Differential inactivation mechanism and covalent adduct formation of ALDH1A1 and ALDH1A2 by WIN18,446

WIN18,446 [N,N′‐1,8‐octamethylenebis‐(2,2‐dichloroacetamide)] was discovered as a reversible male contraceptive, which inhibits spermatogenesis in many species, including humans. The contraceptive activity of WIN18,446 is believed to be due to inhibition of the biosynthesis of the critical regulator of spermatogenesis, intratesticular all‐trans‐retinoic acid ( at RA), via inhibiting aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH1A2. However, the usefulness of WIN18,446 is limited due to its off‐target inhibition of ALDH2, the key enzyme eliminating acetaldehyde. Previous studies have shown that in the presence of NAD + , WIN18,446 is a reversible inhibitor of human ALDH1A1, and a time‐dependent, irreversible inhibitor of ALDH1A2. However, neither the mechanism of ALDH1A2 inactivation by WIN18,446 nor the discrepancy between ALDH1A1 and 1A2 inhibition is understood. Using Q‐TOF MS, we identified an aldehyde metabolite of WIN18,446 formed by ALDH1A2, in the presence of cofactor NAD + . In contrast, the aldehyde metabolite was formed by ALDH1A1 only in the absence of NAD + . Similarly, the inactivation of ALDH1A2 by WIN18,446 was NAD + dependent while ALDH1A1 was only inactivated in the absence of NAD + . Adducts of WIN18,446 with ALDH1A2 were identified with shotgun proteomics and a modified version of Kojak software that allows searching of any mass modified peptides. Using the proteomic MS data, a novel WIN18,446 derived adduct of Cys320 and Cys319 of ALDH1A2 was identified with mass change of 295Da. Based on the identity of this adduct, a mechanism of ALDH1A2 inactivation by WIN18,446 and the pathway to the aldehyde metabolite was proposed (Fig. 1). Interestingly, the proposed mechanism of inactivation does not require NAD + suggesting that NAD + acts as an allosteric modulator affecting WIN18,446 binding orientation within the ALDH1A active site instead of contributing to the catalysis. This interpretation is consistent with existing knowledge of ALDH family enzyme structure‐function. In conclusion, using novel MS proteomics methods we have identified the inactivation mechanism of ALDH1A by WIN18,446, which provides important information to guide development of novel male contraceptive inhibitors of ALDH1A enzymes and to assess structure‐function of ALDH1A inhibitors. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .