Identification of diphenyl furan derivatives via high throughput and computational studies as ArgA inhibitors of Mycobacterium tuberculosis.

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 µM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.

Heterocyclic Diamidine DNA Ligands as HOXA9 Transcription Factor Inhibitors: Design, Molecular Evaluation, and Cellular Consequences in a HOXA9-Dependant Leukemia Cell Model.

Most transcription factors were for a long time considered as undruggable targets because of the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multitarget HOX/PBX protein/protein interaction inhibitors has been developed. As an attractive alternative by inhibiting the DNA binding, we selected a series of heterocyclic diamidines as efficient competitors for the HOXA9/DNA interaction through binding as minor groove DNA ligands on the HOXA9 cognate sequence. Selected DB818 and DB1055 compounds altered HOXA9-mediated transcription in luciferase assays, cell survival, and cell cycle, but increased cell death and granulocyte/monocyte differentiation, two main HOXA9 functions also highlighted using transcriptomic analysis of DB818-treated murine Hoxa9-transformed hematopoietic cells. Altogether, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model.

G-quadruplex-binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo.

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA-binding proteins and through toxic dipeptide repeat proteins generated by repeat-associated non-ATG translation. GGGGCC repeat RNA folds into a G-quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G-quadruplex RNA We investigated their effect in C9orf72 patient iPSC-derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat-expressing Drosophila Therefore, small molecules that target GGGGCC repeat G-quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS These data provide proof of principle that targeting GGGGCC repeat G-quadruplexes has therapeutic potential.

Phenotypic evaluation and in silico ADMET properties of novel arylimidamides in acute mouse models of Trypanosoma cruzi infection.

Arylimidamides (AIAs), previously termed as reversed amidines, present a broad spectrum of activity against intracellular microorganisms. In the present study, three novel AIAs were evaluated in a mouse model of Trypanosoma cruzi infection, which is the causative agent of Chagas disease. The bis-AIAs DB1957, DB1959 and DB1890 were chosen based on a previous screening of their scaffolds that revealed a very promising trypanocidal effect at nanomolar range against both the bloodstream trypomastigotes (BTs) and the intracellular forms of the parasite. This study focused on both mesylate salts DB1957 and DB1959 besides the hydrochloride salt DB1890. Our current data validate the high activity of these bis-AIA scaffolds that exhibited EC50 (drug concentration that reduces 50% of the number of the treated parasites) values ranging from 14 to 78 nM and 190 to 1,090 nM against bloodstream and intracellular forms, respectively, also presenting reasonable selectivity indexes and no mutagenicity profile predicted by in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET). Acute toxicity studies using murine models revealed that these AIAs presented only mild toxic effects such as reversible abdominal contractions and ruffled fur. Efficacy assays performed with Swiss mice infected with the Y strain revealed that the administration of DB1957 for 5 consecutive days, with the first dose given at parasitemia onset, reduced the number of BTs at the peak, ranging between 21 and 31% of decrease. DB1957 was able to provide 100% of animal survival, while untreated animals showed 70% of mortality rates. DB1959 and DB1890B did not reduce circulating parasitism but yielded >80% of survival rates.

Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System.

The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP(C)) into the accumulating, disease-associated isoform (PrP(Sc)). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP(Sc) accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 µM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP(C) and PrP(Sc) were examined. While inhibition of total PrP(C) was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP(C) misfolding to PrP(Sc) Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.

A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.

Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

Structure-dependent inhibition of the ETS-family transcription factor PU.1 by novel heterocyclic diamidines.

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.

CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement.

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine CN to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.

In vitro and in vivo activities of dicationic diguanidino compounds against Echinococcus multilocularis metacestodes.

Alveolar echinococcosis (AE) is a disease predominantly affecting the liver, with metacestodes (larvae) of the tapeworm Echinococcus multilocularis proliferating and exhibiting tumor-like infiltrative growth. For many years, chemotherapeutical treatment against alveolar echinococcosis has relied on the benzimidazoles albendazole and mebendazole, which require long treatment durations and exhibit parasitostatic rather than parasiticidal efficacy. Although benzimidazoles have been and still are beneficial for the patients, there is clearly a demand for alternative and more efficient treatment options. Aromatic dications, more precisely a small panel of di-N-aryl-diguanidino compounds, were screened for efficacy against E. multilocularis metacestodes in vitro. Only those with a thiophene core group were active against metacestodes, while furans were not. The most active compound, DB1127, was further investigated in terms of in vivo efficacy in mice experimentally infected with E. multilocularis metacestodes. This diguanidino compound was effective against AE when administered intraperitoneally but not when applied orally. Thus, thiophene-diguanidino derivatives with improved bioavailability when administered orally could lead to treatment options against AE.

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines.

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.

Evaluation of arylimidamides DB1955 and DB1960 as candidates against visceral leishmaniasis and Chagas' disease: in vivo efficacy, acute toxicity, pharmacokinetics, and toxicology studies.

Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels.

The adaptive potential of a survival artist: characterization of the in vitro interactions of Toxoplasma gondii tachyzoites with di-cationic compounds in human fibroblast cell cultures.

