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D W Boykin

Publications and source records attributed to D W Boykin.

At least 19 recordsLinked to original sources
PubMed2006

Antiparasitic activity of aromatic diamidines is related to apoptosis-like death in Trypanosoma cruzi.

Two aromatic diamidines, furamidine (DB75) and its phenyl-substituted analogue (DB569), which exhibit trypanocidal activity, were assayed against Trypanosoma cruzi and were found to induce apoptosis-like death characteristics such as nuclear DNA condensation and fragmentation, decreased mitochondrial membrane potential and phosphatidylserine exposure. DB569 displays superior trypanocidal activity compared to furamidine and also had higher ability to induce apoptosis-like death in treated parasites. The present results showing apoptosis-like death in T. cruzi after treatment with both DB75 and DB569 make important contributions to the understanding of the mechanisms of the aromatic diamidines, which represent promising trypanocidal compounds.

Animals↗
PubMed2005

In vitro metabolism of an orally active O-methyl amidoxime prodrug for the treatment of CNS trypanosomiasis.

A new aza-analogue of furamidine, 6-[5-(4-amidinophenyl)-furan-2-yl]nicotinamidine (DB820), has potent in vitro antitrypanosomal activity; however, it suffers from poor oral activity because of its positively charged amidine groups. The dimethoxyamidine prodrug of DB820, N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine (DB844), has potent oral activity in mouse models of both early-stage and CNS African trypanosomiasis. Metabolism of DB844 in human liver microsomes (HLM) was investigated using liquid chromatography-mass spectrometry (LC-MS/MS). The metabolism of DB844 in HLM was NADPH-dependent and resulted in the production of eight metabolites over a 90?min incubation. O-Demethylation and N-dehydroxylation reactions resulted in the metabolic conversion of DB844 to its active DB820 metabolite. Chromatographic conditions used for LC-MS analysis allowed for the separation and identification of all metabolites including positional isomers. Demethylation of either the phenyl or pyridine side of DB844 (DB844 m/z 366.2) resulted in the production of two metabolites (M1A, M1B), each with a molecular ion of m/z of 352.3 and MS(2) fragments of 288.1, 305.2, 321.2 and 335.2. However, the intensities of the MS(2) fragments were different among the two isomeric metabolites, and comparison to an authentic standard allowed for the structural determination of each metabolite. The isomeric metabolites M2A and M2B, resulting from amidoxime reductions of M1A and M1B, were also chromatographically separated and had distinguishable MS(2) profiles that allowed for their structural assignments when compared to an authentic standard. The di-amidoxime product resulting from O-demethylation of either side of DB844 was also identified as an abundant metabolite during microsomal incubations. The active antitrypanosomal metabolite, DB820, was the last metabolite to be formed and thus provides evidence that DB844 may effectively be metabolized to its active metabolite in vivo.

Administration, Oral↗
PubMed2005

Aromatic diamidines as antiparasitic agents.

Parasitic infections are widespread in developing countries and frequently associated with immunocompromised patients in developed countries. Consequently, such infections are responsible for a significant amount of human mortality, morbidity and economic hardship. A growing consensus has identified the urgent need for the development of new antiparasitic compounds, mostly due to the large number of drug-resistant parasites and the fact that currently available drugs are expensive, highly toxic, require long treatment regimens and frequently exhibit significantly reduced activity towards certain parasite strains and evolutive stages. In this context, the activity of aromatic diamidines has been explored against a widespread range of micro-organisms, and the authors' present aim is to review the current status of chemotherapy with these compounds against human parasitic infections.

Animals↗
PubMed2004

Phenyl substitution of furamidine markedly potentiates its anti-parasitic activity against Trypanosoma cruzi and Leishmania amazonensis.

