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Reversible Covalent Binding to Cardiac Troponin C by the Ca2+-sensitizer Levosimendan Ian M. Robertson, Sandra E. Pineda-Sanabria, Ziqian Yan, Thomas Kampourakis, Yin-Biao Sun, Brian D. Sykes, and Malcolm Irving Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00758 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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Biochemistry

Reversible Covalent Binding to Cardiac Troponin C by the Ca2+-sensitizer Levosimendan

Ian M. Robertson1,2, Sandra E. Pineda-Sanabria2, Ziqian Yan1, Thomas Kampourakis1, Yin-Biao Sun1, Brian D. Sykes2, and Malcolm Irving1

1

Randall Division of Cell and Molecular Biophysics and British Heart Foundation Centre of

Research Excellence, New Hunt’s House, Guy’s Campus, King’s College London, London, SE1 1UL, UK 2

Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada

Running Title: Reaction between cTnC and Levosimendan

To whom correspondence should be addressed: Prof. Malcolm Irving, Randall Division of Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, New Hunt’s House, Guy’s Campus, King’s College London, London, SE1 1UL, UK, [email protected]

Keywords: Troponin C, Levosimendan, Ca2+-sensitizer, Heart Failure, Thioimidate

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Abstract The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca2+, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca2+ (Ca2+-sensitizers) have therapeutic promise. The only Ca2+-sensitizer used regularly in clinical settings is levosimendan. While the primary target of levosimendan is thought to be cTnC, the molecular details of this interaction are not well understood. In this study we used mass spectrometry, computational chemistry, and nuclear magnetic resonance spectroscopy to demonstrate that levosimendan reacts specifically with cysteine 84 of cTnC to form a reversible thioimidate bond. We also showed that levosimendan only reacts with the active, Ca2+-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca2+-sensitizing function of levosimendan is due to stabilization of the Ca2+-bound conformation of cTnC.

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Some of the best known drugs bind their target protein by covalent modification; examples include aspirin (covalently modifies cyclooxygenase)1 and the penicillin class of antibiotics (react with bacterial DD-transpeptidase)2. Despite these and more recent examples of successful covalent modifiers3, concerted development of covalent drugs has been held back by concerns of off-target reactivity and associated toxicity3, 4. In fact, many of the covalent drugs currently in use were identified as covalent modifiers only after their discovery3. In this study, we provide evidence that levosimendan ((R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridaznyl)phenyl]hydrazone]-propanedinitrile)5, 6 (Scheme 1), a Ca2+-sensitizer used in the treatment of heart failure (trade name: Simdax, Orion Corporation), forms a reversible covalent bond with one of its therapeutic targets, troponin C (TnC), the Ca2+ regulatory protein in the contractile filaments of heart muscle. Systolic heart failure is characterized by a decrease in the ability of the heart to supply the body’s organs with an adequate amount of blood. One current treatment strategy for heart failure is pharmacological enhancement of heart muscle contractility. Heart contractility is regulated by Ca2+ binding to the thin filament protein cardiac troponin C (cTnC). cTnC is an EF-hand protein containing a C-terminal domain (cCTnC) and an N-terminal domain (cNTnC). cCTnC is thought to be predominantly structural, anchoring cTnC to the thin filament, whereas cNTnC is the primary site for the modulation of contraction. When Ca2+ binds to cNTnC, it undergoes a small conformational change to expose a hydrophobic cleft that binds the switch region of troponin I


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(cTnI147-163). cTnI147-163 binding to cNTnC pulls the inhibitory regions of cTnI off actin, which releases muscle inhibition, thereby triggering contraction7, 8. Given the central role cNTnC plays in modulating contraction, enhancing its activity represents an attractive pharmacotherapy for heart failure. One approach is to indirectly increase the cytosolic Ca2+ concentration through inhibition of phosphodiesterase (PDE) 3 or activation of β-adrenergic receptors. This leads to an increase in the number of cTnC molecules activated and therefore an enhancement of contraction. Molecules such as milrinone (PDE3 inhibitor) and dobutamine (β-agonist) work in this manner, but are also associated with severe deleterious effects such as abnormal contraction (arrhythmia and tachycardia) and increased mortality9. Direct enhancement of the activity of the contractile proteins may therefore offer a more promising therapy10. Examples include the newly discovered myosin activator, omecamtiv mecarbil11, or cTnC activators (also known as Ca2+-sensitizers), such as levosimendan 12, 13. Ca2+sensitizers could be designed to enhance the Ca2+-affinity of cNTnC, stabilize the open conformation of cNTnC, and/or enhance the cTnI147-163 affinity of cNTnC 9, 14-16. While a number of small molecules have been shown to modulate the function of cTnC in vitro

14, 17

,

levosimendan is the only Ca2+-sensitizer commonly prescribed for the treatment of heart failure. Levosimendan has been shown to enhance the Ca2+ affinity of cTnC sensitivity of cardiac muscle fibres

5, 18-21

and cardiomyocytes

22

12, 13

as well as the Ca2+-

. A structure-activity study of

levosimendan showed a strong correlation between its ability to bind cTnC and its enhancement of muscle contraction

23

. NMR studies using chemical shift mapping indicated that, although

levosimendan binds both domains of cTnC 24, 25, it is selective for cNTnC when cTnI is present 26, making cNTnC its most likely in vivo binding site. Although there is strong evidence that levosimendan targets cTnC, its binding mode and molecular mechanism have remained elusive. This information is crucial because, whilst levosimendan is primarily thought of as a Ca2+sensitizer, other pharmacological effects such as PDE3 inhibition and ATP-dependent K+ channel


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activation contribute to its in vivo activity

27-29

which limit its use as a therapeutic

30

. A more

thorough understanding of the molecular details of the activation of cTnC by levosimendan will help to design more specific compounds.

Materials and Methods Protein preparation – Human cTnC and cTnC(C35S, C84S) were cloned as previously described31. The DNA from cTnC(C35S, C84S) was used as a template for the preparation of the monocysteine mutants C84S or C35S using a site-directed mutagenesis kit. The purity of the proteins were verified by reverse-phase HPLC and electrospray ionization Mass Spectrometry (ESI-MS). Masses were: cTnC: 18401.53 ± 1.17 Da (expected: 18402.5 Da), cTnC(C35S, C84S): 18369.71 ± 0.67 Da (expected: 18370.39 Da), cTnC(C35S): 18386.09 ± 0.75 Da (expected: 18386.45 Da), cTnC(C84S): 18385.79 ± 0.77 Da (expected: 18386.45 Da). Protein concentration was determined with Non-InterferingTM Protein Assays (G-Biosciences) and by tyrosine absorbance at 280 nm determined for cTnC 32. NMR Spectroscopy – Most of the NMR experiments were run on a Bruker 500-MHz or Bruker 700-MHz spectrometer equipped with a z-axis pulsed field gradient triple-resonance probe. Topspin (Bruker) was used for the analysis of the data. The 13C NMR data of 13C-labelled levosimendan were collected on a Varian Inova 500 MHz spectrometer with a 10mm

