WO2026006502A1 - High-throughput drug screening platform for rapid and efficient identification of compounds that modulate mitochondrial function - Google Patents

Abstract

An automated high throughput screening platform for identification of compounds and genomic constructs having mitochondrial function modulating activity and compounds so identified are provided. Use of such compounds for modulating mitochondrial function are also disclosed. A high-throughput screening method of determining genetic mechanisms of action for therapeutic or toxic agents in genetic or pharmacologic models of disease are also provided.

Description

High-throughput Drug Screening Platform for Rapid and Efficient Identification of Compounds that Modulate Mitochondrial Function

By

Marni J. Falk

Neal D. Matthew

Kelsey Keith

Cross-Reference to Related Application

This application claims priority to US Provisional Patent No. 63/664,649, filed June 26, 2024, the entire disclosure being incorporated herein by reference as though set forth in full.

Grant Statement

This invention was made with government support under grant number R35-GM134863, R01GM115730, and T32NS007413 awarded by the National Institutes of Health. The US government has certain rights in the invention.

Incorporation-by-Reference of Material Submitted in Electronic Form

The contents of the electronic sequence listing (CHOP-157-PCT.xml; Size: 1,996 bytes; and Date of Creation: June 26, 2025) are incorporated herein by reference as though set forth in full.

Field of the Invention

This invention relates to the fields of mitochondrial function and high throughput drug or genomic library screening platforms. More specifically, a robust preclinical screening assay and automated analytic system is provided for rapid, and efficient identification of agents in compound or genomic libraries which enhance or inhibit at least one mitochondrial function, selected from activity, stress resilience, and stability. The system also facilitates simultaneous elucidation and characterization of the agent’s effects on whole organism health. Methods of use of agents so identified for the treatment of mitochondrial dysfunction are also provided. A high- throughput screening method of determining genetic mechanisms of action for therapeutic or toxic agents in genetic or pharmacologic models of disease is also provided. Background of the Invention

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Mitochondrial diseases are a group of highly heterogenous diseases with no specifically approved therapies. Primary mitochondrial disease (PMD) has a prevalence of 1 in 4,300 individuals (Muraresku 2018), not including individuals with secondary mitochondrial dysfunction who may also benefit from treatments for mitochondrial dysfunction (Niyazov 2016). PMD treatment relies mainly on addressing symptoms, with minimal interventions available to target the cause or consequences of the disease directly and are mainly limited to attempting to supplement biomolecules like vitamins, cofactors, and amino acids to improve limited functionality or replace missing factors (Camp 2016). In addition to lacking treatment options, PMD patients are more vulnerable both to environmental toxicants and to drugs that may otherwise be considered safe in the general population. Indeed, many drugs have known effects on mitochondrial activity. Clearly, the need for new treatment modalities and identification of mito-toxic compounds are urgently needed for the management and treatment of PMD and other disorders having secondary mitochondrial disruption and symptoms related to impaired mitochondrial function. Rapid and efficient identification of compounds that disrupt mitochondrial function and cell or organismal health is itself also desirable in conditions ranging from cancer to infectious disease.

Summary of the Invention

In accordance with the present invention, a system for rapid, semi-automated high throughput screening (HTS) and analysis of compounds that modulate at least one of mitochondrial function, organismal health, or cellular health is provided. An exemplary method entails dispensing i) a plurality of genetically altered C. elegans worm strains, said genetic alteration impacting a gene associated with mitochondrial function, regulation, stress resilience, or stability, and ii) wild-type C. elegans, lacking said genetic alteration, into multi- well plates with an semi- or fully-automated dispensing instrument; said worms comprising at least a first and second detectable marker; generating daughter plates in DMSO buffer control harboring a plurality of members from a compound library, and pinning said daughter plate onto the worm containing plates described above, followed by incubation of the worms for a suitable time period for said compound to exert an effect, an integrated metric of worm mass and activity levels is then determined using WormScan, along with mitochondrial stress induction using a high content imaging platform for focusing i) a red fluorescence channel with a 20% exposure sufficient to detect each worm expressing a first red fluorescent selectable marker in the well; ii) a green fluorescence channel with an exposure setting of 40% for detection of levels of a second green fluorescent detectable marker in the well; and ii) an exposure setting of 10% for brightfield image, wherein red fluorescence levels are used to count the number of worms per well, green fluorescence levels correlating with mitochondrial physiology are set to a threshold of 1800 pixel intensity and, applying spot total intensity to measure total green fluorescence in each well. Quality control is then performed on every well and on each plate by i) removing any well with a green channel value 1.5-fold greater than the interquartile range of the other wells for that given compound; ii) removing any well with a red channel count of C. elegans less than 5 or greater than 90, and iii) removing any compound where 3 out of 4 replicate wells failed each plate. The dynamic range for the green channel between conditions worms in step a) when compared to untreated control worms, is assessed by determining a Strictly Standardized Mean Difference (SSMD) calculated as Formula I: and discarding any plate having an SSMD of less than -2. Activity is then normalized by dividing the WormScan score by the number of C. elegans in the well as measured by the red fluorescence channel and normalizing green channel signal from UPRmt (or other genetic or fluorescent indicator of a specific mitochondrial function or parameter or each compound) as the percent reduction in stress relative to the plate controls, thereby allowing for plate-to-plate comparison; background, healthy animal green fluorescence is then subtracted from all values and the percent change between the median disease fluorescence and the control well calculated using Formula II: and selecting hits from library HTS based on a percent reduction in mitochondrial stress greater than 80% and a WormScan score greater than 50. In certain embodiments, the hit is confirmed as a modulator of mitochondrial function in a zebrafish (D. rerio) model. In other embodiments, the hit is confirmed as a modulator of mitochondrial function in a human cell. In other embodiments, the hit is confirmed in C. elegans, D.rerio and H. sapiens cells. The at least two first and second detectable labels are interchangeable, and can be selected from molecules having red, green, blue, or yellow fluorescence.

The hit can be a toxicant or a compound that inhibits mitochondrial function and/or animal survival in a mitochondrial deficient animal, such as those listed in Appendix I. The hit can alternatively be a compound that enhances mitochondrial function and, or reduces mitochondrial stress. The system can also comprise determining whether said agent alters an organismal health or cellular parameter associated with mitochondrial function in C. elegans comprising said genetic alteration relative to wild-type C. elegans; agents which alter said parameter in said genetically altered C. elegans being identified as modulators of mitochondrial function. Cellular parameters to be determined include, without limitation, fecundity, egg hatching rate, development, lifespan, stressor survival, healthspan, animal activity, animal mass, animal size, swimming capacity, pharyngeal pumping rate, mitochondrial oxidant burden, cellular’ oxidant burden, antioxidant capacity, complex I (CI) enzyme activity, CI enzyme assembly, citrate synthase activity, pyruvate dehydrogenase activity, oxygen consumption capacity, ATP production, ATP levels, nicotinamide dinucleotide (NADH and NAD+) levels, (NADH and NAD+) ratio, NAD metabolism, mitochondrial membrane potential, mitochondrial content, mitochondrial structure, mitochondrial ultrastructure, mitochondrial unfolded protein response, mitochondrial stress resilience, integrated stress response, mitochondrial import, mitophagy, autophagy, apoptosis, necrosis, ferroptosis, iron levels, iron metabolism, cell death pathway induction, cytosolic translation activity, nutrient-sensing signaling profile, unfolded protein response activation, lysosomal number, lysosomal activity, proteasome number or activity, transcriptome- wide signaling, amino acid pathway profiles, intermediary metabolic flux rates, steady state metabolism of intermediary metabolites, electrolyte levels or flux such as involving potassium, calcium, sodium, chloride, phosphorous, or magnesium, metal levels such as iron, ferritin, immune markers, amino acid levels, organic acid levels, ammonia levels, and glycoprotein production, cellular proliferation, cell growth, cell death pathways such as autophagy, apoptosis, necrosis, ferroptois, lactic acid level, pyruvate level, glycolysis, cellular redox levels, cell pH, lactate/pyruvate ratio, or target gene expression level.

In certain approaches, the C. elegans comprises a mutation in a gene that modulates one or more of mitochondrial structure, content, proliferation, destruction, stress resilience, and function.

Libraries to be screened include without limitation those selected from a chemical library, a genetic library, an RNA interference (RNAi) library, an siRNA library, a cDNA library, a gDNA library, an mRNA library, a preclinical drug library, a chemical combinatorial library, a CNS penetrant compound library, a commercially available library, a custom proprietary library, a natural product compound library, an FDA approved drug library, a clinical trial tested compound library, a bacterial library, a bacterial product library, or an environmental toxicology compound library.

In certain aspects of the system, the worms comprise a first selectable myo2::mcheny genetic marker for determining worm number per well and a second selectable hsp6::gfp genetic marker for quantifying t7PR”’ⁿnduction. One or more steps, e.g., the pinning step in the system can be automated. In yet another approach, a mitochondrial modulating agent selected from a pharmacologic agent, an acute toxicant and a chronic toxicant is dispensed into the multi-wells of step a) i), to assess activity in the presence of a stressor.

Also provided is composition for the negative modulation of mitochondrial function, comprising an effective amount of at least one agent selected compounds listed in Appendix I. The invention also provides a composition for the positive modulation of mitochondrial function, comprising an effective amount of at least one agent selected from cisplatin; benzalkonium chloride hydrate; cetrimonium bromide; phentolamine HC1; bromindione; polymyxin sulfate; mitoxantrone HC1; or mefloquine HC1 in a pharmaceutically acceptable carrier for administration separately or in combination. The positive modulating composition can further comprising an effective amount of steroids, MAPK-modulators, membrane stabilizers, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, immune modulators, CD4 modulators, CD8 modulators, NK cell modulators, B cell modulators, inhibitors of IgG isotype switching or IgG synthesis, 0-agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines. Also described is a combination therapy comprising at least two agents listed in Appendix I, said agents acting synergistically to negatively modulate mitochondrial function when combined. In yet another aspect, combination therapy comprising at least two agents selected from cisplatin; benzalkonium chloride hydrate; cetrimonium bromide; phentolamine HC1; bromindione; polymyxin sulfate; mitoxantrone HC1; or mefloquine HC1 one or more of N-acetylcysteine, nicotinic acid (niacin), niacinamide, nicotinamide riboside, nicotinamide mononucleotide, probucol, glucose, 2-deoxyglucose, lipoic acid, resveratrol, thiamine, riboflavin, leucine, uridine, 3 -methyladenine, hemin, trypterin, (+) epicatechin, (-) epicatechin, 11 -hydroxyprogesterone, 11- hydroxypregnenolone, cycloheximide, rapamycin, lithium chloride, creatine, dichloroacetate, omaveloxolone, elamipretide, dimethylfumarate, a deubiquitalase (DUB) modulating agent, a PPAR alpha modulating agent, a PPAR gamma modulating agent, a PPAR delta modulating agent, an AMPK modulating agent, a sirtuin modulating agent, a mTORC 1 modulating agent, a MAPK modulating agent, a PGCl-a modulating agent, a HIF1 -alpha modulating agent, a mitochondrial biogenesis modulating agent, an antioxidant, a phosphodiesterase modulator, sildenafil, doxycycline, tetracycline, trapidil, a calcium channel modulator, flunarizine, folinic acid, metformin, a nucleoside, valproic acid, dexamethasone, etoposide, vorinostat, quercitin, hydralazine, pfithrin-a, ginsenoside, sulfonsuccinimidyl oleate, carnitine, levocarnitine, AICAR, GSK2578215A, bezafibrate, vatiquinone, coenzyme Q10, alpha-tocopherolquinone, a coenzyme Q10 analog, ubiquinone, ubiquinol, cytochrome C, 5 I-2-(4-hydroxyphenyl)-ethenyl] benzene-1,3 diol, flavin mononucleotide, urolithin A, idebenone, latrepirdine, 2',3',5'-tri-O-acetyluridine, olesoxime, thiamine diphosphate, vitamin C, vitamin D, vitamin E, lipoic acid, delta- aminolevulinic acid, magnesium, calcium, phosphate, membrane phospholipid, unsaturated fatty acid, pyruvate, nicotinamide adenine dinucleotide, cysteamine bitartrate, acipimox, glutathione, a redox-modulating agent, curcumin, schisandrin, triheptanoin, an inhibitory nucleic acid targeting an upregulated gene associated with mitochondrial dysfunction, an activating genetic construct for increasing expression of a mitochondrial disease modulator protein, a nucleic acid/protein complex for genetic and/or base editing suitable for replacement therapies for primary mitochondrial diseases, a viral or non-viral vector gene therapy targeting a mitochondrial disease mutation, or a combination of any 2, 3, 4, 5 or 6 of the agents listed in a pharmaceutically acceptable carrier for administration, said agents acting synergistically to improve mitochondrial function when combined is disclosed. Preferred combinations for this purpose include glucose and mitoxantrone HC1 or glucose and phcntolaminc HO.

