Isothiocyanates and the NRF2 transcriptional pathway as a potential therapeutic for

Duchenne Muscular Dystrophy

Jordan Gladman5/5/2015

A disease of multiple dysfunctional biological pathways

Multiple Pathways disrupted by loss of Dystrophin

• Inflammation– Nuclear Factor Kappa B (NF-kB)– Tumor Necrosis Factor-alpha (TNF-

alpha)• Intracellular calcium balances• Fibrosis & Muscle Regeneration• Free Radical, Redox Dysfunction &

ROS– Reduction in nNOS/NO– Increased Levels of SOD-1

• Apoptosis & Necrosis– p38 MAPK

Duchenne Muscular Dystrophy Organ System Involvement

Skeletal Muscle Involvement Organ System Involvement

Nrf-2/Phase II genes and Isothiocyanate activation

Osburn and Kensler (2008) Mutation Research 659: 31-39

Phase II genes regulate the expression of detoxifying proteins that protect against oxidative damage and are anti-inflammatory

• Isothiocyanate is the chemical group –N=C=S• Naturally abundant in cruciferous vegetables

• Broccoli: Glucoraphanin SFN (Sulforaphane)• Wasabi: Sinigrin AITC (Allyl Isothiocyanate)• Watercress: Gluconasturtiin PEITC (Phenethyl isothiocyanate)

• Capable of forming a chemical bond with cellular proteins

Nrf2 is ubiquitously expressed with the highest concentrations in the kidney, muscle, lung, heart, liver, and brain

Disruption of Nrf2 signaling promotes cell degradation in skeletal muscle

Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1822, Issue 6, 2012, 1038 - 1050

Loss of NRF2 results in skeletal myopathy

Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1822, Issue 6, 2012, 1038 - 1050

Additional data in exercise induced muscle damage found that increasing NRF2 levels lead to increased levels of ARE target proteins and improved muscle histology

• Glucoraphanin, Sulforaphane’s precursor, is naturally abundant in broccoli and cauliflower

• Identified as a Cancer Chemopreventive• Multiple labs are making sulforaphane derivatives

Glucoraphanin (Glucosinolate precursor)

The Isothiocyanate Sulforaphane

Sulforaphane

myrosinase

Sulforaphane

Sulforaphane and DMD

• The affects of Sulforaphane – p38 MAPK– NF-κB– Myostatin

• Sulforaphane treatment of MDX Mice• Potential negative aspects • Future Steps

P38 MAPK myofiber death

• Wissing et al. (2014) Hum Mol Genet. P38α MAPK underlies muscular dystrophy and myofiber death through a Bax-dependent mechanism.– P38α is over expressed in DMX

– Over-activation of p38α causes severe muscle-wasting pathology in mice

• P38 expression inhibits Nrf2 by keeping it bound to KEAP1

Aurelian (2015) CROSS TALK OF SIGNALING AND APOPTOTIC CASCADES IN THE CNS: TARGET FOR VIRUS MODULATION Frontiers in Bioscience 10, 2776-2787

Expression of p38 MAPK in DMD.

Erin R. Wissing et al. Hum. Mol. Genet. 2014;23:5452-5463

P38 MAPK myofiber death

• SFN suppressed activation of p38 MAPK isoforms by blocking phosphorylation of MKK3/6– Keum et al. (2006) Cancer Res.

Aurelian (2015) CROSS TALK OF SIGNALING AND APOPTOTIC CASCADES IN THE CNS: TARGET FOR VIRUS MODULATION Frontiers in Bioscience 10, 2776-2787

SB731445 SulforaphaneP38 activation has been show to increase Utrophin so it may be incompatible with some forms of DMD therapy

McKinley and Willoughby (2014) Effectiveness of Antioxidant Nutraceuticals in Attenuating Canonical NF-κB Signaling in Human Skeletal Muscle Resulting From Exercise-Induced Inflammation and Oxidative Stress. Journal of Nutritional Health & Food Engineering

The NF-κB pathway and cross talk in DMD

Survival, Proliferation, Inflammation, Immune Regulation

Catabasis, CAT-1004 is SMART linker conjugate of salicylate and docosahexaenoic acid (DHA) designed to enhance the activity of salicylate and DHA in modulating the NF-kB pathway.

Guttridge Lab, NBD delivery improves the disease phenotype of the golden retriever model of Duchenne muscular dystrophy. Skelet Muscle. 2014.

