Complexation of norfloxacin with DNA in the presence of caffeine

1H NMR spectroscopy (500 MHz) has been used to quantify the complexation of the antibacterial antibiotic Norfloxacin (NOR) with DNA in the presence of Caffeine (CAF). Separate studies have been made for the self-association of NOR, its hetero-association with CAF and complexation with a model self-complementary DNA tetramer, 5′-d(TpGpCpA), in order to determine the equilibrium parameters (induced chemical shifts, association constants, enthalpy and entropy) of the two-component mixtures to aid the analysis of the three-component systems. Investigations of the self-association of NOR and its hetero-association with CAF show that the aggregation of NOR molecules and association with CAF in solution are driven by the stacking of aromatic chromophores. The complexation of NOR with d(TGCA) has been analysed in terms of intercalation with the double-stranded form and non-intercalative binding with the single-stranded form of DNA. Investigations of the competitive binding of NOR and CAF with DNA show that at physiological concentrations of NOR (μM) and CAF (mM) the dominant mechanism influencing the affinity of NOR with DNA is the displacement of bound NOR molecules from DNA due to CAF–DNA complexation (i.e. the protector action of Caffeine)

CONTRIBUTIONS OF TM5, ECL3 AND TM6 OF HUMAN BCRP TO ITS OLIGOMERIZATION ACTIVITIES AND TRANSPORT FUNCTIONS

Indiana University-Purdue University Indianapolis (IUPUI)Human BCRP is one of the major ATP-binding cassette transporters involved in the development of multidrug resistance in cancer chemotherapy. Overexpression of BCRP in the tumor cell plasma membrane and apical membrane of the gastrointestinal tract leads to decreased intracellular accumulation of various anticancer drugs as well as reduced drug bioavailability. BCRP has been shown to exist on the plasma membrane as higher forms of homo-oligomers. In addition, the oligomerization domain of BCRP has been mapped to the carboxyl-terminal TM5-ECL3-TM6 and this truncated domain, when co-expressed with the full-length BCRP, displays a dominant inhibitory activity on BCRP function. Thus, the oligomerization of BCRP could be a promising target in reversing multidrug resistance mediated by BCRP. To further dissect the oligomerization domains of human BCRP and test the hypothesis that TM5, ECL3, and TM6 each plays a role in BCRP oligomerization and function, we engineered a series of BCRP domain-swapping constructs with alterations at TM5-ECL3-TM6 and further generated HEK293 cells stably expressing wild-type or each domain-swapping construct of BCRP. Using co-immunoprecipitation and chemical cross-linking, we found that TM5, ECL3, and TM6 all appear to partially contribute to BCRP oligomerization, which are responsible for the formation of oligomeric BCRP. However, only TM5 appears to be a major contributor to the transport activity and drug resistance mediated by BCRP, while ECL3 or TM6 is insufficient for BCRP functions. Taken together, these findings suggest that homo-oligomeric human BCRP may be formed by the interactions among TM5, ECL3 and TM6, and TM5 is a crucial domain for BCRP functions and BCRP-mediated drug resistance. These findings may further be used to explore targets for therapeutic development to reverse BCRP-mediated drug resistance and increase the bioavailability of anti-cancer drugs for better treatment of multidrug resistant cancers

Dynamics and oligomerisation of ABCG2 investigated using various fluorescence techniques

