Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC 50 ], <1 M) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC 50 < 0.12 M) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.Leishmaniasis, a neglected tropical disease, is caused by parasitic protozoa of the genus Leishmania, including 20 species that are pathogenic for humans (21). Clinical manifestations of leishmaniasis mainly consist of cutaneous, mucocutaneous, visceral, and post-kala-azar dermal leishmaniasis, with symptoms ranging from skin and mucosal ulceration to systemic infection that is fatal if left untreated (6). An estimated 12 million people are currently infected with Leishmania, and up to 350 million people in 88 countries are at risk of infection (35). Approximately 2 million new cases of leishmaniasis are believed to occur annually, with 1.5 million for cutaneous leishmaniasis and 0.5 million for visceral leishmaniasis (VL). In macrophages, Leishmania amastigotes adapt to thrive in an acidic subcellular compartment, the parasitophorous vacuole (PV; pH ϳ5) (2), where they maintain a neutral intracellular pH within the parasite by an energy-dependent process (9). Multiple layers of membrane barriers (i.e., host macrophage plasma membrane, phagolysosomal membrane, and Leishmania amastigote plasma membrane) presumably present a formidable challenge for chemotherapeutic agents to target Leishmania parasites in mammalian hosts.Current chemotherapies for leishmaniasis have many limitations, including resis...

ETS transcription factors mediate a wide array of cellular functions and are attractive targets for pharmacological control of gene regulation. We report the inhibition of the ETS-family member PU.1 with a panel of novel heterocyclic diamidines. These diamidines are derivatives of furamidine (DB75) in which the central furan has been replaced with selenophene and/or one or both of the bridging phenyl has been replaced with benzimidazole. Like all ETS proteins, PU.1 binds sequence specifically to 10-bp sites by inserting a recognition helix into the major groove of a 5′-GGAA-3′ consensus, accompanied by contacts with the flanking minor groove. We showed that diamidines target the minor groove of AT-rich sequences on one or both sides of the consensus and disrupt PU.1 binding. Although all of the diamidines bind to one or both of the expected sequences within the binding site, considerable heterogeneity exists in terms of stoichiometry, site–site interactions and induced DNA conformation. We also showed that these compounds accumulate in live cell nuclei and inhibit PU.1-dependent gene transactivation. This study demonstrates that heterocyclic diamidines are capable of inhibiting PU.1 by targeting the flanking sequences and supports future efforts to develop agents for inhibiting specific members of the ETS family.

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.

DNA condensation is a ubiquitous phenomenon in biology, yet the physical basis for it has remained elusive. Here, we have explored the mechanism of DNA condensation through the protamine-DNA interaction, and by examining on it the influence of DNA binding drugs. We observed that the DNA condensation is accompanied by B to Ψ-DNA transition as a result of DNA base pair distortions due to protamine binding, bringing about the formation of toroidal structure through coil-globule transition. The binding energetics suggested that electrostatic energy, bending energy and hydration energy must play crucial roles in DNA condensation. EtBr intercalation interferes with the protamine-DNA interaction, challenging the distortion of the DNA helix and separation of DNA base pairs by protamine. Thus, EtBr, by competing directly with protamine, resists the phenomenon of DNA condensation. On the contrary, netropsin impedes the DNA condensation by an allosteric mechanism, by resisting the probable DNA major groove bending by protamine. In summary, we demonstrate that drugs with distinct binding modes use different mechanism to interfere with DNA condensation.

Structural results with minor groove binding agents, such as netropsin, have provided detailed, atomic level views of DNA molecular recognition. Solution studies, however, indicate that there is complexity in the binding of minor groove agents to a single site. Netropsin, for example, has two DNA binding enthalpies in isothermal titration calorimetry (ITC) experiments that indicate the compound simultaneously forms two thermodynamically different complexes at a single AATT site. Two proposals for the origin of this unusual observation have been developed: (i) two different bound species of netropsin at single binding sites and (ii) a netropsin induced DNA hairpin to duplex transition. To develop a better understanding of DNA recognition complexity, the two proposals have been tested with several DNAs and the methods of mass spectrometry (MS), polyacrylamide gel electrophoresis (PAGE) and nuclear magnetic resonance spectroscopy in addition to ITC. All of the methods with all of the DNAs investigated clearly shows that netropsin forms two different complexes at AATT sites, and that the proposal for an induced hairpin to duplex transition in this system is incorrect.

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.

Background: ETS family transcription factors recognize DNA via structurally conserved DNA-binding domains that share limited amino acid homology. Results: DNA recognition by the ETS domains of Ets-1 and PU.1, two extreme sequence-divergent paralogs, was compared. Conclusion: Preferential hydration differentiates DNA recognition by Ets-1 and PU.1. Significance: Preferential hydration represents a potential mechanism for PU.1 regulation and its activity as a pioneer transcription factor in vivo.

Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.