FN14

TWEAK

The tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) has a wasting effect on skeletal muscle cells both in-vivo and in-vitro

The NF-κB pathway is regulated by Nrf2 and Sulforaphane

• Nrf2 overexpression is capable of inhibiting the NF-κB – Cuadrado A J et al. (2014) Biol Chem. Transcription factors NRF2 and NF-κB are

coordinated effectors of the Rho family, GTP-binding protein RAC1 during inflammation.

• NFκB could directly repress Nrf2 transcription.– Liu GH et al. (2008) Biochim Biophys Acta. NF-kappaB/p65 antagonizes Nrf2-ARE

pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK.

• SFN treatment was found to be able to inhibit IKK/IκB phosphorylation and p65 NFκB subunit nuclear translocation– Xu C et al. (2005) Oncogene. Suppression of NF-kappaB and NF-kappaB-regulated

gene expression by sulforaphane and PEITC through IkappaBalpha, IKK pathway in human prostate cancer PC-3 cells.

• SFN prevented NF-κB binding to its consensus DNA sequence– Elke Heiss et al. (2001) J Biol Chem. Nuclear factor kappa B is a molecular target for

sulforaphane-mediated anti-inflammatory mechanisms.

Sulforaphane

Sulforaphane

Sulforaphane represses Myostatin mRNA levels

• Sulforaphane acts as a histone deacetylase (HDAC) inhibitors

• SFN treatment significantly represses MSTN expression, in porcine myoblast cells

• Protein levels were not examined

Burks and Cohn (2011) Role of TGF-β signaling in inherited and acquired myopathies. Skeletal Muscle 1:19

Fan et al. (2012) Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells. Epigenetics 7:12

• Myostatin is a secreted TGF beta family member that inhibits muscle differentiation

• Animals lacking myostatin or animals treated with substances that block the activity of myostatin have significantly larger muscles

• Beneficial in MDX mouse model

Sulforaphane alleviates muscular dystrophy in mdx mice

Chengcao Sun , Cuili Yang , Ruilin Xue , Shujun Li , Ting Zhang , Lei Pan , Xuejiao Ma , Liang Wang , Dejia Li (2015) Journal of Applied Physiology. Vol. 118 no. 2, 224-237

• Examined the effects of Sulforaphane in mdx mice– Weight of various tissues– Grip strength and run endurance– Plasma Creatine Phosphokinase and Lactate Dehydrogenase Levels– Muscle integrity and histology– Nrf2 genes– Inflammation

Sulforaphane (SFN) increased muscle weight and improved myocardial hypertrophy in mdx mice.

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

• C57BL/10ScSn-Dmdmdx/NJU• 2 mg per kg body weight per day in DMSO/Corn oil by oral gavage

• Data not normalized to body weight

Increase of the forelimb grip and running distance in SFN-treated mdx mice after 8 wks of treatment.

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

• Data not normalized to body weight

SFN alleviated the oxidative stress of mdx mice.

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

Assessing Muscle Damage:

SFN activated (Nrf2)/(ARE) signalling pathway.

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

Assessing Muscle oxidative stress:

Reduction in the levels of Evans blue staining in skeletal muscles from SFN-treated mdx mice.

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

EBD -Marker of muscle integrity

Reduction in central nucleation and fiber size variability in SFN-treated mdx muscle (TA) fibers.

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

Chengcao Sun et al. J Appl Physiol 2015;118:224-237

©2015 by American Physiological Society

SFN ameliorates inflammation (CD45) in mdx muscle fibers

CD45 was used to detect all lymphocytes

Potential negative aspects • Activation of NRF2 may promote the development of de novo

cancerous tumors as well as the development of atherosclerosis by raising plasma cholesterol levels and cholesterol content in the liver

• Sulforaphane in the literature– Mechanisms of action are based on cancer cell lines and often not

separated from activation of NRF2– Studies are descriptive rather than mechanistic in the literature– uM levels used however there is evidence this is obtainable in vivo by oral

administration.

• Sulforaphane may be a therapeutic option for DMD but it may be difficult to commercialize– Many Supplements available– Use of a sulforaphane derivative may be a better choice but no

therapeutic data is available

Collaboration is possible

• Evgen Pharma– Sulforadex® , a novel, synthetic and stabilized version

of the naturally occurring compound sulforaphane– Pipeline: SFX-01: Prostate cancer and Multiple

sclerosis – SFX-02 and SFX-03 in development

• Multiple labs are making sulforaphane derivatives

• Other Isothiocyanates may provide equal or better results

Next Steps

• Develop a collaboration with Evgen Pharma or a laboratory that produces a sulforaphane derivative or possibly other Isothiocyanates

• Additional rigorous studies need to be done in animal models and patient cell lines

• Toxicology and optimal dosing should be investigated