Evaluating p97 Inhibitor Analogues for Their Domain Selectivity and Potency against the p97–p47 Complex

We previously found that p97 ATPase inhibitors 2‐(2‐amino‐1H‐benzo[d]imidazol‐1‐yl)‐N‐benzyl‐8‐methoxyquinazolin‐4‐amine ( ML240 ) and 2‐(2H‐benzo[b][1,4]oxazin‐4(3H)‐yl)‐N‐benzyl‐5,6,7,8‐tetrahydroquinazolin‐4‐amine ( ML241 ) specifically target the D2 domain of wild‐type p97. In addition, one of the major p97 cofactors, p47, decreases their potencies by ～50‐fold. In contrast, N²,N⁴‐dibenzylquinazoline‐2,4‐diamine ( DBeQ ) targets both the D1 and D2 domains and shows only a four‐ to sixfold decrease in potency against the p97–p47 complex. To elucidate structure–activity relationships for the inhibitors, we screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of either or both of the D1 or D2 domains, as well for their effects on p47 potency. The selectivity of 29 of these compounds was further examined by eight‐dose titrations. Four compounds showed modest selectivity for inhibiting the ATPase activity of D1. Eleven compounds inhibited D2 with greater potencies, and four showed similar potencies against D1 and D2. p47 decreased the potencies of the majority of the compounds and increased the potencies of five compounds. These results highlight the possibility of developing domain‐selective and complex‐specific p97 inhibitors in order to further elucidate the physiological roles of p97 and its cofactors.