Manoj Munde

Novel arylimidamides for treatment of visceral leishmaniasis.

ABSTRACT Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC50], <1 μM) 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 (IC50 ≤ 0.12 μM) 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.

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.

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.

Novel linear triaryl guanidines, N-substituted guanidines and potential prodrugs as antiprotozoal agents

A series of triaryl guanidines and N-substituted guanidines designed to target the minor groove of DNA were synthesized and evaluated as antiprotozoal agents. Selected carbamate prodrugs of these guanidines were assayed for their oral efficacy. The linear triaryl bis-guanidines 6a,b were prepared from their corresponding diamines 4a,b through the intermediate BOC protected bis-guanidines 5a,b followed by acid catalyzed deprotection. The N-substituted guanidino analogues 9c-f were obtained in three steps starting by reacting the diamines 4a,b with ethyl isothiocyanatoformate to give the carbamoyl thioureas 7a,b. Subsequent condensation of 7a,b with various amines in the presence of EDCI provided the carbamoyl N-substituted guanidine intermediates 8a-f which can also be regarded as potential prodrugs for the guanidino derivatives. Compounds 9c-f were obtained via the base catalyzed decarbamoylation of 8a-f. The DNA binding affinities for the target dicationic bis-guanidines were assessed by DeltaT(m) values. In vitro antiprotozoal screening of the new compounds showed that derivatives 6a, 9c and 9e possess high to moderate activity against Trypanosoma brucei rhodesiense (T.b.r.) and Plasmodium falciparum (P.f.). While the prodrugs did not yield cures upon oral administration in the antitrypanosomal STIB900 mouse model, compounds 8a and 8c prolonged the survival of the treated mice.

Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures

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.

Synthesis and activity of azaterphenyl diamidines against Trypanosoma brucei rhodesiense and Plasmodium falciparum

A series of azaterphenyl diamidines has been synthesized and evaluated for in vitro antiprotozoal activity against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) and in vivo efficacy in the STIB900 acute mouse model for T. b. r. Six of the 13 compounds showed IC(50) values less than 7 nM against T. b. r. Twelve of those exhibited IC(50) values less than 6 nM against P. f. and six of those showed IC(50) values 0.6 nM, which are more than 25-fold as potent as furamidine. Moreover, two of them showed more than 40-fold selectivity for P. f. versus T. b. r. Three compounds 15b, 19d and 19e exhibited in vivo efficacy against T. b. r. much superior to furamidine, and equivalent to or better than azafuramidine. The antiparasitic activity of these diamidines depends on the ring nitrogen atom(s) location relative to the amidine groups and generally correlates with DNA binding affinity.

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.

Mechanistic Heterogeneity in Site Recognition by the Structurally Homologous DNA-binding Domains of the ETS Family Transcription Factors Ets-1 and PU.1

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. ETS family transcription factors regulate diverse genes through binding at cognate DNA sites that overlap substantially in sequence. The DNA-binding domains of ETS proteins (ETS domains) are highly conserved structurally yet share limited amino acid homology. To define the mechanistic implications of sequence diversity within the ETS family, we characterized the thermodynamics and kinetics of DNA site recognition by the ETS domains of Ets-1 and PU.1, which represent the extremes in amino acid divergence among ETS proteins. Even though the two ETS domains bind their optimal sites with similar affinities under physiologic conditions, their nature of site recognition differs strikingly in terms of the role of hydration and counter ion release. The data suggest two distinct mechanisms wherein Ets-1 follows a “dry” mechanism that rapidly parses sites through electrostatic interactions and direct protein-DNA contacts, whereas PU.1 utilizes hydration to interrogate sequence-specific sites and form a long-lived complex relative to the Ets-1 counterpart. The kinetic persistence of the high affinity PU.1·DNA complex may be relevant to an emerging role of PU.1, but not Ets-1, as a pioneer transcription factor in vivo. In addition, PU.1 activity is critical to the development and function of macrophages and lymphocytes, which present osmotically variable environments, and hydration-dependent specificity may represent an important regulatory mechanism in vivo, a hypothesis that finds support in gene expression profiles of primary murine macrophages.

Probing the Electrostatics and Pharmacological Modulation of Sequence-Specific Binding by the DNA-Binding Domain of the ETS Family Transcription Factor PU.1: A Binding Affinity and Kinetics Investigation

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

Minor Groove to Major Groove, an Unusual DNA Sequence-Dependent Change in Bend Directionality by a Distamycin Dimer

DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.