Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin–actin interaction

Figures

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  Figure 1. Actin (PFA) binding to and dissociation from myosin S1. The equilibrium constant for dissociation, KA, was determined from the ratio of the rate constants k−A/k+A (Equation 9).

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  Figure 2. ATP (T)-induced dissociation of the actin (A)–myosin (M) complex. The strongly bound conformation of the myosin head is shown in green and the weakly bound or detached conformation is shown in red. Kα is the association equilibrium constant for the isomerization of actin–myosin from the closed (A.M) to open (A.M′) conformation of the nucleotide-binding site. K1 (Equation 10) is the association equilibrium constant for ATP binding to myosin that is strongly bound to actin (sometimes referred to as KT), k+2 (Equation 11) is the forward rate constant for isomerization of the collision complex (A.M.T) to the weakly bound ternary complex (A∼M.T), and kdissoc is the rate constant for dissociation of that complex. This paper focuses on the effects of compounds on K1 and k+2 described before the dashed line in the scheme.

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  Figure 3. Chemical structures for ten actin-binding compounds discovered from an HTS FRET assay. The drugs are categorized by their intended application for treatment of human disease (see text for usage and warnings). The drugs will be referred to below by the indicated abbreviations.

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  Figure 4. Effects of S1 binding to pyrene-labeled F-actin (PFA) in the absence and presence of nucleotides.A, fluorescence spectra of 2 μM PFA (black) as a function of [S1] in the absence (green) and presence (red) of 1 mM Mg.ATP. B, inhibition of PFA fluorescence (at 407 nm) as a function of [S1] () and restoration by 1 mM MgATP (•). We also show the effects of forming the Mg.ADP.VO4 () and Mg.PPi () complexes in equimolar PFA-S1 (2 μM). The PFA fluorescence of S1:Actin (0.7:1 M ratio) is shown as a green data point ().

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  Figure 5. Effects of myosin-binding drugs on pyrene-labeled F-actin (PFA) fluorescence. About 5 μM of omecamtiv mecarbil (OM) (purple) and mavacamten (Mava) (pink) on the fluorescence of 2 μM PFA (left, gray) and 2 μM PFA.S1 in the absence (green, middle) and presence (red, right) of 1 mM MgATP.

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  Figure 6. Effects of compounds on the fluorescence of PFA (2 μM).A, dose response of compounds on PFA fluorescence. B, the hyperbolic fits of the fraction of actin with bound compound (fb, Equation 1) versus [compound] for some of the compounds. C, the dissociation equilibrium constant, Kd determined from Hill's plots (Equation 3). D, fbversus [Mit] showing the hyperbolic dependence on Mit in the absence () and presence () of 200–500 μM MgATP.

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  Figure 7. The effects of compounds on pyrene-labeled F-actin (PFA) fluorescence. Normalized fluorescence of 2 μM PFA in the absence (dark gray) and presence (gray) of 100 μM compound. Inset, possible conformational changes in actin because of the compound.

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  Figure 8. The effects of S1 binding to the actin–compound complex in the presence and absence of MgATP. Normalized fluorescence of 2 μM pyrene-labeled F-actin (PFA) (gray) with S1 (0.7 S1/actin) only (pink). A, calculated (using Equation 5, hatched bars) and experimental (green) fluorescence of PFA.compound.S1 complex. B, the effect of 0.5 mM MgATP (peach) on the PFA.compound.S1 complex (green). [Compound] was 100 μM except for Flu and Dan (50 μM) and Mit (10 μM).

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  Figure 9. The effects of rigor binding of S1 to the actin–compound complex.A, normalized fluorescence transients of varying [PFA] mixed with 0.02 μM S1 in the absence of compounds. B, effects of compounds on dissociation equilibrium constant KA for PFA binding to S1. The molar ratio of compound to PFA was 5 for Mit and 25 for Flu and Dan.

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  Figure 10. ATP-induced dissociation of the actin–S1 complex.A, transient kinetics of dissociation of the complex of 0.05 μM PFA +0.05 μM cardiac myosin S1 by 25 μM MgATP. B, kobsversus [MgATP] in the absence (green) and presence (pink) of 0.25 μM Mit. Fast and slow rate constants are kobs1 (filled circles) and kobs2 (open circles). Nonlinear fit to a hyperbolic equation yields k+2 (Table 1).

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  Figure 11. The effects of compounds on MgATP binding and the subsequent isomerization step in the actin–S1 complex.A, normalized equilibrium constant K1 (Fig. 2) for ATP binding to S1 in the rigor PFA.S1 complex (0.05 μM) in the absence (Con) and presence of each compound. B, normalized rate constant k+2, for the isomerization to the weak-binding state. Before normalization, the Con values are K1 = 2.4 ± 0.03 nM−1 and k+2 = 1620 ± 80 s−1 (Table 1). The effects for K1 and k+2 are categorized as mild (blue, <1.5-fold), moderate (violet, 2–3-fold), or severe (pink, >3-fold). [Compound] was 100 μM, except Mit was 5-fold and Flu and Dan were 25-fold over [PFA].