a concentration-dependent manner, with a minimum effective dose of 30 nM (
To confirm the specificity of formoterol, cells were pretreated for 1 hr with the β-AR antagonist propranolol (5 nM) and the β2-AR inverse agonist ICI 115,881 (3 and 10 nM) prior to a 24 h treatment with the formoterol (30 nM). As seen in
Formoterol, nisoxetine, and tomoxetine were aligned in first two and then three dimensions based on the presence of consensus chemical features within 100% of the compounds. This alignment resulted in a pharmacophore containing six features of conserved chemical similarity (
The 6-point pharmacophore model was used to search the ChemBridge DIVERSet™ for similar compounds. This library contains 50,080 unique drug-like compounds that represent a wide range of chemical diversity. These compounds were used to create a 1,420,467 entry conformer library. An in silico search identified 16 compounds containing all six chemical features with a RMSD<1 Å. No compounds with an RMSD>1 Å were identified, but this was expected as each feature is only ˜1 Å in diameter and six features were absolutely required leading to a minimum possible RMSD. Note that the TC search and pharmacophore search of ChemBridge have no overlapping compounds.
RPTC were exposed to the 16 identified ChemBridge compounds (5 μM) for 24 hr and examined for changes in FCCP-OCR. This assay identified three novel compounds (CB1-3) that increase FCCP-OCR above the vehicle control (Table 2). 480 random compounds were tested from the ChemBridge DIVERSet™ on the XF-96. From these 480 random compounds, thirteen were determined to be biogenic (unpublished data). The inventors extrapolate that the spontaneous hit rate of the entire data set would be approximately 2.7%. The pharmacophore performed with a hit rate of 18.8% of the compounds it chose. Using the enrichment factor equation from Pearlman and Charifson (2001), the pharmacophore selected a subset of the database 6.9 times richer in biogenic compounds than the original database.
EF=(HITSsampled-set/Nsampled-set)/(HITStotal-database/Ntotal-database)
RPTC were exposed for 24 h to 10-3000 nM of CB1, CB2, and CB3 to determine potency in increasing FCCP-OCR. CB2 and CB3 increased FCCP-OCR in RPTC with maximal effects at 30 and 100 nM respectively, while CB1 was not effective at this concentration range (
Virtual screening and drug development for β2-AR agonists have been explored utilizing classic SAR in conjunction with X-ray structures and known agonists (de Graff et al. 2008; Renolds et al. 2009; Procopiou et al. 2009). The use of cheminformatics including docking, fragment and pharmacophore based approaches has also been applied to study β2-AR Abrahamian et al. 2003; Glossop et al. 2010; Tasler et al. 2010). Provided herein is a means of combining pharmacophore-based screening with a phenotypic endpoint and quantitative structure activity relationship modeling (QSAR) to identify specific β2-AR agonists that stimulate MB. Furthermore, the resulting compounds were rationalized to the crystal structure of β2-AR.
Materials and Methods:
Isolation of proximal tubules Female New Zealand white rabbits (1.5-2.0 kg) were purchased from Myrtle's Rabbitry (Thompson Station, Tenn., USA). Rabbit renal tubules were isolated using the iron oxide perfusion method as described in Nowak et al. 1995. The resulting proximal tubules were plated on 100-mm tissue culture-grade plastic Petri dishes constantly swirled on an orbital shaker at 80 rpm. The culture medium was a 50:50 mixture of Dulbecco's modified Eagle's essential medium and Ham's F12 (lacking glucose, phenol red, and sodium pyruvate; Gibco BRL) supplemented with and supplemented with HEPES (15 mM), glutamine (2.5 mM), pyridoxine HCl (1 μM), sodium bicarbonate (15 mM), and lactate (6 mM). Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 μg/ml), bovine insulin (10 nM), and
RPTC respirometric assay The OCR measurements were performed using a Seahorse Bioscience XF-96 instrument according to the protocol outlined in Beeson et al. 2010. Each experimental plate was treated with vehicle controls (DMSO<0.5%), a positive control (SRT1720, 10 μM), blank controls, and the appropriate concentration of the compound of interest. The XF96 protocol consists of five measurements of basal OCR (1 measurement/1.5 min), injection of FCCP (0.5 μM), and three measurements of uncoupled OCR (1 measurement/1.5 min). The consumption rates were calculated from the continuous average slope of the O2 partitioning among plastic, atmosphere, and cellular uptake (Ferrick et al. 2008). Quality control evaluations considered the basal and uncoupled rates of the vehicle control, positive control (SRT1720), and variances between duplicate treatment wells. Based on preliminary studies the biogenic threshold value was <1.15 for the mean ratio of (chemical treatment FCCP-OCR/vehicle control FCCP-OCR). This threshold is ≧1 S.D. above the historic mean for the vehicle control.
