Novel stereoselective bufadienolides reveal new insights into the requirements for Na+, K+-ATPase inhibition by cardiotonic steroids

Cardiotonic steroids (CTS) are clinically important drugs for the treatment of heart failure owing to their potent inhibition of cardiac Na+, K+-ATPase (NKA). Bufadienolides constitute one of the two major classes of CTS, but little is known about how they interact with NKA. We report a remarkable stereoselectivity of NKA inhibition by native 3β-hydroxy bufalin over the 3α-isomer, yet replacing the 3β-hydroxy group with larger polar groups in the same configuration enhances inhibitory potency. Binding of the two 13C-labelled glycosyl diastereomers to NKA were studied by solid-state NMR (SSNMR), which revealed interactions of the glucose group of the 3β- derivative with the inhibitory site, but much weaker interactions of the 3α- derivative with the enzyme. Molecular docking simulations suggest that the polar 3β-groups are closer to the hydrophilic amino acid residues in the entrance of the ligand-binding pocket than those with α-configuration. These first insights into the stereoselective inhibition of NKA by bufadienolides highlight the important role of the hydrophilic moieties at C3 for binding, and may explain why only 3β-hydroxylated bufadienolides are present as a toxic chemical defence in toad venom.


Outline of the supporting information
Methods 1. General methods.

Details of the bufadenolide inhibition experiments
13. Details of the kinetic analysis of inhibition.
14. Sample preparation for solid-state NMR

General procedures
Solution-state NMR spectra were obtained on a Bruker AV-400 spectrometer.

Isolation of the starting material bufalin (1)
The dried and powdered venom (1.0 kg) of Bufo bufo Gargarizans was extracted by 95% ethanol under ultrasonic condition (30 min, 40 C) four times (each half hour). The combined extract was concentrated under reduced pressure to provide a residue (280 g), which was subsequently partitioned between methylene dichloride (CH 2 Cl 2 ) and water. The CH 2 Cl 2 solution was evaporated to give a residue (130 g), which was then subjected to silica gel (200 -300 mesh) chromatography, eluted with gradient cyclohexane-acetone to give five fractions (Fr.1-5). Fr. 3 was separated by silica gel chromatography again eluted with cyclohexane-ethyl acetate gradients gradient (20:80 to 90:10) to afford bufalin (4.65 g).

Synthesis of 3R-bufalin (1)
Bufalone (76.8mg, 0.2mmol) was dissolved in anhydrous tetrahydrofuan (5 ml) in a round bottom flask. Sodium borohydride (NaBH 4 ) was added, stirring constantly to completely dissolve. The solution was stirred for one hour at room temperature. Then 5ml water was added slowly to the reaction solution.

X-ray analysis of 5
X-ray diffraction data were collected on an Gemini S ultra spharrie CCD diffractometer using graphite monochromated radiation ( = 1.54178 Å) at room temperature. The crystal structures were elucidated by direct methods using SHELXS-97 and refined by full-matrix least-squares method on F 2 using SHELXS-97. In the structure refinement, non-hydrogen atoms were refined anisotropically. Hydrogen atoms bonded to carbons were placed at their geometrically ideal positions. Hydrogen atoms bonded to oxygen were located by employing the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors.

Preparation and activity assay of Na + ,K + -ATPase
Na + ,K + -ATPase from pig kidney microsomal membranes was purified by differential centrifugation. 27 The specific Na,K-ATPase activity was determined from the difference in the amount of phosphate released in the absence and presence of 1 mM ouabain in a solution containing 130 mM NaCl, 20 mM KCl, 4 mM MgCl 2 and 3 mM ATP. The specific activity of the enzyme preparation was approximately 30 µmol ATP hydrolysed/mg protein per min at 37°C. 27 The phosphorylation capacity is about 2.9 nmol/mg protein and is equal to the high-affinity ouabain binding capacity. 10, 27

Details of the kinetic analysis of inhibition (1) Experimental details of the bufadenolide inhibition
Stock solutions of the inhibitors, typically 10 mM, were prepared in DMSO.
The inhibitory effects of bufadienolides on NKA were determined essentially as previously reported 9, 10 . In brief, NKA is preincubated at 37 o C for 2 hours in the presence of 3 mM MgCl 2 , 3 mM Na-phosphate and 40 mM Tris (pH 7.0) with increasing concentrations of inhibitor. The residual NKA activity is subsequently determined by 40-fold dilution into a standard ATPase assay medium in triplicate, see section SI 11. For each inhibitor concentration the residual activity was determined in two or three independent experiments and the error bars in Fig. 4 indicate the standard deviation. Data in Fig. 4 are given as percent of the NKA activity in the absence of inhibitor (see legend to Fig. 4 for details).
The maximal DMSO concentration of 2.5% after dilution into the NKA incubation medium was 2.5 %. Control experiments showed that incubation with 2.5% DMSO for 2 hours at 37 o C leads to less than 10% inhibition of Na + , K + -ATPase activity. (

