Patent Application
20240009202
The present invention relates to a pharmaceutical composition for preventing or treating nontuberculous mycobacterium (NTM) infectious diseases, containing, as an active ingredient, a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
The present invention claims priority based on the filing date Aug. 19, 2020, application number 10-2020-0103849, title “Mitochondria-targeted antioxidant as agent for pathological inflammation caused by Mabc-R infection”, the entire contents of which are incorporated herein as part of the present invention.
Mycobacterium abscessus (Mabc) is a nontuberculous mycobacterium that causes numerous human infections, usually associated with lung diseases (References [1, 2]). Mycobacterium abscessus is a subspecies of the Mabc complex that includes Mycobacterium abscessus, Mycobacterium massiliense and Mycobacterium bolletii, and has major clinical implications due to antibiotic resistance (References [2, 3]). Mabc often causes pulmonary infections in patients with immune deficiencies, such as cystic fibrosis, and may threaten immunocompetent individuals (References [4, 5]). Mabc is divided into two morphological types, smooth and rough variants, depending on the colony phenotype (References [6, 7]). The Mabc-rough (Mabc-R) variant is more virulent and invasive compared to Mabc smooth forms. Mabc-R has very minimal glycopeptidolipids (GPLs), which mask the underlying phosphatidyl-myo-inositol mannosides and induce innate immune and inflammatory responses (Reference [8]).
Although several risk factors (for example, old age, immune-modulating agents, underlying lung diseases) related to NTM diseases have been reported (References [5, 9]), the host factors that are associated with pathogenesis during Mabc infection are largely unknown.
Mitochondria are essential intracellular organelles for oxidative energy metabolism, adenosine triphosphate (ATP) production, and calcium homeostasis (Reference [10]). In mitochondria, numerous proteins/enzymes associated with respiratory chain reaction, dynamics, and energy metabolism are regulated by lysine acetylation (Reference [10]).
Meanwhile, sirtuin 3 (SIRT3) is a major deacetylase in mitochondria (Reference [11]) and is known to affect mitochondrial functions through nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylation (Reference [12]). The function of SIRT3 in metabolic tissues has been studied and accumulated data implies that SIRT3 plays a crucial role in non-metabolic cells and tissues, including the lungs and immune cells (References [10 and 12 to 15]).
The present inventors confirmed through recent studies that SIRT3 is required for the host's innate defense against Mycobacterium tuberculosis (Mtb) infection. Importantly, SIRT3 contributes to mitochondrial homeostasis and xenophagy activation in macrophages during Mtb infection (Reference [16]). However, observations were inconsistent regarding the function of SIRT3 in host defenses against various bacterial and fungal infections, including Escherichia coli and Klebsiella pneumoniae infections (Reference [15]). In addition, recent studies showed that SIRT3/5 double knockout (KO) mice showed improved resistance to Listeria infection (Reference [17]). Accordingly, to date, the putative role of SIRT3 in NTM infection is unknown. Furthermore, recent advances and emerging data have revealed SIRT3 modulators, which may ultimately be used as promising drugs for control of numerous human disorders (Reference [12]). In this regard, the therapeutic potential of a SIRT3-targeted therapeutic agent could be validated as a potential drug target for the treatment of Mabc infection in vivo.
An object of the present invention is to provide a pharmaceutical composition for use in the prevention or treatment of nontuberculous mycobacterium infectious diseases, containing, as an active ingredient, a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
Under the above-described background, the present inventors confirmed whether mitochondrial homeostasis is important for host protection against Mabc infection and whether SIRT3-mediated, mitochondria-targeted therapy is beneficial for controlling Mabc infection in vivo. As a result, the present inventors confirmed that Mabc infection led to a decrease in expression of SIRT3, mitochondrial reactive oxygen species (ROS) generation, and mitochondrial dysfunction in macrophages, and SIRT3 deficiency increased bacterial loads, mitochondrial ROS and dysfunction, and pathological inflammation in response to Mabc infection.
Furthermore, it was confirmed that the compound represented by Chemical Formula 1, which is a mitochondrial ROS scavenger, has a therapeutic effect against Mabc infection through amelioration of pathological inflammation and suppression of mitochondrial ROS. More specifically, in an exemplary embodiment, it was confirmed that the compound represented by Chemical Formula 1 has an effect of improving the SIRT3 mRNA level, which has decreased due to nontuberculous mycobacterium infection.