13

C

broadband probe. The solid-state 13C CPMAS NMR spectra were acquired on a Varian Inova 600 MHz spectrometer equipped with a 3.2mm T3 HXY probe. The temperature was for the solidstate experiment was 15 °C and the spinning speed was 10 KHz. All solution NMR experiments were collected at 30 °C. All aqueous samples (90% H2O/10% D2O) were dissolved in NMR buffer containing: 100 mM KCl, 10 mM Imidazole, and ~0.1 mM 2,2-dimethyl-2-silapentane-5sulfonate sodium salt (DSS). The 1H chemical shifts were referenced to DSS or DMSO (for 13C


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experiments). Unless otherwise mentioned, the pH for aqeuous NMR samples was maintained between 6.8 and 7.0 and was determined by the 1H chemical shift of imidazole33. Levosimendan

assignment

–

A

stock

of

((4-[(4'R)-4-Methyl-6-oxo-1,4,5,6-

tetrahydropyridazin-3-yl]phenyl) hydrazono) propanedinitrile (Levosimendan) (MW 280.28 g/mol) was dissolved in deuterated dimethyl sulfoxide (DMSO). An aliquot of levosimendan stock was added to the NMR buffer (see above). One-dimensional 1H and

13

C spectra of

levosimendan were acquired. The two-dimensional 13C-natural abundance 1H-13C HSQC and 1H13

C HMBC experiments were acquired for the assignment of 1H-attached

13

C nuclei and

13

C

nuclei two-three bonds away from the 1H. The 1JHC was assumed to be 145 Hz and the 2,3JHC = 10 Hz. The assignments and coupling constants are shown in Supplementary Figures 1 and 2 and Supplementary Tables 1 and 2 and were compared to those previously reported 34. Most of the 1H and 13C assignments were made by use of the 1H,13C-HSQC and 1H,13CHMBC spectra. The lack of a correlation between H4b and C6 (and the presence of a strong peak between H4a and C6) in the 1H,13C-HMBC spectrum strongly suggested that H4a is the proton cis to C7 and H4b is trans to C7. To support this conclusion, the structure of levosimendan was optimized in Gaussian (see below). The structure predicted an H4a – C4 – C5 – C6 dihedral angle of ~171° and an H4b – C4 – C5 – C6 dihedral angle of ~ -70°, which is consistent with our assignments. Furthermore, the chemical shifts and coupling constants were also calculated (see below) for the optimized structure and were found to correlate with our assignments. The assignments of C16, C17 and C19 were first based on that predicted by theory and then confirmed experimentally. 1D 13C spectra of 13C-labelled 13C16, 13C17, 13C19 levosimendan (see synthesis below) revealed that there were three couplings: 117.0 Hz, 93.6 Hz, and 9.4 Hz. The 9.4 Hz coupling was between 13C19 and 13C17 (2JCC) and the couplings of 117 and 93.6 Hz were from 1

JCC between 13C16 and either 13C19 or 13C17. The assignment of 13C16 was confirmed by the 1D

and 2D 13C INADEQUATE experiments (1JCC = 100 Hz). 6 ACS Paragon Plus Environment

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Acid dissociation constant of levosimendan –

The pKa of levosimendan was measured

by titrating 1M HCl or 1M NaOH into the sample. The pH titration points were: 5.92, 6.26, 6.29, 6.42, 6.49, 6.65, 6.68, 6.81, 6.96, 7.16, and 7.4. The points were acquired by first going down to 5.92 from 6.68 and then up to 7.4. Hence several points were near one another, which served as a good means to verify the change in salt concentration did not have a large effect on the pKa of levosimendan. The pKa was fit to the NMR data with the following equation:

= +

Δ

1 + 10 ( )

where δobs is the observed chemical shift at a given pH, δHA is the chemical shift of the fully protonated species of levosimendan, ∆δ is the total chemical shift change over the course of the pH titration, and n is the Hill parameter, which describes the cooperativity of the fit. Synthesis and NMR of 13C-labeled levosimendan –

(R)-6-(4-aminophenyl)-4,5-

dihydro-5-methyl-3(2H)-pyridazinone (OR-1855) (MW 203.24 g/mol) (Toronto Research Chemicals) was mixed with 13C-labelled Malononitrile (~69.06 g/mol). 40.4 mg of OR-1855 (0.2 mmoles) were dissolved in 5 mL of dH2O to which 166 µL of 37% HCl was added. The solution was a brownish colour. 0.69 g of NaNO2 (69.00 g/mol) was dissolved in 100 mL of dH2O. 2 mL of NaNO2 solution (0.2 mmoles) was added dropwise to the ice-cold solution (0-5 °C) of OR1855. Colour turned yellow. The reaction mixture was stirred for 5-10 minutes. The diazo solution was next added drop wise to a solution of 5 mL of 20 mg (0.29 mmoles)

13

C-malononitrile (69.06 g/mol) and 0.83 g (10 mmoles) NaOAc (82.03 g/mol). The

reaction mixture was stirred on ice for 30 min. Then it was taken off the ice and mixed for an additional 30 min to evaporate excess, unreacted NO2. The product immediately precipitated, 7 ACS Paragon Plus Environment

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which was purified by filtration and multiple acid washes. The product was dried overnight and weighed. 46 mg (0.16 mmol) of product (yellow powder) was recovered (~80% yield). The product was verified by LC-MS (284.10) and NMR spectroscopy. Levosimendan and thioimidate optimization – ((4-[(4'R)-4-Methyl-6-oxo-1,4,5,6tetrahydropyridazin-3-yl]phenyl)hydrazono) propanedinitrile (Levosimendan) contains a highly conjugated hydrazone moiety that in neutral conditions would be mostly negatively charged35. Based on the structural similarity of levosimendan with the Carbonyl Cyanide Phenylhydrazones (CCP), the titratable group is the amino group of the hydrazone adjacent to the phenyl36. Therefore, the structure of levosimendan was optimized with a formal charge of -1. The Gaussian files were prepared and log files viewed using Avogadro 37. The structure of levosimendan was optimized in Gaussian 09

38

with b3lyp/6-31G(d). Although the structure of levosimendan is not

known, the X-ray structure of the p-Nitro congener of CCP (NCCP) was solved in its neutral and anionic state 39. Furthermore, the metabolite of levosimendan, OR-1855 has also been determined 40

. The bond lengths determined in the study on NCCP were compared with those calculated for

levosimendan and the ring torsion angle in OR-1855 was compared with that calculated for levosimendan. The theoretical structure closely agreed with those other related structures. The structure of levosimendan with C19 reacted with methanethiol (simple thioimidate model) was optimized in the same manner as levosimendan. Calculated NMR parameters of levosimendan and thioimidate – The optimized structure of levosimendan and the simple thioimidate model were used to calculate the NMR chemical shifts and, in the case of levosimendan, also the coupling constants. The level of theory for the chemical shift calculation was nmr=giao b3lyp/6-31+G(d,p) scrf=(solvent=water)

41

. The

coupling constants of levosimendan were calculated by the method outlined by Bally and Rablen 42

.

