Origin of differences of inhibitory potency of cardiac glycosides in Na+/K+-transporting ATPase from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle

Abstract The inhibitory potency of altogether 95 steroidal compounds (including cardenolides, bufadienolides and their glycosides) on the Na/K-ATPases (Na + /K + -transporting ATPases, EC 3.6.1.37) from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle was compared to probe the complementary chemotopology of the inhibitor binding site areas on the three enzyme variants. The changes of potency, resulting from systematic variations of the geometry of steroid skeleton and the character as well as the structure of side chains at C3 or/and C17 of steroid backbone, allowed the following major conclusions. With the human cardiac and cerebral enzyme forms, the paired K 0.5 ( K ′ D ) values for 77 steroid derivatives, covering seven orders of ten, were highly correlated. On an average, the total of compounds showed a 1.5-fold higher affinity to the cardiac enzyme. This tiny differentiation did not appear to be connected with an important difference in the chemotopology of the complementary subsites for steroid nucleus binding on the two enzyme forms. With the human and guinea-pig cardiac enzyme variants, the K 0.5 values for 69 steroid derivatives, covering six orders of ten, were determined. For 41 5β,14β-androstane derivatives only, the paired K 0.5 values showed a close correlation. Here, the human enzyme variant exhibited 27-fold higher affinity. However, the paired K 0.5 values determined on both enzymes for 28 steroid derivatives of differing structural features were but poorly correlated. Essentially, the geometries of the steroid nucleus determined the differential contributions of the side chains at C3 and C17 to the integral inhibitory potency on the two enzyme variants. Thus, the species differences in the potency of cardiac glycosides were traced to species differences in the complementarity of the steroid binding subsites. Hence, estimates of the potency of new steroidal compounds obtained on the guinea-pig cardiac enzyme can be neither quantitatively nor qualitatively easily extrapolated to the human cardiac enzyme. The extrathermodynamic analysis of the data opened major new insights in the structure-activity relationships concerning the role of C14β-OH, the character of the lead structure in cardioactive steroid lactones, and the significance of the configuration of A/B ring junction.