Methods for modulating a cellular parameter associated with mitochondrial function or dysfunction, comprising administration of the composition of one or more of the compounds described above, to a patient in need thereof are also provided. In certain embodiments, mitochondrial dysfunction is present and symptoms include one or more of muscle weakness, exercise intolerance, chronic fatigue, gastrointestinal dysmotility, cognitive decline, autism spectrum disorder, impaired balance, peripheral neuropathy, metabolic strokes, dysautonomia, vision loss, eye muscle or eyelid weakness, hearing loss, tinnitus, glomerular’ or tubular renal disease, endocrine dysfunction, dyslipidemia, cardiomyopathy, arrhythmia, cardiac conduction block, anemia, failure to thrive, over or underweight, developmental delay, neurodevelopmental regression, cognitive decline and memory impairment, Parkinsonism, mood disorder, dystonia, liver dysfunction or failure, infertility, metabolic instability, stressor-induced acute decompensation, mitophagy disorders, mitochondrial lipid biogenesis disorders, mitochondrial cofactor disorders, primary mitochondrial disease, and secondary mitochondrial disorders including but not limited to resulting from toxins, drugs, natural products, infections, cancer, age, prescribed or illicit medications, smoking, alcohol, environmental exposures, obesity, and genetic disorders that secondarily impair mitochondrial function, structure, or activities. In certain aspects the method can also comprise performance of a compression assay.

The mitochondrial symptoms can be caused by a disorder selected from the group consisting of Complex I disease, Complex II disease, Complex III disease, Complex IV disease, Complex V disease, multiple respiratory chain complex disease, adenine nucleotide translocase deficiency, pyruvate dehydrogenase deficiency, mitochondrial depletion disease, single large- scale mtDNA deletion disease, multiple mitochondrial DNA deletions disease, mitochondrial DNA maintenance defects, mitochondrial translation defects, mitochondrial nucleotide import disease, Friedreich's ataxia, Leber's Hereditary Optic Neuropathy, Autosomal Dominant Optic Atrophy, Kearns-Sayre Syndrome, Pearson Syndrome, Mitochondrial Myopathy, Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-Like Episodes, Myoclonic epilepsy and ragged red fibers, Neurogenic Ataxia and Retinitis Pigmentosa, Mitochondrial Neuro- gastrointcstinal cnccphalomopathy, maternally inherited diabetes and deafness, FBXL4 mitochondrial encephalomyopathy, primary lactic acidosis, Leigh syndrome, Leigh-like syndrome, and multi-system mitochondrial disease.

Also provided is method for inhibiting growth or survival of a cancer cell or tumor comprising administration of an effective amount of one or more mitotoxicants listed in Appendix 1, thereby inhibiting growth of, or killing said cell. In an alternative embodiment, a method for inhibiting growth of an infectious disease-causing virus, bacteria, parasite, or fungal cell comprising administration of an effective amount of one or more mitotoxicants listed in Appendix 1, thereby inhibiting growth of, or killing said infected cell.

Finally, genetic alterations can be present in nuclear DNA in one or more genes selected from AARS2, ABCB7, ABCC8, ACAD8, ACAD9, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ACO2, ADCK3, ADRB2, ADRB3, AFG3L2, AGK, AGRP, AIFM1, AK2, AKAP10, AKT2, ALAS2, ALDH2, ALDH4A1, ALDH6A1, AMACR, AMT, APOPT1, APTX, ARMS2, ATP5AI, ATP5E, ATP5FIA, APT5F1D, ATP5F1E, ATPAF2, AUH, BAX, BCAT2, BCKDHA, BCKDHB, BCL2, BCS1L, BOLA3, C8orf38, C10orf2, C12o 62, C12o 65, C19orfl2, C20orJ7, C1QBP, CAPN10, CARS2, CARTPT, CDH23, CDKAL1, CHCHD10, CHKB, CISD2, CLRN1,COA5, COA7, COAIO, COQ2, COQ4, COQ6, COQ7, COQ8A, COQ9, COXIO, COX14, C0X15, COX20, C0X412, C0X6B1, C0X8A, CPS1, CPT1A, CPT2, CRAT, CYB5R3, CYC1, CYC5, CYCS, CYP11A1, CYP1161, CYP1162, CYP24A1, CYP27A1, CYP2761, D2HGDH, DARS2, DBT, DECR1, DFNB31, DGUOK, DHODH, DIABLO, DLD, DLAT, DMGDH, DNA2, DNAJC19, DNM1L, EARS2, ECHS1, ELAC2, ENPP1, ETFA, ETFB, ETFDH, ETHEl, FARS2, FASTKD2, FBXL4, FOXRED1, FH, FXN, GA TM, GCDH, GCGR, GCK, GCSH, GDAP1, GFER, GFM1, GHRL, GJB2, GJB3, GJB6, GK, GLDC, GLRX5, GLUD1, GPD2, GPR98, GTPBP3, HADH, HADHA, HADHB, HARS2, HCCS, HIBCH, HK1, HLCS, HMGA1, HMGCS2, HMGCL, HNF1A, HNF1B, HNF4A, H0GA1, USD 17610, HSPD1, HTRA1, HTRA2, IBA57, IDH2, IDH3B, IGF2BP2, IL6, INSR, IRS1, IRS2, ISCA1, ISCA2, ISCU, IVD, KANK1, KARS, KCNJ11, KIF1B, L2HGDH, LARS2, LEPR, LIAS, LIPC, LRPPRC, LRRK2, LYRM4, LYRM7, MAO A, MAPK8IP1, MAR52, MC4R, MCCC1, MCCC2, MCEE, ME2, MFF, MFN2, MGME1, MIPEP, MLYCD, MMAA, MMAB, MMADHC, MPC1, MPV17, MRAP2, MRPL3, MRPL44, MRPS2, MRPS7, MRPS16, MRPS22, MRPS34, MST01, MTFMT, MT01, MTPAP, MET, MY07A, NAGS, NARS2, NDUFA1, NDUFA2, NDUFA6, NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFA 13, NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, NDUFB3, NDUFB8, NDUFB9, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS5, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NEURODI, NF2, NFU1, NGLY1, NR0B2, NUBPL, NUP62, OAT, OGDH, 0PA1, 0PA3, OTC, 0XCT1, PANK2, PARK2, PARK7, PAX4, PC, PCCA, PCCB, PCDH15, PCK2, PDHA1, PDHB, PDHX, PDP1, PDSS1, PDSS2, PDX1, PET100, PINK1, PNKD, PNPT1, POLG, P0LG2, POMC, PPARG, PPARGC1B, PPDX, PPP1R3A, PUS1, PYCR1, PYY, RARS2, REEP1, RMRP, RMND1, RNASEH1, RRM2B, SACS, SARDH, SARS2, SC01, SC02, SC03, SDHA, SDHAF1, SDHAF2, SDHB, SDHC, SDHD, SERAC1, SFXN4, SIM1, SLC2A2, SLC2A4, SLC22A5, 5LC25A1, 5LC25A3, 5LC25A4, 5LC25A12, 5LC25A13, 5LC25A15, 5LC25A19, 5LC25A20, 5LC25A22, 5LC25A26, 5LC25A38, SLC30A8, 50D2, SPG7, SPG20, STAR, SUCLA2, SUCLG1, SURF1, TAC01, TARS2, TAX, TDF7L2, TEAM, TIMM8A, TIMMDC1, TK2, TMEM70, TMEM126, TMEM126B, T0P3A, TRIT1, TRMT10C, TRMT5, TRMU, TRNT1, TSFM, TTC19, TUFM, TWNK, UCP1, UCP2, UCP3, UNG, UQCC2, UQCC3, UQCRB, UQCRC2, UQCRQ, USMG5, USH1C, USH1G, USG2A, VARS2, WARS2, WFS1, WWOX, XPNPEP3, and YARS2.

Brief Description of the Drawings

Figures 1A -IB. Fig. 1A) Theoretical reasons for mitochondrial sensitivity to exposures to environmental chemicals. From Meyer et al. (2013). Fig. IB) Perturbation of the folding environment in mitochondria up-regulates the expression of nuclear' genes encoding mitochondrial chaperones causing mitochondrial stress as evidenced by the mitochondrial unfolded protein response (UPR" '). A model for the signal transduction pathway of the UPR^mt is shown.

Figures 2A -2F. A customized, automated, analytic system was used for high-throughput screening of a 2,560-compound library of FDA-approved drugs and natural compounds (MicroSource) in 3 mutant C. elegans strains, including missense mutants for NDUFS2 ^/' (gas- l(fc21)) and OPA1' ' (eat-3(R289Q), “RQ”), as well as a heteroplasmic single large-scale mitochondrial DNA deletion model (SLSMD, uaDf5). (Figs. 2A-2C) The number of compounds surviving at each screening stage for the given C. elegans strain are shown, including (1) Total compound number screened, (2), hits that met initial threshold level, (3) hits that replicated at same dose in the same assay, and (4) hits that showed a dose-dependent response in a multi-point dose response curve in same assay. (Figs. 2D-2F) Percent reduction in mitochondrial stress for the best compounds ranked in order of increasing efficacy from each strains’ independent screen along with respective controls: untreated animals, animals treated with ivermectin (antihelminthic compound) as a negative control, animals treated with our previously identified effective therapy (/V-acctylcystcinc (NAC) or thiamine) as a positive control, along with untreated healthy animals. (Fig. 2D) NDUFS2' ' gas-l(fc21) HTS was completed with hits at both 0.4 pM and 40 pM concentrations, where each lead compound only hit at a single concentration. Only 40 pM concentration was tested in RQ and uaDf5 worm strains HTS. From the 2,560 compounds we found 8 reproducible hits, a rate of 0.3%, which were validated with further lower-throughput phenotypic outcome measures in the gas-1 (fc21 ) model and in the ndufs2~^l~ zebrafish model.

Figures 3A - 3C. Toxicity results from the NDUFS2-/- gas-l(fc21) screen. Cutoffs for toxicity were determined by examining the controls’ mitochondrial stress (Fig. 3 A) and WormScan -based quantitative measurement of C. elegans neuromuscular swimming activity, normalized by the number of animals present (Fig. 3B), where a drug that had either a low reduction/increase in mitochondrial stress or low worn activity was considered as indicative of potential toxicity. (Fig. 3C) Reduction in mitochondrial stress vs worm activity is plotted at the two concentrations tested in the screen (0.4 pM and 40 pM), with potentially toxic compounds highlighted based on mechanism (stress, activity, or both) based, on which they were called as toxic.

Figures 4A - 4D. Graphical flow chart showing steps in high throughput screening (HTS) of the library of potential mitochondrial function modulators in Figure 1. (Fig. 4 A) Visualization of suitable equipment for plating worms, e.g., a MultiFlo FX (1) and manual drug pinning equipment (2). Note automated platforms such as the Agilent Bravo Liquid Handling platform are also suitable for this purpose) to evaluate 2,560 compound library in 384 -well plate format. (Fig 4B) HTS flow chart of the physical experimental steps. For experimental set up and treatment, worms at L4 larval stage were dispensed into a 384 well-plate at 1 worm I pL, with the MultiFlo FX Multimode Dispense (BioTck Instruments). Daughter plates were generated at (0.1 mM and) 10 mM) in DMSO from the Spectrum Collection (MicroSource Discovery Systems, Inc.) that includes 32 individual 96-well plates containing 2,560 compounds at 10 mM in DMSO. These daughter plates were then pinned into 384-well plate already containing worms, media, and drug. After incubation for a suitable time period, worm activity was determined using WormScan and mitochondrial UPR^mt stress induction was determined using a Cclllnsight CX5 (Thermo Scientific) by modifying a protocol adapted from previous C. elegans HTS (Gosai 2010). (Fig 4C). Overview of HTS automated data quality control and normalization pipeline: Quality control was performed on every well and on each plate, where any plate not satisfying predetermined threshold levels described herein below were discarded. (Fig 4D) Overview of HTS automated data normalization and hit identification: Activity was normalized by dividing the WormScan score by the number of C. elegans in the well as measured by the red channel. Background, both in random expression of fluorescence by the healthy control and in the background level of stress experienced by the disease model, varied for each plate so the UPRmt for each compound was normalized as the percent reduction in stress relative to the plate controls to allow for plate-to-plate comparison.

Figures 5A - 5D. Hit selection of drug library compounds screened. Fig 5A) Graph shows significant mitochondrial stress modulators identified in the high throughput screening (HTS) assay evaluated at two concentrations (0.4 pM and 40 pM). Results are shown for concurrent screening in NDUFS2-/- gas-l(fc21) on metrics of worm activity (y-axis) and reduction of mitochondrial stress induction (x-axis), with screen controls colored: untreated animals, animals treated with ivermectin (anti-helminthic compound) as a negative control, animals treated with our previously identified effective therapy (N-acetylcysteine (NAC) or thiamine) as a positive control, along with untreated healthy animals. Fig 5B-C) Graph depicts results control compounds from all plates, positive (N-acetylcysteine, NAC), negative (ivermectin), control treatments in genetic mutant (gas-l(fc21)), and control (N2) worms at same concentration on (Fig. 5B) mitochondrial stress reduction and (Fig. 5C) worm activity. The distribution of the controls was used to select the threshold for a “hit” compound, with the lines on B and C giving the threshold for the given metric. Fig 5D). Graph depicts lead HTS library hit compound names identified on initial screen to reduce mitochondrial stress in gas-1 as compared to wild-type (N2) and positive (NAC) and negative (ivermectin) control compounds when tested at 0.4 pM (circle) or 40 pM (triangle) concentration. Leads are ranked by effect strength in initial screen.