The human ABCG2 (second member of ABC transporter G-subfamily) is an important ATP-dependent exporter in the body with broad substrate specificity including xenobiotics (e.g. anticancer agents) and endogenous compounds (e.g. sterols and lipids). ABCG2 was first discovered in a multidrug resistant breast cancer cell line and it is suggested to cause resistance to chemotherapy in certain cancers such as acute myeloid leukaemia and small cell lung cancer. Physiologically, ABCG2 is found in the protective sanctuary sites of the body, for instance the gut and blood-brain-barrier, affecting pharmacokinetics and treatment efficacies of small molecule drugs. Structurally, the polypeptide chain of ABCG2 contains a single nucleotide binding domain and a single transmembrane domain, which is half the number of domains required for a fully functional ABC transporter. Although many have suggested that ABCG2 function as dimer or higher order oligomer, studies so far have been unable to convincingly address the oligomeric state of ABCG2. We aim to bridge this knowledge gap by resolving the oligomerisation of ABCG2 using fluorescence techniques in mammalian cells. The expression and function of fluorescent proteins tagged ABCG2 were verified using confocal imaging and fluorescence accumulation assays, prior to the fluorescence studies. As the membrane dynamics of ABCG2 are unknown, we first measured the diffusion of ABCG2 in live HEK293T cells using fluorescence recovery after photobleaching (FRAP) microscopy, in comparison to membrane localised fluorescent proteins and a full length (i.e 4 domain) ABC transporter (ABCC4). We also demonstrated oligomerisation of ABCG2 by measuring a specific increase in fluorescence resonance energy transfer (FRET) efficiency between CFP- and YFP-tagged ABCG2 expressed in live HEK293T cells in comparison to non-specific control interactions, including with the adenosine A3 receptor. Subsequently, we employed high resolution and single particle fluorescence techniques to resolve the oligomeric organisation of ABCG2. First, fluorescence fluctuations of tagged ABCG2 within a confocal volume, positioned on the upper plasma membrane, were measured using fluorescence correlation spectroscopy (FCS) at “single molecule” resolution. Photon counting histogram (PCH) analysis of the FCS measurements was performed to determine the molecular brightness of the fluorescent species detected within the confocal volume. Using CD86 and CD28 as monomer and oligomer controls respectively, PCH analysis demonstrated higher order oligomer formation of ABCG2, with increased brightness (up to 4-fold) observed for both ABCG2 and CD28, compared to CD86. For validation of the oligomeric organisation of ABCG2, we acquired a series of single particle photobleaching images of cells expressing fluorescent protein tagged ABCG2 using total internal reflection fluorescence (TIRF) microscopy at the lower plasa membrane, and employed a step detection algorithm to identify the number of photobleaching steps within the distinguished fluorescent spots. Statistical modelling of the photobleaching step frequency histogram provided credible evidence of tetrameric organisation of ABCG2 in the plasma membrane. The findings and methodology presented in this study have provided further insights into the membrane dynamics and oligomerisation of ABCG2. This could lead to future studies to explore new pharmacological avenues that target the oligomerisation interfaces of ABCG2

Dynamics and oligomerisation of ABCG2 investigated using various fluorescence techniques