Therefore, the present invention provides a pharmaceutical composition for use in the prevention or treatment of nontuberculous mycobacterium infectious diseases, containing, as an active ingredient, a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
In the present specification, the compound represented by Chemical Formula 1,5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine is a compound disclosed through International Patent Publication No. WO2009-025477, and is a substance known to exhibit preventive, therapeutic and ameliorative effects on necrosis and related diseases.
According to WO2009-025477, the compound of Chemical Formula 1 is known to exhibit preventive or therapeutic and ameliorative effects on necrosis and related diseases. According to WO2009-025477, the necrosis and related diseases include acute/chronic liver diseases (for example, hepatitis, hepatic fibrosis, and liver cirrhosis), neurodegenerative diseases (for example, dementia, Parkinson's disease, and Huntington's disease), ischemic heart disease, reperfusion injury (Korean Patent No. 10-1325272), ischemic stroke or ischemic injury, pancreatitis, bacterial/viral sepsis, diabetes mellitus or diabetes complications, diabetic vascular disease [in particular, these types of diabetes are caused by pancreatic cell-destroying substances, and mediated by viruses, hyperglycemia, fatty acids, diet, toxins, streptozotocin, and the like], necrotizing proctitis, cystic fibrosis, rheumatoid arthritis, degenerative arthritis, nephropathy, bacterial infection, viral infection (for example, HIV), multiple sclerosis, leukemia, lymphoma, neonatal respiratory distress syndrome, asphyxia, tuberculosis, endometriosis, angiasthenia, psoriasis, frostbite, steroid side effects, gangrene, tenderness, hemoglobinuria, burns, hyperthermia, Crohn's disease, celiac disease, compartment syndrome, spinal cord injury, glomerulonephritis, muscular dystrophy, inherited metabolic disease, mycoplasma disease, anthrax, Andersen's disease, congenital mitochondrial disease, phenylketonuria, placental infarction, syphilis, aseptic necrosis, and the like. In addition, necrosis and associated diseases caused by drugs and toxic substances are selected from the group consisting of necrosis associated with alcoholism, the exposure to, and/or administration and/or self-administration of, cocaine, drugs (for example, paracetamol), antibiotics, anti-cancer agents, Adriamycin, puromycin, bleomycin, NSAID, cyclosporine, toxic chemicals (for example, carbon tetrachloride, cyanide, methanol, and ethylene glycol), poison gas, agrochemicals, heavy metals (for example, lead, mercury, and cadmium), or injury due to the exposure to radioactivity/UV and associated necrosis thereof.
Furthermore, the compound of Chemical Formula 1 is expected to additionally exhibit preventive or therapeutic and ameliorative effects on acute/chronic kidney disease, traumatic brain injury, amyotrophic lateral sclerosis which is a neurodegenerative disease, necrotizing colitis, viral infection (for example, SARS-CoV), skin diseases including psoriasis and allergic dermatitis, organ preservation/organ transplantation (see Korean Patent Nos. 10-10983 and 10-1941004), and the like among necrosis and related diseases.
Further, a pharmaceutical composition including the compound of Chemical Formula 1 has a function of regulating intracellular calcium, and may ameliorate ER stress and mitochondrial dysfunction caused by abnormal intracellular calcium levels. Therefore, the pharmaceutical composition including the compound of Chemical Formula 1 is expected to exhibit preventive or therapeutic and ameliorative effects on related diseases. Related diseases are as follows.
Inflammatory pulmonary diseases including acute lung injury syndrome/acute pulmonary disease, pneumonia, tuberculosis, asthma, pulmonary arterial hypertension, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and cystic fibrosis (see mitochondrial dysfunction in fibrotic diseases. cell Death Discov. 2020 Sep. 5; 6:80. Mitochondrial dysfunction in lung aging and diseases. Eur Respir Rev. 2020 Oct. 15; 29(157):200165, and see Korean Patent No. 10-1636563)
Demyelinating diseases including demyelination and amyotrophic lateral sclerosis (ALS), hypertension including pulmonary artery hypertension, stroke, prion disease, epilepsy, ataxia, migraines, cognitive decline, seizures, tremors, mental illness (for example, depression) (see Neuronal and glial calcium signaling in Alzheimer's disease. Cell Calcium. October-November 2003; 34(4-5):385-97. Mitochondrial disorders: challenges in diagnosis & treatment. Indian J Med Res. 2015 January; 141(1): 13-26.)