Figures 6A-6C. gas-l(fc21 ) Screen Validation. (Fig. 6A) Replicates of drags that met our cut off for hit, at the same hit concentration, either 0.4 or 40 pM, looking at reduction in mitochondrial stress as measure by hsp-6::gfp. (Fig. 6B) Dose curves for hits where replicates showed the same activity, again looking at reduction in mitochondrial stress. (Fig. 6C) Confirmation of HTS library hits in orthogonal assay at the level of worm neuromuscular thrashing activity. Lead hits were evaluated at a 3-point concentration curve to evaluate ability to rescue worm activity (body bends per second). Each dot indicates one well per condition. Improved NDUFS2-/- gas-1 worm activity toward that of wild-type (N2) worms was seen with Benzalkonium chloride hydrate at 4 and 40 pM, bromindione at 4 pM, cisplatin at 40 pM, mefloquine hydrochloride at 4 pM, mitoxantrone at 4 pM and 40 pM, and polymyxin B sulfate at 4 pM. Tested in R by ANOVA with a post-hoc Tukey test which includes correction for multiple testing, p < 0.05.

Figure 7. Confirmation of HTS pipeline hits pre-clinical efficacy in Zebrafish mitochondrial complex I NDUFS2-/- disease model with low-dose acute mitochondrial complex I toxic inhibition with rotenone (35 nM) are rescued by Cisplatin. NDUFS2-/- mutants had reduced swimming activity in dark cycles (Zebrabox, Viewpoint), which was further exacerbated by acute rotenone exposure for 3 hours on 7 days post fertilization (dpf). Pre-treatment with cisplatin (40 uM) identified on the worm complex I gas-1 (NDUFS2-/-) mutant HTS significantly improved zebrafish swim activity. Each dot indicates a single animal, with 3 biological replicate experiments completed.

Figure 8. Confirmation of HTS pipeline 8 hit compounds identified in C. elegans mitochondrial stress and worm activity screen demonstrating pre-clinical efficacy in Zebrafish mitochondrial complex I NDUFS2-/- disease model. The 8 lead compounds identified in NDUFS2-/- (gas-1) worm HTS were evaluated at the level of dark-period swimming activity in NDUFS2-/- zebrafish lar vae. Zebrafish media was changed to 10 mM Tris E3 pH 7.2 on 5 dpf and animal treatment with compounds was initiated on 6 dpf for -24 h. Swimming activity was quantified by Zebrabox (Viewpoint) analysis following 20 min light acclimation time during four 10 min dark/light cycles. One biological replicate experimental trial was analyzed by averaging activity across all four dark cycles. Each data point indicates an individual 7 dpf larvae. n= 12 larvac/condition except n=ll for DMSO control and for drug 7. All drugs showed promising effects, with drugs 1, 2, 3, 4, and 7 significantly rescuing NDUFS2-/- zebrafish swimming activity. Drugs: 1. Cisplatin; 2. Benzalkonium chloride hydrate; 3. Cetrimonium Bromide; 4. Phentolamine HC1; 5. Bromindione; 6. Polymyxin; 7. Mitoxantrone HC1; 8. Mefloquine HC1. Figure 9. Combination treatments. C. elegans neuromuscular thrashing assay testing our three best hits at 4 pM in combination with 10 mM glucose. Glucose and all drugs individually show rescue, and phentolamine hydrochloride and mitoxantrone hydrochloride show significantly increased rescue in combination with glucose over either glucose or drug treatment alone. Tested in R by ANOVA with a post-hoc Tukey test which includes correction for multiple testing, p < 0.05. (BCH = Benzalkonium Chloride Hydrate, PH = Phentolamine Hydrochloride, MH = Mitoxantrone Hydrochloride)

Figures 10A-10B. Compression Screening. (Fig. 10A) Schematic diagram of a compression screening assay. The simple colored shapes (blue circles, magenta rhombuses, and yellow triangles) represent three individual drugs. All the individual shapes are then added to a single compressed plate containing two drugs per well. The drugs are distributed such that the individual drugs, for example the dark blue circles shown in wells A 1-4, are tested in four unique combinations. Using this approach, the drug represented by the yellow equilateral triangle is identified as the agent responsible for the observed biological effect since it is the only shape common to all wells that showed an altered stress response. (Fig. 10B) Results from compression screen in DARS2-/- C. elegans. Percent reduction in mitochondrial stress in on the x-axis, WormScan activity is on the y-axis, and each combination of drugs is plotted, along with controls. There is a 75% reduction in the number of wells, screened; compare to Fig. 5A.

Detailed Description of the Invention

Mitotoxicants act through multiple mechanisms, including not only impairment of electron transport chain enzymatic activity but also through modulation of mitochondrial Ca²⁺ channel activity, pH changes, dysfunction arising in nuclear crosstalk, and more as conveyed in Fig. 1 (Meyer 2018). Both individuals’ mitochondrial disease status (Cohen 2010) as well as their mtDNA variants (Pereira 2012) may affect their response to various drugs and environmental toxicants. However, the effects of environmental toxicant effects on mitochondria remains understudied (Meyer 2018).

Described here is a high-throughput screening (HTS) assay of drug and/or genomic scale libraries in C. elegans, utilizing the mitochondrial unfolded protein response (UPR^mt) as a quantitative fluorescence reporter of mitochondrial stress induction (Durieux 2011) combined with concurrent analysis of agents’ effect on worm activity. When errors in mitochondrial function result in mis-folded mitochondrial proteins or abnormalities in electron transport chain complexes activity or integrated function, expression increases of mitochondrial-associated protein chaperones, including heat shock protein 6 (hsp6, which is the worm orthologue of human HSP70) with induction of the UPR^mt (Yoneda 2004). This HTS assay can be used with any genetic model of PMD or other genetic disorders or pharmacologic agents that may secondarily disrupt mitochondrial function, even with otherwise non-viable mutations through knockdown of the gene by using feeding RNAi. Effects on worm activity are also concurrently measured in the same animal populations and plates using WormScan (Matthew 2016). Using integrated analysis of the combination of changes in mitochondrial stress and worm activity, compounds can be rapidly screened simultaneously both for their efficacy and toxicity in PMD, secondary mitochondrial dysfunction, or potential efficacy in disorders where mitochondrial inhibition is a therapeutic goal ranging from cancer to infectious disease. Further, performing genomic library screen in a compound-treated genetic disease model is a powerful strategy to reverse and thereby rapidly identify molecular mechanisms underlying a selected compound’s therapeutic or toxic effects in a given genetic or toxic disorder.

Definitions

As used herein, the term “activity” refers to a biological activity.

As used herein, the term “pharmacological activity” refers to the inherent physical properties of a compound described herein. These properties include but are not limited to halflife, solubility, and stability and other pharmacokinetic properties.

The term “hit” refers to a test compound that shows desired properties in an assay. The term “test compound” refers to a chemical to be tested by one or more screening method(s) as a putative modulator. A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a nucleic acid, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof. Usually, various predetermined concentrations of test compounds are used for screening, such as 0.1 nanomolar (nM), 1 nanomolar, 4 nanomolar, 0.01 micromolar (pM), 1 micromolar, 10 micromolar, 40 micromolar, 100 micromolar, and 1 millimolar (mM). Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target. The terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, c.g., as compared to a control. The terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.

The term “modulate” as used herein refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase or decrease as compared to controls in the absence of these compounds. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. A compound that increases a known activity is an “agonist”. One that decreases, or prevents, a known activity is an “antagonist”.

An “inhibitory mitochondrial function modulator” may be useful for the treatment of disorders where death of a target cell provides a therapeutic benefit.

A modulator which “activates or stimulates mitochondrial function or stability” may be useful for treatment of a disorder characterized by mitochondrial dysfunction.

The term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition or activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99, or 100%.

The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.

The term “library” refers to a large collection of diverse molecules, peptides, small molecules, pharmacological agents, drugs, and nucleic acids, etc. which can be screen to adi in the discovery of new and novel substances for pharmaceutical applications. Libraries can comprise 100s, 1000s or 100,000s or millions of molecules.

A “mitochondrial toxicity assay” provides the means to measure mitochondrial dysfunction due to the toxic effect of a test compound as described herein. Such assays can be used to assess toxicity of small molecule formulations comprising pharmaceuticals, industrial chemicals and consumer products.

As used herein, “subject” includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The subject can be an invertebrate, e.g., C. elegans, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

By “treatment” and "treating" is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, ameliorization, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

A cell can be in vitro and be maintained as a primary or immortalized cell line.. Alternatively, a cell can be in vivo and can be found in a subject. A “cell” can be a cell from any organism including, but not limited to, a bacterium.

By the term “effective amount” of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to improve mitochondrial function, thereby providing therapeutic benefit to the subject. In another approach, an effective amount of a mitochondrial toxicant is provided to target cells (e.g., cancer cells or infected cells etc,) for growth inhibition or cell death.

The term “synergy” or “synergistic” refers to the interaction or cooperation of two or more substances, or other agents to produce a combined effect greater than the sum of their separate effects. In certain embodiments, the combinations provided herein act synergistically.

By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

"Sample" is used herein in its broadest sense. A sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.

A "genetic or protein alteration" as used herein, includes without limitation, naturally occurring mutations, chemically induced mutations, genetic alterations generated via introduction of siRNA, RNAi, mRNA, gDNA, cDNA, antisense oligonucleotides and CRISPR- CAS9 targeted gene constructs. Protein alterations can be generated via pharmacological inhibition or modification of proteins involved in mitochondrial respiratory chain function.

The terms "agent" and "compound" are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, pcptidc/DNA complexes, and any nucleic acid-bascd molecule which exhibits the capacity to modulate the activity of a mitochondrial disease associated gene.

An inhibitory nucleic acid can reduce expression of a protein encoded by a gene selected from atg-9, dct-1, pink-1, sqst-1, hrdl-1, mul-1, pdr-1, plastin-1, siah-1, unc-51, herein after referred to as mitophagy modulator proteins. The inhibitory nucleic acid can reduce expression of an mRNA sequence encoding the mitophagy modulator proteins or genomic DNA encoding the mRNA.

As used herein, "mitochondrial related disorders" related to disorders which are due to abnormal mitochondria structure or function, such as for example, a mitochondrial genetic mutation, enzyme pathways, etc. Examples of disorders include and are not limited to: loss of motor control, muscle weakness and pain, gastrointestinal disorders and swallowing difficulties, poor growth, cardiac disease, liver disease, diabetes, respiratory complications, seizures, visual/hearing problems, lactic acidosis, developmental delays and susceptibility to infection. The mitochondrial abnormalities give rise to "mitochondrial diseases" which include, but not limited to: AD: Alzheimer's Disease; ADPD: Alzheimer's Disease and Parkinson’s Disease; AMDF: Ataxia, Myoclonus and Deafness, CIPO: Chronic Intestinal Pseudo-obstruction with myopathy and Opthalmoplegia; CPEO: Chronic Progressive External Ophthalmoplegia; DEAF: Maternally inherited Deafness or aminoglycoside-induced Deafness; DEMCHO: Dementia and Chorea; DMDF: Diabetes Mellitus & Deafness; Exercise Intolerance; ESOC: Epilepsy, Strokes, Optic atrophy, & Cognitive decline; FBSN: Familial Bilateral Striatal Necrosis; FICP: Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy; GER: Gastrointestinal Reflux; KSS Kearns Sayre Syndrome LDYT: Leber's hereditary optic neuropathy and Dystonia; LHON: Leber Hereditary Optic Neuropathy; LIMM: Lethal Infantile Mitochondrial Myopathy; MDM: Myopathy and Diabetes Mellitus; MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes; MEPR: Myoclonic Epilepsy and Psychomotor Regression; MERME: MERRF/MELAS overlap disease; MERRF : Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM: Maternally Inherited Hypertrophic CardioMyopathy; MICM: Maternally Inherited Cardiomyopathy; MILS: Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Mitochondrial Encephalomyopathy; MM: Mitochondrial Myopathy; MMC: Maternal Myopathy and Cardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NARP: Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; alternate phenotype at this locus is reported as Leigh Disease; NIDDM: Non-Insulin Dependent Diabetes Mellitus; PEM: Progressive Encephalopathy; PME: Progressive Myoclonus Epilepsy; RTT: Rett Syndrome; SIDS: Sudden Infant Death Syndrome.