The human ABCG2 (second member of ABC transporter G-subfamily) is an important ATP-dependent exporter in the body with broad substrate specificity including xenobiotics (e.g. anticancer agents) and endogenous compounds (e.g. sterols and lipids). ABCG2 was first discovered in a multidrug resistant breast cancer cell line and it is suggested to cause resistance to chemotherapy in certain cancers such as acute myeloid leukaemia and small cell lung cancer. Physiologically, ABCG2 is found in the protective sanctuary sites of the body, for instance the gut and blood-brain-barrier, affecting pharmacokinetics and treatment efficacies of small molecule drugs. Structurally, the polypeptide chain of ABCG2 contains a single nucleotide binding domain and a single transmembrane domain, which is half the number of domains required for a fully functional ABC transporter. Although many have suggested that ABCG2 function as dimer or higher order oligomer, studies so far have been unable to convincingly address the oligomeric state of ABCG2. We aim to bridge this knowledge gap by resolving the oligomerisation of ABCG2 using fluorescence techniques in mammalian cells. The expression and function of fluorescent proteins tagged ABCG2 were verified using confocal imaging and fluorescence accumulation assays, prior to the fluorescence studies. As the membrane dynamics of ABCG2 are unknown, we first measured the diffusion of ABCG2 in live HEK293T cells using fluorescence recovery after photobleaching (FRAP) microscopy, in comparison to membrane localised fluorescent proteins and a full length (i.e 4 domain) ABC transporter (ABCC4). We also demonstrated oligomerisation of ABCG2 by measuring a specific increase in fluorescence resonance energy transfer (FRET) efficiency between CFP- and YFP-tagged ABCG2 expressed in live HEK293T cells in comparison to non-specific control interactions, including with the adenosine A3 receptor. Subsequently, we employed high resolution and single particle fluorescence techniques to resolve the oligomeric organisation of ABCG2. First, fluorescence fluctuations of tagged ABCG2 within a confocal volume, positioned on the upper plasma membrane, were measured using fluorescence correlation spectroscopy (FCS) at “single molecule” resolution. Photon counting histogram (PCH) analysis of the FCS measurements was performed to determine the molecular brightness of the fluorescent species detected within the confocal volume. Using CD86 and CD28 as monomer and oligomer controls respectively, PCH analysis demonstrated higher order oligomer formation of ABCG2, with increased brightness (up to 4-fold) observed for both ABCG2 and CD28, compared to CD86. For validation of the oligomeric organisation of ABCG2, we acquired a series of single particle photobleaching images of cells expressing fluorescent protein tagged ABCG2 using total internal reflection fluorescence (TIRF) microscopy at the lower plasa membrane, and employed a step detection algorithm to identify the number of photobleaching steps within the distinguished fluorescent spots. Statistical modelling of the photobleaching step frequency histogram provided credible evidence of tetrameric organisation of ABCG2 in the plasma membrane. The findings and methodology presented in this study have provided further insights into the membrane dynamics and oligomerisation of ABCG2. This could lead to future studies to explore new pharmacological avenues that target the oligomerisation interfaces of ABCG2

Application of computational methods for predicting protein interactions

Protein interactions with other proteins or small molecules are critical to most physiological processes. These interactions may be characterized experimentally, but this can be time consuming and expensive; computational methods for predicting how two proteins interact, or which regions of a protein are most favorable for binding, are thus valuable tools for understanding how proteins of interest function, and have applications in drug discovery and identifying proteins of therapeutic interest. The ClusPro and FTMap algorithms for docking or solvent mapping, respectively, model protein-protein and protein-small molecule interactions, and can be used to identify the most likely orientations of a protein complex or the regions on a protein surface with the greatest propensity for binding. Here we describe three applications of ClusPro and FTMap. ClusPro was used to develop a method for determining whether a protein-protein interface is biologically relevant, by docking the proteins and comparing the results to the given interface; a larger number of near-native structures--which have interfaces similar to that of the given complex--was found to correspond to a greater probability that an interface is biological. In another project, ClusPro was used to predict whether a mutation in a multimeric complex would trigger the formation of a supramolecular assembly, based on how often that mutated residue appeared in the interfaces of the docking results; if a mutation caused such a residue to be present in the docked interfaces more often, in comparison to those of the wild-type structure, then it was likely to induce self-assembly. FTMap was used to detect and analyze the druggability of potential allosteric sites in kinases, with mapping performed on all available kinase structures to identify and determine the potential binding affinity of binding hot spots located outside of the active site. Discrimination of proteins as dimers or monomers was implemented as an addition to the ClusPro server, ClusPro-DC, and the results of the druggability analysis of kinases were organized into an online resource, the Kinase Atlas.2019-02-20T00:00:00