Furamidine (DB75) and related unfused aromatic diamidines have proven useful for the treatment of parasitic infections. These compounds were primarily developed to combat infections by Pneumocystis carinii and African trypanosomes but they are also active against other parasites. Here we have investigated the in vitro effects of DB75 and its phenyl-substituted analog DB569 on two kinetoplastid haemoflagellates Trypanosomatidae: Trypanosoma cruzi and Leishmania (L) amazonensis. The phenyl-amidine compound DB569 has equivalent DNA binding properties compared to DB75 but it was selected on the basis of its distinct tumor cell distribution properties. We found that DB569 is significantly more potent than DB75 at reducing the proliferation of the parasites, using either isolated parasites in cultures or with cardiomyocyte and macrophage host cells. DB569 is effective towards the intracellular forms of T. cruzi (IC(50) in the low-micromolar range) and it exhibits trypanocidal dose-dependent effects against trypomastigote forms of T. cruzi parasites obtained from the Y strain and Dm28c clone, which belong to two different biodemes. Fluorescence microscopy experiments indicated that both diamidines were mostly localized in the nucleus of the mammalian host cells and within the nuclei and kinetoplast of the parasites. Electron microscopy studies showed that the treatment of the parasites with DB75 and DB569 induces important alterations of the parasite nucleus and kinetoplast, at sites where their DNA target is localized. Altogether, the data suggest that the phenyl-substituted furamidine analogue DB569 is a potential new candidate for the treatment of the Chagas' disease and Leishmaniasis.

Animals↗
PubMed2003

Synthesis and antitumor evaluation of some new substituted amidino-benzimidazolyl-furyl-phenyl-acrylates and naphtho[2,1-b]furan-carboxylates.

The multistep synthesis of a series of substituted amidino-benzimidazolyl-furyl-phenyl-acrylic acid's esters and substituted amidino-benzimidazolyl-naphtho[2,1-b]furan-carboxylic acid's esters is described starting from corresponding 3-(2-furyl)-2-phenyl-acrylic acids. The new compounds were tested on the cytostatic activities against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), colon carcinoma (HT 29), melanoma (HBL), and human fibroblasts cell line (WI38). All compounds inhibited the proliferation of tumor cell lines. Inhibitory effect of examined compounds depended on concentration, but without significant difference among the type of tumor cells. The compounds 2 and 5 exerted very low inhibitory effect on the growth of human fibroblasts. Unsubstituted derivative 8 has not inhibited any tested cell lines.

Acetates↗
PubMed2001

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove.

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.

Antifungal Agents↗
PubMed2001

Novel dications with unfused aromatic systems: trithiophene and trifuran derivatives of furimidazoline.

We report the synthesis, interaction with DNA, topoisomerase II inhibition, and cytotoxicity of two novel unfused aromatic dications derived from the antimicrobial agent furimidazoline. The central diphenylfuran core of furimidazoline has been replaced with a trithiophene (DB358) or a trifuran (DB669) unit and the terminal imidazoline groups were preserved. The strength and mode of binding of the drugs to nucleic acids were investigated by complementary spectroscopic techniques including spectrophotometric, surface plasmon resonance, circular and linear dichroism measurements. The trifuran derivative forms intercalation complexes with double-stranded DNA, whereas the mode of binding of the trithiophene derivative varies depending on the drug/DNA ratio, as independently confirmed by NMR spectroscopic studies performed with (A-T)7 and (G-C)7 oligomers. Two-dimensional NMR data provided a molecular model for the binding of DB358 within the minor groove of the AATT sequence of the decanucleotide d(GCGAATTCGC)(2). DNase I footprinting experiments confirmed the sequence-dependent binding of DB358 to DNA. The trithiophene derivative interacts preferentially with AT-rich sequences at low concentrations, but can accomodate GC sites at higher concentrations. DNA relaxation assays revealed that DB358 stimulated DNA cleavage by topoisomerase II, in contrast to DB669. The substitution of N-alkylamidines for the imidazoline terminal groups abolished the capacity of the drug to poison topoisomerase II. At the cellular level, flow cytometry analysis indicated that DB358, which is about six times more cytotoxic than the trifuran analogue, induced a significant accumulation of HL-60 human leukemia cells in the G2/M phase. The incorporation of thiophene heterocycles appears as a convenient procedure to limit the strict AT selectivity of dications containing an extended unfused aromatic system and to design cytotoxic DNA intercalating agents acting as poisons for human topoisomerase II.