“Mitochondrial DNA (mtDNA) deletion syndromes (e.g., Single Large-Scale mtDNA Deletion Syndrome (SLSMDS)” predominantly comprise three overlapping phenotypes that are usually simplex (i.e., a single occurrence in a family), but rarely may be observed in different members of the same family or may evolve from one clinical syndrome to another in a given individual over time. The three classic phenotypes caused by mtDNA deletions are Kearns-Sayre syndrome (KSS), Pearson syndrome (PS), and chronic progressive external ophthalmoplegia (CPEO). Activation of mitophagy is efficacious for amelioration of symptoms for this mitochondrial disease.

KSS is a progressive multisystem disorder defined by onset before age 20 years, pigmentary retinopathy, and CPEO; additional features include cerebellar ataxia, impaired intellect (intellectual disability, dementia, or both), sensorineural hearing loss, ptosis, oropharyngeal and esophageal dysfunction, exercise intolerance, muscle weakness, cardiac conduction block, and endocrinopathy.

Pearson syndrome (PS) is characterized by sideroblastic anemia and exocrine pancreas dysfunction, often with lactic acidosis, and may be fatal in infancy without appropriate hematologic management.

PEO is characterized by ptosis, impaired eye movements due to progressive paralysis of the extraocular muscles (ophthalmoplegia), oropharyngeal weakness, and variably severe proximal limb weakness with exercise intolerance.

Rarely, a mtDNA deletion, especially when at high heteroplasmy levels, can manifest as Leigh syndrome.

Pharmaceutical Formulations

Where clinical applications are contemplated pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions (e.g., expression vector) that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to render drugs stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the drug dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the ail. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.

The active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal, as well as through nasal feeding tubes or gastrostomy or jejunal ports and tubes that are commonly needed in primary mitochondrial disease patients. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.

The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

Therapies In another embodiment, treatment of mitochondrial disease is contemplated. The treatment can comprise administration of a single effective agent or one or more agents in combination. Combinations may be achieved by treating patients with a single composition or pharmacological formulation that includes two or more agents, or by treating the patient with distinct compositions or formulations, at the same time, wherein each composition includes a distinct agent. Alternatively, the various agents may be given in a staggered fashion ranging from minutes, to hours, to weeks. In such embodiments, one would generally ensure that the period of time between each delivery was such that the agents would still be able to exert an advantageously combined effect on the cell or subject. In such instances, it is contemplated that one would typically contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

By way of illustration, where benzalkonium chloride hydrate is "A", cisplatin is B," and a third agent is "N-acetyl-cysteine, “C”, the following permutations are exemplary; [ A/B/C B/A/C A/C/B B/C/A C/A/B C/B/A Other combinations wherein multiple administrations of one or more agents are likewise contemplated.

Furthermore, multiple administrations of the cocktail itself are contemplated, such as in an ongoing or chronic basis. The administrations may be twice daily, daily, twice weekly, weekly, every other week, or monthly. They may also be administered for therapeutic purposes to mitochondrial disease patients who are acutely decompensating on a continual or more frequent basis in an acute medical setting (emergency department, intensive care unit, etc).

In another aspect, the present disclosure provides compositions comprising one or more of compounds as described above and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinar y uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.

When used to treat or prevent such diseases, the compounds described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases. As noted above, compounds Drugs 1, 2, 3, 4, and 7 displayed some level of rescue. The numbers in the figures correspond to the following tested compounds: Drugs: 1. Cisplatin; 2. Benzalkonium chloride hydrate; 3. Cetrimonium Bromide; 4. Phentolamine HC1; 5.

Bromindione; 6. Polymyxin; 7. Mitoxantrone HC1; 8. Mefloquine HC1.

The compounds may also be administered in mixture or in combination with agents useful to treat other disorders or maladies, such as steroids, MAPK- modulators, membrane stabilizers, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgG isotype switching or IgG synthesis, P-agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to name a few. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.

Pharmaceutical compositions comprising the compound(s) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically. The compounds may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, oral, topical, ocular, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophore.TM. or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known.

For topical administration, the compound(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the ait. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation or insufflation, the compound(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

For ocular administration, the compound(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.

For prolonged delivery, the compound(s) can be formulated as a depot preparation for administration by implantation or intramuscular' injection. The compound(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).

Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethylsulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The compound(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.

The amount of compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compound(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.

Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.

Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active metabolite compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and/or active metabolite compound(s) which arc sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and/or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

The following materials and methods are provided to facilitate practice of the present invention.

C. elegans strains and maintenance

C. elegans wild-type N2 Bristol worms, SJ4100 (zcls 13 [hsp-6p: : gfp+ lin-35(+)]'), VS21 (hjSi2() [myo-2p: :mCherry: :unc-54 3'UTRJ) and CW152 (gas-l(fc21f) were obtained from the Caenorhabditis Genetics Center (CGC). Two C. elegans fluorescent knock-in strains were made, MJF2/N2 (hsp-6p::gfp + myo-2p: :mCherry) and MJF3 (gas-l fc21) (hsp-6p::gfp + myo- 2p::mCherry)). Animals were maintained at 20 °C on nematode growth media (NGM) OP50 plates (10X concentration of OP50 E. coli on a 150 mm diameter plate) unless stated otherwise.

Plate setup for high content imaging analysis

Setup and treatment. In preparation for sorting, animals were washed off standard NGM OP50 plates and transferred into 15 mL conical tubes for egg prep and then allowed to grow onto standard NGM OP50 plates until L4. Worms at L4 stage were dispensed into a 384 well-plate at 1 worm / pL, with the MultiFlo FX Multimode Dispense (BioTek Instruments) using a 10 pL comb in OP50 E. coli ODeoo of ~0.7 with a final volume of 50 pL and about 50 worms per well. Daughter plates were generated at (0.1 mM and) 10 mM in DMSO from the Spectrum Collection (MicroSource Discovery Systems, Inc.) that includes 32 individual 96-well plates containing 2,560 compounds at 10 mM in DMSO. These daughter plates were then pinned into 384- well plate already containing worms, S-medium and OP50 E. coli using a VP 408 A pinner (V&P Scientific, Inc), giving a final concentration of (~0.4 pM or) -40 pM. These 384-well plates were returned to the incubator in a box lined with a damp paper towel to maintain 100% humidity and prevent evaporation of the C. elegans in liquid media and incubated for 24 hoursl

Activity and quantification of (UPR^mt) and quality control parameters:

( ii ) Activity and quantification of(UPR"“): Worm activity analysis for each plate was undertaken using WormScan, as previously described (Matthew 2016). After activity analysis was measured, 50 pL of NaN₃0.1 M with 0.01% Tween-20 was added to each well to bring the final concentration to 50 mM. Worms were allowed to settle to the bottom of each well for 10 minutes. Mitochondrial stress induction was measured using a Cellinsight CX5 (Thermo Scientific) using a protocol adapted from previous C. elegans HTS (Gosai 2010). Briefly, SpotDetector was used to focus the red channel with a 20% exposure sufficient to detect all the worms in the wells, an exposure setting of 40% was used for the green channel and 10% was used for the brightfield image. The red channel was used to count the number of worms per well, the green channel was set to a threshold of 1800 pixel intensity and, using spot total intensity, was used to measure the total green fluorescence per well.

Quality control was performed on each well and each plate. Any well with a green channel value 1.5-fold greater than the interquartile range of the other wells for that given compound was removed. If the red channel count of C. elegans in any well was less than 5 or greater than 90, the well was removed from further analysis. Any compound where 3 out of 4 replicate wells failed was removed and repeated. For each plate, the dynamic range was assessed for the green channel between healthy and disease animals, to ensure adequate ability to detect true hits using the Strictly Standardized Mean Difference (SSMD) calculated as:

A SSMD of less than -2 was required for a plate to pass QC. Any plates that did not pass were repeated.

Normalization and hit identification: Activity was normalized by dividing the WormScan score by the number of C. elegans in the well as measured by the red channel. Background, both in random expression of fluorescence by the healthy control and in the background level of stress experienced by the disease model, varied for each plate so the UPR^I11Lfor each compound was normalized as the percent reduction in stress relative to the plate controls to allow for plate-to-plate comparison. Background, healthy animal fluorescence, was subtracted from all values and then percent change between the median disease fluorescence and the well was calculated for each well. Hits were selected based on a percent reduction in mitochondrial stress greater than 80% and a WormScan score greater than 50. These cutoffs were based on inflection points in the given distributions.

C. elegans thrashing

C. elegans were exposed to drags from L4 larval stage overnight and then washed off using S- basal from the standard NGM plates that contained drugs at the concentration stated and placed on the plate lid. A 10 second video was collected using a Basler USB Camera (model #108014) with a KOWA industrial lens 75mm/F2.5. The FIJI (PMID: 22743772) plugin wrMTrck (PMID: 25591151) was used to measure the body bends per second of each individual C. elegans and for three biological replicates (PMID: 33871460).

C. elegans lifespan

Neuromuscular activity, and lifespan were quantified in the C. elegans gas-1 (fc21) ndufs2’^/_ mutants. A semi-automated screening method (Mathew et al, 2016) was used to test drug treatment effects on an integrated C. elegans health endpoint of fecundity, brood size, and behavior.

Zebrafish activity

Neuromuscular- swimming activity analysis in zebrafish larvae. Swimming activity was assessed by monitoring larval zebrafish movement after transition between light and dark periods at 7 dpf using the Zebrabox tracking system and ZebraLab software (ViewPoint Life Sciences, Montreal, Canada). The larvae were placed individually into wells of a 96 square well plate (Whatman 7701-1651) with Tris-buffered (10 mM Tris-HCl, pH 7.2) embryo E3 media and allowed to acclimate under light (100% light power) in the Zebrabox system for 20 min prior to experimentation. The experiment consisted of four cycles of a 10 min dark period (0% intensity) followed by a 10 min light-on period (100% intensity, -1880 Lux). Technical replicate each included at least 12 control larvae ndufs2^+/+,+/'' and 12 ndufs2'^/' larvae. Three biological replicate experiments were performed per condition. Under the curve analysis was performed to analyze the zebrafish activity of larvae during the dark cycles using GraphPad Prism 9.

Determination of mitochondrial oxidant burden. Mitochondrial oxidant burden (MitoSOX Red), membrane potential (tetramethylrhodamine ethyl ester, TMRE), and mitochondrial content (MitoTrackcr Green FM, MTG) were performed in C. elegans at 20°C. using in vivo terminal pharyngeal bulb relative fluorescence microscopic quantitation. Briefly, synchronous populations of Day 0 young adults were moved to 35 mm NGM plates spread with OP50 E. coll, a desired drug treatment (e.g., different concentrations of cisplatin in combination with other agents (galactose) or buffer control (S-basal/water for all other drugs) was performed on NGM plates. Simultaneously with the drug treatments, worms were treated with either 10 mM MitoSOX Red (matrix oxidant burden), 100 nM TMRE (mitochondrial membrane potential), or 2 pM MitoTracker Green FM (mitochondria content) for 24 h. The next day, worms were transferred with a pick onto 35 mm agar plates spread with OP50 E. coli without dye for 1 h to allow clearing of residual dye from the gut. Worms were then paralyzed in situ with 5 mg/ml levamisole. Photographs were taken in a darkened room at 160. times, magnification with a Cool Snap cf2 camera (Nikon, Melville, N.Y.). A CY3 fluorescence cube set (MZFLIII, Leica, Bannockburn, Ill.) was used for MitoSOX and TMRE. A GFP2 filter set (Leica) was used for MitoTracker Green FM. Respective exposure times were 2 s, 320 ms, and 300 ms for each of MitoSOX, TMRE, and MitoTracker Green FM. The resulting images were background subtracted, and the nematode terminal pharyngeal bulb was manually circled to obtain mean intensity of the region by using Fiji Is Just ImageJ. Fluorescence data for each strain were normalized to its same day control to account for day-to-day variation. A minimum of 3 independent experiments of approximately 50 animals per replicate were studied per strain per dye. The significance of the difference in the mean fluorescence intensity between strains under different experimental conditions was assessed by mixed-effect ANO VA, which analyzes potential batch effect due to samples being experimentally prepared, processed, and analyzed on different days by including a batch random effect in the model. A statistical significance threshold was set at P<0.05. All statistical analyses were performed in SAS 9.3.

Modulation of mitochondrial function in human cells

Human fibroblasts can be studied from subjects harboring a 1067 del (p.Gly356Alafs*15) nonsense mutation in the maternal FBXL4 allele and a C.1790A>C (p.GLn597Pro) missense mutation in the paternal FBXL4 allele (Gal et al, 2013). Fibroblasts were cultured in DMEM (1 g/L glucose, 0.8 g/L L-Glutamine, 110 mg/L Sodium Pyruvate). Light, fluorescence, confocal microscopy and transmission electron microscopy (TEM, Lavorato et al, 2017) methods were used to analyze proband fibroblasts and mitochondrial morphology at baseline and following metabolic stress induced by incubating cells for 48 hours in glucose/uridine-free media. Mitotracker green was used for fluorescence microscopy, Tom20 Antibody (Santa Cruz) and DAPI was used for confocal microscopy.