Chemical Informatics Modeling: Modeling, simulations and visualizations were performed using MOE Version 2010.10 (CCG). Simulations were performed on a Dell E8500 with an Intel Core 2 Duo @3. 16 GHz using a Windows XP OS, all other computational procedures were performed using a Dell XPS M1530 with an Intel Core2 Duo processor T8300@2.40 GHz w 2GB RAM using a Windows Vista OS. Compound similarity was measured and visualized using the Tanimoto coefficient metric based on MACCS structural keys using ChemMine (Backman et al. 2011).
Pharmacophore Modeling: Structures of all screened compounds were analyzed using MOE software. Using tables of inhibitors incorporated as Simplified Molecular Input Line Entry Specification (SMILES) strings (which allow for the avoidance of ambiguity in the specification of a chemical structure via a text string) were imported into MOE as a mdb database. The pharmacophore model was generated by manual alignment of compounds and rigid refinement. Molecules were added to the pharmacophore with successive adjustment of the alignment and modification of the features. A generalized pharmacophore was created by generating features that encompassed the set of corresponding features from each individual pharmacophore. If there was overlap in a feature of type X in pharmacophores A, B, and C (features XA, Xb, and Xc, respectively), a feature of type X would be created for the generalized pharmacophore and its sphere would be expanded to contain XA, XB, and Xc to give feature Xgen. For a set of pharmacophores developed from drugs acting upon the same receptor, there may exist features that are not shared by all pharmacophores. If a molecule significantly and adversely affected the alignment (i.e., loss of multiple features), then it was not included in the pharmacophore. Molecules were rigidly aligned manually, and then subjected to MOE flexible body refinement (configuration limit 100, alpha 1, gradient test 0.01, RMSD tolerance 0.5, maximum steps 500). Initial alignments were performed using the molecules' respective energy minima. However, as the subset of tested molecules increased, less energetically favorable conformations were incorporated to account and allow for new information. In these cases, the alignment was performed by hand and the alignment was rigidly refined to prevent thermodynamic considerations from disrupting the pharmacophore. Consensus pharmacophores were calculated using a distance parameter (tolerance) maintained at the default value of 1.2 Å and a threshold of 100%. Pharmacophore feature projections including Pi rings (PiN), H-bond acceptors (Don2), and H-bond acceptors (Acc2) were not included in the analysis.
Pharmacophore virtual database searching: The ChemBridge DIVERSet was searched using pharmacophore models. Using tables of the compounds represented as Simplified Molecular Input Line Entry Specification (SMILES), strings were imported into MOE as a mdb database. The 5 point pharmacophore model was used to search the Chembridge DIVERSet. The DIVERSet database contained 50,080 unique compounds representing diverse drugs designed for primary drug discovery. The chemical graphs of the DIVERSet were used to create a 2,500,000 compound conformer library, using MOE conformation import and allowing a maximum of 250 conformers per compound and a strain limit of 4 kcal. Using the five point CB pharmacophore model conformers were matched to the model which required all features to be present. In order to match all five features an RMDS threshold of <1{acute over (Å)} was a functional upper boundary due to the radius (max=1.2 {acute over (Å)}, avg=0.72 {acute over (Å)}) of each feature.
