Diamidines are a class of compounds that target the minor groove of DNA and have antiparasitic and antimicrobial properties. Their mechanism of action has not been fully elucidated, but may include changes in DNA topology. In this study we have investigated such changes using methods of gel electrophoresis including ligation ladders and cyclization assays. We found that topology changes were sequence dependent. Compounds typically caused non-anomalously migrating ATATA sequences to migrate as if they were bent, while A5 sequences that normally migrated anomalously became less so in the presence of certain diamidines. Select compounds induced changes in cyclization efficiency that were also sequence dependent; DB75 significantly abolished cyclization in A5 containing sequences but enhanced it in sequences containing ATATA sites.

SHAPE-DEPENDENT MOLECULAR RECOGNITION OF SPECIFIC SEQUENCES OF DNA BY HETEROCYCLIC CATIONS by YI MIAO Under the Direction of Dr. W. David Wilson ABSTRACT DB921 and DB911 are biphenyl-benzimidazole-diamidine isomers with a central para- and meta-substituted phenyl group, respectively. Unexpectedly, linear DB921 has much stronger binding affinity with DNA than its curved isomer, DB911. This is quite surprising and intriguing since DB911 has the classical curved shape generally required for strong minor groove binding while DB921 clearly does not match the groove shape. Several biophysical techniques including thermal melting (Tm), circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) have been utilized to investigate the interactions between these compounds and DNA. The structure of the DB921-DNA complex reveals that DB921 binds to DNA with a reduced twist of the biphenyl for better fit of DB921 into the minor groove. A bound water molecule complements the curvature of DB921 and contributes for tight binding by forming H-bonds with both DNA and DB921. Structure-affinity relationship studies of a series of DB921 analogs show that the benzimidazole group is one of the key groups of DB921 for its strong binding to the minor groove. Thermodynamic studies show that the stronger binding of DB921 is due to a more favorable binding enthalpy compared to DB911 even though the complex formation with DNA for these compounds are all predominantly entropically driven. DB921 also has more negative heat capacity change than DB911. The initial studies of inhibition of the interaction between an AT hook peptide of HMGA proteins and its target DNA by a set of diamidine AT-minor groove binders using biosensor-SPR technique show that the inhibitory ranking order is consistent with that of binding affinity and linear-shaped DB921 still has excellent inhibitory effects. These heterocyclic cations rapidly inhibit the binding of DBD2 peptide to the DNA and may only block the specific AT binding of the peptide without hindering the non-specific binding interaction. The results of this project have shown that DB921 represents a new novel effective minor groove binder that does not fit the traditional model and is a potential inhibitor for DNA/protein complexes. INDEX WORDS: Molecular recognition, DNA binding, Minor groove binding, Linear shape, Compound curvature, Binding affinity, Binding kinetics, Thermodynamics, Surface plasmon resonance, Isothermal titration calorimetry, Inhibition

DNA minor groove binding compounds have had limited therapeutic uses, in part due to problems with sequence specificity. A two-site model has been developed to enhance specificity, in which compounds bind to two short AT sites separated by one or two GC base pairs. Using thermal melting, heterocyclic dications with this capability were tested with various oligonucleotides for binding affinity and specificity. Compounds of interest were further probed using circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling. Several compounds were found to “jump” a GC base pair, binding to AT sites in the minor groove of DNA with a two-site recognition mode. One compound was also found to recognize a single intervening GC base pair. Compounds with terminal, non-polar amidine extensions were found to have increased DNA binding compared to analogs with terminal amidines. This unique, two-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.

PURPOSE: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. EXPERIMENTAL DESIGN: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. RESULTS: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. CONCLUSIONS: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.

Recently, there has been increasing focus toward the development of small molecules designed to target a specific sequences of double stranded DNA for therapeutic purposes1. Minor groove binding compounds have been shown to be capable of selectivity target GC sites in AT tract DNA2. In this research, binding selectivity was investigated using absorption, fluorescence and circular dichroic properties of selected DB minor groove binders in the presence of two unique DNA sequences. Further insight was gained by comparing the electrostatic potential maps and optimized structures of the compounds of interest. Using the results presented, potential selective minor groove binders can be selected for further investigation and kinetic studies.

