(A) Structure of the optimized hybrid compound, ML282-86 (SMAL), which combines two optimized motifs: the methylated terminal phenyl group was adopted from LR340-006 and the rigid benzyl linker to the tetrazole heterocycle was adopted from ML282-56. (B) SMAL inhibits SV40 T Ag ATPase activity in a dose-dependent manner. SMAL was titrated into a steady state ATPase assay with T Ag. Data represent the means of three independent experiments, +/− SD. (C) SMAL is the only compound amongst the examined MAL2-11B tetrazole derivatives that modulates the ATPase activity of human Hsp70. Steady state ATPase assays were used to quantitate the turnover number (ATP molecules hydrolyzed by Hsp70 per minute) in the presence of the indicated compounds at a final concentration of 30μM versus a DMSO control. Data represent the averages of three independent experiments, +/− SD. (D) SMAL inhibits Hsp70 ATPase activity in a dose-dependent manner. SMAL was titrated into a steady state ATPase assay with human Hsp70. Data represent the means of three independent experiments, +/− SD.