2D-QSAR modeling and screening: A 2D-QSAR models were built that showed significant predictive ability. QSAR modeling correlated activity as a binary function to the calculated 2D descriptors. Due to the large divergence in chemical space the original compound list was pruned to 19 compounds, representing 12 active and 7 inactives. In total 186 2D descriptors were calculated. These were trimmed though integrative modeling and removal of low predictive descriptors to a final set of 13 descriptors. The final cross-validated model was saved as a .fit file and used to search 2D chemical mdb libraries.
in silico receptor interaction studies: The interaction of ligands from the compound library were analyzed for molecular interactions. The consensus pharmacophore model was used as a placement strategy. The consensus pharmacophore was aligned manually within the β2-AR Xray structure (pdb 3NYA (Wacker et al. 2010)). The alignment was guided by overlaying the model on top of alprenolol. After pharmacophore placement, alprenolol was removed from the system and modeled ligands placed according to the conformer from the pharmacophore model. The ligand-receptor dimer system was then energy minimized using OPLA-AA. Some modest manual adjustment was required to prevent van der Walls coincidence that would negatively affect the energy minimization. The minimized system was then analyzed by visualization of key interactions and ligand interaction maps.
To expand understanding of β2-agonist-stimulated MB, the inventors tested β2-AR agonists and similar ligands. Chemotype evaluation revealed that ten compounds contain a phenethylamine core common to many biogenic amine ligands that are β-AR specific (
There were three types of concentration-response phenotypes. Five of the twelve compounds tested were very potent, had Michaelis-Menten type concentration-response curves and included fenoterol, formoterol, procaterol, nisoxetine and tomoxetine (
Due to the discrepancy in the ability of high affinity and selective β2-AR ligands to stimulate MB, the inventors interrogated the ligand structure using chemical similarity clustering (
Cluster 1 was further explored using pharmacophore modeling because their chemical similarity did not explain their biological effect. Procaterol, formoterol and fenoterol were aligned by hand based on steric considerations. The alignment was then refined using MOE's flexible alignment tool. The final pharmacophore model generated had 100% consensus features with a maximum feature radius of 1.2 Å (
Features F1, F3, F4, and F5 represent the phenethylamine core found in many sympathomimetic compounds as well as the endogenous sympathetic agonists. Features F6 and F7 are an expansion in the hydrophobic space occupied by this core scaffold. Given that neither epinephrine nor norepinephrine elicit the MB nor contain these features, these features occupy necessary space for MB. Features F1, F3, F4, and F5 represent the phenylethanamine core found in many sympathomimetic compounds as well as the endogenous sympathetic agonists. Features F6 and F7 are an expansion in the hydrophobic space occupied by this core scaffold. Given that neither epinephrine nor norepinephrine elicit the MB nor contain these features, these features occupy necessary space for MB. However, because non-biogenic compounds (e.g., isoproterenol) contain these features, they are not sufficient for mitochondrial biogenesis. Feature F2, although present in all the fully biogenic β2 agonists, is present in non-biogenic agonists and is not present in clusters 2 or 3; therefore, it is neither necessary nor sufficient in itself for mitochondrial biogenesis.
Cluster 2 contained nisoxetine and tomoxetine, which are characterized as norepinephrine reuptake inhibitors (NRIs) and represent non-classical but chemically similar compounds to β2-AR agonists that stimulate MB. Nisoxetine and tomoxetine were aligned in first two and then three dimensions. A high quality model with seven features and tight spheres was generated (
Features F3, F4, F5, F6, and F7 in this pharmacophore represent the phenethylamine core. However, unlike the Cluster 1 pharmacophore, there were no further hydrophobic substituents on the carbon alpha to the nitrogen hydrogen bond donor group. This indicates that features F1 and F2 (hydrophobic acceptor and aromatic, respectively), absent from the cluster 1 pharmacophore, are sufficient to stimulate MB when coupled with the phenethylamine core. However, due to its absence in cluster 3, F2 alone may not be sufficient to generate a biogenic phenotype.