Insulin resistance, hyperlipidemia, atherosclerosis, inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis, Various cancers and cancer metastases (see reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal. 2014 Jul. 20; 21(3):396-413.)
Visual impairment-related diseases (for example, retinitis pigmentosa, optic neuropathy, cataract, and glaucoma), anemia, cholestasis, hypoparathyroidism, pancytopenia, pancreatic disorder, lactic acidosis, lactic acidemia, hearing loss, short stature, ileus, cardiac conduction defects, cardiomyopathy, endometriosis, infertility, and premature menopause (see Mitochondrial diseases: the contribution of organelle stress responses to pathology. Nat Rev Mol Cell Biol. 2018 February; 19(2):77-92. Seminars in medicine of the Beth Israel Hospital, Boston. Mitochondrial DNA and disease. N Engl J Med. 1995 Sep. 7; 333(10): 638-44. Mitochondrial injury and dysfunction in hypertension-induced cardiac damage. Eur Heart J. 2014 Dec. 7; 3258-3266.)
Muscular dystrophy including limb girdle/Becker muscular dystrophy (LGMD/BMD) and Duchenne muscular dystrophy (DMD) (see Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition. Biochim Biophys Acta Mol Basis Dis. 2020 May 1; 1866(5): 165674.)
Aging and aging-related diseases (see Interrelation between ROS and Ca2+ in aging and age-related diseases. Redox Biology 2020; 6:101678.)
According to WO2009-025477, the pharmaceutical composition including the compound of Chemical Formula 1 not only exhibits liver protection and liver function improving effects, but also a preventive or therapeutic effect on acute and chronic liver diseases such as chronic hepatic diseases such as fatty liver, hepatic fibrosis and hepatocirrhosis, and hepatitis caused by viruses or drugs. As a result, hepatic disease complications such as portal hypertension may be prevented or treated, but the disease is not limited thereto. More specifically, the pharmaceutical composition according to the present invention is also effective for the treatment or prevention of hepatic diseases selected among liver transplantation, alcoholic or non-alcoholic fatty liver (see Korean Patent No. 10-2006247), hepatic fibrosis, hepatocirrhosis, hepatitis caused by viruses or drugs, and effective for alcoholic acute and chronic hepatic diseases. Further, the composition according to the present invention is effective for the treatment or prevention of fatty liver derived from fatty acids, or acute and chronic hepatic diseases derived from fatty liver.
According to Korean Patent No. 10-1852304, the compound of Chemical Formula 1 may enhance differentiation efficiency and maturity of stem cell-derived cardiomyocytes by comprising the step of culturing stem cells.
In addition, according to WO2016-072692, the compound of Chemical Formula 1 can also be used for the prevention and treatment of mucositis.
The present invention also provides a method for preventing or treating nontuberculous mycobacterium infectious diseases, the method comprising: administering the pharmaceutical composition according to the present invention to a subject in need thereof.
As used herein, the nontuberculous mycobacteria (NTM) refers to all mycobacteria except for the Mycobacterium complex and M. leprae, and may be, for example, at least one selected from the group consisting of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium fortuitum and Mycobacterium abscessus, preferably Mycobacterium abscessus, but is not limited thereto.
Specifically, in the present invention, nontuberculous mycobacterium infectious diseases may comprise one or more selected from the group consisting of lung diseases, lymphadenitis, skin/soft tissue/bone infections and disseminated diseases, but is not limited as long as it is a symptom that appears due to nontuberculous mycobacterium infection.
In the related art, it is known that two injection drugs need to be used together with one of amikacin, imipenem, and cefoxitin together with clarithromycin against lung diseases caused by a Mycobacterium abscessus strain.
Therefore, the compound represented by Chemical Formula 1 of the present invention or a pharmaceutically acceptable salt thereof may be used in combination with clarithromycin; and one or more drugs selected from the group consisting of amikacin, imipenem and cefoxitin.
As used herein, “treatment” refers to stopping or delaying the progress of the disease when used for a subject who shows the symptoms of the onset of the disease, and “prevention” refers to stopping or delaying the symptoms of the onset of the disease when used for a subject that does not show, but is at risk of, the symptoms of the onset of the disease.