The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

Example 1

High Throughput Screening Assay for Identifying Compounds which Modulate Mitochondrial Function in Target Cells

Mitochondrial disease is a highly heterogeneous, systemic disease with varying presentations, age of onset, and severity. Symptoms range from mild and tolerable to severe and progressive leading to early death. Primary mitochondrial disease has a prevalence of 1 in 4,300 individuals (PMID 30393588), not including individuals with diseases that include secondary mitochondrial dysfunction as a symptom who would also benefit from treatments for mitochondrial dysfunction (PMID: 27587988). There are no approved or specific treatments for mitochondrial disease, with current treatment strategies relying on addressing symptoms and trying to alleviate metabolic dysfunction by supplementing missing metabolites and by treating with antioxidants to address the high levels of mitochondrial reactive oxygen species that are often a feature of the disease (PMID: 27665271, PMID: 24985754, PMID: 24116962). Given that the cost of developing a novel drug through regulatory approval is estimated to exceed $2.5 billion dollars (PMID: 26928437) and that it takes on average 9 years from the first human trials to FDA approval in the US (PMID: 34759309), the repurposing of existing FDA-approved drugs could greatly reduce both the cost associated with drug development and accelerate the time to treatment for patients (PMID: 29343694).

Given the wide variation in symptom presentation and the incomplete understandings of the physiological mechanisms underpinning mitochondrial dysfunction, we were interested in designing a screen that is unbiased, phenotypically accurate, and high throughput (PMID: 33641258). Phenotypically, drug screening using cells has limitations as cells do not recapitulate the highly heterogeneous whole-organism features of mitochondrial disease (PMID: 22068913). Live animal screenings arc not generally amenable to high-throughput assays due to the complexity of working with them but using Caenorhabditis elegans (C. elegans) with their small size, transparency, quick generation time, and high homology with humans makes the problem tractable. Others have taken advantage of these features (PMID: 21103396, PMID: 33589689, PMID: 27579370, PMID: 34299431, PMID: 27755544), ) and here we extend them to specific mitochondrial diseases.

Working with C. elegans, in combination with mitochondrial disease models in other species, we have previously shown that the complex I-deficient gas-l(fc21 ) C. elegans model, which harbors an autosomal recessive missense mutation in the ortholog encoding the NDUFS2 gene, (PMID: 15178135), can both recapitulate features of human mitochondrial disease and be used to identify treatments that alleviate those features (PMID: 30668749, PMID: 33640978). Building on that work, we have developed a Danio rerio model harboring a 16 base-pair (AUCGUAUCUGGAAGAA) deletion that causes a frame shift mutation and a premature stop codon near the NDUFS2 point mutation in the C. elegans (see supplemental figure SI). This mutation in NDUFS2 results in: (i) a decrease in mitochondrial CI activity by -80%, (ii) a -50% decrease in swimming activity (a proxy for neuromuscular function), (iii) a failure of the swim bladder to inflate, and (iv) a grey and round liver phenotype.

Screen overview

When assessing the efficiency and accuracy of the screen, a library of 2,560 FDA- approved therapies and natural compounds were tested as shown in Figure 4. Fluorescent knock- ins were created for both healthy and disease animals N2 (Jisp-6p::gfp + myo-2p: nnCherry) and gas-l(fc21) (hsp-6p::gfp + myo-2p: : inCherry) with mCherry marking heads and gfp controlled by the hsp-6 p) promoter which increases activity when the mitochondria/cells are stressed and activate UPR^mt. For the screen C. elegans were plated on a 384-well plate, testing 80 compounds from our library with 4 replicates each. After incubation with the drug, toxicity was assessed using a measure of activity, WormScan, as previously described (pubmed ID: 22457766 and 27755544). Plates were then scanned with spots counted on the red channel to detect the number of C. elegans present in the well and mitochondrial stress measured on the green channel (Figure. 4). We screened the full library twice, testing the compounds each once at 0.4pM and at 40 pM to see if there was an advantage to testing multiple doses. In addition to untreated healthy and disease animals we used ivermectin at 40 pM, a known toxic anthelmintic as a negative drug treatment control and N-acetyl cysteine (NAC) at 25 mM, a known moderately effective treatment for mitochondrial disease as a positive drug treatment control.

Taking the fluorescence data from the screen, semi- automated quality control removes individual wells with abnormal numbers of animals or abnormal fluorescence. Compounds with too few replicates are removed and the dynamic range between healthy and disease animals on the plate is measured using Strictly Standardized Mean Difference (SSMD) to ensure a large enough signal-to-noise ratio to enable detection of true hits.

For data analysis and hit selection, activity and mitochondrial stress were analyzed in parallel, with percent reduction in mitochondrial stress the primary outcome and normalized activity used to filter for toxicity (Figure. 3). As a negative control, Ivermectin, the known anthelminthic, (Figure. 3), reduces activity. Mitochondrial stress was background normalized by removing the fluorescent signal detected in the healthy animals and then normalized relative to the disease by calculating the percent reduction in mitochondrial stress compared to untreated disease control (Methods). Activity was normalized by dividing the WormScan score by the number of animals in the well (Methods). Hits were selected by plotting the percent reduction in stress both of all compounds and of controls and picking the inflection point where mitochondrial stress reduction approached normal levels. Activity of the controls was also plotted and used to determine a threshold below which the lack of activity indicated toxicity based on the Ivermectin replicates (Figure. 3).

Figure 4 provides a schematic showing high throughput screening (HTS) of the library of potential mitochondrial function modulators identified. The details of the set up and treatment of C. elegans are described above and shown in Fig. 4A. Activity was quantified using WormScan and UPR^mt was quantified using the Thermo Fisher CX5 High Content Imager. (Fig 4C). Overview of HTS automated data quality control and normalization pipeline: Quality control was performed both on every well and each plate. Any well with a green channel value 1.5-fold greater than the interquartile range of the other wells for that given compound was removed. If the red channel count of C. elegans in any well was less than 5 or greater than 90, the well was removed from further analysis. Any compound where 3 out of 4 replicate wells failed was removed and repeated. For each plate, the dynamic range was assessed for the green channel between healthy and disease animals, to ensure adequate ability to detect true hits using the Strictly Standardized Mean Difference as described herein below.

Figure 5 shows the criteria used for hit selection of drug library compounds screened. Fig 5 A is a graph showing significant mitochondrial function modulators identified in the high throughput screening assay evaluated at two concentrations (0.4 pM and 40 pM) as evidenced by concurrent screening metrics of worm activity (y-axis) and reduction of mitochondrial stress induction (x-axis) Figs. 5B and 5C show data depicting results of replicate analyses of lead hits and control compounds, relative to positive (A-acctylcystcinc, NAC) and negative (ivermectin) control treatments in genetic mutant (gas-1) and control (N2) worms at same concentration on (5B) mitochondrial stress reduction and (Fig. 5C) worm activity. Fig 5D). Graph depicts lead HTS library hit compound names identified on initial screen to reduce mitochondrial stress in gas-1 as compared to wild-type (N2) and positive (NAC) and negative (ivermectin) control compounds when tested at 0.4 pM (circle) or 40 pM (triangle) concentration.

To confirm HTS library hits, first we repeated them at the same concentration they were screened at, either 0.4 or 40 pM (Fig. 6A). For the 8 compounds that reproduced the screen results, we tested a dose curve (Fig. 6B). We also confirmed HTS library hits in orthogonal assay as a function of worm thrashing activity levels. Lead hits were evaluated at a 3-point concentration curve to evaluate ability to rescue worm activity. Each dot indicates one well per condition. Improved gas-1 worm activity toward that of wild-type (N2) worms was seen with Benzalkonium chloride hydrate at 4 and 40 pM, bromindione at 4 pM, cisplatin at 40 uM, mefloquine hydrochloride at 4 pM, mitoxantrone at 4 pM and 40 pM, and polymyxin B sulfate at 4 pM (Fig. 6C). We wanted to test our three strongest hits, benzalkonium chloride hydrate, phentolamine hydrochloride, mitoxantrone hydrochloride, in combination with glucose. All four individual treatments showed a significant rescue as shown previously, and phentolamine hydrochloride and mitoxantrone hydrochloride show significantly increased rescue in combination with glucose over either glucose or drug treatment alone. (Fig. 9).

Figure 7 shows confirmation of HTS pipeline hits having pre-clinical efficacy in Zebrafish mitochondrial complex I NDUFS2'^/' disease model with low-dose acute mitochondrial complex I toxic inhibition with rotenone (35 nM) which can be rescued by Cisplatin. NDUFS2' ' mutants had reduced swimming activity in dark cycles (Zebrabox, Viewpoint), which was further exacerbated by acute rotenone exposure for 3 hours on 7 days post fertilization (dpf). Pre- trcatmcnt with cisplatin (40 uM) identified on the worm complex I gas-1 (NDUFS2~'~) mutant HTS significantly improved zebrafish swim activity. Each dot indicates a single animal, with 3 biological replicate experiments completed.

The 8 lead compounds identified in NDUFS2^/' (gas-1) worm HTS were evaluated in NDUFS2 ^ zebrafish larvae. Zebrafish media was changed to 10 mM Tris E3 pH 7.2 on 5 dpf and animal treatment with compounds was initiated on 6 dpf for ~24 h. Swimming activity was quantified by Zebrabox (Viewpoint) analysis following 20 min light acclimation time during four 10 min dark/light cycles. One biological replicate experimental trial has successfully been performed and the data was analyzed by taking the average activity during all four dark cycles. Each data point is an individual 7 dpf larvae. n= 12 larvae/condition except n=ll for DMSO control and for drug 7. Drugs 1, 2, 3, 4, and 7 displayed some level of rescue. Drugs: 1. Cisplatin; 2. Benzalkonium chloride hydrate; 3. Cetrimonium Bromide; 4. Phentolamine HC1; 5. Bromindione; 6. Polymyxin; 7. Mitoxantrone HC1; 8. Mefloquine HC1.

Finally, a drug compression screen in a DARS2 ^/_ C. elegans model was performed. Instead of screening single agents at a time, multiple compounds are screened simultaneously in the same well. Compounds are distributed among wells in unique combinations so that when wells “hit” the unique combination of wells can narrow it down to 1-2 compounds (Figure 10A). This greatly increases the speed of screening; using our 32-plate FDA and natural compound library as an example, we did four-fold compression, combining four plates into one which drops the number of plates need to screen 2,560 compounds down to 8 from 32. We completed a screen using the compressed plates in a DARS2 C. elegans model, the gene responsible for catalyzing the formation of the mitochondrial t-RNA for the amino acid aspartate (Fig. 10B).

Conclusion

Treatment of mitochondrial disease relies mainly on addressing symptoms with minimal interventions available to target the cause or consequences of the disease directly, mainly limited to attempting to supplement biomolecules like vitamins, cofactors, and amino acids to improve limited functionality or replace missing factors. In addition to lacking treatment options, mitochondrial disease patients are more vulnerable both to environmental toxicants and to drugs that may otherwise be safe. Described herein is a high-throughput screening platform using C. elegans that can be used for any genetic model of mitochondrial disease and can screen for both compounds that arc potentially therapeutic for mitochondrial disease and well as for toxic compounds.

Screening is done in C. elegans, to allow for assessing toxicity in a whole organism with multiple cell types as opposed to a single cultured cell type. We have made many C. elegans fluorescent knock-in strains (hsp-6p::gfp + myo-2p: :mCherry) where mCherry marks the head of all the animals for simple detection and hsp-6p is the reporter for the assay. The outcome is reduction in mitochondrial stress; hsp-6p is induced as part of the mitochondrial unfolded protein response (UPR^MT) (Fig. IB), which is upregulated in response to stress there. Animals are plated on a 384-well plate where we can test 80 compounds at a time with 4 replicates of each drug in individual wells, incubated with the drug for 24 hours, then scanned in the appropriate channels. Before scanning, to assess toxicity, animal activity is assessed using WormScan. Post-assay, we have also developed a semi-automated data analysis and reporting pipeline where the data is quickly and automatically filtered for quality control, including number of animals, fluorescence, missingness, and plate dynamic range, normalized as percent reduction in fluorescence, then checked for hits. Potentially beneficial hits show a high percent reduction in fluorescence, while potentially negative hits show a low percent reduction in fluorescence. Activity is used as a negative fdter only; any potentially beneficial hit with low activity is removed, while low activity is sufficient for a potentially toxic hit on its own.

We have screened three different models of mitochondrial disease, an NDUFS2 knockout model, gas-l(fc21 ), an OPA1 knockout model, OPA R289Q). and a single large-scale mitochondrial deletion model (SLSMD), uaDf5, using N2 Bristol worms as a wild-type control in a 2,560 compound library of FDA-approved drugs with some natural compounds. Have taken 8, 16, and 10 number of reproducible potentially therapeutic hits forward to validation in each of the models respectively. Additionally, we have screened the same 2,560 compound library in a DARS2‘^/_ model of mitochondrial disease, reducing the number of plates needed from 32 to 8, greatly speeding up the screening process. On the other hand, we have recently identified about 200 potentially toxic drugs in mitochondrial disease using the same data from the gas-l(fc21 ) screen. See Appendix I.