The potential overlap of cluster 1 and cluster 2 was then determined (
Because the above models and clusters only provide information relevant to the phenethylamine core, the inventors expanded on the discovery of the MB compounds CB2 and CB3 (
To further limit the chemical space being tested, the results of the CB pharmacophore search were refined using a discrete 2D-QSAR model. A preliminary 2D-QSAR model was built from all of the tested compounds using a pharmacophore-based on the cluster 3 pharmacophore. Binary QSAR modeling was used to account for the inability to determine IC50 measurements or perform linear QSAR with OCR data. The cross-validated predicative statistics for the QSAR model were 86% for actives and 100% for inactive with a p-value <0.001. If a random hit rate of 3% is assumed, this process gave an enrichment of 3.5× over random. When used together the pharmacophore model and 2D-QSAR yielded 1196 from the 2810 discrete compounds. To further limit the test set the inventors further applied an RMSD filter of <0.3 Å to the pharmacophore results to further refined and narrowed the results to 48 chemicals.
The 48 compounds predicted by pharmacophore and QSAR modeling were tested for their ability to induce MB at 30 nM in RPTC. Five of the 48 compounds (10%) produced MB, resulted in the expansion of the original chemogram, shows that the newly characterized compounds diverge from classical β2-AR chemical space, and demonstrates how directed chemical modeling can explore new chemical clusters with similar biological properties. A workflow for modeling for combined QSAR and pharmacophore modeling for virtual screening was used.
Finally, the inventors investigated whether there were conserved features (
In the consensus pharmacophore, F3, F4, F6, and F7 represent the phenethylamine core. F5 is a donor feature in the meta position to the ethanamine portion of the core and is unique to the Cluster 1 pharmacophore. Because it is not found in the other clusters and is also present in non-biogenic compounds, it is neither necessary nor sufficient for mitochondrial biogenesis. However, it may play a role in stabilizing receptor interactions of individual compounds to generate a conformational change that induces biogenesis. F1 and F2 represent the aromatic feature and hydrophobic acceptor features from cluster 2, respectively. F1 is present in clusters 2 and 3 and is sufficient to cause biogenesis when added to the core. F2 is only present in cluster 2 and is therefore neither necessary nor sufficient. Likewise, the donor feature F5 is present only in cluster 1 and is also neither necessary nor sufficient. The hydrophobic feature F8 is present in all clusters and is therefore a necessary addition to the phenethylamine core in stimulating mitochondrial biogenesis.
The MB ligands were analyzed for predicted interactions with the β2-AR X-ray structure (Ghanouni et al. 2001; Suryanarayana et al. 1992; Rasmussen et al. 2011; Rosenbaum et al. 2007; Wacker et al. 2010). Using the structure of the β2-AR bound to the antagonist alprenolol (pdb: 3NYA (Wacker et al. 2010)), the consensus pharmacophore was utilized as a placement strategy to identify potential high-likelihood interactions (Audet et al. 2008). Alprenolol was chosen over other available structures due to the number of chemical similarities of the antagonist to the pharmacophore models. The inventors observed overlap between the donor feature from the cluster 1 pharmacophore and Phe289 or Phe290 (depending on the ligand placement) within the ligand binding pocket without overlapping with any receptor amino acids indicating the pocket has room for the multiple conformations predicted by modeling (FIGS. 16A-F). The specific conformer of each biogenic ligand was placed within the ligand binding site of the receptor. The final agonist-receptor complex was energy minimized and analyzed for potential interactions.