In the present invention, the pharmaceutical composition may contain a pharmaceutically acceptable carrier together with the compound of the present invention, if necessary.
The compound of Chemical Formula 1 according to the present invention may be administered in various oral and parenteral dosage forms, and during formulation, the compound of Chemical Formula 1 according to the present invention is prepared using a diluent or an excipient, such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant, which are commonly used.
A solid formulation for oral administration comprises a tablet, a pill, a powder, a granule, a capsule, a troche, and the like, and the solid formulation is prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like with one or more compounds of the present invention. Further, in addition to a simple excipient, lubricants such as magnesium stearate and talc are also used. A liquid preparation for oral administration corresponds to a suspension, a liquid for internal use, an emulsion, a syrup, and the like, and the liquid preparation may comprise various excipients, for example, a wetting agent, a sweetener, an aroma, a preservative, and the like, in addition to water and liquid paraffin, which are commonly used simple diluents.
A preparation for parenteral administration comprises an aqueous sterile solution, a non-aqueous solvent, a suspension solvent, an emulsion, a freeze-dried preparation, a suppository, or the like. As a non-aqueous solvent and a suspension solvent, it is possible to use propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an injectable ester such as ethyl oleate, and the like. As a base of the suppository, it is possible to use Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerol, gelatin, and the like.
In addition, the effective dosage of the compound of Chemical Formula 1 of the present invention for the human body may vary depending on the patient's age, body weight, sex, administration form, health condition, and severity of disease, and is generally about 0.001 to 100 mg/kg/day, preferably 0.01 to 35 mg/kg/day. Based on an adult patient weighing 70 kg, the dosage is generally 0.07 to 7000 mg/day, preferably 0.7 to 2500 mg/day, and the compound of the present invention may be administered once or in several divided doses a day at regular time intervals according to the judgment of a doctor or pharmacist.
In the present invention, when the pharmaceutical composition is formulated as a drug, reference may be made to the content disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton PA, which is incorporated herein by reference.
The term “subject” of the present invention includes animals such as horses, sheep, pigs, goats, camels, antelopes, and dogs, or humans, which have a nontuberculous mycobacterium infectious disease whose symptoms can be alleviated by administration of the pharmaceutical composition according to the present invention. By administering the pharmaceutical composition for use in the prevention or treatment according to the present invention to a subject, nontuberculous mycobacterium infectious diseases may be effectively prevented or treated.
As used herein, the “administration” refers to introduction of a predetermined material to a human or animal by any appropriate method, and for the route of administration of the therapeutic composition according to the present invention, the preventive or therapeutic composition according to the present invention may be orally or parenterally administered via any general route, which may reach a target tissue.
Numerical values set forth herein should be construed to comprise a range of equivalents, unless otherwise specified.
The compound represented by Chemical Formula 1 according to the present invention or a pharmaceutically acceptable salt thereof can be used for use in the prevention or treatment of nontuberculous mycobacterium (NTM) infectious diseases.
The benefits and features of the present invention, and the methods of achieving the benefits and features will become apparent with reference to experimental examples and preparation examples to be described below in detail. However, the present invention is not limited to the experimental examples and the preparation examples to be disclosed below and may be implemented in various other forms, and the present invention is provided for rendering the disclosure of the present invention complete and for fully informing the scope of the present invention to a person with ordinary skill in the art to which the present invention pertains.
In the following Examples, the compound represented by Chemical Formula 1 according to the present specification is described as MIT-001.
Materials and Experimental Methods
1. Mycobacterial Strains and Inoculum Preparation for Infection
In the present invention, the smooth-morphotype Mabc ATCC19977 WT (Mabc-S) strain and the isogenic rough type (Mabc-R) strain were used. This genetically identical rough type of Mabc ATCC19977 strain was obtained through continuous anaerobic passage of the WT strain and has previously been used to study Mabc-R virulence factors. The bacteria were incubated at 37° C. using an orbital shaker (Middlebrook 7H9) in Middlebrook 7H9 medium containing 10% oleic albumin dextrose catalase (BD, Franklin Lakes, NJ) until the mid-log phase (OD600 nm=0.6).