The

functional

and

basis

sets

were

b3lyp/6-31G(d,p)


nmr=(fconly,readatoms)

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iop(3/10=1100000),

selecting

for

the

proton

couplings

only

(for

details

see:

http://cheshirenmr.info/Recommendations.htm). Nitrile electrophilicity – In order to characterize whether levosimendan would be likely to react with a thiol, we repeated the protocol outlined by Oballa et al.

43

. Levosimendan was

initially optimized with the b3lyp/6-311G(d,p) level of theory followed by a single-point energy calculation in water (b3lyp/6-311G(d,p) scrf=(pcm,solvent=water) scf=tight). The same protocol was done for methanethiol and the thioimidate adducts. Finally, the reactivity index (in Kcal/mol), which is equal to Ethioimidate - Elevosimendan - Emethanethiol, was calculated. The more negative the reactivity index, the stronger the electrophile. The reactivity of levosimendan was assessed experimentally following the protocol proposed by MacFaul et al. 44. 110 µM and 55 µM levosimendan (from a 25 mM stock in DMSO) was mixed with 5 mM reduced L-glutathione (GSH) and 1 mM EDTA at pH 7.4. The mixture was incubated at 37 °C. The sample mixture was monitored at regular intervals by HPLC (Agilent 1200 series HPLC). The chromatography was run starting with 60% Buffer A (HPLC grade H2O + 0.1 % Trifluoroacetic acid (v/v)) and 40% Buffer B (80% Acetonitrile + 0.1 % Trifluoroacetic acid with HPLC grade H2O (v/v)) and finishing with 40% Buffer A and 60% Buffer B. The rate calculations were done by monitoring the area of the levosimendan peak with time. The reaction with glutathione to form the thioimidate intermediate is complete within the first point for HPLC (1 hour), followed by further reaction of levosimendan to a thiazoline and other products (see Figure S7). The half-life for 110 µM levosimendan was calculated as 22.5 ± 1.7 hours and for 55 µM levosimendan it was 6.4 ± 1.4 hours. Liquid Chromatography-Mass Spectrometry – All mass spectrometry was done on a Hewlett Packard (Agilent) 1100 Series LC/MSD using the electrospray ionization method and detected in positive mode. The Chromatography was run starting with 60% Buffer A (LC-MS


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water + 0.1 % Formic Acid (v/v)) and 40% Buffer B (80% Acetonitrile/water + 0.1% FA (v/v) and finishing with 10% Buffer A and 90% Buffer B. Levosimendan (in DMSO to a final concentration of 2%) was mixed with 1 mg/ml cTnC(wt), cTnC(C35S,C84S), cTnC(C35S), or cTnC(C84S) in 0.2 mM CaCl2, 100 mM KCl, and 10 mM Imidazole, pH 7.0 for 90 min (cTnC(C35S,C84S) was incubated for an extra 30 min to test for any non-specific interaction non-cysteines). Following incubation, 5 µL of the reaction mixtures were injected into the columns and the masses were analyzed. Preparation of levosimendan-cysteine file – The levosimendan-cysteine adduct was optimized as described for levosimendan (see above). C19 was chosen to be the site of the thioimidate since it is predicted to be slightly more reactive than C17. Furthermore, the intensity of

13

C19 and

13

C17 signals are equivalent in the absence of cTnC, whereas in the presence of

cTnC, the intensity of 13C19 is reduced compared to 13C17 (Figure 3), indicating that it is likely the primary reaction site. Following optimization, Avogadro 37 was used to convert the output file from Gaussian into protein data bank (PDB) format. The program xplo2d was used to generate the topology, parameter, and structure files that define the bond lengths, bond angles, atomic radii, masses, and charges of the molecule

45

. The parameters for C84 were then incorporated

from those for cysteine using the buildit.py script provided by Clore. The parameter and topology files were manually edited to include output from xplo2d. Charges calculated by Gaussian for the levosimendan-cys adduct were then incorporated (Cysteine charges were adjusted to make the overall charge of the adduct -1). The structure of levosimendan was restrained using improper angles from the structure of levosimendan calculated in Gaussian. The angles surrounding the thioimidate bond were compared to other X-ray structures involving nitrile inhibitors bound to cysteine proteases 46-49. The pyridazinone ring and phenyl ring were allowed to rotate with respect to one another and the nitrile/hydrazone kept planar.


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Preparation of troponin file – The NMR structure of cChimera (the covalent complex of cNTnC, cTnI and i9 was used for the docking (PDB code: 2N7L)). The NMR structure was edited so that the His tag, cTnI and i9 were removed from the file. C84 was modified to the covalently-bound levosimendan pseudoresidue (containing the angles and bond lengths calculated by Gaussian) using the XPLOR-NIH 50 script buildtag.py from M. Clore’s group. The parameter, topology, and structure files for the calcium ion in the N-terminal domain of cNTnC have been previously described elsewhere 51. Structure calculation – Xplor-NIH was used to calculate the structure of cNTnClevosimendan50, 52. The protocol followed was modified from 53 to only use NOEs in the structure calculation. Initially, levosimendan was fused to the C84 of cTnC(C35S) as outlined above. Then, the structure was minimized using the NOE restraints, keeping the backbone of cNTnC rigid. A semi-rigid simulated annealing protocol was used 53. The backbone atoms for cNTnC were kept rigid during the calculation, but the all the side chains were permitted to rotate. A conformational database potential was used for the side-chain dihedral angles 54. Intermolecular NOEs were used to determine the orientation of levosimendan when it is bound to cTnC(C35S). The packing of the protein complex was improved by including a potential for the expected radius of gyration for the complex 55. The final kPRE was 60 kcal mol-1 Å-2. The ensemble presented in this study represents the 10 lowest energy conformations. For a detailed description of the semi-rigid annealing calculation see Clore, 2000 53. Paramagnetic relaxation enhancements (PREs) were employed to help validate the NOEdriven docked model of cNTnC-levosimendan. The experiment was very similar to that previously outlined for the structure of dfbp-o bound to cNTnC-cTnI144-163

56

.