Common residue interactions were found across the full biogenic agonists (
In the first cluster of classical β2 agonists (cluster 1), formoterol had extensive hydrogen bonding interactions, with its alkyl hydroxyl group interacting with both Asp113 and Asn312. The p-OH group interacted with the backbone of residues Trp286 and Phe289. Hydrophobic interactions occurred between Tyr308 and the formamide hydrogen and between Phe193 and the methoxyphenyl ring. These interactions, and especially that of Asp113, differ greatly from the other ligands due to the extra space occupied by the formamide chain. This is further demonstrated by the interactions between the receptor and fenoterol. The resorcinol hydroxyl groups on fenoterol interact with the receptor (Asn312 and the backbone of Trp286). Asn312 also interacts with the amine on the alkyl chain, as does Asp113. Asp113 in turn interacts with the hydroxyl of the ethanolamine core. The p-OH distal to the phenethylamine core interacts with Cys191. Finally, the phenyl ring of the phenethylamine core is in proximity to Phe289, suggesting the potential for a it-stacking interaction. Despite structural similarities to formoterol, the small differences result in distinct conformational changes of both the receptor and the ligand. A similar phenomenon arises with procaterol. Although it lies in the same chemical cluster as fenoterol and formoterol, its interactions include a it-stacking interaction between Phe289 and the quinoline system, a hydrophobic interaction between Tyr208 and the quinoline ring, a hydrophobic interaction between Phe193 and the N-isopropyl system, and a hydrogen bond between the amine and Asp113. This π-stacking interaction is conserved among MB compounds and is facilitated in procaterol due to the size of its aromatic system.
The second cluster containing nisoxetine and tomoxetine conserved the Asp113 interactions with their secondary amines. Tomoxetine also had a hydrophilic interaction with Ser203 and the methoxy group and a hydrophobic interaction between Trp109 and the N-methyl group, while the phenyl rings of tomoxetine had a hydrophobic interaction with Phe193 and a π-stacking interaction with Trp286. Despite the remarkable similarity of these compounds, the methoxy group on nisoxetine allows for hydrophilic interactions that place it in a certain area of the binding pocket. This allows for the difference in interactions between two compounds that are so similar.
The NRI's represent a vast chemical space, but nisoxetine and tomoxetine represent a distinct and limited class of NRI's (Civitarese et al. 2008). These drugs have less use due to the recognition of the promiscuity in mechanism, although they have only recently been proposed to potentially stimulate the β2-AR (Wills et al. 2012). Other NRIs lack the features described here found necessary for supporting β2-AR mediated MB. One example is desipramine, which has a tail secondary amine; however it is missing all the alkoxy and hydroxy moieties including the classical ethanolamine and more specifically the acceptor feature (F3) from the models (
Both compounds of novel cluster 3 have interactions between Asn312 and the tertiary amine. CB2 has interactions between two of its CH2 components with Phe193. CB3, however, has a hydrophobic interaction between residues Phe193 and Phe289 and the phenyl system. It also has a hydrophilic interaction between Tyr308 and its amide carbonyl group. Once again, despite the chemical similarity based on Tc, these compounds differ greatly in their possible 3D interactions with the receptor.
The partial biogenic compounds were ambiguous, requiring multiple alignments to achieve a conformation sampling the canonical residues (i.e., Asp113 and Asn312) (Suryanarayana et al. 1992; Rasmussen et al. 2011; Wacker et al. 2010; Audet et al. 2008; Vilardaga 2006; Suryanarayana et al. 1993). These data reveal that MB compounds can transiently and rapidly alter conformations, resulting in their partial biogenic phenotype. In the case of CPB, the cyclopentyl group makes the ligand too large to rapidly alter its conformation preventing the transient interactions with the residues necessary for MB. One of the potential conformations had a it-stacking interaction between Phe289 and the CPB phenyl group. This is similar to the full agonist procaterol, which also potentially interacts with Phe290. The hydrophobic interactions between the cyclopentyl group and the binding pocket may aid in dynamic receptor conformation sampling as it tries to find an appropriate minimal energy conformation. This is in contrast to isoetharine, which has an isopropyl group rather than a cyclopentyl group. The lack of volume distal to the phenyl group prevents the ligand from being pressed deeper into the binding pocket, thereby preventing the necessary biogenic interactions with the receptor.
