Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyses the removal of 3’ and 5’-DNA phosphodiester adducts.  It also confers resistance to the anticancer type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA by catalyzing the hydrolysis of the phosphodiester bond between the Y723 residue of TOP1 and the 3’-phosphate of DNA. Blocking TDP1 function is an attractive approach for enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. TDP1 inhibitors could potentially play important therapeutic roles in anti-cancer therapy by synergizing with TOP1 inhibitors. Previously, using an X-ray crystallographic screen of more than 600 fragments, we have reported small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited TDP1 inhibitory potencies. We now report our analysis of a 21,000-member library of drug-like small molecules in microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar <i>N</i>,2-diphenylimidazo[1,2-<i>a</i>]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton by applying one-pot Groebke-Blackburn-Bienayme multicomponent reactions (GBBR) using a diversity of aldehyde, amine and isocynide fragments. In this way, we arrived at analogs having micromolar inhibitory potencies. Solving crystal structures of a subset of compounds bound to TDP1 showed their binding in the TDP1 catalytic site where they mimicked aspects of substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand-protein interactions containing highly similar elements. Importantly our inhibitors show competitive inhibition against TDP1, are active in whole cell extracts with native TDP1 and synergize with the canonical TOP1 inhibitor camptothecin. Crystallographic analyses show they access the catalytic phosphate-binding pocket while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. Our current work will serve as a foundation upon which a new genre of potent multivalent TDP1 inhibitors can be built. Given the current state of TDP1 inhibitor development, this achievement is significant in its implications for future development of high TDP1 affinity and selectivity ligands and drugs.

Tyrosyl-DNA phosphodiesterase 1 inhibitors that simultaneously access the catalytic pocket and two substrate binding sites identified from small molecule microarray

ACS Spring Meeting 2021

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