Prior to

determining PREs between Gd3+ and levosimendan, it was essential to characterize the binding of lanthanides to cTnC, which are expected to bind to the Ca2+ binding sites of cTnC. Lanthanum, a diamagnetic lanthanide, was titrated into cTnC-Mg2. Since the C-domain of cTnC is unstructured 11 ACS Paragon Plus Environment

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in the absence of Ca2+ (or Mg2+) 57, Mg2+ was added to cTnC so that the C-domain chemical shifts could be assigned (Figure S9) and therefore monitored throughout the La3+ titration. Moreover, Mg2+ is not expected to compete significantly with La3+-binding since the dissociation constant (KD) of the C-domain for Mg2+ is in the 10-3 M range58 while the KD for the lanthanides are in the range of 10-7 - 10-9 M59. The titration of La3+ into cTnC-Mg2 indicates that it preferentially binds to the C-domain before binding to the N-domain (Figure S10). This suggests that Gd3+ would also bind in the same order of preference. It has been shown that in the absence of the anchoring region of cTnI (residues 34-71; cTnI34-71), levosimendan binds to both the N- and C-domains of cTnC

26

; therefore, in order to distinguish PREs between Gd3+ bound to the N-domain and

levosimendan with those involving the C-domain, cTnI34-71 was added to cTnC-Gd3 prior to titrating it into levosimendan. A solution of levosimendan was titrated with a 0.27 mM solution of cTnC(C35S) bound to 0.54 mM cTnI34-71 and 0.82 mM Gd3+. At each titration point 1D 1H spectra were acquired and line widths at half peak height (∆ν) were measured to estimate T2 (∆ν = 1/(πT2)). The average change in ∆ν was calculated for each peak from levosimendan and was fit as a function of cTnCGd3-cTnI34-71 concentration to calculate dissociation constant (KD) of levosimendan to the equation:

=

[] []!!"

=

[#$ %(%&'()]!!" ) *[]!!"

, where fbound is the fraction of levosimendan bound to cTnC(C35S), [levo]total and [cTnC(C35S)] are the total concentrations of levosimendan or cTnC(C35S), and [levo]bound is the concentration of levosimendan bound to cTnC(C35S). This equation as been derived previously 60. Once the KD


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had been calculated, the fraction of levosimendan bound to cTnC-Gd3-cTnI34-71 was plotted versus 1/T2. The slope of this plot is the increase in relaxation rates caused by Gd3+ (1/T2m) 61, which can be used by the Solomon-Bloembergen equation

62-64

to estimate the distance between

the nuclei and Gd3+. Using the simplifications outlined in 65, we solved for the distance between Gd3+ and each proton nucleus (r) from levosimendan using the following equation:

1 . , / , 0, 1(1 + 1) 37# = 547# + ; 4 +,153 1 + 9:, 7#,

, where ϒ is the magnetogyric ratio of 1H, β is the Bohr magneton, ωI is the nuclear procession frequency of 1H, g is the Landé factor, τc is the correlation time (estimated to be 5 ns (as previously calculated for the N-domain of cTnC66), and J is the total angular momentum for the Gd3+ ion. Finally, the quantity

< = > = ?= @(@*A) A'

has a value of 2.583 x 1017 s-2 Å6 (for further

discussion see 67).

Results Levosimendan reacts with thiols to form a thioimidate bond – Prior to investigating the interaction between levosimendan and cTnC, we characterized the chemical state of levosimendan. 1H and 13C chemical shifts of levosimendan were assigned using two-dimensional 1

H,13C HSQC and HMBC experiments (Figure S1 and S2 and Tables S1 and S2). Most of the 1H

chemical shifts of levosimendan in aqueous solvent were not significantly perturbed compared to


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those in DMSO34; however, there were several large

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13

C chemical shift changes (Figure S3).

Several of these shifts were due to solvent effects68; however, other changes may also be caused by a change in its ionization state. We measured the pKa of levosimendan with 1H NMR (Figure S4) to be 6.19, which is close to the previously reported value of 6.2635. We also found that levosimendan became insoluble as the pH was lowered, which is consistent with the transition from a charged to a neutral form. Therefore, at physiological pH (pH 7.4) levosimendan is mostly negatively charged. A theoretical study by Cohen et al.

69

predicted that the likely ionization site

is N14 (in the hydrazone functional group). The optimized structure of negatively charged levosimendan at N14 is shown in Figure S1. The observation that cysteine 84 (C84) of cTnC is required for levosimendan binding23, 24, suggests that the malononitrile group of levosimendan might react with the thiol of C84 to form a thioimidate bond (Scheme 1). Thioimidate bonds have been seen in covalent complexes between nitrile inhibitors and cysteine proteases70,

71

. Therefore, provided the malononitrile of

levosimendan is sufficiently reactive, it might function via a similar mechanism. The reaction energy of levosimendan with methanethiol to form a thioimidate was calculated as outlined by Oballa et al.

43

. The malononitrile group was predicted to be electronegative, with C-19 being

slightly more reactive than C-17 (-4.86 kcal/mol vs. -4.18 kcal/mol). To validate this predicted reactivity, 13C3-labeled levosimendan (13C-16, 13C-17 and 13C19) was synthesized (Figure S5) and combined with reduced glutathione (GSH) in DMSO (Figure S6). The 1D 13C spectrum of 10 mM 13C3-levosimendan was acquired in the absence and presence of 100 mM GSH at a series of time points. Multiple peaks appeared rapidly in the 13C spectrum. The 13C chemical shifts of unreacted levosimendan had almost entirely disappeared by the final time point (22.5 hrs). The chemical shifts for the new peaks at 168 ppm, 117 ppm and 105 ppm are in good agreement with the predicted chemical shifts of a thioimidate bond formed between levosimendan and methanethiol (180.7, 113.7, and 106.6 ppm, respectively). 14 ACS Paragon Plus Environment

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Biochemistry

One of the limitations of the above experiment is that it was done in DMSO. Since reaction rates can be quite different in different solvents68, we repeated the GSH experiment in aqueous solvent. 2 mM

13

C3-levosimendan was treated with 100 mM GSH. Immediately

following the addition of GSH the resonances from unreacted levosimendan were replaced by three new peaks at similar chemical shifts as the product formed between levosimendan and GSH in DMSO (Figure 1). This suggests that levosimendan reacts faster with GSH in aqueous solvent, although differences in the GSH:levosimendan ratio probably also contribute to the increased reaction rate. Due to low signal-to-noise ratio and the low solubility of levosimendan, the spectrum was acquired over approximately 3 hrs, making the kinetics of this reaction impossible to establish by NMR. Interestingly, this product was not stable. 1D

