Structural and inhibition studies on Human Flap Endonuclease

Flap endonuclease 1 (FEN1) is a paradigm of structure-specific metallonuclease family that selectively catalyzes the removal of single-stranded 5ʹ-flaps, occurring in vivo primarily in Okazaki fragment maturation and DNA repair. Enzymatic malfunction of FEN1 has dramatic consequences for eukaryotic genome stability. Since overexpression of FEN1 has been reported in multiple cancers, FEN1 inhibition is proposed to have a therapeutic potential. Without any clinical trials related to FEN1 inhibition registered to date, small molecule inhibitors are now recognized as possible leads for the development of novel anticancer drugs. Aiming at the identification of small molecule FEN1 inhibitors at an in silico stage, a dual approach of virtual and physical screening was followed. Manipulation of active site metal charges led to a well-performing docking strategy under +2 active site metal charges that can effectively predict the great majority of in vitro confirmed hits and used for cost-effective FEN1 inhibitor identification. In addition, following a collaborative hit expansion study an inhibitor of HsFEN1 with IC50 of ∼1.7 μM was identified for future inhibitor development studies, expanding the reported to date range of FEN1 inhibitors. Crystallographic analyses also led to two HsFEN1:product DNA complex structures supporting the dsDNA substrate’s ability to bend 100°, “trapping” of the 1-nt long 3ʹ-flap and positioning of 5ʹ-flap towards the active site because of HsFEN1 binding at the DNA bend. The presence of CaCl2 allowed some enzymatic activity and facilitated crystallization of a product complex whose structure was determined at 2.3 Å. Whilst this project focuses on human FEN1, microbial FEN inhibitors identified by the University of Sheffield (UoS) spin-out DeFENition Ltd primarily for antibiotics development were examined within the context of the current thesis for their induced cytotoxicity. Although structure-activity relationships fell outside the scope of the current thesis, this first ever collected cytotoxicity dataset provided key information for future toxicity studies and downstream compound development.