The impact of di-cationic pentamidine-analogues against Toxoplama gondii (Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites of T. gondii Rh and T. gondii Me49 with an IC(50) of 0·11 and 0·13 µM, respectively. Pre-incubation of fibroblast monolayers with 1 µM DB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5-6 days of drug exposure, T. gondii tachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2 µM. Out of a set of 32 di-cationic compounds screened for in vitro activity against T. gondii, the arylimidamide DB745, exhibiting an IC(50) of 0·03 µM and favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adapted T. gondii (IC(50)=0·07 µM). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adapted T. gondii tachyzoites, but not on DB750-adapted T. gondii. Adaptation of T. gondii to DB745 (up to a concentration of 0·46 µM) was much more difficult to achieve and feasible only over a period of 110 days. In cultures infected with DB750-adapted T. gondii seemingly intact parasites could occasionally be detected by TEM. This illustrates the astonishing capacity of T. gondii tachyzoites to adapt to environmental changes, at least under in vitro conditions, and suggests that DB745 could be an interesting drug candidate for further assessments in appropriate in vivo models.

Di-cationic arylimidamides act against Neospora caninum tachyzoites by interference in membrane structure and nucleolar integrity and are active against challenge infection in mice.

Neospora caninum is considered to be the main cause of bovine abortion in Europe and the USA, leading to considerable financial impact. Losses are caused directly by abortions or indirectly through breeding of calves with impaired viability. Due to the lack of effective chemotherapy against bovine neosporosis, there is a need to develop new anti-protozoal compounds, which would either eliminate the parasite or avoid its transmission. In order to identify compounds of interest, the in vitro activities of 41 di-cationic pentamidine derivatives were studied employing a transgenic N. caninum clone expressing beta-galactosidase as a reporter gene. The arylimidamide DB745, previously shown to be highly active against Leishmania donovani in vitro and in vivo, appeared as the most promising compound, with an IC50 of 80 nM in 3-day growth assays and severely affecting both host cell invasion as well as intracellular proliferation. TEM of intracellular tachyzoites identified distinct alterations related to the nucleolus and the nuclear and cellular membrane. Long-term growth assays showed that DB745 acted parasiticidal upon the Nc-Liv isolate, but not against the Nc-1 isolate of N. caninum. In vivo studies in N. caninum (Nc-1 isolate) infected mice showed that daily intraperitoneal application of DB745 for a period of 14 days resulted in a decreased number of clinically affected animals, and lower cerebral parasite burdens in DB745-treated mice compared to non-treated mice. These results illustrate the potential of dicationic arylimidamides for the treatment of N. caninum infections.

The efficacy of novel arylimidamides against Trypanosoma cruzi in vitro.

The present study aimed to determine the in vitro biological efficacy and selectivity of 7 novel AIAs upon bloodstream trypomastigotes and intracellular amastigotes of Trypanosoma cruzi. The biological activity of these aromatic compounds was assayed for 48 and 24 h against intracellular parasites and bloodstream forms of T. cruzi (Y strain), respectively. Additional assays were also performed to determine their potential use in blood banks by treating the bloodstream parasites with the compounds diluted in mouse blood for 24 h at 4°C. Toxicity against mammalian cells was evaluated using primary cultures of cardiac cells incubated for 24 and 48 h with the AIAs and then cellular death rates were determined by MTT colorimetric assays. Our data demonstrated the outstanding trypanocidal effect of AIAs against T. cruzi, especially DB1853, DB1862, DB1867 and DB1868, giving IC50 values ranging between 16 and 70 nanomolar against both parasite forms. All AIAs presented superior efficacy to benznidazole and some, such as DB1868, also demonstrated promising activity as a candidate agent for blood prophylaxis. The excellent anti-trypanosomal efficacy of these novel AIAs against T. cruzi stimulates further in vivo studies and justifies the screening of new analogues with the goal of establishing a useful alternative therapy for Chagas disease.

Experimental treatment of Neospora caninum-infected mice with the arylimidamide DB750 and the thiazolide nitazoxanide.

The cationic arylimidamide DB750 and the thiazolide nitazoxanide had been shown earlier to be effective against Neospora caninum tachyzoites in vitro with an IC(50) of 160nM and 4.23µM, respectively. In this study, we have investigated the effects of DB750 and nitazoxanide treatments of experimentally infected Balb/c mice, by applying the drugs either through the oral or the intraperitoneal route. In experiment 1, administration of DB750 (2mg/kg/day) and nitazoxanide (150mg/kg/day) started already 3 days prior to experimental infection of mice with 2×10(6) tachyzoites. Following infection, the drugs were further administrated daily for a period of 2 weeks, either orally or intraperitoneally. Intraperitoneal injection of DB750 was well tolerated by the mice, but treatment with nitazoxanide resulted in death of all mice within 3 days. Upon intraperitoneal application of DB750, the cerebral parasite load was significantly reduced compared to all other groups, while oral application of DB750 and nitazoxanide were not as effective, and resulted in significant weight loss. In experiment 2, mice were infected with 2×10(6) tachyzoites and at 2 weeks post-infection, DB750 (2mg/kg/day) was applied by intraperitoneal injections for 14 days. In the DB750-treated group, only 2 out of 12 mice succumbed to infection, compared to 7 out of 12 mice in the placebo-group. DB750 treatment also resulted in significantly reduced cerebral parasite burden, and reduced numbers of viable tachyzoites. Our data suggest that DB750 exerted its activity also after crossing the blood-brain barrier, and that this class of compounds could be promising for the control of N. caninum-associated disease.