13

C experiments over ~24

hours showed the appearance of a new peak at ~160 ppm linked to the disappearance of the peak at 170 ppm (Figure S7). This is not unexpected for highly reactive nitriles in aqueous solvents44. To aid in identifying the products formed by this reaction, we also characterized the reactivity by HPLC. 110 µM levosimendan was incubated with 5 mM GSH 44 (Figure S7). Consistent with the NMR data, we observed the immediate formation of a species with a molecular weight equivalent to a thioimidate adduct and the slower formation of a thiazoline product. Taken together, these data not only demonstrate that levosimendan is electronegative, but also indicate that its metabolism through adduct formation with GSH 72, 73 can occur spontaneously. Levosimendan forms a covalent bond with cTnC – Having established the reactivity of levosimendan towards thiol groups, we next investigated whether it reacted with cTnC, which contains two cysteines: C35 and C84. We incubated 1 mg/ml (~55 µM) wild-type cTnC or cTnC(C35S, C84S) with 160 µM levosimendan at pH 7.0 in the presence of 200 µM Ca2+ for 90 minutes at room temperature (Figure 2). The mass of wild-type cTnC was 18401.5 ± 1.2 Da (S.D.) (expected 18402.5 Da). Following the incubation with levosimendan, two species were observed: unreacted cTnC with a measured mass of 18402.2 ± 1.0 Da, and a new species with a


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mass of 18681.6 ± 0.8 Da. The difference between the two masses is 279.4 ± 1.3 Da, which is in agreement with that of levosimendan (280.3 Da). No double-reacted cTnC was observed and no degradation products (e.g. thiazoline) were observed. It has been shown previously by HPLC that levosimendan can react with cTnC(C35S,C84S) in a 24 hour incubation at 40 °C 74. However, we did not observe a new species following incubation of cTnC(C35S,C84S) with levosimendan under our conditions (mass before 18369.7 ± 0.7 Da, after 18370.1 ± 0.5 Da; expected mass 18370.4 Da). Our results therefore provide strong evidence that levosimendan can form a covalent bond with either C35 or C84 of cTnC. Levosimendan reacts with Cys 84 of cTnC – To determine which of these cysteines are involved, we prepared cTnC with either C35 or C84 mutated to serine. Prior to incubation with levosimendan C84S had a mass of 18385.8 ± 0.8 Da (expected, 18386.5 Da) and C35S had a mass of 18386.1 ± 0.8 Da (expected, 18386.5 Da). Following incubation, cTnC(C84S) remained as a single species with mass 18385.8 ± 0.5 Da (Figure 2c), whereas with cTnC(C35S) two species were observed with masses 18386.0 ± 1.0 Da and 18666.2 ± 1.7 Da, with a difference (280.2 ± 2.0 Da) corresponding to the mass of levosimendan (Figure 2d). The relative abundance of the unreacted and reacted species was ~1:1 for both cTnC(C35S) and cTnC(WT). We incubated cTnC(C35S) with varying concentrations of levosimendan and acquired LC-MS data at several time points (Figure 3). We found that, as expected, the fraction of cTnC(C35S) that reacted with levosimendan increased as the concentration of levosimendan was increased. We also found that the rate of the reaction increased as the concentration of levosimendan increased. No species with a mass greater than the mass expected for levosimendan+cTnC(C35S) was identified, even following the 24 hr incubation of a levosimendan:cTnC(C35S) mixture at a ratio of ~30:1. These results indicate that levosimendan reacts specifically with C84 of cTnC in a dose-dependent manner. The finding that levosimendan specifically targets C84 of cTnC also has implications for its isoform specificity since fast-twitch 16 ACS Paragon Plus Environment

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Biochemistry

skeletal TnC does not contain a cysteine in its regulatory domain. The reaction of levosimendan with cTnC is Ca2+-dependent and reversible – In its Ca2+free state, cNTnC is in a closed conformation, unable to bind to cTnI147-163 to activate contraction. Once Ca2+-binds to cNTnC, the domain enters an equilibrium between closed and open conformations75, cNTnC

77

76

. cTnI147-163 binds to, and stabilizes the open conformation of Ca2+-bound

. Like cTnI147-163, the binding of levosimendan to cTnC has been shown to be Ca2+-

dependent 12 18; therefore we expected that levosimendan would not react with cTnC(C35S) in its Ca2+-free state. 160 µM of levosimendan was incubated with 1 mg/ml cTnC(C35S) in the presence of 5 mM EGTA (to chelate Ca2+) for 90 min (Figure S8a). Less than 10% of the cTnC(C35S) reacted versus ~45-50% when Ca2+ is present (Figures 2a, d), showing that levosimendan binding to cTnC is indeed Ca2+-dependent. shown that the effects of levosimendan are reversible

Physiological 18

studies

have

. To investigate whether the covalent

adduct is reversible, we incubated 1 mg/ml cTnC(C35S) with 1.2 mM levosimendan and 1 mM CaCl2 (no EGTA) for ~3.5 hours at room temperature to ensure most of the cTnC(C35S) reacted with levosimendan (Figure S8b). After an overnight dialysis in levosimendan-free buffer at 4 °C, the major species had an average mass corresponding to that of unreacted cTnC(C35S), and the reacted species was less than 10% (Figure S8c). As thioimidate bonds can undergo an elimination reaction to a thiol and nitrile 78, the observation that the cTnC(C35S)-levosimendan complex is reversible is consistent with the formation of a thioimidate. Levosimendan forms a thioimidate bond with cTnC – To provide further evidence of the formation of a thioimidate bond, 3.7 mM

13

C3-levosimendan was incubated with 1.3 mM

cTnC(C35S) and 8 mM Ca2+ (Figure 4). Following incubation, a resonance at 106 ppm appeared, which is in excellent agreement with the theoretical 13C chemical shift for C-16 of levosimendan in a thioimidate bond, 106.6 ppm (see above). The absence of a peak at the same chemical shift in the 13C NMR spectrum of 0.5 mM 13C-labeled cTnC in the presence of 8 mM Ca2+ indicates that 17 ACS Paragon Plus Environment

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the resonance at 106 ppm must be associated with levosimendan and not with naturally abundant 13

C resonances from cTnC. This is especially evident when the spectrum from 13C-labelled cTnC

is scaled to the level of naturally abundant 13C-labelled cTnC that would be present in Figure 4b (Figure 4d). It was not possible to distinguish the other expected resonances for the thioimidate bond from those expected for naturally abundant

13

C-cTnC. In summary, the computational

chemistry, LC-MS and NMR spectroscopy data all indicate that levosimendan forms a thioimidate covalent bond with cTnC. NMR-based structural model of levosimendan bound to cTnC – No

high-resolution

structure of levosimendan bound to cNTnC has been described, probably as a result of exchange broadening of NMR spectra, the presence of multiple peaks, and protein-drug complex instability24 as discussed above. Several NMR studies have indicated, however, that levosimendan most likely binds in the hydrophobic pocket of cNTnC (between helices A, B, C, and D)13,