2) Detailed explanation of the kinetic analysis of inhibition
The interaction between bufalins (I) and Na + ,K + -ATPase (E) is interpreted as a two-step process: After equilibrium is attained by preincubation of the enzyme and inhibitor, the residual Na + ,K + -ATPase activity is measured by a 40-fold dilution into the assay medium for 2 min. It is assumed that the dilution leads to an immediate dissociation of E•I into E + I, and it is also assumed that the conformational change E•I ↔ E•I* is slow on the 2-minutes time scale of the functional assay.
The observed decrease in the Na + ,K + -ATPase activity is thus an estimate of the amount of E•I* present at a given inhibitor concentration in the preincubation media, and the observed concentration dependence of inactivation is related to K i as well as K C .
The data are fitted by a non-linear least squares function relating the NKA activity (Activity(I)) at a given concentration of inhibitor [I] to a sum of two hyperbolic terms containing the parameters for the high-affinity as well as the low-affinity components of inhibition:

Activity(I) = 100% -F high * [I]/( K Diss,high + [I]) -F low * [I]/( K Diss,low + [I])
The two components have magnitudes of F high and F low (in %). The dissociation constant derived from the curve fitting (K Diss,high ) is related to K i and K C through the equation K Diss,high = K i /(K c + 1).
Following the above analysis, the relative magnitude of the high-and low-affinity component is a measure of K C . Taking compound 1α as an example (see Table 1), we observe that 85% of activity (F high ) is associated with high affinity inhibitor binding (and thus F low = 15% with low affinity), which gives K C = 85%/15% = 5.67. For 1α a dissociation constant K i for the initial binding of inhibitor to E is K i = K Diss,high • (K c + 1) = 77 µM.
The "low-affinity" component determined in the Na + ,K + -ATPase assay reflects the sensitivity of the free enzyme (E) in the assay medium to the inhibitor carried over from the preincubation medium (and 40-fold diluted). It reflects inhibitor binding under ATP-hydrolysis conditions, which are very different from the preincubation medium with phosphate (and no ATP) present.

The eight inhibitors have widely different kinetic properties, and it is
illustrative to consider three of them in detail.
Compound 5α inactivates NKA with a single component (F low = 0, Table 1) which is similar to the inactivation mode of ouabain 10 . In terms of the model above there is virtually no E•I at equilibrium, all is displaced towards E•I* (K c is very large, 49, see Table 1). The initial binding step is described by K i = 1920 µM , indicating a much weaker initial binding than for compound 1α (see above).
Note that when the preincubated enzyme is diluted into the ATPase assay medium the activity determined as a function of the inhibitor concentration is proportional to the remaining free enzyme (E).
Compound 4β inactivates the Na,K-ATPase with about 86% (=F high ) of the activity being very sensitive towards the inhibitor and the remaining activity is not affected by further increase in inhibitor concentration (K Diss,low > 1 mM, see Table 1). The simplest interpretation of this finding is that the equilibrium between E•I and E•I* is displaced 6.14 fold (= K c ) towards E•I*, which represents 86% of the enzyme. K i is about 0.31µM, indicating a much stronger initial binding of this compound than for compounds 1α or 5α. Upon 40-fold dilution into the ATPase assay medium E•I (equal to 14%) is dissociated towards E + I, and we determine the about 14% (=F low ) of Na,K-ATPase activity originating from the E•I-complex in the preincubation medium. The E•I* complex dissociates extremely slowly on the 2-min time scale of the ATPase assay (see ref. 10). For the 4β compound it is observed that there is virtually no low-affinity inactivation in the concentration ranges studied here. It seems that 4β lacks inactivating potency in the ATPase assay medium (K Diss,low is very large).
Compound 5β, on the other hand, displays a composite inactivation pattern with a high-affinity inactivation as well as an observable low-affinity inactivation (in contrast to that described above for compound 4β). K i is about 0.26 µM, similar to that of compound 4α. About 22% of the ATPase activity is lost with a K Diss,low of about 68 µM, see Table 1. We interpret this as reflecting the inactivating potency of 5β in the ATPase assay medium. In the preincubation situation about 22% of the enzyme is in the E•I-form, and in the ATPase assay medium this is dissociated towards E + I. Compound 5β is thus potent enough to inactivate the free enzyme E in the ATPase assay medium.
In control experiments without preincubation we have observed that addition of Compound 5β to the ATPase assay medium directly indeed inactivates the Na,K-ATPase activity and with the same potency as observed in Figure 4C (data not shown).
It should be noted that the errors in the values for K Diss,low in the present experiments (see Table 1) are too large to allow meaningful correlation between the low-affinity inhibitory potency and the conformation of the inhibitor.

Sample preparation for solid-state NMR
Na + ,K + -ATPase membranes (13 nmol protein) were prepared as a pellet by centrifugation at 100,000 g for 30 min at 4°C, resuspended in 1 ml incubation medium, and incubated with 16 nmol of labeled inhibitor (5α and 5β) for 60 min at 25°C. The suspension was centrifuged (100,000  g at 4°C) for 30 min, and the pellet was transferred to a 4-mm external diameter zirconia MAS rotor fitted with Kel-F inserts to confine the sample to the center of the rotor. In ouabain pre-incubation experiments, the membranes were first incubated with ouabain for 1h, then the same amount of 5β was added and further incubated for 1h.