Anti-HIV Agents↗
PubMed2001

Diguanidino and "reversed" diamidino 2,5-diarylfurans as antimicrobial agents.

Dicationic 2,5-bis(4-guanidinophenyl)furans 5a-5f, 2,5-bis[4-(arylimino)aminophenyl]furans 6a-6b and 6e-6k, and 2,5-bis[4-(alkylimino)aminophenyl]furans 6c-6d have been synthesized starting from 2,5-bis[tri-n-butylstannyl]furan. Thermal melting studies with poly dA*dT and the duplex oligomer d(CGCGAATTCGCG)2 demonstrated high DNA binding affinities for a number of the compounds. The binding affinities are highly dependent on structure and are significantly affected by substituents both on the phenyl rings of the 2,5-diphenylfuran nucleus and on the cationic centers. Of the 17 novel dicationic compounds synthesized, six (6a, 6b, 5b, 6f, 6h, 6i) exhibited MICs of 2 microg/mL or less versus Mycobacterium tuberculosis. Of the compounds screened against Candida albicans, three gave MICs of 2 microg/mL or less (5b, 6h, 6i), and two (5b, 6i) were fungicidal, unlike a standard antifungal drug fluconazole, which was fungistatic. In addition, one of the tested compounds (6i) exhibited a MIC of <1 microg/mL against Aspergillus fumigatus, while also being a fungicidal against this organism. Finally, when evaluated against an expanded fungal panel, compound 6h showed good activity against Cryptococcus neoformans and Rhizopus arrhizus.

Amidines↗
PubMed2001

Evaluation of the influence of compound structure on stacked-dimer formation in the DNA minor groove.

The Human Genome Project as well as sequencing of the genomes of other organisms offers a wealth of DNA targets for both therapeutic and diagnostic applications, and it is important to develop additional DNA binding motifs to fully exploit the potential of this new information. We have recently found that an aromatic dication, DB293, with an amidine-phenyl-furan-benzimidazole-amidine structure can recognize specific sequences of DNA by binding in the minor groove as a dimer [Wang, L., Bailly, C., Kumar, A., Ding, D., Bajic, M., Boykin, D. W., and Wilson, W. D. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 12-16]. The dimer binding is strong, highly cooperative and, in contrast to many closely related heterocyclic dications, has both GC and AT base pairs in the minor groove binding site. The aromatic heterocycle stacked dimer is quite different in structure from the polyamide-lexitropsin type compounds, and it is a dication while all lexitropsin dimers are monocations. The heterocyclic dimer represents only the second small molecule class that can recognize mixed sequences of DNA. To test the structural limits on the new type of complex, it is important to probe the influence of compound charge, chemical groups, and structural features. The effects of these compound molecular variations on DNA complex formation with several DNA sequences were evaluated by DNase I footprinting, CD and UV spectroscopy, thermal melting, and quantitative analysis with surface plasmon resonance biosensor methods. Conversion of the amidines to guanidinium groups does permit the cooperative dimer to form but removal of one amidine or addition of an alkyl group to the amidine strongly inhibited dimer formation. Changing the phenyl of DB293 to a benzimidazole or the benzimidazole to a phenyl or benzofuran also inhibited dimer formation. The results show that formation of the minor groove stacked-dimer complex is very sensitive to compound structure. The discovery of the aromatic dimer mode offers new opportunities to enhance the specificity and expand the range of applications of the compounds that target DNA.

Amidines↗
PubMed2001

A heterocyclic inhibitor of the REV-RRE complex binds to RRE as a dimer.