In conclusion, we have developed a robust and flexible platform for addressing any number of questions in any mitochondrial disease in a high-throughput manner. This work has been expanded to cells, to RNAi C. elegans and zebra fish to allow for screening of even lethal genetic mutants, to larger libraries as well as increasing throughput with increased automation.

APPENDIX I

MITOCHONDRIAL TOXICANTS IDENTIFIED IN SCREEN assay_ drug median_percent median group concentration_uM change wormscan

0.4 16(17)-EPOXY-5- -47.20802981 29 activity_only PREGNENOLONE

0.4 2',4'-DIHYDROXY-6'- -78.84130307 0.4166666666666667 both METHOXY ACETOPHENONE

0.4 2', 6’- -16.25225475 31.866666666666667 activity_only

DIMETHOXYACETOPHENONE

0.4 2-HYDROXY-4-METHOXY- -67.62583764 119.11627906976744 fluorescence_only

BENZOPHENONE

0.4 5 -METHYLHYDANTOIN -9.85458182 27.727249747219414 activity _only

0.4 5alpha-CHOLESTAN-3beta-OL- -80.43428952 110.6046511627907 fluorescence_only 6-ONE

0.4 7 -HYDROXYFLAVONE -139.5417393 274.0394736842105 fluorescence_only

0.4 ALANYL-dl-PHENYLALANINE -65.93993843 104.5 fluorescence_only

0.4 ANTIMYCIN A (Al shown) -105.0622591 68.10864661654136 fluorescence_only

0.4 AVERMECTIN Ala 62.423167104583314 36.08825283243888 activity_only

0.4 BENZANTHRONE -79.38584763 94.94521963824289 fluorescence_only

0.4 BRAZILEIN -2446.241026 198.72727272727272 fluorescence_only

0.4 CARBIMAZOLE 31.185591298269117 42.595238095238095 activity _only

0.4 CEFACLOR -225.0006809 149.58660130718954 fluorescence_only

0.4 CHLORMIDAZOLE -29.73470474 45.51971544715447 activity _only

0.4 CHLORTHALIDONE -1.026799821 40.590042374999996 activity _only

0.4 CHLORZOXAZONE 3.349483378351046 44.2962963 activity _only

0.4 CICLOPIROX OLAMINE 16.64099064120603 45.33870968 activity_only

0.4 CINOXACIN -4.331732106 43.50471932 activity _only

0.4 CLIDINIUM BROMIDE 3.5349644374429636 47.85576923 activity _only

0.4 CYAN1D1N CHLORIDE -65.89944751 96.53846153846152 fluorescence_only

0.4 DESLORATADINE -70.99171764 79.41093366093367 fluorescence_only

HYDROCHLORIDE

0.4 DIBENZOYLMETHANE -88.18028776 89.89223839223838 fluorescence_only

0.4 DIHYDROROTENONE -182.8879908 21.224400871459697 both

0.4 DULOXETINE -65.49950005 122.62648083623694 fluorescence_only

HYDROCHLORIDE

0.4 EMEDASTINE DIFUMARATE -63.94705791 125.56060606060606 fluorescence_only

0.4 EPIGALLOCATECHIN-3- -64.70295243 126.02857142857144 fluorescence_only

MONOGALLATE

0.4 EPIRUBICIN -1811.96405 102.69655797101449 fluorescence_only

HYDROCHLORIDE

0.4 ERDOSTEINE 16.709584579256685 43.70967741935484 activity _only

0.4 ESTRADIOL-3-SULFATE, -23.18209654 1.226190476190476 activity _only

SODIUM SALT ETHIONINE 6.850009179161784 0.19444444444444445 activity _only ETHOXYQUIN -19.51589683 29.06451612903226 activity_only HAEMATOXYLIN -78.35249324 125.3888888888889 fluorescence_only HAEMATOXYLIN -291.3883522 187.40725806451613 fluorescence_only

PENTAACETATE HARMALINE -185.9000234 75.17562984496124 fluorescence_only IODOFORM -86.99697594 84.96129032258065 fluorescence_only ISOROTENONE -110.1790977 87.14921062764728 fluorescence_only LANOSTEROL ACETATE 57.95946994463091 49.341711956521735 activity _only LAPACHENOL 16.705872606223107 0 activity_only LUFENURON 0.12507051393049418 49.75 activity_only LYNESTRENOL -79.69115065 192.03571428571428 fluorescence_only MOMETASONE FUROATE -87.37628307 94.37767857142856 fluorescence_only MOXIDECTIN 72.84516730611412 31 activity_only OXIBENDAZOLE -90.8491032 157.7589134125637 fluorescence_only PHENYLACETOHYDROXAMIC -64.89796841 95.28923076923076 fluorescence_only

ACID PUTRESCINE -100.3427937 122.8695652173913 tluorescence_only

DIHYDROCHLORIDE PYRROMYCIN -376.0297227 90.66666666666669 fluorescence_only PYRVINIUM PAMOATE -66.62393065 64.81181318681318 fluorescence_only QUINIZARIN -225.7227571 170.21428571428572 fluorescence_only RESVERATROL 37.29200414955636 47.55911330049261 activity_only RIBOFLAVIN 5-PHOSPHATE -652.4988621 89.39534884 fluorescence_only SODIUM ROTENONE -374.6871994 1.6923076923076923 both SALICYL ALCOHOL -63.76636132 82.82492997 fluorescence_only SECURININE -3.860249751 43.81142857142857 activity_only SULINDAC -61.35597472 97.34130434782608 fluorescence_only THIMEROSAL -74.37578498 103.48736097067744 fluorescence_only TRANYLCYPROMINE -66.39048557 111.31744271988174 fluorescence_only SULFATE TRYPTOPHAN (L) -63.65944056 77.02439024390245 fluorescence_only USNIC ACID -88.51008826 54.19618366129994 fluorescence_only XANTHONE -153.2834706 77.58012821 fluorescence_only alpha-CYANO-3- -14.74278774 0.15217391304347827 activity_only

HYD ROXY CINNAMIC ACID alpha-CYANO-4- -70.93364794 150.240625 fluorescence_only

HYDROXYCINNAMIC ACID 1,4-NAPHTHOQUINONE -198.3843443 115.84033613445378 fluorescence_only 2’-HYDROXYCHALCONE -267.3575724 74.84 fluorescence_only 2,3-DIHYDROXY-6,7- -128.2203778 97.36265969802557 fluorescence_only

DICHLOROQUINOXALINE 2.4-DIMETHOXY-2'- -75.31880459 102.45972644376899 fluorescence_only HYDROXYCHALCONE 2-ACETAMIDOFLUORENE -28.74746977 10.848484848484848 activity_only 2-HYDROXY-4-METHOXY- -102.4960654 78.2375 fluorescence_only BENZOPHENONE

3,7-DIMETHOXYFLAVONE -98.63103729 59.04825581395349 fluorescence_only

4’-HYDROXYCHALCONE -198.9970496 129.03484848484848 fluorescence_only

4'-METHOXYCHALCONE -249.63119 74.34628543499511 fluorescence_only

4,6-DIMETHOXY-5- 1.7653586398777326 30.47619047619047 activity_only METHYLTSOFLAVONE

4-HYDROXYCHALCONE -403.4216433 53.735703812316714 fluorescence_only

4-METHYLESCULETIN -82.3174422 61.107070707070704 fluorescence_only

5-HYDROXY-2',4',7,8- -71.01059918 105.4 fluorescence_only

TETRAMETHOXYFLAVONE 6,2’-DLMETHOXYFLAVONE -74.39163985 136.15257352941177 fluorescence_only

6-(3,4- -361.0052875 142.48925667828107 fluorescence_only

METHYLENEDIOXYSTYRYL)- alpha-PYRONE 6-AZA-2-THIOTHYMINE -71.61219967 115.84776785714286 fluorescence_only

ACACETIN -588.7262624 77.91681818181817 fluorescence_only

ACEDAPSONE -63.23500134 96.81161745827984 fluorescence_only

ACETYL-DIHYDRO-7- -91.98877273 158.8243398392652 fluorescence_only

EPIKHIVORIN AGELASINE -80.46572767 46.882539091586004 both

ALAN YL-dl-LEUCLNE -38.56927724 49.648648648648646 activity _only

ALIZARIN -226.043417 103.39910714285713 fluorescence_only

ALTRENOGEST -71.73962521 117.3632862644416 fluorescence_only

ANABASINE -10.49022501 46.32551020408163 activity _only

HYDROCHLORIDE

ANTHRACENE- 1 ,4-DIONE -197.4059827 73.08888888888889 fluorescence_only

ARTEMETHER -65.12168173 61.652882205513784 fluorescence_only

ASPARTAME -82.86361956 102.99526515151516 fluorescence_only

ASTEMIZOLE -87.15914025 106.76068376068375 fluorescence_only

AVERMECTIN Ala 29.72394727663468 29.923076923076923 activity_only

AVOCADYNE -388.0459182 88.50999999999999 fluorescence_only

AVOCADYNE ACETATE -476.9651941 4.364130434782608 both

AVOCATIN A -496.2234612 30.425630252100838 both

BENZANTHRONE -1736.906282 99.47619047619048 fluorescence_only

BIFONAZOLE -114.9237918 103.95575723199093 fluorescence_only

CANTHARIDIN 22.443817108405742 46.90196078431372 activity _only

CAPTAN -94.3199967 83.95 fluorescence_only

CEFDITORIN PIVOXIL -76.26121068 100.0936507936508 fluorescence_only

CHAULMOOGRIC ACID 28.872708717857314 45.25437651494748 activity _only

CHLORMID AZOLE -180.0874916 68.35438596491227 fluorescence_only

CHLOROTHALONIL -285.6539396 62.893410852713174 IIuorescence_only

CHLOROXINE -830.2083357 74.11610772357724 fluorescence_only

CHLORZOXAZONE 3.9885250567040327 46.19712919 activity _only

CHRYSAROBIN -472.3136495 71.70242681047765 fluorescence_only CHRYSENEQUINONE -239.5704483 73.39697802197801 fluorescence_only CHRYSOPHANOL -133.5748422 114.54862579281182 tluorescence_only CLIDINIUM BROMIDE 12.005092149893933 48.88094035 activity_only CLINDAMYCIN 0.429632303 48.98833334 activity _only HYDROCHLORIDE CLOMIPHENE CITRATE 51.36889160220888 46.63636364 activity_only CLONIDINE 9.025692744 49.50815218 activity_only

HYDROCHLORIDE CLOSANTEL -64.55556476 94.20820669 fluorescence_only CURCUMIN -551.5973021 86.00260104041615 fluorescence_only CYCLOBENZAPRINE -74.22584912 98.9 tluorescence_only HYDROCHLORIDE CYCLOSPORINE -82.25271119 112.25675675675676 fluorescence_only CYTIDINE 18.221959021892957 49.61893939 activity _only DEACETYLGEDUNIN -64.89686373 78.22739541160594 fluorescence_only DEGUELIN(-) -177.7512461 102.05967741935484 fluorescence_only DIBENZOTHIOPHENE -48.67998215 20.574025974999998 activity _only DIBENZOYLMETHANE -72.65411059 95.27556818181819 fluorescence_only DICLORALUREA -77.84487684 108.6 fluorescence_only DIHYDROCELASTROL -201.6513514 71.17021276595744 fluorescence_only DIMETHADIONE -59.83486229 26.79204546 activity _only DIOXYBENZONE -243.8950683 176.7556561 fluorescence_only DORAMECTIN -123.9439995 28.516660881638323 both EMODIN -590.2662732 130.21212121212122 fluorescence_only EPICATECHIN -80.51864968 75.90217391304347 fluorescence_only PENTAACETATE ETHACRIDINE LACTATE -296.8912064 82.86303827751196 fluorescence_only ETHOPROPAZINE -68.02889327 81.49222222222222 fluorescence_only HYDROCHLORIDE EXALAMIDE -191.5319739 104.85 fluorescence_only FENTHION -69.96099794 90.61 fluorescence_only FLAVONE -349.2328952 77.39085623678648 tluorescence_only FLUPHENAZINE -102.0919198 5.333333333333333 both HYDROCHLORIDE FLUTAMIDE -160.9278365 105.86086226203808 fluorescence_only FLU VOXAM1NE MALEATE -94.17526072 69.15909091 fluorescence_only GEDUNOL -85.52573229 74.44025735294117 fluorescence_only GLUCOSAMINE -165.4416034 42.55072463768116 both HYDROCHLORIDE GLYCERIN -89.55128044 7.196356275303644 both GOSSYPIN -94.80736573 499.8333333333333 tluorescence_only HARMALINE -268.5912555 60.128365384615385 fluorescence_only HEXACHLOROPHENE -153.0613348 168.46172248803828 fluorescence_only HYDROLYSIS PRODUCT OF -284.1935398 241.66666666666663 fluorescence_only BUSSEIN IDRAMANTONE 13.564996635584757 48.54 activity_only IODOFORM -110.55968 97.44453044375643 fluorescence_only