DESIGN AND SYNTHESIS OF NATURAL COMPOUNDS ANALOGUES WITH CYTOTOXIC ACTIVITY

This Ph.D. work was carried out as a part of project focusing on the search of new naturally-derived topoisomerase I inhibitors as potential antitumor compounds. Topoisomerase I is the target of anticancer drug campthotecin (CPT). First generation analogues of CPT are already approved as anticancer agents in human therapy, and several second and third generation derivatives are well advanced in clinical trials. Nevertheless, identification of new campthotecins or alternative molecular scaffolds endowed with similar properties is highly desirable because CPT derivatives were shown to be worth of improvement, above all for the unstable \uf061-hydroxylactone moiety. First, new CPT derivatives were planned and synthesized, in an effort to decrease the toxicity and improve the E ring stability of the natural alkaloid. The study was undertaken to explore new structural changes at the E ring, while keeping a lactone moiety in this portion of the molecule. Thus, new analogues with an inverted lactone ring were designed and synthesized. The compounds retained a good cytotoxic activity on human non-small cancer cells H460. At the same time, the search of new scaffolds showing topoisomerase I inhibitory activity was developed. Attention focused on a new class of marine alkaloids called lamellarins that have been recently studied as Topo I inhibitors. In this Ph.D. work new lamellarin analogues were designed. Molecular models of the ternary complex formed between the DNA-Topo I ensemble and the new derivatives were built, and allowed to optimize the scaffold structure. Thus the synthesis of a number of new compounds endowed with cytotoxicity in the low micromolar range was accomplished

Deciphering the Details of RNA Aminoglycoside Interactions: From Atomistic Models to Biotechnological Applications

Aminoglycosides are a class of antibiotics functioning through binding to 16S rRNA A-site and inhibiting the bacterial translation. However, the continuous emergence of drug-resistant strains makes the development of new and more potent antibiotics necessary. Aminoglycosides are also known to interact with various biologically crucial RNA molecules other than 16S rRNA A-site and inhibit their functions. As a result, they are considered as the single most important model to understand the principles of RNA small molecule recognition. The detailed understanding of these interactions is necessary for the development of novel antibacterial, antiviral or even anti-oncogenic agents. In our studies, we have studied both the natural aminoglycoside targets like Rev responsive element (RRE), trans-activating region (TAR) of HIV-1 and thymidylate synthase mRNA 5\u27 untranslated (UTR) region as well as the in vitro selected neomycin, tobramycin and kanamycin RNA aptamers. By this way, we think we have covered a variety of binding pockets to figure out the critical nucleic acid residues playing essential role in aminoglycoside recognition. Along with all these RNAs, we studied more than 10 aminoglycoside ligands to pinpoint the chemical groups in close contact with RNAs. To determine thermodynamic parameters for these interactions, we utilized isothermal titration calorimetry (ITC) assay by which we found that the majority of these interactions are enthalpy driven. More specifically, RNA aminoglycoside interactions are mainly derived by electrostatic and hydrogen binding interactions. Our studies indicated that the amino groups on the first ring of the aminoglycosides are essential for high affinity binding whereas having bulky groups on ring II sterically eliminate their interactions with RNAs. RNA binding trend of aminoglycosides are as follows: neomycin-B \u3e ribostamycin \u3e kanamycin-B \u3e tobramycin \u3e paromomycin \u3e sisomicin \u3e gentamicin \u3e kanamycin-A \u3e geneticin \u3e amikacin \u3e netilmicin. Aminoglycoside binding to the aptamer was shown highly buffer dependent. This phenomenon was analyzed in five different buffers and found that cacodylate-based buffer changes the specificity of the aptamer. In addition to ITC, we have used molecular docking to specifically find out the chemical groups in these interactions. We have specified the nucleic acid residues interacting with aminoglycosides. In parallel, molecular dynamics (MD) simulations of neomycin RNA aptamer with neomycin-B in an all-atom platform in GROMACS were carried out. The results showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin-B aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Also, A16 was found to be flexible, which was confirmed by 2AP fluorescence studies. In this analysis, the buffer dependence was also confirmed against neomycin-B, ribostamycin and paromomycin. One of the challenges in therapeutics is the emergence of resistant cells. They become reistant to the drugs via changing the target site, or enzymatically modifying the drug, or producing drug pumps to export the drugs. To overcome the very last challenge, we are utilizing RNA-aminoglycoside partners to keep high intracellular drug concentration and increase the efficacy of aminoglycosides against bacteria. We called the system as DRAGINs (Drug binding aptamers for growing intracellular numbers). We express these RNAs in bacteria and detect their growth rate in order to evaluate their response to different concentration of aminoglycosides. In this study, we found that we could successfully decrease the IC50 values by 2 to 5 fold with the help of aminoglycoside-binding RNA aptamers. Finally, we are mathematically modeling the effect of aptamers on IC50 values of drugs with the use of four-compartment model. In our research group, we are utilizing these RNA-aminoglycoside partners to develop tags for detecting RNA in vivo and in real time. We called this system as intracellular multiaptamer genetic tags (IMAGEtags)