As part of a search for organic compounds that selectively target RNA, we found that specific diphenylfuran derivatives, which are related to compounds that bind to the DNA minor groove, bind very strongly to RNA in a manner very sensitive to the structure of the compounds. In extended development of the diphenylfuran series, we found that a tetracationic heterocycle containing a phenyl-furan-benzimidazole unfused aromatic system, DB340, exhibits pronounced selectivity for the RRE RNA stem-loop from HIV-1. We report here RNA footprinting, spectroscopic analysis, affinity determinations, and initial NMR structural results of the complex. The results indicate that DB340 binds to RRE in a highly structured and cooperative complex at a 2:1 DB340 to RRE ratio. Overlap in the NMR spectra prevents detailed description of binding interactions at this time, but we are able to place DB340 in the RNA minor groove. Additionally, footprinting results and studies with mutant RRE sequences indicate that the internal loop of RRE is required for specific binding of DB340 as with the Rev protein. These results provide exciting new ideas for rational drug design with RNA as is now common with DNA and proteins.

Amino Acid Sequence↗
PubMed2001

Inhibition of the HIV-1 rev-RRE complex formation by unfused aromatic cations.

RNA viruses cause a wide range of human diseases. Development of new agents to target such viruses is an active area of research. Towards this goal, a series of diphenylfuran cations as potential inhibitors of the Rev-RRE complex have been designed and synthesized. Analysis of the interaction of the diphenylfurans with RRE and TAR RNA model systems by gel shift assays indicates that they exhibit both sequence and structure-dependent binding modes. Our results show a strong interaction between the diphenylfuran ring system and RRE bases, while the TAR interactions are much weaker with the compounds that are the best inhibitors of Rev-RRE.

Aminoglycosides↗
PubMed2000

DNA minor groove interactions and the biological activity of 2,5-bis.

2,5-Bis-[4-(N-cyclobutyl-amidino)phenyl] furan and 2,5-bis-[4-(N-cyclohexyl-amidino)phenyl] furan have activity against Pneumocystis carinii and also show cytotoxicity against several tumour cell lines. These activities are correlated with DNA-binding abilities; the crystal structures of complexes with the DNA sequence d(CGCGAATTCGCG) is reported here. Interactions with, and effects on, the DNA minor groove, are found to be factors in the biological properties of these compounds.

Cell Line↗
PubMed2000

A thermodynamic and structural analysis of DNA minor-groove complex formation.

As part of an effort to develop a better understanding of the structural and thermodynamic principles of DNA minor groove recognition, we have investigated complexes of three diphenylfuran dications with the d(CGCGAATTCGCG)(2) duplex. The parent compound, furamidine (DB75), has two amidine substituents while DB244 has cyclopentyl amidine substituents and DB226 has 3-pentyl amidines. The structure for the DB244-DNA complex is reported here and is compared to the structure of the DB75 complex. Crystals were not obtained with DB226 but information from the DB75 and DB244 structures as well as previous NMR results on DB226 indicate that all three compounds bind in the minor groove at the AATT site of the duplex. DB244 and DB75 penetrate to the floor of the groove and form hydrogen bonds with T8 on one strand and T20 on the opposite strand while DB226 forms a complex with fewer interactions. Binding studies by surface plasmon resonance (SPR) yield -delta G degrees values in the order DB244>DB75>DB226 that are relatively constant with temperature. The equilibrium binding constants for DB244 are 10-20 times greater than that for DB226. Isothermal titration calorimetric (ITC) experiments indicate that, in contrast to delta G degrees, delta H degrees varies considerably with temperature to yield large negative delta Cp degrees values. The thermodynamic results, analyzed in terms of structures of the DNA complexes, provide an explanation of why DB244 binds more strongly to DNA than DB75, while DB266 binds more weakly. All three compounds have a major contribution to binding from hydrophobic interactions but the hydrophobic term is most favorable for DB244. DB244 also has strong contributions from molecular interactions in its DNA complex and all of these factors combine to give it the largest-delta G degrees for binding. Although the factors that influence the energetics of minor groove interactions are varied and complex, results from the literature coupled with those on the furan derivatives indicate that there are some common characteristics for minor groove recognition by unfused heterocyclic cations that can be used in molecular design.

Base Sequence↗
PubMed2000

Specific molecular recognition of mixed nucleic acid sequences: an aromatic dication that binds in the DNA minor groove as a dimer.

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

Amidines↗
PubMed1999

Prodrugs for amidines: synthesis and anti-Pneumocystis carinii activity of carbamates of 2,5-bis(4-amidinophenyl)furan.

