The Myomatrix Reloaded: ECM and integrin therapies for the treatment of muscular dystrophy

1. The Myomatrix Reloaded: ECM and integrin therapies for the treatment of muscular dystrophy Dean J Burkin, Ph.D. Professor of Pharmacology Center for Molecular Medicine University of Nevada School of Medicine dburkin@medicine.nevada.edu

2. Disclosure statement: The University of Nevada, Reno (UNR) has been issued a patent on the therapeutic use of laminin, laminin derivatives, and their compositions. UNR has licensed this technology to Prothelia Inc. and has a small equity share in this company. UNR has patents pending on the therapeutic use of integrin enhancing small molecules and Galectin-1, their derivatives, and compositions. UNR has licensed these technologies to StrykaGen Corp. which is owned by Dr. Dean Burkin and Dr. Ryan Wuebbles.

3. The a7b1 integrin ● Laminin receptor in skeletal, cardiac and vascular smooth muscle. ● Mediates “inside-out” and “outside-in” cell signaling. ● Mutations in the a7 integrin gene cause congenital myopathy. ● Major modifier of disease progression in other muscle diseases (e.g. MDC1A and DMD). Laminin-111, 211, 221 a7b1 integrin ILK FAK Talin Vinculin Cell Signaling

4. Duchenne Muscular Dystrophy Normal DMD From: A Kornberg Common human X-linked neuromuscular disease DMD patients suffer from severe, progressive muscle degeneration and die in their second or third decade from heart and/or respiratory failure There is no cure for this devastating neuromuscular disease DMD patients and the mdx mouse model have mutations in the gene encoding dystrophin

5. Major laminin-binding complexes in skeletal and cardiac muscle Merosin Deficient Congenital Muscular Dystrophy Limb-Girdle Muscular Dystrophy a7 integrin congenital myopathy ILK FAK Duchenne Muscular Dystrophy Hypothesis: the a7b1 integrin is a modifier of disease progression in DMD

6. Transgenic overexpression of the a7 integrin in muscle restores viability to a mouse model of DMD Burkin et al., (2001) Journal of Cell Biology 152

7. Loss of the a7 integrin and dystrophin causes severe muscular dystrophy in mice

8. Development of a muscle cell based assay to report a7 integrin expression 7 integrin 7+/- myoblast cell line 7 promoter Lac Z Screen Drug libraries Hit validation FDG-based fluorescence assay Validate in mouse and human muscle cells

9. FDG-based fluorescence assay was used to detect b-galactosidase in 7bmyo+/- myogenic cells Fluorescein Fluorescein mono galactopyranoside (FMG) Fluorescein di galactopyranoside (FDG) Abs/Em = 490/514

10. Integrin Enhancing Compounds (IEC) • Libraries Screened: • Prestwick Chemical and Microsource Spectrum Libraries (BioFocus DPI, Leiden Netherlands with facilities in UK, Basel, Heidelberg) (Overington et al., 2006). • 2) DIVERSet library (Chembridge Corp., San Diego, CA) and compounds from the ChemDiv library. • 3) LOPAC library (Sigma-RPI) consists of 1280 Pharmaceutically active compounds. • 4) MLSMR-Molecular Libraries Small Molecule Repository. • Total screened: ~430,000 compounds

11. SU9516 acts in a dose-dependent manner and promotes myogenic differentiation Sarathy et al., Submitted

12. SU9516 increases a7 integrin in mouse and human DMD myogenic cells Sarathy et al., Submitted

13. SU9516 exhibits on-target in vivo activity to increase the a7b1 integrin Sarathy et al., Submitted

14. SU9516 increases diaphragm muscle contractility in mdx mice Sarathy et al., Submitted

15. SU9516 improves diaphragm muscle contractions evoked by phrenic nerve stimulation

16. SU9516 improves neuromuscular kinetics of the mdx diaphragm muscle Sarathy et al., Submitted

17. SU9516 improves muscle regeneration and reduces pathology in the diaphragm of the mdx mouse Sarathy et al., Submitted

18. SU9516 inhibits the activation of p65 and NF-kB in the diaphragm muscle of mdx mice Sarathy et al., Submitted

19. Identifying the mechanism of action of SU9516 in DMD myogenic cells KiNativ assay ● Patient myotubes were treated with SU9516 or vehicle for 48 hrs ● Cells were treated with KiNativ probe (biotinylated acyl phosphates of ATP and ADP) ● Proteins extracted and subjected to LC-MS/MS to identify the molecular signature of biotin labeled proteins Sarathy et al., Submitted

20. The WNK1 inhibitor STOCK1S506 promotes a7 integrin expression in DMD myotubes

21. Model for the action of SU9516 in DMD myogenic cells

22. LAMA2-related Congenital Muscular Dystrophy ● Caused by mutations in the LAMA2 gene resulting in a complete or partial absence (rare) of laminin-211 and 221 ● Characterized by severe muscle weakness from birth, delayed motor milestones, peripheral neuropathy and changes in brain white matter. From: Carsten Bonnemann, MD ● There is no known treatment or cure for LAMA2-CMD

23. Normal Muscle MDC1A Muscle Laminin-411, 511, galectin, fibronectin Laminin-211, 221 a7b1 integrin ILK ILK FAK FAK Talin Talin Vinculin Vinculin Cell Signaling Hypothesis: Increased a7 integrin can prevent muscle disease progression in laminin-a2 deficient muscle

24. Transgenic overexpression of the a7 integrin in muscle restores viability to a mouse model of MDC1A

25. SU9516 increases a7 integrin in the muscle of the dyW mouse model of LAMA2-CMD __________ * Vehicle SU9516 Vehicle SU9516

26. The a7 integrin enhancing small compound SU9516 improves activity in a mouse model of LAMA2-CMD

27. Conclusions ● Using a novel muscle cell based assay we have identified biologics and small molecules that increase a7 integrin in mouse and patient muscle cells ● SU9516 represents a first-in-class a7 integrin enhancing drug that can be used to treat DMD regardless of the mutation. ● SU9516 improves muscle pathology and functional preclinical outcome measures in mouse models of DMD and LAMA2-CMD ● SU9516 can be used as a molecular probe to dissect pathways that regulate the a7b1 integrin in muscle ● Integrin enhancing therapeutics may be developed individually or in combination as potential therapies for the treatment of muscular dystrophy

28. Thanks University of Nevada School of Medicine Burkin lab Ryan Wuebbles, PhD Pam Van Ry, PhD Paul Brewer, PhD Pamela Barraza-Flores Tatiana Fontelonga Apurva Sarathy Ashley Tachione Rebecca Evans Andreia Nunes Vivian Cruz Jordan Tice Tyler Van Ry Murdoch Children’s Research Institute, Victoria, Australia Kathryn North, MD National Center for Advancing Translational Sciences (NCATS) Juan Marugan, PhD Marc Ferrer, PhD Noel Southall, PhD Lesley Matthews-Griner, PhD Support Muscular Dystrophy Association Parent Project Muscular Dystrophy CureCMD & SAM NIH/NINDS: R21NS58429 (+supplemental) NIH/NIAMS: R21AR060769 NIH/NIAMS: R01AR053697 NIH/NIAMS: R01AR064338