The metaproterenol analysis underwent multiple conformations, all sampling at least one of the canonical residues. This indicates that the partial agonist activity of metaproterenol results from being too small to adequately remain in the binding pocket. Its small size therefore allows for it to easily sample the necessary residues for biogenesis, but it does not allow it to stably and simultaneously sample them. Compared to isoproterenol, which has a catechol ring rather than a resorcinol ring, metaproterenol has twice as many conformations available to it that sample the same residues. Since it has two m-OH groups rather than an m-OH group and a p-OH group, it can more easily sample the it-stacking interactions with F289. This structural difference helps to explain the differences in receptor-ligand interactions (and therefore the phenotypic differences) between isoproterenol and metaproterenol.
In this study a recently developed phenotypic respirometric assay was utilized to screen for MB agents in primary RPTC. A phenotypic assay has the major advantage of linking specific ligand-mediated receptor activation (i.e. β2-AR) to a distinct cellular function. Although FCCP-OCR is a one dimensional parameter it is reflective of a complex process and ideal for elucidating signaling bias. Furthermore, using primary cultures of RPTC have more scientific and clinical relevance than similar assays in cell lines, due to the reliance of RPTC on aerobic respiration.
17 MB agents were identified, three major chemical clusters, three cluster specific pharmacophore models, a QSAR model and a consensus pharmacophore model. Three distinct OCR phenotypes were observed, including full biogenic, partial biogenic, and non-biogenic activities amongst the agonists and similar compounds. The inventors rationalized the chemical structures of β2-AR ligands with their ability to regulate MB, and determined that the reported Kd of these compounds are not in agreement with the ability to produce MB. The inventors rationally expanded the chemical space available for MB to include defined NRIs and uncharacterized compounds. The consensus pharmacophore model derived from three discrete pharmacophore models allows for the precise spatial orientation of the alternate chemical moieties that potentially extend the core model towards a biased model. Furthermore, the model reveals there are at least three discrete interactions that potentially create the biased effect.
There is still insufficient detail on the structural rationale behind the design of biased agonists. Molecular docking has been used in the discovery of β2 agonists and antagonists using the GPCR ligand binding site, but the atomic basis is still an area of active investigation (Baloyannis et al. 2004; Ktritch et al. 2009). While the inventors initial thought was that the MB potential of the compounds would correlate with their reported affinity, there appears to be no relationship between Kd and MB potential (
The inventors elucidated a simple binary 2D-QSAR model consisting of 13 descriptors. Binary QSAR leads to a marginal decrease in absolute predictive power, while still allowing an orthogonal methodology compared to pharmacophore modeling. The fact that the developed model was refined to only 13 descriptors indicated that there are limited and specific sets of requirements. Binary QSAR also identified the conserved electronic chemical features consistent with bioactivity. Analysis of the top descriptors used in the 2D-QSAR model provides insight into the chemical properties that lead to biogenic capability (Peterson et al. 2009). The top descriptor was the number of oxygen atoms indicating dependence on a proton acceptor. The number of aliphatic hydroxyl groups (avg=2.18 per molecule) and nitrogen atoms (avg=1.36) was also necessary indicating a need for protein donors (avg=1.5). The HBD and HBA descriptors, which represent counts of hydrogen bond donors and hydrogen bond acceptors, came up in four forms indicating the necessity for reversible non-covalent interactions to support MB. The remaining descriptors indicated a limit to the number and types of intramolecular bonds: # bonds (avg=41.46), # heavy bonds (avg=20.36), # atoms (avg=40.72), # rotatable bonds (avg=5.72). All these data indicate that the biased MB potential of the ligands is related to the non-covalent and flexible interaction of the drug with its receptor.