24, 79

. Additionally, we have obtained high-resolution structural information of the

binding of several levosimendan analogues, dfbp-o56 and i9 80, to cNTnC and cNTnC-cTnI. Dfbp-o was discovered to bind in the hydrophobic pocket of cNTnC and increase Ca2+sensitivity56. Unfortunately, dfbp-o had a relatively low affinity for cNTnC (~1 mM). Therefore, based on the limitation of dfbp-o as a pharmaceutical lead coupled with preliminary insights into the covalent mechanism of levosimendan, we designed a novel covalent Ca2+-sensitizer called i980. i9 (Figure 5a) shares some chemical features with dfbp-o, such as the difluorophenyl ring, but is able to form a covalent bond with thiol-containing residues, like levosimendan. In contrast to levosimendan, however, i9 forms an irreversible thioether bond with C84. The structure of i9 bound to a hybrid protein containing cNTnC and the switch region of cTnI (cChimera) was solved by NMR spectroscopy. Like dfbp-o, i9 binds to the hydrophobic pocket of cNTnC and increases the Ca2+-sensitivity of cardiac muscle contraction. However, the irreversible nature of the thioether bond between C84 and i9 impairs muscle relaxation, which would limit its 18 ACS Paragon Plus Environment

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Biochemistry

effectiveness as a therapeutic. To model the conformation of levosimendan bound to cNTnC we utilized unambiguous NOEs from the cChimera-i9 complex (PDB: 2N7L)

80

. Levosimendan was docked onto the

structure of cNTnC from the cChimera-i9 structure. The NOEs for the levosimendan structure were (i9 numbering in brackets): H-9/H-13 (H-2/H-6) and the methyl protons of M80; H2 (H-5’) and the methyl protons of V72, V64, and the delta protons of I61; H4a and H4b (H-3’) with the delta methyl protons of I36; and N1 (H-6’) and the methyl protons of V64. The ensemble of the 10 lowest energy structures is shown in Figure 5b. Although levosimendan is bound to the same region of cNTnC across the ensemble of structures, it adopted two discrete conformations (model 1 and model 2; inset in Figure 5b). To determine which of these two conformations is adopted by levosimendan in its complex with cNTnC, we measured paramagnetic relaxation enhancements (PREs) between the paramagnetic lanthanide, Gd3+, and the protons on levosimendan. PREs are dipolar interactions made between the unpaired electrons of Gd3+ and the surrounding nuclei that result in enhancement of the transverse (T2) and longitudinal (T1) relaxation rates. As dipole-dipole interactions are distance dependent, these relaxation enhancements can be used to derive distances81. Furthermore, since PREs can be measured by simply acquiring 1D NMR spectra, many of the difficulties (e.g. long-term complex stability and multiple protein peaks) that have complicated

the

structure

determination

of

cNTnC-levosimendan

are

circumvented.

Unfortunately, since the electrons are distributed symmetrically around Gd3+, analysis of the dipole-dipole interaction only provides distance information and includes no anisotropic information 82. The distances between Gd3+ and H4a, H4b, H7, H9/H13, and H10/H12 protons, measured using PRE (Figure 5c-e), were compared with those between the corresponding protons and Ca2+


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in the two NOE-driven models (Table 1). Model 1 gave a much better fit to the PRE data, so we focused on this conformation for the remainder of the present study. We note however that a limitation of the PRE measurement is that since cTnC is highly negatively charged at neutral pH (pI ~ 4), there may be non-specific binding of Gd3+, which would lead to non-specific broadening. Structure-based insights into the mechanism of levosimendan –Superimposition of the model 1 cNTnC-levosimendan structure on the Ca2+-free closed structure of cNTnC (Figure 6a) shows that there is a significant steric clash between helix B of cNTnC and levosimendan, which suggests that cTnC must be in an open conformation to accommodate levosimendan binding. This explains why levosimendan is less likely to form a covalent bond with cTnC in the absence of Ca2+, and suggests that levosimendan enhances the Ca2+-affinity of cTnC

12, 13

and muscle Ca2+-

sensitivity (5,15-17) by stabilizing the Ca2+-bound conformation of cTnC. Several studies have indicated that in addition to C84, D88 and D87 are important for levosimendan binding to cNTnC13, 23. In the structure of cNTnC-levosimendan, the side chains of D88 and D87 are in close proximity to the thioimidate (Figure 6b), which may stabilize its positively charged state (pKa ~7 78, 83). Although the neutral thioimidate bond is rapidly reversed by a base elimination reaction, the positively charged state is much more stable 78. Therefore, the role of D88 and D87 may be related to their stabilization of the thioimidate bond. Superimposing the structure of cNTnC-levosimendan with that of cChimera-i9 (Figure 6c) indicates that both levosimendan and i9 bind in the hydrophobic pocket. Despite both molecules covalently binding to C84, the orientations of their bonds are quite different (Figure 6c). This may be due to differences in the conformational freedom of the two molecules: i9 contains several rotatable sp3-hybridized carbons, whereas the malononitrile and hydrazone moieties of levosimendan are highly conjugated and therefore relatively rigid. This conformational difference may have mechanistic implications, since i9 decreases cTnI binding to


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Biochemistry

cNTnC via a steric clash between i9 and cTnI at the site of the covalent bond 80. The orientation of levosimendan indicates that, unlike i9, levosimendan would not clash with cTnI (Figure 6d). Indeed, a recent study found that levosimendan does not impair cTnI binding to cTnC84. NMR and molecular dynamics suggest that Ca2+-bound cNTnC is in equilibrium between closed and open states, with the closed state being the predominant form 75, 76 (Figure 7a). It is not until cTnI147-163 is bound that the open state is stabilized

77

. The binding of Ca2+-sensitizers to

cNTnC shifts this equilibrium towards the open conformation of cNTnC

14

(Figure 7b). The

covalent agonist i9 shifts the equilibrium further to favour the open state of cNTnC (Figure 7c) and thereby increases the Ca2+ sensitivity of muscle contraction

80

. Although this enhances

contractility, since i9 does not dissociate from cNTnC it also impairs relaxation, which limits its use as a Ca2+-sensitizer in a clinical setting. In contrast, levosimendan, which forms a reversible covalent bond with cNTnC, has an intermediate effect on the equilibrium of the closed-open transition, and does not impair relaxation18.

