Identification of a small-molecule inhibitor that selectively blocks DNA-binding by Trypanosoma brucei replication protein A1

Abstract

Replication Protein A (RPA) is a broadly conserved complex comprised of the RPA1, 2 and 3 subunits. RPA protects the exposed single-stranded DNA (ssDNA) during DNA replication and repair. Using structural modeling, we discover an inhibitor, JC-229, that targets RPA1 in Trypanosoma brucei, the causative parasite of African trypanosomiasis. The inhibitor is highly toxic to T. brucei cells, while mildly toxic to human cells. JC-229 treatment mimics the effects of TbRPA1 depletion, including DNA replication inhibition and DNA damage accumulation. In-vitro ssDNA-binding assays demonstrate that JC-229 inhibits the activity of TbRPA1, but not the human ortholog. Indeed, despite the high sequence identity with T. cruzi and Leishmania RPA1, JC-229 only impacts the ssDNA-binding activity of TbRPA1. Site-directed mutagenesis confirms that the DNA-Binding Domain A (DBD-A) in TbRPA1 contains a JC-229 binding pocket. Residue Serine 105 determines specific binding and inhibition of TbRPA1 but not T. cruzi and Leishmania RPA1. Our data suggest a path toward developing and testing highly specific inhibitors for the treatment of African trypanosomiasis.