After cultivation, bacterial culture broths were harvested by centrifugation, and a bacterial pellet was washed three times with a PBS buffer solution to completely remove glycerol and BSA. Since these bacteria, particularly Mabc-R, grow into cord-formatted clumps, they were separated into single cells at 3000 rpm for 2 minutes using a tissue homogenizer consisting of a Teflon rod and a glass tube (Wheaton, Millville, NJ, USA). After the separation process, the bacterial single-cell suspensions were aliquoted and stored at −70° C. until just before use. Prior to use, the frozen bacterial stocks were thawed in ice and immersed in an ultrasonic bath (As-one, Osaka, Japan) to prevent re-clumping and were used for the infection procedure.
Mabc CIP 104536T R and S strains were kindly provided by Dr. Laurent Kremer (Universite de Montpellier, Montpellier, France). Mabc CIP 104536T R and S morphotypes carrying a pMV262-mWasabi plasmid that enables the expression of mWasabi were used for the evaluation of bacterial dissemination of ZF. Mid-log phase Mabc strains were harvested and declumped before preparing a frozen stock using a 26-gauge needle and sonication was performed at 40 kHz for 30 seconds three times (Branson CPX3800, Danbury, CT, USA). Prior to injection, the numbers of viable bacteria were enumerated by plating serially diluted stocks on 7H10 agar. For ZF infection, the inoculum was diluted with phosphate-buffered saline-Tween 20 (PBST) 0.05% Tween 80 and resuspended in phenol red 0.085% to obtain 130 CFU/mL.
2. Mycobacterial Infection of Mice
Mycobacterial infection of SIRT3 WT and KO mice was performed as previously described. Groups of male 6 to 8-week-old mice were infected intranasally with Mabc-R (1×106 or 107 CFU/mouse) or Mabc-S (1×107 CFU/mouse) for 5 to 7 days. At 24 hours post-infection, the numbers of bacteria in the lungs of at least three mice were determined to confirm the Mabc-R and Mabc-S inoculum, and represent the average inoculum of mice in each group.
3. Maintenance of Mice and Isolation of Macrophages
SIRT3 WT and KO mice were kindly provided by Dr. Hyun Seok Kim (Ewha Womans University, Seoul, Korea) and maintained under specific-pathogen-free conditions at the Chungnam National University School of Medicine. The mice used in all experiments were 6 to 8 weeks old and sex-matched. All mice were bred and housed for experiments in accordance with the guidelines of the Chungnam National University School of Medicine. Mice experimental protocols were approved by the Institutional Animal Care and Use Committee of Chungnam National University (CNUA-18-0117).
Primary mouse bone marrow-derived macrophages (BMDMs) were isolated and cultured as previously described. BMDMs were isolated and differentiated after culture for 4 to 5 days in medium containing 25 ng/mL macrophage colony-stimulating factor (R&D Systems, Minneapolis, MIN, USA). Peritoneal macrophages (PMs) were isolated 2 to 3 days after injection with 1 mL of PBS supplemented with 3% thioglycollate (Sigma-Aldrich, St. Louis, MO, USA). Prior to isolation, PMs in the mouse abdominal cavity were collected with pre-chilled PBS supplemented with 10% FBS. BMDMs and PMs were cultured in a medium consisting of DMEM supplemented with 10% FBS, and penicillin-streptomycin-amphotericin B in a 5% CO2 atmosphere at 37° C.
4. Antibodies and Chemicals
MIT-001 (C24H29N3O3S; Patent No. KR2008-0080519) was provided by MitoImmune Therapeutics, Inc. (Seoul, Korea). Antibodies against SIRT3 (5490S) and ACTB (sc-47778) were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. Antibodies against NDUFA9 (ab14713) and UQCRC2 (ab14745) were purchased from Abcam (Cambridge, UK), antibodies against SDHA (5839) and COX4 (4844) were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against ATPSA (459240) were purchased from Thermo Fisher Scientific. A propidium iodide (PI) solution (P3566) and MitoSOX™ Red (M36008) were purchased from Invitrogen (Carlsbad, CA, USA). 3-TYP (S8628) was purchased from Selleckchem. Resveratrol was synthesized from 1-ethynyl-3,5-dimethoxybenzene, as described in Reference [20].