Discussion It is remarkable that 30 years after the identification of Ca2+-sensitizers as a therapeutic class

85

and 20 years since the discovery of levosimendan

12, 13

, levosimendan remains the sole

Ca2+-sensitizer in wide-spread clinical use for the treatment of heart failure. Part of the barrier to the discovery of new Ca2+-sensitizers has been our incomplete understanding of how levosimendan targets cTnC. In this study we have shown that the malononitrile group of levosimendan is capable of forming a covalent bond with thiols. We used mass spectrometry and NMR spectroscopy to show that levosimendan forms a reversible thioimidate ester specifically with C84 located at the edge of the hydrophobic pocket of cTnC. We also showed that the binding of levosimendan to C84 21 ACS Paragon Plus Environment

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requires the presence of Ca2+ which indicates that levosimendan only binds to the active, open state of cTnC. Finally, we used PREs and molecular modelling to predict the conformation of levosimendan when it is bound to cTnC. Previous NMR studies showed that levosimendan is capable of binding to both wild-type and C35S,C84S variants of cTnC

24, 79

, but the binding mode is different for the two isoforms,

involving fast exchange for cTnC(C35S,C84S)79 and slow exchange for wild-type cTnC (and cTnC(C35S))

24

, indicating that the binding mode depends on the presence of C84. The

malononitrile group of levosimendan is also critical for its binding to cTnC and the associated enhancement of the Ca2+ affinity of TnC and Ca2+-sensitization in myofibrils 23, in contrast with the negligible effects produced by modification of the pyridazinone ring. These results reinforce the present conclusion that levosimendan forms a covalent bond with C84 of cTnC and that this binding mechanism is responsible for its enhancement of Ca2+-sensitivity in cardiac muscle. Despite the above findings, controversy remains about levosimendan’s mechanism of action

28, 8687

. Although originally identified as a Ca2+-sensitizer, levosimendan has since been

shown to be a PDE3 inhibitor and an ATP-dependent K+ channel activator27-29. It is important to note that those alternative mechanisms of action could not be responsible for the observed Ca2+sensitization by levosimendan in permeabilized muscle fibres

5, 18-21

, in which the sarcoplasmic

reticulum and cell membrane are demembranated and the Ca2+ concentration is controlled directly at the level of the sarcomeric proteins. The mechanism of action of levosimendan in vivo remains less clear however. An early study measuring Ca2+-transients in intact cardiac papillary muscle and ventricular cardiomyocytes concluded that levosimendan increased contractility directly by its action on cTnC in addition to increasing peak systolic [Ca2+] as a result of its inhibition of PDE3 88. A more recent study of the effects of the PDE3 inhibitor cilstamide and the cardiotonic agent EMD57033 concluded that


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Biochemistry

levosimendan’s positive inotropic activity in ventricular muscle strips could be explained solely by its PDE3 inhibition 28, but this conclusion has been challenged on the basis that EMD57033 does not act directly on cTnC (ref 55). Parallel measurements of contractility and calcium transients remain the best way to separate the contributions of Ca2+ sensitization and/or PDE3 inhibition to levosimendan’s activity 87, 88. Given the wealth of evidence that levosimendan binds to cTnC 12, 13 and increases Ca2+-sensitivity in permeabilized muscle fibres (5, 18-21) and in intact muscle fibres 88, it is most likely that levosimendan’s biological and therapeutic activity is due to a combination of both PDE3 inhibition and Ca2+-sensitization of cTnC. The present conclusion that levosimendan forms a covalent bond with cTnC may help explain its pharmacodynamics, and in particular the effects observed for 24 hours after infusion despite its relatively short half-life (~1-2 hours) 89. Although levosimendan’s pharmacologically active metabolite OR-1896 has a much longer half-life (~80 hours)90, its plasma concentration is also very low in these conditions. The present results suggest a novel explanation for the sustained effects of levosimendan following infusion based on the slow reversal of its covalent reaction with cTnC. The present results offer more general insights into the pharmacological modulation of cTnC. The observation that levosimendan is able to form a specific, reversible covalent bond with C84 provides unique opportunities for the design of novel Ca2+-sensitizers that are more specific for cTnC and less specific for PDE3. For example, many PDE3 inhibitors contain pyridazinone chemical groups, which form crucial dipolar interactions with PDE3

91

. The structure-activity

study that indicated that the malononitrile group is critical for levosimendan’s activity 23 provides evidence that the pyridazinone group could be altered to enhance levosimendan’s selectivity for cTnC over PDE3.


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Finally, the present results illuminate the binding properties of an optimized Ca2+sensitizer for cTnC. Molecules that bind cTnC too tightly are likely to impair relaxation, and therefore not be a useful cardiotonic drug. The irreversible covalent activator, i9 is an example of this class of compound80. An optimal drug for cTnC would balance calcium sensitization at a clinically useful level in vivo without a significant effect on the rate of relaxation. It is possible that levosimendan achieves this balance because it is able to form a reversible covalent bond with its target. The thioimidate bond would form during systole and break during diastole. The result is that a small fraction of levosimendan would be covalently bound at steady-state, and that this would be enough to enhance contractility in situ. This may help explain why, despite the low affinity of levosimendan for cTnC measured here (100-200 µM), the maximal increase in cardiac output could be achieved at about 50 nM in isolated perfused guinea pig hearts 92. Furthermore, at low concentrations (< 100 nM), levosimendan acts predominantly as a Ca2+-sensitizer rather than a PDE inhibitor 93. In conclusion, the discoveries presented in this study not only elucidate the molecular mechanism of levosimendan, but also outline a novel approach of pharmacologically targeting cTnC, which would be valuable because of the known off-target effects of levosimendan. The new molecular insights into the interaction between levosimendan and cTnC presented in this manuscript will aid the rational design of novel, more specific Ca2+-sensitizing agents for the treatment of heart failure.

Acknowledgements: NMR experiments were produced in part using the facilities of the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trust. We thank Andrew Atkinson for assistance with these experiments. The authors are grateful


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Biochemistry

to Robert Boyko for maintaining NMR spectrometers at the University of Alberta. The authors also would like to thank Andrea Knowles, Craig Markin, David Trentham, and Luca Fusi for technical assistance and discussion on the manuscript.

Supporting Information Available: The NMR spectra and chemical shift assignments of levosimendan; measurement of the pKa of levosimendan; synthesis and assignment of 13C-labeled levosimendan; reaction of levosimendan with glutathione (GSH); Ca2+-dependence and reversibility of the reaction between cTnC(C35S) and levosimendan; assignment of backbone amide chemical shifts of cTnC; La3+ titration into cTnC-2Mg2+; chemical shift assignments of levosimendan; theoretical coupling constants compared to experimental values.

References: [1] Warner, T. D., and Mitchell, J. A. (2002) Cyclooxygenase-3 (COX-3): Filling in the gaps toward a COX continuum?, Proc. Natl. Acad. Sci. U. S. A. 99, 13371-13373. [2] Waxman, D. J., and Strominger, J. L. (1983) Penicillin-Binding Proteins and the Mechanism of Action of Beta-Lactam Antibiotics, Annu. Rev. Biochem. 52, 825-869. [3] Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The resurgence of covalent drugs, Nat. Rev. Drug Discovery 10, 307-317. [4] Potashman, M. H., and Duggan, M. E. (2009) Covalent Modifiers: An Orthogonal Approach to Drug Design, J. Med. Chem. 52, 1231-1246. [5] Haikala, H., Nissinen, E., Etemadzadeh, E., Linden, I. B., and Pohto, P. (1992) Levosimendan Increases Calcium Sensitivity without Enhancing Myosin Atpase Activity and Impairing Relaxation, J. Mol. Cell. Cardiol. 24, S97-S97.