The partial and non-biogenic compounds have classical architecture incorporating both ethanolamine and either catechol or resorcinol moieties while none of the full biogenics have a catechol. From a chemistry perspective isoetharine and procaterol are bioisosteres of one another, with procaterol having a quinoline system rather than a catechol. However, procaterol is strongly biogenic, while isoetharine is not, indicating that the space occupied by the quinoline ring is also important for the MB phenotype. Furthermore, CPB exhibits a partial biogenic response while differing from isoetharine by only two carbons. This indicates that, in addition to the stronger hydrogen bond interactions with the receptor, weaker hydrophobic interactions play an important role in differentiating phenotypic responses in the β2-AR. There were more hydrophobic interactions in the full biogenics in these simulations compared to the other two classes of compounds. It is interesting to note that no simulated interactions with Tyr316, a member of the canonical polar triad for agonist binding, was observed. It is possible this triad is responsible for the activity of full endogenous agonists and during ligand simulations the inventors repeatedly found that Tyr316 formed a stabilizing bond with Asp113, which has been previously been associated with agonist activity (Katritch et al. 2009). However, the prevalence of the phenethylamine core indicates that hydrogen bonding interactions is still critical in ligand binding in the receptor pocket to allow slower hydrophobic interactions to occur.
Understanding of GPCR pluridimensional signaling has led to the conception of manipulating pharmaceutical design for functional selectivity (Galandrin et al. 2006; Kenakin et al. 2010; Luttrell et al. 2011; Stallaert et al. 2012). The mechanism of MB and indeed of signaling bias is not yet known and not generalizable to a particular chemical class or chemotype. It is possible the three MB phenotypes represent three distinct states of the receptor that the ligands are able to bias. The ability of different orthosteric ligands to induce a spectrum of receptor conformations allows for multiple signaling states of GPCRs. In such a model, the ligand receptor complex, not solely the receptor, specifies the activation state and is therefore the minimal pharmacological interpretation unit. In the case of the MB β2-AR agonists, the non-overlapping features were inferred to allow for biased agonism by stabilizing different conformations of the receptor (Reynolds et al. 2009; Audet et al. 2008; Sauliere et al. 2012; Seifert et al. 2001). By allowing for the stabilization of multiple conformations with a single compound, it should be possible to sample multiple pathways with a given binding event and thereby potentiate MB. Furthermore, by utilizing this same process of compound development for other desired effects, it should be possible to attenuate negative effects in addition to enhancing desired pathways.
The overall therapeutic paradigm for usage of MB agents may be performed using short term and low dose administration in order to boost mitochondrial function resulting in the stimulation of cellular/organ repair and regeneration. This preliminary structural rationale for MB compounds is not specific to a chemical class, but a binding mode. Not unexpectedly, it is the ligand-receptor complex that dictates signaling. The pharmacophore models can be utilized to develop novel compounds with biogenic potential that minimize the deleterious effects sometimes associated with activation of the β2-AR. This would allow for the use of compounds over a longer time course to treat chronic mitochondrial problems.
Mice were co-injected with either saline or dexamethasone (25 mg/kg, i.p.) followed by a second injection with either saline or formoterol (BD 100/300/ ug/Kg, i.p.) or tomoxetine (“atomoxetine” 100 ug/kg, i.p.) for 7 days. On the 8th day, gastrocnemius muscle was isolated from both the hind limbs and weighed. The average weights were normalized to initial body weights. DEX caused a 15% decrease in gastrocnemius wt. when compared to saline controls. Both formoterol and atomoxetine administration restored atrophied muscle back to normal. Results are shown in
Materials and Methods
I/R model of AKI. Eight-week-old male C57BL6 mice weighing 25-30 g were subjected to bilateral renal pedicle ligation. Briefly, renal artery and vein were isolated and blood flow was occluded with a vascular clamp for 20 min, and mice were euthanized at 24, 72, or 144 h after procedure, at which time kidneys were harvested for molecular analysis. All procedures involving animals were performed with approval from the IACUC in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Assessing renal function. For mice, blood was collected by retro-orbital eye bleed 24 h and 144 h. Creatinine levels were measured using a Quantichrom Creatinine Assay Kit (BioAssay Systems, Hayward, Calif.) according to the manufacturer's protocol.