5. Determination of CFUs from Mabc-Infected Lungs and Macrophages
For in vivo CFU assays, lungs of the infected mice were harvested on day 5 or 7, depending on the experimental design. For the measurement of the bacterial burden, the lungs were homogenized in PBST and serial dilutions of the homogenates were plated on duplicate plates of Middlebrook 7H10 agar.
For the quantification of intracellular bacteria from macrophages, CFU assays were performed as previously described in Reference [21]. Briefly, Mabc-infected BMDMs were washed with PBS, and a fresh medium containing 50 μg/mL gentamicin (Sigma-Aldrich, St. Louis, MO, USA) was added and cell lysis was performed with 0.3% saponin (Sigma-Aldrich) to release intracellular bacteria. Then, infected lysates were vigorously resuspended, transferred to screw-capped tubes, and sonicated in a preheated 37° C. water bath sonicator (Elma, Singen, Germany) for 5 minutes. Aliquots of the sonicated lysates were diluted 5-fold in 7H9 medium and homogenates were plated on duplicate plates of Middlebrook 7H10 agar. Bacterial colonies were counted after 3 to 5 days of incubation at 37° C.
6. Immunohistochemistry (IHC) and PI Staining for Mabc-Infected Lung Tissues
Lungs were harvested from mice infected with Mabc for 10 days. The lungs were fixed in 10% formalin and embedded in paraffin wax. For histopathology, lung paraffin sections (4 μm) were cut and stained with hematoxylin and eosin (H&E) as described in Reference [21]. For analysis of the extent of tissue necrosis, PI staining was performed. For analysis of the extent of tissue necrosis, lung paraffin sections (4 μm) were cut and immunostained with a propidium iodide solution (P3566; Invitrogen, Carlsbad, CA, USA). After initiation, fluorescence images were obtained using a confocal laser-scanning microscope (LSM 710; Zeiss, CLSM, Jena, Germany), with constant excitation, emission, pinhole, and exposure-time parameters. H&E staining was scanned using an Aperio digital pathology slide scanner (Leica) and imaged using an Aperio ScanScope® CS System. To quantify the inflamed area and necrosis, the MFI of the red threshold was determined using FIJI software.
7. RNA Extraction and Quantitative Real-Time PCR (qPCR) Analysis
RNA extraction and qPCR were performed as described in Reference [21]. Briefly, total RNA from BMDMs or lung tissues was isolated using the TRIzol reagent (Thermo Fisher Scientific). cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, 18064). qPCR was performed using cDNA, primers, and SYBR Green PCR Kits (Qiagen, 204074) using a Real-time PCR Cycler Rotor-Gene Q 2plex system (Qiagen GmbH, 9001620, Hilden, Germany). The samples were amplified for 50 cycles as follows: 95° C. for 5 seconds and 60° C. for 10 seconds. To analyze qPCR data, relative quantification was performed using the 2ΔΔCt method using Gapdh as an internal control gene. Data was expressed as a relative fold change. The primer sequences are shown in Table 1.
8. Enzyme-Linked Immunosorbent Assay (ELISA)
The concentration of TNF in lung tissues was measured using a commercially available ELISA kit (BD Biosciences, 558534, San Jose, CA, USA). Experiments were performed according to the protocol provided by the manufacturer.
9. Western Blot Analysis
Tissue homogenates were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid (DOC), 0.1% SDS, and 1 mM PMSF) (ELPIS Bio, Lexington, MA, USA) supplemented with protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein were mixed with 5×SDS sample buffer (ELPIS) and heated for 5 minutes. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked in 5% skim milk in Tris-buffered saline-Tween 20 (TBS-T) at room temperature for 1 hour, and were incubated at 4° C. overnight with the following specific primary antibodies: anti-SIRT3 (5490, Cell Signaling Technology), anti-ACTB (sc-47778, Santa Cruz Biotechnology), anti-NDUFA9 (ab14713, Abcam, Cambridge, UK), anti-SDHA (5839, Cell Signaling Technology), anti-UQCRC2 (ab14745, Abcam), anti-COX4 (4844, Cell Signaling Technology), anti-ATPSA (459240, Thermo Fisher Scientific). After washing with TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1 hour. Blots were imaged using a chemiluminescence assay kit (Millipore) in a UVitec Alliance mini-chemiluminescence device (BioSPX, Abcoude, Netherlands). Band densities were quantified using ImageJ software, and data were normalized to β-actin loading control.