Combined treatment of heterocyclic analogues and benznidazole upon Trypanosoma cruzi in vivo.

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via i.p. route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals.

In vitro efficacy of dicationic compounds and mefloquine enantiomers against Echinococcus multilocularis metacestodes.

The current chemotherapy of alveolar echinococcosis (AE) is based on benzimidazoles such as albendazole and has been shown to be parasitostatic rather than parasiticidal, requiring lifelong duration. Thus, new and more efficient treatment options are urgently needed. By employing a recently validated assay based on the release of functional phosphoglucose isomerase (PGI) from dying parasites, the activities of 26 dicationic compounds and of the (+)- and (-)-erythro-enantiomers of mefloquine were investigated. Initial screening of compounds was performed at 40 µM, and those compounds exhibiting considerable antiparasitic activities were also assessed at lower concentrations. Of the dicationic drugs, DB1127 (a diguanidino compound) with activities comparable to nitazoxanide was further studied. The activity of DB1127 was dose dependent and led to severe structural alterations, as visualized by electron microscopy. The (+)- and (-)-erythro-enantiomers of mefloquine showed similar dose-dependent effects, although higher concentrations of these compounds than of DB1127 were required for metacestode damage. In conclusion, of the drugs investigated here, the diguanidino compound DB1127 represents the most promising compound for further study in appropriate in vivo models for Echinococcus multilocularis infection.

In vitro trypanocidal activity of DB745B and other novel arylimidamides against Trypanosoma cruzi.

OBJECTIVES: As part of a search for new therapeutic opportunities to treat chagasic patients, in vitro efficacy studies were performed to characterize the activity of five novel arylimidamides (AIAs) against Trypanosoma cruzi. METHODS: The trypanocidal effect against T. cruzi was evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by MTT assays against mouse cardiomyocytes. RESULTS: Our data demonstrated the trypanocidal efficacy of these new compounds against bloodstream trypomastigotes and intracellular amastigotes, exhibiting IC50 values ranging from 0.015 to 2.5 and 0.02 to0.2 µM, respectively. One of the compounds, DB745B, was also highly active against a broad panel of isolates, including those naturally resistant to benznidazole. DB745B showed higher in vitro efficacy than the reference drugs used to treat patients (benznidazole IC50= 12.94 µM) and to prevent blood bank infection (gentian violet IC50= 30.6 µM). CONCLUSIONS: AIAs represent promising new chemical entities against T. cruzi and are also potential trypanocidal agents to prevent transfusion-associated Chagas' disease.

DNA minor groove induced dimerization of heterocyclic cations: compound structure, binding affinity, and specificity for a TTAA site.

With the increasing number and variations of genome sequences available, control of gene expression with synthetic, cell-permeable molecules is within reach. The variety of sequence-specific binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this article is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, we made modifications to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine, binds strongly to TTAA as a dimer and has selectivity (K(TTAA)/K(AATT)=6). CD and DNase I footprinting studies confirmed the preference of this compound for TTAA. In summary, (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) -NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves.

Arylimidamide DB766, a potential chemotherapeutic candidate for Chagas' disease treatment.

Chagas' disease, a neglected tropical illness for which current therapy is unsatisfactory, is caused by the intracellular parasite Trypanosoma cruzi. The goal of this work is to investigate the in vitro and in vivo effects of the arylimidamide (AIA) DB766 against T. cruzi. This arylimidamide exhibits strong trypanocidal activity and excellent selectivity for bloodstream trypomastigotes and intracellular amastigotes (Y strain), giving IC(50)s (drug concentrations that reduce 50% of the number of the treated parasites) of 60 and 25 nM, respectively. DB766 also exerts striking effects upon different parasite stocks, including those naturally resistant to benznidazole, and displays higher activity in vitro than the reference drugs. By fluorescent and transmission electron microscopy analyses, we found that this AIA localizes in DNA-enriched compartments and induces considerable damage to the mitochondria. DB766 effectively reduces the parasite load in the blood and cardiac tissue and presents efficacy similar to that of benznidazole in mouse models of T. cruzi infection employing the Y and Colombian strains, using oral and intraperitoneal doses of up to 100 mg/kg/day that were given after the establishment of parasite infection. This AIA ameliorates electrocardiographic alterations, reduces hepatic and heart lesions induced by the infection, and provides 90 to 100% protection against mortality, which is similar to that provided by benznidazole. Our data clearly show the trypanocidal efficacy of DB766, suggesting that this AIA may represent a new lead compound candidate to Chagas' disease treatment.