ISOCONAZOLE NITRATE 84.92757477799435 48.75 activity_only

ISOOSAJIN -99.5615149 73.92307692307692 fluorescence_only

ISOROTENONE -159.6790941 96.92857142857144 fluorescence_only

KAEMPFEROL -64.1593892 110.2162162162162 fluorescence_only

LEVAMISOLE 87.87911191 8.319327731092438 activity_only

HYDROCHLORIDE

LIOTHYRONINE -134.5045306 105.03544494720967 fluorescence_only

LOMERIZINE -28.94060047 48.5 activity _only

HYDROCHLORIDE

MECLOCYCLINE -369.9724454 39.304621848739494 both SULFOSALICYLATE MELPHALAN -174.8030867 87.23397129186603 fluorescence_only

MESOTRIONE -73.16546974 125.65195246179965 fluorescence_only

METHYL 7- -71.55761241 81.58859649122806 fluorescence_only

DESHYDROXYPYROGALLIN-

4-CARBOXYLATE

METHYLBENZETHONIUM 91.16581949538374 25.078947368421055 activity_only CHLORIDE

MEXENEONE -106.4602135 69.88095238095238 fluorescence_only

MEXICANOLIDE 30.73552859479968 46.56603773584906 activity _only

MONOCROT ALINE -61.00578471 95.12903225806453 fluorescence_only

MORIN -65.49966123 82.7827381 fluorescence_only

MOXIDECTIN 98.62728559 35.28030303030303 activity_only

N-ACETYLPROLINE (dl) 6.2704547570525655 5.791614255765199 activity _only

N-METHYL-D-ASPARTIC ACID -19.35944505 0 activity _only

NITROXOLINE -93.53351939 101.00531582238901 fluorescence_only

PALMATINE -258.8031796 60.952260458839405 fluorescence_only

PAZUFLOXACIN MESYLATE -10.36566477 46.54054054054054 activity _only

PENFLURIDOL 102.65838815785716 48.174725274725276 activity_only

PHENYLMERCURIC ACETATE -158.5390001 11.483516483516484 both

PHYSCION -119.7286044 86.46875 fluorescence_only

PLUMBAGIN -107.9147938 71.26111111 fluorescence_only

PODOTOTARIN -67.10966755 107.15384615384616 fluorescence_only

POMIFERIN -64.78636165 138.56890756302522 fluorescence_only

PROCHLORPERAZINE -33.96206316 44.669604863221885 activity _only

EDISYLATE PROGESTERONE -104.9198604 46.20278293135436 both

PROPOFOL -151.1144551 64.33439201451905 fluorescence_only

PURPUROGALLIN-4- -713.1930456 168.9487068965517 fluorescence_only

CARBOXYLIC ACID PYRANTEL PAMOATE -66.92406374 22.293023255813953 both

PYRVINIUM PAMOATE -638.7116835 49.47674418604651 both

RESERPINE -617.0453389 86.17948717948718 fluorescence_only

RESORCINOL -452.2476263 80.11324786324786 fluorescence_only

RETINYL PALMITATE -99.76661139 115.5611954459203 fluorescence_only 40 RETUSIN -73.91518196 124.4090909090909 fluorescence_only

40 ROBUSTIC ACID 22.919510330090212 46.214285714285715 activity_only

40 ROSOLIC ACID -75.17923554 39.26820728291317 both

40 ROTENONE -278.1411469 1.3852357320099256 both

40 SALIDROSIDE -87.70673263 165.82986111111111 fluoresceiice_only

40 SECURININE 26.77552497 49.702127659574465 activity_only

40 TANSHINONE IIA -63.79535829 89.31955128 fluorescence_only

40 TANSHINONE IIA SULFONATE 6.334635293733233 49.509433962264154 activity_only

SODIUM

40 TEBUCONAZOLE -67.51729574 78.31029810298102 fluorescence_only

40 TERFENADINE -77.82972213 116.02857142857142 fluorescence_only

40 TRICLOSAN -238.233489 137.32142857142856 fluorescence_only

40 XANTHONE -174.2448409 99.5 fluorescence_only

40 XANTHYLETIN -130.9034416 73.04890282131662 fluorescence_only

40 alpha-LAPACHONE -61.14097247 108.71052631578948 fluorescence_only

40 trans, trans-FARNESOL -65.38488742 86.22132352941176 fluorescence_only

References

Muraresku C.C., McCormick E.M., and Falk M.J. (2018). Mitochondrial Disease: Advances in clinical diagnosis, management, therapeutic development, and preventative strategies. Curr Genet Med Rep. 6(2), 62-72. doi: 10.1007/s40142-018-0138-9

Niyazov D.M., Kahler S.G., and Frye R.E. (2016). Primary Mitochondrial Disease and Secondary Mitochondrial Dysfuction: Importance of Distinction for Diagnosis and Treatment. Mol Syndromol 7(3):122-37. doi: 10.1159/000446586.

Camp K.M. et al. (2016). Nutritional interventions in primary mitochondrial disorders:

Developing an evidence base. Mol Genet Metab. 19(3): 187-206. doi: 10.1016/j.ymgme.2016.09.002

Meyer J.N., Hartman J.H. and Mello D.F. (2018) Mitochondrial Toxicity. Toxicological Sciences, Volume 162, Issue 1, Pages 15-23, https://doi.org/10.1093/toxsci/kfy008

Cohen B. H. (2010). Pharmacologic effects on mitochondrial function. Dev. Disab. Res. Rev. 16, 189 -199. Review, doi: 10.1002/ddrr,106

Pereira C. V, Oliveira P. J., Will Y, Nadanaciva S. (2012). Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms. Toxicol. Appl. Pharmacol. 264, 167-181. doi: 10.1016/j.taap.2012.07.030

Durieux J., Wolff S., and Dillin A. (2011) The Cell Non- Autonomous Nature of Electron Transport Chain-Mediate Longevity. Cell. 144(1): 79-91. doi: 10.1016/j.cell.2010.12.016 Yoneda T. et al. Compartment- specific perturbation of protein handling activates genes encoding mitochondrial chaperones. (2004). J Cell Sci. 117(Pt 18):4055-66. doi: 10.1242/jcs.01275.

Matthew M.D., Matthew N.D., et al. (2016). PLoS Negl Trop Dis. 10(10):e0005058. doi:

10.1371 /j ournal .pntd.0005058 Gosai S.J., et al. (2010). PLoS One. Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin al-antitrypsin Z 5(ll):el5460. doi: 10.1371/journal.pone.0015460

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A system for rapid, semi-automated high throughput screening (HTS) and analysis of compounds that modulate at least one of mitochondrial function, organismal health, or cellular health comprising: a) dispensing i) a plurality of genetically altered C. elegans worm strains, said genetic alteration impacting a gene associated with mitochondrial function, regulation, stress resilience, or stability, and ii) wild-type C. elegans, lacking said genetic alteration, into multi-well plates with a semi- or fully-automated dispensing instrument; said worms comprising at least a first and second detectable marker; b) generating daughter plates in DMSO buffer control harboring a plurality of members from a compound or genetic library, and pinning said daughter plate onto the worm containing plate of step a) followed by incubation of the worms for a suitable time period for said compound or genetic molecule to exert an effect; c) determining an integrated metric of worm mass and activity levels using WormScan, d) determining mitochondrial stress induction using a high content imaging platform for focusing: i) a red fluorescence channel with an exposure sufficient to detect each worm expressing a first red fluorescent selectable marker in the well; ii) a green fluorescence channel with an exposure setting sufficient for detection of levels of a second green fluorescent detectable marker in the well; and iii) an exposure setting suitable for brightfield image, wherein red fluorescence levels are used to count the number of worms per well, green fluorescence levels correlated with mitochondrial physiology are set to a threshold of 1800 pixel intensity and spot total intensity is applied to measure total green fluorescence in each well; e) performing quality control on every well and on each plate by: i) removing any well with a green channel value 1.5-fold greater than the interquartile range of the other wells for that given compound; ii) removing any well with a red channel count of C. elegans less than 5 or greater than 90, and iii) removing any compound where 3 out of 4 replicate wells failed on each plate, f) assessing the dynamic range for the green channel between conditions of worms in step a) when compared to untreated control worms, by determining a Strictly Standardized Mean Difference (SSMD) calculated as Formula I: and discarding any plate having an SSMD of less than -2; g) normalizing activity by dividing the WormScan score by the number of C. elegans in the well as measured by the red fluorescence channel and normalizing green channel signal (UPRmt or other genetic or fluorescent indicator of a specific mitochondrial function or parameter or each compound as the percent reduction in stress relative to the plate controls), thereby allowing for plate-to-plate comparison; h) subtracting background, healthy animal fluorescence from all values and calculating percent change between the median disease fluorescence and the control well using Formula II: i) selecting hits from library HTS based on a percent reduction in mitochondrial stress greater than 80% and a WormScan score greater than 50.

2. The system of claim 1, wherein said hit is confirmed as a modulator of mitochondrial function in a zebrafish (D. rerio) model.

3. The system of claim 1 or claim 2, wherein said hit is confirmed as a modulator of mitochondrial function in a human cell.

4. The system of any one of claims 1 to claim 3, wherein said hit is one or more compounds that inhibits or improves mitochondrial function and/or animal survival in a mitochondrial deficient animal listed in Appendix I.

5. The system of any one of claims 1 to 4 comprising determining whether said agent alters an organismal health or cellular parameter associated with mitochondrial function in C. elegans comprising said genetic alteration relative to wild-type C. elegans; agents which alter said parameter in said genetically altered C. elegans being identified as modulators of mitochondrial function.

6. The system of claim 5, wherein said cellular parameter is selected from the group consisting of fecundity, egg hatching rate, development, lifespan, stressor survival, healthspan, animal activity, animal mass, animal size, swimming capacity, pharyngeal pumping rate, mitochondrial oxidant burden, cellular oxidant burden, antioxidant capacity, complex 1 (Cl) enzyme activity, Cl enzyme assembly, citrate synthase activity, pyruvate dehydrogenase activity, oxygen consumption capacity, ATP production, ATP levels, nicotinamide dinucleotide (NADH and NAD+) levels, (NADH and NAD+) ratio, NAD metabolism, glutathione levels (GSSG or GSH) or GSH:GSSH ratio, mitochondrial membrane potential, mitochondrial content, mitochondrial structure, mitochondrial ultrastructure, mitochondrial unfolded protein response, mitochondrial stress resilience, integrated stress response, mitochondrial import, mitophagy, autophagy, apoptosis, necrosis, ferroptosis, iron levels, iron metabolism, cell death pathway induction, cytosolic translation activity, nutrient-sensing signaling profile, unfolded protein response activation, lysosomal number, lysosomal activity, proteasome number or activity, transcriptome-wide signaling, amino acid pathway profiles, intermediary metabolic flux rates, steady state metabolism of intermediary metabolites, electrolyte levels or flux such as involving potassium, calcium, sodium, chloride, phosphorous, or magnesium, metal levels such as iron, ferritin, immune markers, amino acid levels, organic acid levels, ammonia levels, and glycoprotein production, cellular proliferation, cell growth, cell death pathways such as autophagy, apoptosis, necrosis, ferroptosis, pyroptosis, lactic acid level, pyruvate level, glycolysis, cellular redox levels, cell pH, lactate/pyruvate ratio, ketone body levels or ratios, or target gene expression level.

7. The system of claim 5, wherein said C. elegans comprising a mutation in a gene that modulates one or more of mitochondrial structure, content, proliferation, destruction, stress resilience, and function.

8. The system of claim 1, wherein said library is selected from a custom library, a chemical library, a genetic library, an RNA interference (RNAi) library, an siRNA library, an shRNA library, a cDNA library, a gDNA library, an mRNA library, a preclinical drug library, a chemical combinatorial library, a CNS penetrant compound library, a commercially available library, a custom proprietary library, a natural product compound library, an FDA approved drug library, a clinical trial tested compound library, a bacterial library, a bacterial product library, or an environmental toxicology compound library.

9. The system of claim 1, wherein said red fluorescence exposure is 20%, said green fluorescence exposure is 40%, said brightfield exposure is 10% and said worms comprise a myo2::mcherry genetic marker for determining worm number per well and a hsp6::gfp genetic marker for quantifying UPRmt induction.

10. The system of claim 1, wherein one or more steps are automated and a mitochondrial modulating agent selected from a pharmacologic agent, an acute toxicant and a chronic toxicant is dispensed into the multi- wells of step a) i).

11. The system of claim 10, where the pinning step is automated.

12. The system of any one of the preceding claims wherein said high content imager is a Cellinsight CX5 high content imaging platform with or without Momentum-based automation.

