for steric and temporal (due to the kinetics of the chemo-mechanical cycle) restrictions in the path(s) leading toward the binding
site, as had earlier been suggested.3,15 In the same study, a ligand-based method using dissimilarity distances between compounds
calculated by extended chemical fingerprints proved able to select for actives.24
Figure 4. Overview of the myosin II ATPase inhibitory potency of (±)-trans-2-(hydroxymethyl)blebbistatin (±)-trans-6, (±)-cis-2-(hydroxymethyl)blebbi-
statin (±)-cis-6 and (±)-2-(allyloxymethyl)blebbistatin (±)-7 (the latter containing an 84:16 mixture of the (±)-trans- and (±)-cis-diastereoisomer), evaluated
in a steady-state ATPase assay against rabbit skeletal-muscle myosin II. The data points represent the mean ± s.d. of at least three samples (N = 1).
Concentrations exceeding 100 µM were not evaluated in this screen. As an approximation, the relative ATPase activity obtained for (S)-blebbistatin (S)-1 at
a concentration of 32.5 µM was used to set the lower asymptote of the 4-parameter logistic curve fitted to (±)-trans-2-(hydroxymethyl)blebbistatin (±)-trans-
6.
In conclusion, the synthesis of a small set of blebbistatin derivatives with C-ring modifications at C2 was attempted in order to (i)
enable an additional hydrogen bonding interaction and (ii) optimize filling of the binding pocket. (±)-Trans-2-
(hydroxymethyl)blebbistatin (±)-trans-6, (±)-cis-2-(hydroxy-methyl)blebbistatin (±)-cis-6 and (±)-2-(allyloxymethyl)-blebbistatin
(±)-7 (an 84:16 mixture of the trans- and cis-diastereoisomer) were synthesized. The myosin II ATPase inhibitory potency of these
analogs with small C-ring modifications was lower than that of parent compound (S)-blebbistatin (S)-1. Potency improvement from
C-ring modification thus seems not straightforward, yet additional C-ring-modified analogs must be evaluated in order to test this
hypothesis. Such compounds should preferentially be designed using ligand-based methods.
Acknowledgments
B.I.R. and S.V. respectively thank the Foundation for Scientific Research – Flanders (FWO-Vlaanderen) and the Special
Research Fund of Ghent University (BOF-UGent) for financial support for this work.
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