Measurement of Mitochondrial ROS and Immunofluorescence
Mitochondrial ROS were measured as described previously in Reference [16]. SIRT3 WT and KO BMDMs were incubated with 3 μM MitoSOX Red Mitochondrial Superoxide Indicator (Invitrogen, M36008). After 20 minutes, the cells were washed and measured using an immunofluorescence assay. Nuclei were stained by incubation with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) at the same time. Immunofluorescence images were obtained using a confocal laser-scanning microscope (Zeiss). The mean fluorescence intensity (MFI) level of mitochondrial ROS was calculated for each sample. Each experiment was performed in triplicate, and at least 200 cells per well were counted.
11. Mitochondrial Oxygen Consumption Rate (OCR) Analysis
The XF24 biosensor cartridge was activated with 1 mL of XF24 calibrant solution (Seahorse Bioscience, Billerica, MA) per well at 37° C. for 24 hours in a non-CO2 incubation system. SIRT3 WT and KO BMDMs infected with Mabc for 18 hours were seeded at 2×104 cells per well and incubated at 37° C. for 24 hours. The cell plate was incubated at 37° C. for 1 hour in a non-CO2 incubation system after the addition of a 590 μl assay medium to each well. ATPase inhibitor oligomycin A (20 μg/mL, Sigma-Aldrich, MO, USA), uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 μM, Sigma-Aldrich, MO, USA), and mitochondrial complex I inhibitor rotenone (20 μM, Sigma-Aldrich, MO, USA) were sequentially added to each well after measurement of basal OCR. The oxygen consumption rate of the entire process was measured using a Seahorse Bioscience XF24 analyzer (Seahorse Bioscience, Billerica, MA).
12. Transmission Electron Microscopy (TEM) Analysis
SIRT3 WT and KO mouse lung tissues were fixed with 2.5% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.2) containing 0.1% CaCl2). After 3 hours, the cells were post-fixed with 1% OsO4 in a 0.1 M sodium cacodylate buffer containing 0.1% CaCl2) for 2 hours. The tissues were rinsed with cold distilled water and slowly dehydrated using an ethanol series and propylene oxide at 4° C. The tissues were embedded in Embed-812 and cured at 60° C. for 30 hours. Ultrathin sections (70 nm) were cut with a diamond knife and an ULTRACUT UC7 ultramicrotome (Leica) and mounted on formvar-coated copper grids. Sections were stained with 4% uranyl acetate for 7 minutes and lead citrate for 7 minutes. TEM-stained sections were scanned using a Bio-High Voltage EM system (JEM-1400 Plus and JEM-1000 BEF; JEOL Ltd., Tokyo, Japan).
13. Statistical Analysis
Statistical analysis was performed in Prism (GraphPad Software, v5.01, 2007). Data is presented as mean±SEM (standard error of the mean). Data was analyzed using a two-tailed Student's t-test or non-parametric test. In the non-parametric test, two conditions were compared using a Mann-Whitney U-Test and three or more conditions were compared using one-way ANOVA with a Dunn's multiple comparison test where appropriate. Specific p values are described in detail in the figure legends. For the ZF survival study, Kaplan-Meier survival curves were generated and analyzed by Gehan-Breslow-Wilcoxon test.