Indiana University-Purdue University Indianapolis (IUPUI) / The DNA minor groove continues to be an important biological target in the development of anticancer, antiviral, and antimicrobial compounds. Among agents that target the minor groove, studies of well-established benzimidazole-based DNA binders such as Hoechst 33258 have made it clear that the benzimidazole-amidine portion of these molecules promotes an efficient, site-selective DNA association. Building on the beneficial attributes of existing benzimidazole-based DNA binding agents, a series of benzimidazole-amino acid conjugates was synthesized to investigate their DNA recognition and binding properties. In this series of compounds, the benzimidazole-amidine moiety was utilized as a core DNA “anchoring” element accompanied by different amino acids to provide structural diversity that may influence DNA binding affinity and site-selectivity. Single amino acid conjugates of benzimidazole-amidines were synthesized, as well as a series of conjugates containing 20 dipeptides with the general structure Xaa-Gly. These conjugates were synthesized through a solid-phase synthetic route building from a resin-bound amino acid (or dipeptide). The synthetic steps involved: (1) the coupling of 4-formylbenzoic acid to the resin-bound amino acid (via diisopropylcarbodiimide and hydroxybenzotriazole); followed by (2) introduction of a 3,4-diaminobenzamidoxime in the presence of 1,4-benzoquinone to construct the benzimidazole ring; and, finally, (3) reduction of the resin-bound amidoxime functionality to an amidine via treatment with 1M SnCl2•2H2O in DMF before cleavage of final product from the resin. The synthetic route developed and employed was simple and straightforward except for the final reduction that proved to be very arduous. All target compounds were obtained in good yield (based upon weight), averaging 73% mono-amino acid and 78% di-amino acid final compound upon cleavage from resin. Ultimately, the DNA binding activities of the amino acid-benzimidazole-amidine conjugates were analyzed using a fluorescent intercalator displacement (FID) assay and calf thymus DNA as a substrate. The relative DNA binding affinities of both the mono- and di-amino acid-benzimidazole-amidine conjugates were generally weaker than that of netropsin and distamycin with the dipeptide conjugates showing stronger binding affinities than the mono-amino acid conjugates. The dipeptide conjugates containing amino acids with positively charged side chains, Lys-Gly-BI-(+) and Arg-Gly-BI-(+), showed the strongest DNA binding affinities amongst all our synthesized conjugates.

No / The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Computer-aided drug discovery involves the application of computer science and programming to solve chemical and biological problems. Specifically, the QSAR (Quantitative Structure Activity Relationships) methodology is used in drug development to provide a rational basis of drug synthesis, rather than a trial and error approach. Molecular dynamics (MD) studies focus on investigating the details of drug-target interactions to elucidate various biophysical characteristics of interest. Infectious diseases like Trypanosoma brucei rhodesiense (TBR) and P. falciparum (malaria) are responsible for millions of deaths annually around the globe. This necessitates an immediate need to design and develop new drugs that efficiently battle these diseases. As a part of the initiatives to improve drug efficacy QSAR studies accomplished the formulation of chemical hypothesis to assist development of drugs against TBR. Results show that CoMSIA 3D QSAR models, with a Pearson’s correlation coefficient of 0.95, predict a compound with meta nitrogens on the phenyl groups, in the combinatorial space based on a biphenyl-furan diamidine design template, to have higher activity against TBR relative to the existing compound set within the same space. Molecular dynamics study, conducted on a linear benzimidazole-biphenyl diamidine that has non-classical structural similarity to earlier known paradigms of minor groove binders, gave insights into the unique water mediated interactions between the DNA minor groove and this ligand. Earlier experiments suggested the interfacial water molecules near the terminal ends of the ligand to be responsible for the exceptianlly high binding constant of the ligand. Results from MD studies show two other modes of binding. The first conformation has a single water molecule with a residency time of 6ns (average) that is closer to the central part of the ligand, which stabilizes the structure in addition to the terminal water. The second conformation that was detected had the ligand completely away from the floor of the minor groove, and hydrogen bonded to the sugar oxygens.

Cell surface saccharides are involved in a variety of essential biological events. Fluorescent sensors for saccharides can be used for detection, diagnosis, analysis and monitoring of pathological processes. The boronic acid functional group is known to bind strongly and reversibly to compounds with diol groups, which are commonly found on saccharides. Sensors that have been developed for the purpose of saccharide recognition have shown great potential. However, they are very hydrophobic and this lack of essential water-solubility makes them useful in biological applications. The first section of this dissertation details the process of developing water-soluble saccharide sensors that change fluorescent properties upon binding to saccharides. The second section of the dissertation focuses on the development of DNA-minor groove binders as antiparasitical agents. Parasitical diseases comprise some of the world’s largest health problems and yet current medication and treatments for these parasitical diseases are often difficult to administer, costly to the patients, and have disruptive side effects. Worse yet, these parasites are developing drug resistance, thus creating an urgent need for new treatments. Dicationic molecules constitute a class of antimicrobial drug candidates that possess high activity against various parasites. The second section details the development of a series of di-cationic agents that were then screened in in vitro activities against parasitical species.

The development of small-molecules which target nucleic acids, more specifically the minor groove of DNA, in a sequence specific manner and control gene expression are currently being investigated as potential therapeutic compounds for the treatment of various diseases, including cancer, as well as viral and bacterial infections. The naturally occurring compounds netropsin and distamycin have been shown to demonstrate antitumor and antibacterial properties. Currently, there are synthetic efforts to create pyrrole and imidazole-containing polyamide derivatives of netropsin and distamycin that show potential as medicinal agents. Synthetic pyrrole and imidazole-containing polyamides are potentially useful for targeting and modulating the expression of genes, including those associated with cancer cell growth. The key challenges that must be overcome to realize this goal of using synthetic polyamides in the treatment of disease are the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. As part of a systematic study within the authors’ laboratory, our goal is to develop polyamides which can be synthesized readily yet possess excellent sequence specificity, stronger binding affinity, high solubility in biological media and enhanced cell penetration and nuclear localization properties. There is a need to develop a library of modified polyamides which target DNA and exhibit improved biological properties. The present study is a systematic examination of the binding properties of various modified synthetic polyamide compounds. The synthetic polyamide derivatives presented have more potential as therapeutic candidates over other synthetic polyamides because of their increased water solubility, smaller molecular weights, and molecular design, thus, allowing them to penetrate into cells and localize in the nucleus.