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Funding Agencies This study was supported by grants from the British Heart Foundation (Y.B.S., FS/09/001/26329 and FS/15/1/31071), the UK Medical Research Council (M.I., G0601065), the Canadian Institutes of Health Research (B.D.S., 37769), by a fellowship from the London Law Trust and CIHR (to I.R.), and by a studentship from KCL BHF Centre of Research Excellence (to T.K.).


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Figure Legends Scheme 1. Proposed reaction scheme between levosimendan (1) and a cysteine (2) to form a thioimidate (3).

Figure 1. Reaction of 13C3-levosimendan with glutathione (GSH) in aqueous solvent. a. The 1D 13

C NMR spectrum of 13C3-levosimendan, b. GSH and of c. 2 mM 13C3-levosimendan with 100

mM glutathione (GSH). The four sharp resonances at 170-180 ppm in b. and c. are from natural abundant 13C carbonyl/carboxyl resonances from GSH. The resonance assignments in c. were based on predicted reactivity and chemical shifts of a thioimidate bond at C-19.

Figure 2. The electrospray ionization mass spectrum of Ca2+-saturated cTnC incubated with levosimendan. 5 µL of 1 mg/ml a. cTnC(wt), b. cTnC(C35S,C84S), c. cTnC(C84S) and d. cTnC(C35S) were incubated with 160 µM of levosimendan for 90 min (left: before the reaction and right: after the incubation). The 17+ charge-state cTnC is labeled in black. The peaks labeled in red in a. and d. represent a new molecule with a molecular weight of a. cTnC(wt) + 279.36 ± 1.25 Da and d. cTnC(C35S) + 280.16 ± 1.99 Da.

Figure 3. The cTnC(C35S)-levosimendan reaction as monitored by Mass Spectrometry. a. 0.5 mM levosimendan was mixed with 1 mg/ml cTnC(C35S) in the presence of 200 µM Ca2+ at pH 7.0 (10 mM imidazole, 100 mM KCl) at room temperature and the reaction was monitored by Liquid Chromatography-Mass Spectrometry (5 µg injection) over time (recorded as time of


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Biochemistry

injection). The +17 charge states of cTnC(C35S)-levosimendan are indicated with a red arrow. b. The plot of the fraction of cTnC(C35S) reacted (based on the absolute absorbance values) fitted with a one phase association model in GraphPad for a range of levosimendan concentrations (0.05 mM - 1.5 mM).

Figure 4. NMR evidence of the formation of a thioimidate bond between levosimendan and cTnC(C35S). The 1D 13C NMR spectrum of levosimendan-13C3 a. in isolation or b. in the presence of cTnC(C84S). The red star above the broad peak in b is assigned to reacted C-16, whereas the arrow near 130 ppm denotes signals assigned to cTnC. c. The 13C-NMR spectrum of 15

N,13C-labeled cTnC is shown to illustrate that the peak in b. is not from cTnC. d. The same 13C

NMR spectrum of 15N,13C-labeled cTnC in c. scaled to the level of naturally abundant 13C cTnC present in b.

Figure 5. Model structure(s) of levosimendan bound to cNTnC. a. The chemical structures of levosimendan (1) and i9 (2). b. The ensemble of structures of levosimendan covalently bound to C84 using restraints from the cChimera-i9 structure. Inset. Close-up of levosimendan with the two main conformations of levosimendan displayed. c. The 1H spectra of levosimendan as a function of cTnC-Gd3-cTnI34-71 concentration (shown on the right of the spectra in µM). d. The fitted dissociation constant of levosimendan for cTnC-Gd3-cTnI34-71 based on the paramagnetic broadening of the 1H signals as a function of cTnC-Gd3-cTnI34-71 concentration. e. The enhanced relaxation rates (T2) of levosimendan signals versus the fraction of levosimendan bound to cTnCGd3-cTnI34-71. The dramatic difference between the internal reference DSS and the representative signals from levosimendan illustrate that the majority of the paramagnetic relaxation


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enhancement is due to specific binding between levosimendan and cTnC-Gd3 and not to nonspecific broadening from free Gd3+.

Figure 6. Comparison of the levosimendan-cNTnC structure with other cNTnC structures. a. Superposition of the lowest energy structure from model 1 of cNTnC-levosimendan with the lowest energy structure of Ca2+-free cNTnC (PDB: 1SPY). Levosimendan is shown in mesh surface representation to highlight its clash with the backbone of Ca2+-free cNTnC. b. Close-up of the residues in close proximity to the thioimidate bond of cNTnC-levosimendan. c, d. Superposition of the lowest energy structures of cNTnC-levosimendan with cChimera-i9 (PDB: 2N7L) in the c. absence and d. presence of cTnI from the core troponin complex (PDB: 1J1D). The arrow indicates the site of a steric clash with cTnI. In all structures the backbone of cNTnC from the cNTnC-levosimendan structure is in grey and levosimendan is in blue.

Figure 7. Proposed model for the modulation of the closed-to-open transition of cNTnC by Ca2+sensitizers. a. The systolic, Ca2+-bound (black circle), state of cNTnC (grey circular sector) undergoes a transition between a mostly closed conformation (top) and a fully open state (bottom) that binds cTnI and triggers muscle contraction. b-d. Ca2+-sensitizers (blue ellipse) activate contraction by shifting the equilibrium towards the open conformation. b. Non-covalent Ca2+sensitizer, such as dfbp-o. c. Irreversible covalent binders, such as i9, also impair relaxation. d. The reversible covalent bond (dotted line) formed by levosimendan has an intermediate effect on the closed-to-open transition and does not impair relaxation.


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Biochemistry

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Scheme 1.


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Figure 1.


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Figure 2.


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Biochemistry

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Figure 3.


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Biochemistry

Figure 4.


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Figure 5.


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Figure 6.


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Figure 7.


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Table 1. Distances between levosimendan and the site II-bound metal measured from experimental PREs or from the NOE-driven models

PRE (Å)1

Model 1 (Å)2

Model 2 (Å)2

H4a

15.1

15.3 ± 0.8

9.1 ± 0.8

H4b

15.6

14.4 ± 0.7

10.7 ± 0.7

H7

15.6

15.4 ± 1.1

7.5 ± 0.5

H10, H12

16.5

15.1 ± 0.4

12.6 ± 0.4

H9, H13

16.7

14.2 ± 0.6

11.1 ± 0.4

1

PRE distances have an estimated error of ± 10%

2

Distances are the mean ± SD


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TOC


Reversible Covalent Binding to Cardiac Troponin ... - ACS Publications

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