13. A composition for the negative modulation of mitochondrial function, comprising an effective amount of at least one agent of selected compounds listed in Appendix I.

14. A composition for the positive modulation of mitochondrial function, comprising an effective amount of at least one agent selected from cisplatin; benzalkonium chloride hydrate; cetrimonium bromide; phentolamine HC1; bromindione; polymyxin sulfate; mitoxantrone HC1; or mefloquine HC1 in a pharmaceutically acceptable carrier for administration separately or in combination.

15. The composition of claim 14 wherein said agent is cisplatin.

16. The composition of claim 14, wherein said agent is benzalkonium chloride hydrate.

17. The composition of claim 14, wherein said agent is cetrimonium bromide.

18. The composition of claim 14, wherein said agent is phentolamine HC1.

19. The composition of claim 14 further comprising an effective amount of glucose, steroids, MAPK-modulators, membrane stabilizers, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, immune modulators, CD4 modulators, CD8 modulators, NK cell modulators, B cell modulators, inhibitors of IgG isotype switching or IgG synthesis, P-agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines .

20. A combination therapy comprising at least two agents listed in Appendix I, said agents acting synergistically to negatively modulate mitochondrial function when combined.

21. A combination therapy comprising at least two agents selected from cisplatin; benzalkonium chloride hydrate; cetrimonium bromide; phentolamine HC1; bromindione; polymyxin sulfate; mitoxantrone HC1; or mefloquine HC1 in a pharmaceutically acceptable carrier for administration, said agents acting synergistically to improve mitochondrial function when combined.

22. The combination therapy of claim 21, wherein said agents are glucose and phentolamine HC1.

23. The combination therapy of claim 21 , wherein said agents are glucose and mitoxantrone HC1.

24. A method for modulating a cellular parameter associated with mitochondrial function or dysfunction, comprising administration of the composition of one or more of the compounds of claim 13 to claim 23 to a patient in need thereof.

25. The method of claim 24, wherein mitochondrial dysfunction is present and symptoms include one or more of muscle weakness, exercise intolerance, chronic fatigue, gastrointestinal dysmotility, cognitive decline, autism spectrum disorder, impaired balance, peripheral neuropathy, metabolic strokes, dysautonomia, vision loss, eye muscle or eyelid weakness, hearing loss, tinnitus, glomerular or tubular renal disease, endocrine dysfunction, dyslipidemia, cardiomyopathy, arrhythmia, cardiac conduction block, cardiac failure, anemia, failure to thrive, over or underweight, developmental delay, neurodevelopmental regression, cognitive decline and memory impairment, Parkinsonism, mood disorder, dystonia, liver dysfunction or failure, infertility, metabolic instability, stressor-induced acute decompensation, mitophagy disorders, mitochondrial lipid biogenesis disorders, mitochondrial cofactor disorders, primary mitochondrial disease, and secondary mitochondrial disorders including but not limited to resulting from toxins, drugs, natural products, infections, cancer, age, prescribed or illicit medications, smoking, alcohol, environmental exposures, obesity, and genetic disorders that secondarily impair mitochondrial function, structure, or activities.

26. The method of claim 24, wherein said mitochondrial symptoms are caused by a disorder selected from the group consisting of Complex I disease, Complex II disease, Complex III disease, Complex IV disease, Complex V disease, multiple respiratory chain complex disease, adenine nucleotide translocase deficiency, pyruvate dehydrogenase deficiency, mitochondrial depletion disease, single large-scale mtDNA deletion disease, multiple mitochondrial DNA deletions disease, mitochondrial DNA maintenance defects, mitochondrial DNA repair system defects, mitochondrial translation defects, mitochondrial nucleotide import disease, Friedreich's ataxia, Leber's Hereditary Optic Neuropathy, Autosomal Dominant Optic Atrophy, Kearns-Sayre Syndrome, Pearson Syndrome, Mitochondrial Myopathy, Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-Like Episodes, Myoclonic epilepsy and ragged red fibers, Neurogenic Ataxia and Retinitis Pigmentosa, Mitochondrial Neuro-gastrointestinal encephalomopathy, maternally inherited diabetes and deafness, FBXL4 mitochondrial encephalomyopathy, primary lactic acidosis, Leigh syndrome, Leigh-like syndrome, Leigh syndrome spectrum, Cockayne syndrome, Aging, Frailty syndrome, cancer, and multi-system mitochondrial disease.

27. The method of claim 24, wherein symptoms of mitochondrial disease are alleviated.

28. The method of claim 24, wherein symptoms of acute mitochondrial toxicity are inhibited or prevented.

29. The method of claim 24, wherein symptoms of acute decompensation from mitochondrial dysfunction are inhibited or prevented.

30. The method of claim 24, wherein symptoms of chronic mitochondrial toxicity arc inhibited or prevented.

31. The method of claim 24, wherein symptoms of chronic decompensation from mitochondrial dysfunction are inhibited or prevented.

32. The system of claim 3, comprising performance of a compression assay.

33. A method for inhibiting growth or survival of a cancer cell or tumor comprising administration of an effective amount of one or more mitotoxicants listed in Appendix 1 , thereby inhibiting growth of, or killing said cell.

34. A method for inhibiting growth of an infectious disease-causing vims, bacteria, parasite, or fungal cell comprising administration of an effective amount of one or more mitotoxicants listed in Appendix 1, thereby inhibiting growth of, or killing said infected cell.

35. The method of claim 24, comprising administration of an agent selected from one or more of N-acetylcysteine, nicotinic acid (niacin), niacinamide, nicotinamide riboside, nicotinamide mononucleotide, probucol, glucose, 2-deoxyglucose, lipoic acid, resveratrol, thiamine, riboflavin, leucine, uridine, 3-methyladenine, hemin, trypterin, (+) epicatechin, (-) epicatechin, 11- hydroxyprogesterone, 11-hydroxypregnenolone, cycloheximide, rapamycin, lithium chloride, creatine, dichloroacetate, omaveloxolone, elamipretide, dimethylfumarate, a deubiquitalase (DUB) modulating agent, a PPAR alpha modulating agent, a PPAR gamma modulating agent, a PPAR delta modulating agent, an AMPK modulating agent, a sirtuin modulating agent, a mTORCl modulating agent, a MAPK modulating agent, a PGCl-a modulating agent, a HIF1 -alpha modulating agent, a mitochondrial biogenesis modulating agent, an antioxidant, a phosphodiesterase modulator, sildenafil, doxycycline, tetracycline, trapidil, a calcium channel modulator, flunarizine, folinic acid, metformin, a nucleoside, valproic acid, dexamethasone, etoposide, vorinostat, quercitin, hydralazine, pfithrin-a, ginsenoside, sulfonsuccinimidyl oleate, carnitine, levocamitine, AICAR, GSK2578215A, bezafibrate, vatiquinone, coenzyme Q10, alphatocopherolquinone, a coenzyme Q10 analog, ubiquinone, ubiquinol, cytochrome C, 5 I-2-(4- hydroxyphenyl)-ethenyl] benzene- 1,3 diol, flavin mononucleotide, urolithin A, idebenone, latrepirdine, 2',3',5'-tri-O-acetyluridine, olesoxime, thiamine diphosphate, vitamin C, vitamin D, vitamin E, lipoic acid, delta-aminolevulinic acid, magnesium, calcium, phosphate, membrane phospholipid, unsaturated fatty acid, pyruvate, nicotinamide adenine dinucleotide, cysteamine bitartrate, acipimox, glutathione, a redox-modulating agent, curcumin, schisandrin, triheptanoin, an inhibitory nucleic acid targeting an upregulated gene associated with mitochondrial dysfunction, an activating genetic construct for increasing expression of a mitochondrial disease modulator protein, a nucleic acid/protein complex for genetic and/or base editing suitable for replacement therapies for primary mitochondrial diseases, a viral or non-viral vector gene therapy targeting a mitochondrial disease mutation, or a combination of any 2, 3, 4, 5 or 6 of the agents listed.

36. The system of claim 1, wherein said genetic alteration is in nuclear DNA in one or more genes selected from AARS2, ABCB7, ABCC8, ACAD8, ACAD9, ACADM, ACADS, ACADSB, ACADVL, ACAT1, AC02, ADCK3, ADRB2, ADRB3, AFG3L2, AGK, AGRP, A1FM1, AK2, AKAP10, AKT2, ALAS2, ALDH2, ALDH4A1, ALDH6A1, AMACR, AMT, APOPT1, APTX, ARMS2, ATP5A1, ATP5E, ATP5F1A, APT5F1D, ATP5F1E, ATPAF2, AUH, BAX, BCAT2, BCKDHA, BCKDHB, BCL2, BCS1L, BOLA3, C8orf38, C10orf2, C12orf62, C12orf65, C19orfl2, C20orf7, C1QBP, CAPN10, CARS2, CARTPT, CDH23, CDKAL1, CHCHD10, CHKB, CISD2, CLRN1,COA5, COA7, COAIO, COQ2, COQ4, COQ6, COQ7, COQ8A, COQ9, COXIO, COX14, COX15, COX20, C0X412, COX6B1, COX8A, CPS1, CPT1A, CPT2, CRAT, CYB5R3, CYC1, CYC5, CYCS, CYP11A1, CYP1161, CYP1162, CYP24A1, CYP27A1, CYP2761, D2HGDH, DARS2, DBT, DECR1, DFNB31, DGUOK, DHODH, DIABLO, DLD, DLAT, DMGDH, DNA2, DNAJC19, DNM1L, EARS2, ECHS1, ELAC2, ENPP1, ETFA, ETFB, ETFDH, ETHE1, FARS2, FASTKD2, FBXL4, FOXRED1, EH, FXN, GA TM, GCDH, GCGR, GCK, GCSH, GDAP1, GFER, GFM1, GHRL, GJB2, GJB3, GJB6, GK, GLDC, GLRX5, GLUD1, GPD2, GPR98, GTPBP3, HADH, HADHA, HADHB, HARS2, HCCS, HIBCH, HK1, HLCS, HMGA1, HMGCS2, HMGCL, HNF1A, HNF1B, HNF4A, H0GA1, HSD17610, HSPD1, HTRA1, HTRA2, IBA57, IDH2, IDH3B, IGF2BP2, IL6, INSR, IRS1, IRS2, ISCA1, ISCA2, ISCU, IVD, KANK1, KARS, KCNJ11, KIF1B, L2HGDH, LARS2, LEPR, LIAS, LIPC, LRPPRC, LRRK2, LYRM4, LYRM7, MAO A, MAPK8IP1, MAR52, MC4R, MCCC1, MCCC2, MCEE, ME2, MFF, MFN2, MGME1 , MIPEP, MLYCD, MMAA, MMAB, MMADHC, MPC1 , MPV17, MRAP2, MRPL3, MRPL44, MRPS2, MRPS7, MRPS16, MRPS22, MRPS34, MST01, MTFMT, MT01, MTPAP, MUT, MY07A, NAGS, NARS2, NDUFA1, NDUFA2, NDUFA6, NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFA13, NDUFAF1, NDUFAF2, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, NDUFB3, NDUFB8, NDUFB9, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS5, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NEUROD1, NF2, NFU1, NGLY1, NROB2, NUBPL, NUP62, OAT, OGDH, OPA1, OPA3, OTC, OXCT1, PANK2, PARK2, PARK7, PAX4, PC, PCCA, PCCB, PCDH15, PCK2, PDHA1, PDHB, PDHX, PDP1, PDSS1, PDSS2, PDX1, PET100, PINK1, PNKD, PNPT1, POLG, POLG2, POMC, PPARG, PPARGC IB, PPDX, PPP1 R3A, PUS I , PYCR1 , PYY, RARS2, REEP1 , RMRP, RMND1 , RNASEH1, RRM2B, SACS, SARDH, SARS2, SC01, SCO2, SCO3, SDHA, SDHAF1, SDHAF2, SDHB, SDHC, SDHD, SERAC1, SFXN4, SIM1, SLC2A2, SLC2A4, SLC22A5, 5LC25A1, 5LC25A3, 5LC25A4, 5LC25A12, 5LC25A13, 5LC25A15, 5LC25A19, 5LC25A20, 5LC25A22, 5LC25A26, 5LC25A38, SLC30A8, 50D2, SPG7, SPG20, STAR, SUCLA2, SUCLG1, SURF1,

TAC01, TARS2, TAZ, TDF7L2, TFAM, TIMM8A, TIMMDC1, TK2, TMEM70, TMEM126, TMEM126B, TOP3A, TRIT1, TRMT10C, TRMT5, TRMU, TRNT1, TSFM, TTC19, TUFM, TWNK, UCP1, UCP2, UCP3, UNG, UQCC2, UQCC3, UQCRB, UQCRC2, UQCRQ, USMG5, USH1C, USH1G, USG2A, VARS2, WARS2, WFS1, WWOX, XPNPEP3, and YARS2.

37. The method of claim 24, wherein said agents are glucose and phentolamine HC1.

38. The method of claim 24, wherein said agents are glucose and mitoxantrone HO.