The classical model of DNA minor groove binding compounds is that they should have a crescent shape that closely fits the helical twist of the groove. Several compounds with relatively linear shape and large dihedral twist, however, have been found recently to bind strongly to the minor groove. These observations raise the question of how far the curvature requirement could be relaxed. As an initial step in experimental analysis of this question, a linear triphenyl diamidine, DB1111 and a series of nitrogen tricyclic analogues were prepared. The goal with the heterocycles is to design GC binding selectivity into heterocyclic compounds that can get into cells and exert biological effects. The compounds have a zero radius of curvature from amidine carbon to amidine carbon but a significant dihedral twist across the tricyclic and amidine-ring junctions. They would not be expected to bind well to the DNA minor groove by shape-matching criteria. Detailed DNaseI footprinting studies of the sequence specificity of this set of diamidines indicated that a pyrimidine heterocyclic derivative, DB1242, has remarkable binding specificity for a GC rich sequence, -GCTCG-. It binds to the GC sequence more strongly than to the usual AT recognition sequences for curved minor groove agents. Other similar derivatives did not exhibit the GC specificity. Biosensor-surface plasmon resonance and isothermal titration calorimetry experiments indicate that DB1242 binds to the GC sequence as a highly cooperative stacked dimer. Circular dichroism results indicate that the compound binds in the minor groove. Molecular modeling studies support a minor groove complex and provide an inter-compound and compound-DNA hydrogen bonding rational for the unusual GC binding specificity and the requirement for a pyrimidine heterocycle. This compound represents a new direction in development of DNA sequence specific agents and it is the first non-polyamide, synthetic compound to specifically recognize a DNA sequence with a majority of GC base pairs.

Transporters play a vital role in both the resistance mechanisms of existing drugs and effective targeting of their replacements. Melarsoprol and diamidine compounds similar to pentamidine and furamidine are primarily taken up by trypanosomes of the genus Trypanosoma brucei through the P2 aminopurine transporter. In standardized competition experiments with [ 3 H]adenosine, P2 transporter inhibition constants (K i ) have been determined for a diverse dataset of adenosine analogs, diamidines, Food and Drug Administration-approved compounds and analogs thereof, and custom-designed trypanocidal compounds. Computational biology has been employed to investigate compound structure diversity in relation to P2 transporter interaction. These explorations have led to models for inhibition predictions of known and novel compounds to obtain information about the molecular basis for P2 transporter inhibition. A common pharmacophore for P2 transporter inhibition has been identified along with other key structural characteristics. Our model provides insight into P2 transporter interactions with known compounds and contributes to strategies for the design of novel antiparasitic compounds. This approach offers a quantitative and predictive tool for molecular recognition by specific transporters without the need for structural or even primary sequence information of the transport protein.Trypanosoma brucei are unicellular trypanosomal parasites that cause African sleeping sickness in humans and nagana in livestock. These trypanosomes are auxotrophic for purines and thus rely entirely on purine supplies salvaged from the host environment. As such, T. brucei brucei expresses a multitude of purine nucleoside and nucleobase transporters (1). One of these, the T. brucei aminopurine P2 transporter, is unusual as a genuine nucleoside-nucleobase transporter in that it equally transports the nucleoside adenosine and the nucleobase adenine but has virtually no affinity for any other natural purines or pyrimidines (1-3). Yet, despite this apparent high level of selectivity, it has been shown that P2 also mediates cellular uptake of the Food and Drug Administration-approved drugs melarsoprol and pentamidine (2, 4, 5), the main veterinary trypanocides diminazene aceturate (6) and possibly isometamidium (7), and various nucleoside drugs (8).The unusual nature of this transporter has led to efforts to exploit it as an efficient conduit for novel trypanocides (9, 10), but this requires the identification of the exact pharmacophore as well as the physical limitations on size and charge distribution of the extracellular binding site of the transporter. From the structural similarities between known P2 substrates, it could be concluded early on that the so-called amidine motif of adenine, i.e. N(1)ϭC(6)-NH 2 (see Fig. 1), was very likely to play a major role in the high affinity interaction with the transporter (3, 11). However, quantitative information or three-dimensional models explaining the high affinity binding, by one transporter, of such di...

Given the increasing significance of diamidines as DNA-targeted therapeutics and biotechnology reagents, it is important to establish the variations in thermodynamic quantities that characterize the interactions of closely related compounds to different sequence AT binding sites. In this study, an array of methods including biosensor-surface plasmon resonance (SPR), isothermal titration microcalorimetry (ITC), circular dichroism (CD), thermal melting (Tm) and molecular modeling have been used to characterize the binding of dicationic diamidines related to DB75 (amidine-phenylfuran-phenyl-amidine) with alternating and nonalternating AT sequences. Conversion of the central furan of DB75 to other similar groups, such as thiophene or selenophene, can yield compounds with increased affinity and sequence binding selectivity for the minor groove. Calorimetric measurements revealed that the thermodynamic parameters (ΔG, ΔH, ΔS) that drive diamidine binding to alternating and nonalternating oligomers can be quite different and depend on both DNA sequence and length. Small changes in a compound can have major effects on DNA interactions. By choosing an appropriate central group it is possible to "tune" the shape of the molecule to match DNA for enhanced affinity and sequence recognition.