Syntheses of several carbamate analogues of 2, 5-bis(4-amidinophenyl)furan (1) under mild conditions and their evaluation as prodrugs against Pneumocystis carinii pneumonia (PCP) in an immunosuppressed rat model are described. Thus, nine new bis-carbamates: methoxycarbonyl (2), 2,2,2-trichloroethoxycarbonyl (3), ethylthiocarbonyl (4), benzyloxycarbonyl (5), (4-methyl-2-oxo-1, 3-dioxol-4-en-5-yl)methoxycarbonyl (6), phenoxycarbonyl (7), 4-fluorophenoxycarbonyl (8), 4-methoxyphenoxycarbonyl (9), and (1-acetoxy)ethoxycarbonyl (10) and a bis-carbonate ethoxycarbonyloxy (11) of the bis-amidine 1 have been synthesized and evaluated. The in vivo results show that the 4-fluorophenyl carbamate 8 and the 4-methoxyphenyl carbamate 9 in this series had the best anti-PCP activity by both intravenous and oral administration at a dosage level of 22 mol and 33 micromol/kg/day, respectively. Compounds 3-7 were also more active than the parent drug (1) on oral administration. The acute toxicity usually exhibited by the parent amidine 1 at a dosage level of 22 micromol/kg/day on intravenous administration has been significantly reduced by the prodrug modifications, with the exception of compound 10 which exhibited some toxicity. This report also describes the synthesis of several aryl-alkyl and aryl-aryl carbonates (12-14, 16-23) as efficient reagents for the preparation of carbamate derivatives from bis-arylamidines.

Administration, Oral↗
PubMed1999

2,4-Diphenyl furan diamidines as novel anti-Pneumocystis carinii pneumonia agents.

Dicationic 2,4-bis(4-amidinophenyl)furans 5-10 and 2, 4-bis(4-amidinophenyl)-3,5-dimethylfurans 14 and 15 have been synthesized. Thermal melting studies revealed high binding affinity of the compounds to poly(dA-dT) and to the duplex oligomer d(CGCGAATTCGCG)2. All of the new compounds were effective against Pneumocystis carinii pneumonia in the immunosuppressed rat model with up to 200-fold increase in activity compared to the control compound pentamidine. No toxicity was noted for 5, 7-10 at the dose of 10 micromol/kg/d; however, the isopropyl analogue 7 showed toxicity comparable to pentamidine at the dosage of 20 micromol/kg/d. Dimethylation of the parent compound on the furan ring resulted in reduced activity and increased toxicity.

Amidines↗
PubMed1999

Inhibition of HIV-1 Tat-TAR interaction by diphenylfuran derivatives: effects of the terminal basic side chains.

A series of four biscationic diphenylfuran derivatives was used to investigate drug binding to the transactivation response element (TAR) RNA. The drugs, which are active against the Pneumocystis carinii pathogen (PCP), differ by the nature of the terminal basic side chains. Furimidazoline (DB60) is more potent at inhibiting binding of the Tat protein to TAR than furamidine (DB75) and the amidine-substituted analogues DB244 and DB226. In vivo studies using the fusion-induced gene stimulation (FIGS) assay entirely agree with the in vitro gel mobility shift data. The capacity of the drugs to antagonize Tat binding correlates with their RNA binding properties determined by melting temperature and RNase protection experiments. Footprinting studies indicate that the bulge region of TAR provides the identity element for the diphenylfurans. Access of the drugs to the major groove cavity at the pyrimidine bulge depends on the bulk of the alkylamine substituents. Experiments using TAR mutants show that the bulge of TAR is critical for drug binding but also reveal that the fit of the drugs into the major groove cavity of TAR does not involve specific contacts with the highly conserved residue U23 or the C x G26-39 base pair. The binding essentially involves shape recognition. The results are also discussed with respect to the known activity of the drug against PCP which is the major cause of mortality in AIDS patients. This study provides guidelines for future development of TAR-targeted anti-HIV-1 drugs.

Amidines↗