Functional and structural analysis of AT-specific minor groove binders that disrupt DNA–protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast

Abstract

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.

Drug resistance in protozoan parasites

“…The drug may not have a single protein target that can be mutated but may bind instead to DNA as intercalator or minor groove binder (e.g. phenanthridines, diamidines [24][25][26]), or e.g. to haem (chloroquine [27]), or be more generally cytotoxic, with multiple targets ( polypharmacology), as may be the case for heavy metal drugs (arsenicals, antimonials) and suramin [28], in which case selectivity depends mostly on selective entry into the parasite rather than into the host cells, through unique transporters or other uptake mechanisms [20,28,29].…”

A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis

“…Two PBDs, SG3199 and SG3552 (ref(36)) (Figure 1B), were used in these experiments; each was used as a toxin-linker derivative, SG3249 and SG3376 respectively (Figure 1B), for antibody conjugation. PBDs are DNA minor groove binding toxins (37–40) and were chosen as trypanosomes have a highly complex mitochondrial genome formed from a network of thousands of concatenated DNA circles and are consequently susceptible to DNA binding toxins. This sensitivity is illustrated by the original patent on ethidium bromide as a treatment for trypanosome infection and ethidium derivatives are still used for animal trypanosomiasis (41, 42).…”

Discovery of Sustainable Drugs for Neglected Tropical Diseases: Cashew Nut Shell Liquid (CNSL)‐Based Hybrids Target Mitochondrial Function and ATP Production in Trypanosoma brucei

“…Transmission electron microscopy assay on T. b. brucei and HFF cells : TEM of bloodstream trypomastigotes (427WT) and of HFF was performed essentially as previously described . Briefly, cell cultures were adjusted to 2.5×10 6 cells mL −1 and incubated in the presence or absence of test compounds at EC 50 .…”

“…[51] Transmission electron microscopy assay on T. b. brucei and HFF cells:T EM of bloodstream trypomastigotes (427WT) and of HFF was performed essentially as previously described. [52] Briefly,c ell cultures were adjusted to 2.5 10 6 cells mL À1 and incubated in the presence or absence of test compounds at EC 50 .C ells were fixed overnight at 4 8Ci n2 .5 %g lutaraldehyde and 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). Samples were washed with 0.1 m phosphate buffer (pH 7.4), post-fixed in 1% osmium tetroxide for 1h on ice, and washed with the phosphate buffer.…”