Targeting receptors and DNA secondary structures with small molecules and calix[4]arene conjugates

This body of research is focused on developing calixarene conjugates targeting i-motif structures and integrin receptors. In Chapter 1, a general background of DNA secondary structures and i-motifs was described, mainly focused on the biological relevance, the experimental techniques and known interacting ligands in i-motif studies. In Chapter 2, a high-throughput i-motif ligand screen method was established, based on fluorescent intercalator displacement. Thiazole orange was used as the fluorescent intercalator in the screen against human telomeric i-motif. Its binding was studied using several spectroscopic techniques. A compound library was used to evaluate the newly developed high-throughput screen method using a plate reader and tobramycin was found as the most valuable hit compound in this screen. In Chapter 3, a family of water soluble, DNA-targeting calixarene conjugates were synthesis and characterised. They were functionalised with and DNA-binding moiety on the lower rim. It was found that two of the calixarene conjugates, 28 and 54, were able to condense G-quadruplex and i-motif forming sequences from human telomere and c-MYC promoter. The calixarene induced condensation was stable under acidic pH, but behave reversible by heating at neutral pH. Chapter 4 discussed the possibility to develop a calixarene based tumour recognising ligand. In order to achieve tumour targeting, a novel cyclic RGD peptide bearing alkyne was made and tested in the ‘click’ reaction. Later on, a route to conjugate the novel cyclic RGD peptide with a calixarene tethered with a fluorescent tag was established. It was found that a linker between calixarene and peptide moiety or the copper (I) catalyst was crucial in making this calixarene-peptide conjugate. Chapter 5 described the experimental procedures used in Chapter 2, 3 and 4. Chapter 6 summarised the key findings in Chapter 2, 3 and 4, as well as proposing the future work for all three chapters

The expression and localisation of membrane transporters and P450 enzymes along the longitudinal and crypt-villus axes of the rat intestine and their response to oral imatinib

PhD ThesisA reduction in oral bioavailability of a wide range of drug compounds may occur following efflux of drug substrates into the intestinal lumen mediated by ATP dependent ABC transporters MDR1, MRP2 and BCRP and/or by enteric metabolism of drug compounds by CYP450 enzymes. Furthermore, inter-individual variability in the expression and function of key drug disposition proteins, which may occur following a regime of drug treatment, has the potential to cause altered pharmacokinetic profiles leading to failure of therapy. This thesis aimed to establish mRNA and protein expression of key drug transporter proteins and CYP450 enzymes both along the length of the intestine and across the crypt-villus axis using NanoString mRNA technology and immunohistochemistry. Key findings include; a) evidence for cytoprotection of the crypt cells distinct from enterocytes, b) a lack of correlation between Mrp2 mRNA and protein expression in the rat small intestine, with a paucity of brush-border protein in all regions, c) using the M70 antibody, Bcrp protein was immunolocalised to the lateral cell membranes of cryptal epithelial in the duodenum, jejunum and ileum with no apparent protein expression above the crypt-villus junction. Oral administration of the tyrosine kinase inhibitor imatinib mesylate was found to cause significant induction of Cyp1a1 mRNA expression with maximal induction in ileum samples showing an average 169-fold increase in expression. Immunohistochemistry indicates an associated increase in Cyp1a1 protein levels. In addition intestinal exposure to imatinib was found to cause significant changes in mRNA expression levels of genes including Mdr1a and Bcrp. This thesis presents key differences with regards to protein localisation of ABC transporters proposed to play an important role in conferring intestinal drug resistance between rat and human tissues and highlights the high levels of complexity associated with oral drug delivery and intestinal absorption