Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy

Figures

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  Figure 1. Dystrophin-actin biosensor, which measures FRET from ABD1-mClover3 (donor, green star) to Alexa 568–labeled actin (acceptor, red star). Actin (yellow)- or ABD1 (gray)-binding compounds (orange pentagon) can act as modulators that enhance the binding of ABD1 to actin, as detected by decreased lifetime, providing a potential therapeutic approach to treat DMD. The fluorescent protein mClover3 (green star) is fused to the C-terminus of hDYS-ABD1/hUTR-ABD1 (gray). ABD, actin-binding domain; DMD, Duchenne muscular dystrophy; hDYS, human dystrophin; hUTR, human utrophin.

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  Figure 2. FRET biosensor detects specific binding of human dystrophin ABD1 (black) and human utrophin ABD1 (red) to actin in a concentration-dependent manner.A, Lifetime and B, FRET. Control experiments with mClover3 (green), the fluorescent protein without ABD1, show that the FRET change resulted exclusively from actin-ABD1 binding (N = 2–8: errors are SD's for points with N = 3 or greater, while the N = 2 data points are simply means of N = 2). ABD, actin-binding domain.

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  Figure 3. A representative LOPAC screen. Hits (red circles) were identified as 4SD (showed as dashed lines) of DMSO-only control. Interfering (fluorescent) compounds were flagged (magenta) as they are not real hits. Eleven hit compounds reproducibly changed FRET in triplicate screens. DMSO, dimethyl sulfoxide; LOPAC, Library of Pharmacologically Active Compounds.

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  Figure 4. Concentration-response curves for LOPAC hit compounds.A, compound CRC (N = 4–8): 1 μM hDYS-ABD1 and 10 μM AF-actin were incubated with each of the hit compounds (0–100 μM) and FRET was calculated. B, actin CRC (N = 3–8): Compounds that had significant effects on CRC were further tested with increasing concentrations of actin. One micromolar hDYS-ABD1 or hUTR-ABD1 was incubated with increasing [AF-actin] using 50 μM compound. Cisplatin and cDPCP both increased the affinity of hDYS-ABD1 for actin, toward the level of hUTR-ABD1. EC50 values for Cisplatin and cDPCP are summarized in Table 1. Errors are reported as SD. ABD, actin-binding domain; AF-actin, Alexa 568 F-actin; CRC, concentration-response curve; hDYS, human dystrophin; hUTR, human utrophin; LOPAC, Library of Pharmacologically Active Compounds.

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  Figure 5. Cisplatin derivatives. All four derivatives of cisplatin increase FRET between actin and hDYS-ABD1 in a concentration-dependent manner and have a wide range of EC50 (Table 1). These are promising tool/lead compounds for future studies. ABD, actin-binding domain; hDYS, human dystrophin.

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  Figure 6. Cosedimentation. hDYS-ABD1 (5 μM) was incubated with increasing concentration (0–80 μM) of F-actin at 100 μM cisplatin or 1% DMSO, centrifuged at high speed. A, representative cosedimentation SDS-PAGE gel. Odd numbered lanes are supernatant and even numbered lanes are pellet. B, bound fraction of hDYS-ABD1 to actin was determined from (A). Gray lines indicate linear fit. Magenta and black lines indicate single hyperbolic fit with Bmax fixed to 1.0 (ns not significant, ∗p < 0.0224, ∗∗p < 0.0011, ∗∗∗p < 0.0005). Errors are reported as SD, (N = 3–4). ABD, actin-binding domain; DMSO, dimethyl sulfoxide; hDYS, human dystrophin.

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  Figure 7. Selectivity of tool compound (N = 2–8). Effect of cisplatin is much less on hUTR-ABD1 compared with hDYS-ABD1, indicating that cisplatin acts selectively on actin–DystrophinABD1 complex (N = 2–8: errors are SD's for points with N = 3 or greater, while the N = 2 data points are simply means of N = 2). ABD, actin-binding domain; hDYS, human dystrophin; hUTR, human utrophin.

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  Figure 8. Concentration-response curves for the promising SELLECK hit compounds.A, compound CRC (N = 3): 1 μM hDYS-ABD1 and 10 μM AF-actin were incubated with each of the hit compounds (0–100 μM) and FRET was calculated. B, actin CRC (N = 2–8): Compounds were further tested with increasing concentrations of actin. One micromolar hDYS-ABD1 or hUTR-ABD1 was incubated with increasing [AF-actin] using 50 μM compound. All four SELLECK compound increased the affinity of hDYS-ABD1 for actin, toward the level of hUTR-ABD1. EC50 values are summarized in Table 2 (errors are SD's for points with N = 3 or greater, while the N = 2 data points are simply means of N = 2). ABD, actin-binding domain; AF-actin, Alexa 568 F-actin; CRC, concentration-response curve; hDYS, human dystrophin; hUTR, human utrophin.

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  Figure 9. Concentration response effects of the most promising compounds on mClover3 (fluorescent protein, no ABD1,green), mClover3-ABD1 (Donor only, blue), mClover3-ABD1 +Alexa 568 actin (Donor + Acceptor, magenta), and Alexa 568 actin (acceptor only, red). Fluorescent protein control, donor, and donor+acceptor samples were excited at 473 nm and acceptor only sample was excited at 532 nm. A, Cisplatin. B, Bacitracin Zinc. C, Hexachlorophene. (N = 4: Errors are reported as SD). ABD, actin-binding domain.