John O. Trent

Discovery and Development of the G-rich Oligonucleotide AS1411 as a Novel Treatment for Cancer

Certain guanine-rich (G-rich) DNA and RNA molecules can associate intermolecularly or intramolecularly to form four stranded or quadruplex structures, which have unusual biophysical and biological properties. Several synthetic G-rich quadruplex-forming oligodeoxynucleotides have recently been investigated as therapeutic agents for various human diseases. We refer to these biologically active G-rich oligonucleotides as aptamers because their activities arise from binding to protein targets via shape-specific recognition (analogous to antibody-antigen binding). As therapeutic agents, the G-rich aptamers may have some advantages over monoclonal antibodies and other oligonucleotide-based approaches. For example, quadruplex oligonucleotides are non-immunogenic, heat stable and they have increased resistance to serum nucleases and enhanced cellular uptake compared to unstructured sequences. In this review, we describe the characteristics and activities of G-rich oligonucleotides. We also give a personal perspective on the discovery and development of AS1411, an antiproliferative G-rich phosphodiester oligonucleotide that is currently being tested as an anticancer agent in Phase II clinical trials. This molecule functions as an aptamer to nucleolin, a multifunctional protein that is highly expressed by cancer cells, both intracellularly and on the cell surface. Thus, the serendipitous discovery of the G-rich oligonucleotides also led to the identification of nucleolin as a new molecular target for cancer therapy.

Stability and kinetics of G-quadruplex structures

In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence.

Antiproliferative Activity of G-rich Oligonucleotides Correlates with Protein Binding

Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotide-binding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.

Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth

6-Phosphofructo-1-kinase, a rate-limiting enzyme of glycolysis, is activated in neoplastic cells by fructose-2,6-bisphosphate (Fru-2,6-BP), a product of four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes (PFKFB1-4). The inducible PFKFB3 isozyme is constitutively expressed by neoplastic cells and required for the high glycolytic rate and anchorage-independent growth of ras-transformed cells. We report herein the computational identification of a small-molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which suppresses glycolytic flux and is cytostatic to neoplastic cells. 3PO inhibits recombinant PFKFB3 activity, suppresses glucose uptake, and decreases the intracellular concentration of Fru-2,6-BP, lactate, ATP, NAD+, and NADH. 3PO markedly attenuates the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC50, 1.4-24 μmol/L) and is selectively cytostatic to ras-transformed human bronchial epithelial cells relative to normal human bronchial epithelial cells. The PFKFB3 enzyme is an essential molecular target of 3PO because transformed cells are rendered resistant to 3PO by ectopic expression of PFKFB3 and sensitive to 3PO by heterozygotic genomic deletion of PFKFB3. Importantly, i.p. administration of 3PO (0.07 mg/g) to tumor-bearing mice markedly reduces the intracellular concentration of Fru-2,6-BP, glucose uptake, and growth of established tumors in vivo. Taken together, these data support the clinical development of 3PO and other PFKFB3 inhibitors as chemotherapeutic agents. [Mol Cancer Ther 2008;7(1):110–20]

Molecular Docking of Intercalators and Groove-Binders to Nucleic Acids Using Autodock and Surflex

The molecular docking tools Autodock and Surflex accurately reproduce the crystallographic structures of a collection of small molecule ligands that have been shown to bind nucleic acids. Docking studies were performed with the intercalators daunorubicin and ellipticine and the minor groove binders distamycin and pentamidine. Autodock and Surflex dock daunorubicin and distamycin to their nucleic acid targets within a resolution of approximately 2 A, which is similar to the limit of the crystal structure resolution. However, for the top ranked poses, Autodock and Surflex both dock ellipticine into the correct site but in a different orientation compared to the crystal structure. This appears not only to be partly related to the symmetry of the target nucleic acid, as ellipticine is able to dock from either side of the intercalation site, but also due to the shape of the ligand and docking accuracy. Surflex docks pentamidine in a symmetrically equivalent orientation relative to the crystal structure, while Autodock was able to dock this molecule in the original orientation. In the case of the Surflex docking of pentamidine, the initial rmsd is misleading, given the symmetrical structure of pentamidine. Importantly, the ranking functions of both of these programs are able to return a top pose within approximately 2 A rmsd for daunorubicin, distamycin, and pentamidine and approximately 3 A rmsd for ellipticine compared to their respective crystal structures. Some docking challenges and potential pitfalls are explored, such as the importance of hydrogen treatment on ligands as well as the scoring functions of Autodock and Surflex. Overall for this set of complexes, Surflex is preferred over Autodock for virtual screening, as although the results are comparable, Surflex has significantly faster performance and ease of use under the optimal software conditions tested. These experiments show that molecular docking techniques can be successfully extended to include nucleic acid targets, a finding which has important implications for virtual screening applications and in the design of new small molecules to target therapeutically relevant morphologies of nucleic acids.

