Selective targeting of human TREX1 exonuclease by small molecule inhibitors is mediated by a conformational switch.

Three-prime Repair EXonuclease 1 (TREX1) is a 3'-to-5' exonuclease that degrades cytoplasmic DNA to prevent aberrant innate immune and inflammatory responses. Loss of TREX1 function results in the accumulation of cytosolic DNA and autoimmune disorders in humans, whereas in cancer cells, TREX1 inhibition can promote antitumor immunity triggered by tumor-derived cytoplasmic DNA. Here, we show selective inhibition of human TREX1 (hTREX1) by small molecule inhibitors and reveal their mechanism of target engagement by X-ray crystallography. The structures show that these inhibitors competitively block the binding of the 3'-terminal nucleotide of DNA substrates and make a hydrogen bond with a conserved Glu residue in the active site. Furthermore, binding of the inhibitors stabilizes Tyr129 of hTREX1 in an alternative conformation incompatible with DNA binding, generating a deep hydrophobic pocket that accommodates a key aromatic moiety of the inhibitors. The structural observations provide the basis for high selectivity of these inhibitors for hTREX1 over the closely related hTREX2 or other viral DEDDh-family nucleases. Our studies demonstrate that the conformational flexibility of the hTREX1 active site plays a crucial role in its selective inhibition, informing future design of hTREX1-selective ligands through this structural framework.