Oligonucleotide Delivery Systems for Camptothecins

Camptothecin drugs are stabilized in their antitumor active lactone form by complexation with an oligonucleotide including RNA or catalytic RNA. The oligonucleotide-camptothecin drug complex may be incorporated within a macromolecular assembly including both viral and non-viral oligonucleotide vectors. The invention allows combination gene and camptothecin drug therapy

Ligand Selectivity in the Recognition of Protoberberine Alkaloids by Hybrid-2 Human Telomeric G-Quadruplex: Binding Free Energy Calculation, Fluorescence Binding, and NMR Experiments

The human telomeric G-quadruplex (G4) is an attractive target for developing anticancer drugs. Natural products protoberberine alkaloids are known to bind human telomeric G4 and inhibit telomerase. Among several structurally similar protoberberine alkaloids, epiberberine (EPI) shows the greatest specificity in recognizing the human telomeric G4 over duplex DNA and other G4s. Recently, NMR study revealed that EPI recognizes specifically the hybrid-2 form human telomeric G4 by inducing large rearrangements in the 50-flanking segment and loop regions to form a highly extensive four-layered binding pocket. Using the NMR structure of the EPI-human telomeric G4 complex, here we perform molecular dynamics free energy calculations to elucidate the ligand selectivity in the recognition of protoberberines by the human telomeric G4. The MM-PB(GB)SA (molecular mechanics-Poisson Boltzmann/Generalized Born) Surface Area) binding free energies calculated using the Amber force fields bsc0 and OL15 correlate well with the NMR titration and binding affinity measurements, with both calculations correctly identifying the EPI as the strongest binder to the hybrid-2 telomeric G4 wtTel26. The results demonstrated that accounting for the conformational flexibility of the DNA-ligand complexes is crucially important for explaining the ligand selectivity of the human telomeric G4. While the MD-simulated (molecular dynamics) structures of the G-quadruplex-alkaloid complexes help rationalize why the EPI-G4 interactions are optimal compared with the other protoberberines, structural deviations from the NMR structure near the binding site are observed in the MD simulations. We have also performed binding free energy calculation using the more rigorous double decoupling method (DDM); however, the results correlate less well with the experimental trend, likely due to the difficulty of adequately sampling the very large conformational reorganization in the G4 induced by the protoberberine binding

Structure of a two-G-tetrad intramolecular G-quadruplex formed by a variant human telomeric sequence in K+ solution: insights into the interconversion of human telomeric G-quadruplex structures

Human telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. The telomeric sequence shows intrinsic structure polymorphism. Here we report a novel intramolecular G-quadruplex structure formed by a variant human telomeric sequence in K+ solution. This sequence forms a basket-type intramolecular G-quadruplex with only two G-tetrads but multiple-layer capping structures formed by loop residues. While it is shown that this structure can only be detected in the specifically truncated telomeric sequences without any 5′-flanking residues, our results suggest that this two-G-tetrad conformation is likely to be an intermediate form of the interconversion of different telomeric G-quadruplex conformations

NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region

BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. Here we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K(+) solution. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the experimental mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to determine the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G(3)NG(3) sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design

Structure of the Hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K+ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K+ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres

Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K+. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel–antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5′- and 3′-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K+. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K+. The unique human telomeric G-quadruplex structure formed in K+ suggests that it can be specifically targeted for anticancer drug design

I-Motif Structures Formed in the Human c-MYC Promoter Are Highly Dynamic–Insights into Sequence Redundancy and I-Motif Stability

The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3′ consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine+–cytosine base pairs are not always necessary for an intramolecular I-motif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III1 sequence and appears to provide stability to the c-MYC I-motif

High-resolution NMR spectroscopic analysis of anticancer drugs, DNA and their interactions: 1. Platinum anticancer compounds - DNA interactions. 2. Anthracycline drugs - DNA interactions and modified DNA