Immunoblot analysis. Renal cortical tissue was lysed in RIPA buffer containing cocktail protease and phosphatase inhibitors. Total protein content was measured by the BCA assay. Fifty micrograms of total protein were loaded into SDS-PAGE gels and immunoblots were performed as previously described (26). Antibodies used for immunoblot studies were obtained from the following vendors: caspase 3 (Enzo Life Sciences), GAPDH (Fitzgerald Antibodies), COX I and NDUFB8 (MitoSciences), KIM-1 (R&D Systems), and PGC-1α (Calbiochem).
mRNA analysis. Total RNA was isolated from renal cortical tissue with TRIzol (Invitrogen) according to the manufacturer's protocol and cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Fermentas). PCR reactions were performed using 3:1 diluted cDNA product. Primers sequences for NDUFB8 and COX I were used.
Formoterol Restores Kidney Function After I/R Injury
Initially, to identify the most clinically relevant and translatable time of dose for formoterol to restore kidney function the inventors defined the time of maximal injury, as determined by serum creatinine (SCr), after I/R induced AKI. Maximal injury was observed at 24 h, SCr was approximately six fold higher than that of sham animals. It was at this time that administration of formoterol was initiated. After five doses the inventors observed complete recovery in SCr, 0.29 mg/dL. However, in the absence of formoterol SCr remained persistently elevated by approximately three fold higher compared to the control group at 144 h after I/R injury (
Formoterol Rescues Proximal Tubules from Damage After I/R Injury
Kidney injury molecule-1 (Kim-1) is a marker specific to tubular injury and is ultimately undetectable in healthy kidneys (65,66). Therefore, the inventors evaluated if administration of formoterol after I/R injury was capable of decreasing the KIM-1 protein expression. It was discovered that administration of formoterol after I/R injury restored expression of KIM-1 at 144 h post injury to the level of sham vehicle. In contrast, the I/R vehicle group showed a three-fold increase, over vehicle, in KIM-1 protein expression out to 144 h (
Effect of Formoterol on Expression of PGC-1α and ETC Components after I/R Injury
Previous in vivo and in vitro research has identified depletion of mitochondrial function and abundance in models of hypoxia correlated with dysfunctional kidney function. Furthermore, the overexpression of PGC-1α, the master regulator of mitochondrial biogenesis, has been shown to recover cellular respiration and increase ATP production after hypoxic injury. Recently, formoterol has been identified as a pharmacological agent capable of stimulating mitochondrial biogenesis in the renal cortical tissue of naïve mice. Therefore, the inventors tested if formoterol was capable of restoring the gene and protein expression of components for the mitochondrial electron transport chain and PGC-1α at the peak of kidney injury, as determined by serum creatinine (SCr), which was 24 hours. Gene transcription of total PGC-1α, COX I (nuclear-encoded), and NDUFB8 (nuclear-encoded) was significantly down regulated in the I/R vehicle group at 144 h (
Formoterol Restores Mitochondrial Function After I/R Injury
Previous models of hypoxic injury to renal proximal tubule cells have reported decreased mitochondrial function to be correlated with of tubular injury. In combination with previous findings indicative of mitochondrial biogenesis, the inventors evaluated if treatment with formoterol after I/R injury is capable of restoring mitochondrial function. Mitochondrial function was measured ex vivo by isolating renal mitochondria and determining state 4 (basal respiration) and state 3 (ADP-stimulated respiratory rate). Treatment with formoterol after I/R injury was observed to completely restore mitochondrial respiration. In contrast, treatment with vehicle after I/R was significantly decreased compared to all other groups, indicative of decreased mitochondrial function (
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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This application claims the benefit of U.S. Provisional Patent Application No. 61/621,896, filed Apr. 9, 2012, the entirety of which is incorporated herein by reference.
This invention was made with government support under T32 CA119945-04, F32 ES020103-01, DK062028, ES012878, DK071997, GM084147, and C06 RR-015455 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61621896 | Apr 2012 | US |
