Antiparasitic compounds that target DNA

Abstract

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite. With time the compounds are also generally seen in the cell nucleus but are not significantly observed in the cytoplasm. The kinetoplast decays over time and disappears from the mitochondria of treated cells. At this point the compounds begin to be observed in other regions of the cell, such as the acidocalcisomes. The cells typically die in 24-48h after treatment. Active compounds appear to selectively target extended AT sequences and induce changes in kinetoplast DNA minicircles that cause a synergistic destruction of the catenated kinetoplast DNA network and cell death.

Figure 1

Pentamidine, furamidine and analogs along with and -AATT- DNA hairpin are shown. All compounds are dications.

Figure 2

A. A view of the crystal structure of pentamidine bound to the -AATT- site of d(CGCGAATTCGCG)₂ is shown [25]. The view on the left is into the minor groove and the view on the right shows how the compound fits into the groove. In this conformation the amidines are H-bonded to TO2 groups and the compound makes contacts with the groove floor and walls. B. A view into the minor groove of the DB75 crystal structure with the same binding site and DNA sequence is shown at the left [53]. The DNA contacts are similar to those with pentamidine but the DB75 structure is preorganized to bind in this orientation. The view on the right is from the major groove through the base pairs to DB75. The red dots identify the amidine to TO2 H-bond interactions. The compound curvature allows DB75 to slide deeply into the groove in AT sequences and make excellent contacts with the bases and groove walls.

Figure 2

A. A view of the crystal structure of pentamidine bound to the -AATT- site of d(CGCGAATTCGCG)₂ is shown [25]. The view on the left is into the minor groove and the view on the right shows how the compound fits into the groove. In this conformation the amidines are H-bonded to TO2 groups and the compound makes contacts with the groove floor and walls. B. A view into the minor groove of the DB75 crystal structure with the same binding site and DNA sequence is shown at the left [53]. The DNA contacts are similar to those with pentamidine but the DB75 structure is preorganized to bind in this orientation. The view on the right is from the major groove through the base pairs to DB75. The red dots identify the amidine to TO2 H-bond interactions. The compound curvature allows DB75 to slide deeply into the groove in AT sequences and make excellent contacts with the bases and groove walls.

Figure 3

S427 Trypanosoma brucei brucei trypanosomes were incubated in vitro with 500 nM of DB351 for various time points between 1 hour and 24 hours. Trypanosomes were washed and resuspended in freshly isolated mouse blood. Dry smears were prepared and examined using a Nikon Microphot microscope with a UV2A cube. Nuclei are found in the center of trypanosomes, and the kinetoplast is found at the end of trypanosomes.

Figure 4

SPR sensorgrams for binding of DB351 and DB820 with an -AATT- site, as in Figure 2, in an immobilized hairpin DNA (Figure 1). The compound concentrations were 10 nM to 1 μM from bottom to top. The experiments were carried out in Tris buffer at 25 °C.

Figure 5

Binding curves from the SPR results in Figure 4 for the DB75 derivatives are shown with r values plotted against the unbound compound concentration, C_f (flow solution). The data were fitted to a two site model using equation 1 and all compounds have one strong binding site in the AATT sequence and some much weaker secondary binding.

Figure 6

(A) Sequence of trans and cis oligomers used in the gel electrophoresis experiments. (B) Native PAGE of trans (lanes 2 and 4) and cis ligation ladders (lanes 1 and 3) and 20-bp marker (M) in the presence of DB351. Circular products (C) and linear ligation products (L) are marked, where L2 corresponds to the 42-bp dimer, L3 to the 63-bp, and so forth. (C) Plot of relative mobility (where R_L= apparent length/actual length) versus actual length (L_act) for trans (red circle) and cis (red triangle) ligation ladders without DB351 and trans (blue circle) and cis (blue triangle) with DB351. Only linear products were used to calculate R_L, and missing points correspond to linear ligation products indistinguishable from circular products.

Figure 7

The structures of diamidine prodrugs are shown for a number of different heterocycles.

Figure 8

Plot of log (1/IC₅₀) vs ΔTm and log K for the compounds of Figure 1. The correlation between antitrypanosomal activity and binding to AT DNA for this set of compounds can be seen.