Functional and structural analysis of AT-specific minor groove binders that disrupt DNA-protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast
Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)-biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida. ; Spanish Ministerio de Economia y Competitividad [SAF2015-66690-R to C.D.]; Spanish Ministerio de Ciencia e Innovaci ´on [BFU2009-10380 to J.L.C.]; Generalitat de Catalunya [project SRG2009-1208 to J.L.C.]; Spanish Ministerio de Educaci´on, Cultura y Deporte [FPU12/04824, EST14/00449 to C.R.M.]; CONACYT [312212 to C.R.M.]; Short Term Scientific Mission COST Action CM1307 [COST-STSM-CM1307–35281 to C.R.M.]; PhD studentship from the Tertiary Education Trust Fund of the Federal Government of Nigerian [TETFUND-2011/2012 to G.U.E.]; Wellcome Centre for Molecular Parasitology is supported by core funding from theWellcome Trust [085349, L.L.]. Funding for open access charge: SpanishMinisterio de Economia y Competitividad [Grant SAF2015-66690-R]. Conflict of interest statement. None declared. ; Peer Reviewed