Chemotherapy with anticancer drugs is one of the main method of cancer treatment. The exploitation of the stereochemical interactions between anticancer drugs and DNA is of great importance for the ultimate clinical advances of cancer chemotherapy, which needs the detailed structural knowledge of DNA, drugs, and their interactions. Cisplatin is one of the most effecient and widely used anticancer drugs in the world. Extensive effort has been devoted to designing the new better anticancer platinum compounds. The structural studies on interactions of two anticancer platinum compounds, cisplatin and the third-generation bisplatinum compound 1,1/t,t, with DNA are described in this thesis. The structure of an intrastrand cisplatin-crosslinked didentate DNA duplex consisting of d(CCTG\rm\sp*G\sp*TCC) and its complement d(GGACCAGG) is determined by NMR spectroscopy. The refined duplex is unwound (\sim{-}21\sp\circ) and kinked ({\sim}58\sp\circ) toward the major groove at the \rm G\sp*G\sp* site and the minor groove is significantly widened. The stability of the major intrastrand cisplatin-G\rm\sp*pG\sp* adduct has been studied and this intrastrand cisplatin-crosslinked adduct appears to be converted into an interstrand crosslink adduct. Three palindromic DNA oligonucleotides, each having a single intrastrand cisplatin crosslinked at GpG site, have also been studied by NMR spectroscopy. The structural consequence of the incorporation of the \rm G\sp*G\sp* lesions into palindromic sequences is dependent on the location of the lesion sites in the sequence. Such alternative structural distortions may be relevant in understanding the protein recognition of the cisplatin-induced lesions. A new anticancer bisplatinum compound 1,1/t,t exhibits excellent cytotoxicity towards cisplatin-resistant cancer cells. The structure of the interstrand adduct of 1,1/t,t with a palindromic DNA oligomer CATGCATG has been determined by NMR spectroscopy. Upon platination by 1,1/t,t, the DNA octamer forms a novel hairpin structure with the platinated G\sb4 residue adopting a syn conformation and with the guanine base in the minor groove. Two such hairpins stack end-over-end and are linked together by the butanediamine tether to form a dumbbell structure. Such unusual structural distortion induced by the bisplatinum compound is completely different from that of the anticancer drug cisplatin-DNA adduct and may provide clues to explain the distinct biological activities of the two compounds.Anthracycline antibiotics are important anticancer intercalative drugs. The solution structures of anticancer anthracycline drugs aclacinomycin A and B, nogalamycin and disnogalamycin, complexed to a DNA hexamer have all been determined by high resolution NMR spectroscopy. Structural modification of DNA through covalent interactions have significant functional consequences and/or anticancer activities. Structural analysis of the C\sp2-methyl-hypoxanthine:Cytosine base pair and O\sp6-ethyl-Guanine:Cytosine base pair in B-DNA help understand their biological functions.U of I OnlyETDs are only available to UIUC Users without author permissio

Molecular Recognition of the Hybrid-Type G-Quadruplexes in Human Telomeres

G-quadruplex (G4) DNA secondary structures formed in human telomeres have been shown to inhibit cancer-specific telomerase and alternative lengthening of telomere (ALT) pathways. Thus, human telomeric G-quadruplexes are considered attractive targets for anticancer drugs. Human telomeric G-quadruplexes are structurally polymorphic and predominantly form two hybrid-type G-quadruplexes, namely hybrid-1 and hybrid-2, under physiologically relevant solution conditions. To date, only a handful solution structures are available for drug complexes of human telomeric G-quadruplexes. In this review, we will describe two recent solution structural studies from our labs. We use NMR spectroscopy to elucidate the solution structure of a 1:1 complex between a small molecule epiberberine and the hybrid-2 telomeric G-quadruplex, and the structures of 1:1 and 4:2 complexes between a small molecule Pt-tripod and the hybrid-1 telomeric G-quadruplex. Structural information of small molecule complexes can provide important information for understanding small molecule recognition of human telomeric G-quadruplexes and for structure-based rational drug design targeting human telomeric G-quadruplexes

Optimization of Growth Conditions in Minimal Media for Production of Nucleolin Protein

Class of 2016 AbstractObjectives: The purpose of this experiment is to compare NUC1234 expression levels from E. coli cultures by manipulating cell cultures according to the optical density (OD595) at which protein expression is induced, and the harvest time after induction. Methods: E. coli BL21(DE3) cells transformed with DNA to produce NUC1234 were plated and then grown in minimal media for protein expression. They were induced at a variety of optical densities and harvested at different times post-induction. Protein quantities from each sample were then compared using a protein determination assay and an SDS-PAGE gel. Results: The sample induced at an OD595 of 0.5 yielded a lower concentration of protein (0.874409mg/ml) compared to other samples; however, it visualized as a stronger band in the SDS-PAGE gel. The sample harvested at 6 hours yielded the largest protein concentration (1.347215mg/ml) among all active samples and appeared as bold as, or bolder than, those harvested at other times. Conclusions: The results of the protein assay study and gel visualization suggest that the optimal conditions for the production of NUC1234 are growth to an OD595 of 0.5 before induction of protein expression and a harvest time of 6 hours after induction.This item is part of the Pharmacy Student Research Projects collection, made available by the College of Pharmacy and the University Libraries at the University of Arizona. For more information about items in this collection, please contact Jennifer Martin, Librarian and Clinical Instructor, Pharmacy Practice and Science, [email protected]