Results
1. SIRT3 is Required for Host Defense Against Mabc Infection
Given the present inventors' previous findings of SIRT3 during Mtb infection (Reference [16]), the present inventors first investigated whether Mabc infection decreased the levels of SIRT3 in macrophages and in vivo. The present inventors found that Mabc-R infection significantly decreased the expression of SIRT3 in macrophages in a time-dependent manner (
Further, the SIRT3 KO mice had enhanced lung pathology at 5 dpi using Mabc-R (that is, granulomatous and inflammatory lesions in the lungs) compared to the SIRT3 WT mice (
2. SIRT3 is Required for the Amelioration of Pathological Inflammation and Control of Mitochondrial Damage During Mabc-R Infection
Since the Mabc-R variant is more virulent and active in the induction of inflammation than Mabc-S(Reference [8]), the present inventors next compared the lung inflammatory responses between SIRT3 WT and SIRT3 KO mice using the Mabc-R strain. To examine this, the present inventors collected lung tissues at 1 and 3 dpi, and performed qRT-PCR analysis for the mRNA levels of proinflammatory cytokines/chemokines. As illustrated in
It is well established that SIRT3 is essential in mitochondrial homeostasis and functions to protect various cells and tissues from stress-induced cell death (References [22 to 25]). In addition, the present inventors reported that SIRT3 is required for the maintenance of mitochondrial homeostasis during Mtb infection (Reference [16]). When the present inventors next performed ultrastructural analysis between SIRT3 WT and KO lungs after infection, the TEM data showed that SIRT3-deficient lungs had a marked accumulation of damaged mitochondria, as represented by swollen and disrupted cristae, when compared with SIRT3 WT mice at 5 dpi after Mabc-R infection (
3. SIRT3 Deficiency Increases Inflammatory Responses and Mitochondrial Oxidative Stress in Macrophages During Mabc-R Infection
The present inventors next compared the mRNA expression of inflammatory cytokines in BMDMs of SIRT3 WT and SIRT3 KO mice after Mabc-R infection. Mabc-R-mediated mRNA generation of Tnf, Il6, and Cxcl2 was significantly increased in BMDMs of SIRT3 KO mice compared to SIRT3 WT mice after infection in a time-dependent manner (
Previous studies demonstrated that mitochondrial ROS generation is regulated by SIRT3 in various cells (References [26 to 29]). Therefore, mitochondrial redox status was compared between SIRT3 WT and SIRT3 KO BMDMs using MitoSOX Red, a highly selective fluorescent probe for the detection of mitochondrial O2− (Reference [30]). The present inventors first examined whether Mabc-R generated more mitochondrial ROS in WT BMDMs than Mabc-S. The present inventors infected BMDMs with either Mabc-R or Mabc-S and found that Mabc-R led to more mitochondrial ROS production at 2 hours after infection than Mabc-S did. In addition, the present inventors found that mitochondrial O2− generation was significantly increased in SIRT3 KO BMDMs, when compared with SIRT3 WT BMDMs, after Mabc-R infection (
4. SIRT3 is Essential for the Maintenance of Oxidative Phosphorylation Function and Blockade of Exaggerated Cell Death During Mabc-R Infection
The mitochondrial oxidative phosphorylation (OXPHOS) system is essential for energy production and cellular homeostasis (Reference [31]). The present inventors further determined the levels of major mitochondrial proteins in SIRT3 WT and KO lungs during Mabc-R infection. The present inventors found that the protein expression levels of OXPHOS were dramatically suppressed in the SIRT3 KO lungs, when compared with those of SIRT3 WT lungs, at 3 dpi after Mabc-R infection (I: NDUFA9, II: SDHA, III: UQCRC2, IV: COX4, V: ATP5A) (
Given the findings that SIRT3 deficiency results in increased mitochondrial defects and ROS production, and reduced OXPHOS activity, the present inventors next examined whether cell death was upregulated in the lungs of SIRT3 KO mice compared to those of SIRT3 WT mice. The present inventors previously showed that Mabc-R variants induced greater cell death in RAW264.7 cells than the smooth strain did (Reference [18]). Therefore, the present inventors elucidated whether Mabc-R infection induced the activation of PI-positive cell death in infected lungs of mice. As illustrated in
5. Blockade of Excess Mitochondrial ROS Enhances the Antimicrobial Response and Ameliorates Pathological Inflammation During Mabc-R Infection
The mitochondrial ROS scavenger MIT-001 (previously, NecroX-7) removes mitochondrial ROS, calcium, and reactive nitrogen species (References [32 and 33]). As expected, treatment of BMDMs with MIT-001 remarkably suppressed the generation of mitochondrial ROS in response to Mabc-R infection (
We next examined the in vivo effects of MIT-001 on pathological inflammatory responses during Mabc-R infection. The mRNA expression of Tnf and Il6 was dramatically increased in the mouse lungs after Mabc-R infection (
To further analyze the mitochondrial damage in vivo, the present inventors performed TEM analysis of lung tissues of Mabc-R-infected mice regardless of MIT-001 treatment. After treatment with MIT-001, there were considerably fewer damaged mitochondria with disrupted cristae in the alveolar cells of lung tissues of Mabc-R-infected mice than in vehicle control mice (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0103849 | Aug 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/010982 | 8/18/2021 | WO |