Structure and Stability of Higher-Order Human Telomeric Quadruplexes

G-quadruplex formation in the sequences 5-(TTAGGG)(n) and 5(TTAGGG)(n)TT (n = 4, 8, 12) was studied using circular dichroism, sedimentation velocity, differential scanning calorimetry, and molecular dynamics simulations. Sequences containing 8 and 12 repeats formed higher-order structures with two and three contiguous quadruplexes, respectively. Plausible structures for these sequences were determined by molecular dynamics simulations followed by experimental testing of predicted hydrodynamic properties by sedimentation velocity. These structures featured folding of the strand into contiguous quadruplexes with mixed hybrid conformations. Thermodynamic studies showed the strands folded spontaneous to contain the maximum number contiguous quadruplexes. For the sequence 5(TTAGGG)(12)TT, more than 90% of the strands contained completely folded structures with three quadruplexes. Statistical mechanical-based deconvolution of thermograms for three quadruplex structures showed that each quadruplex melted independently with unique thermodynamic parmameters. Thermodynamic analysis revealed further that quadruplexes in higher-ordered structures were destabilized relative to their monomeric counterparts, with unfavorable coupling free energies. Quadruplex stability thus depends critically on the sequence and structural context.

A novel, macrophage migration inhibitory factor suicide substrate inhibits motility and growth of lung cancer cells

Although chemokine and growth factor receptors are attractive and popular targets for cancer therapeutic intervention, structure-based targeting of the ligands themselves is generally not considered practical. New evidence indicates that a notable exception to this is macrophage migration inhibitory factor (MIF). MIF, an autocrine- and paracrine-acting cytokine/growth factor, plays a pivotal role in both the initiation and maintenance of neoplastic diseases. MIF possesses a nonphysiologic enzymatic activity that is evolutionarily well-conserved. Although small molecule antagonists of MIFs enzymatic active site have been reported to inhibit biological activities of MIF, universally high IC(50)s have limited their clinical appeal. Using a computational virtual screening strategy, we have identified a unique small molecule inhibitor that serves as a suicide substrate for MIF, resulting in the covalent modification of the catalytically active NH(2)-terminal proline. Our studies further reveal that this compound, 4-iodo-6-phenylpyrimidine (4-IPP), is approximately 5x to 10x times more potent in blocking MIF-dependent catalysis and lung adenocarcinoma cell migration and anchorage-independent growth than the prototypical MIF inhibitor, ISO-1. Finally, using an in silico combinatorial optimization strategy, we have identified four unique congeners of 4-IPP that exhibit MIF inhibitory activity at concentrations 10x to 20x lower than that of parental 4-IPP.

The Tail of the Telomere

The structure of a higher-order G-quadruplex structure for human telomeric DNA is presented. The structure was determined by a novel integrated approach in which molecular dynamics simulations were used to produce a stable structure, from which specific experimentally accessible properties were predicted. These properties were tested by sedimentation velocity and steady-state fluorescence measurements. The structure that emerges is a dimeric structure with two quadruplex units, each with a different structure. The interface between the quadruplex units is stabilized by specific stacking interactions of loop nucleotides. The interface is a unique structure and a unique target for drug design.

Resolution and characterization of the structural polymorphism of a single quadruplex-forming sequence

The remarkable structural polymorphism of quadruplex-forming sequences has been a considerable impediment in the elucidation of quadruplex folds. Sequence modifications have commonly been used to perturb and purportedly select a particular form out of the ensemble of folds for nuclear magnetic resonance (NMR) or X-ray crystallographic analysis. Here we report a simple chromatographic technique that separates the individual folds without need for sequence modification. The sequence d(GGTGGTGGTGGTTGTGGTGGTGGTGG) forms a compact quadruplex according to a variety of common biophysical techniques. However, NMR and chromatography showed that this oligonucleotide produces at least eight monomeric quadruplex species that interconvert very slowly at room temperature. We have used a combination of spectroscopic, hydrodynamic and thermodynamic techniques to evaluate the physicochemical properties of the mixture and the individual species. These species have almost identical thermodynamic, hydrodynamic and electrophoretic properties, but significantly different NMR and circular dichroism (CD) spectra, as well as kinetic stability. These results demonstrate that simple standard low-resolution techniques cannot always be used for quadruplex fold determination or quality control purposes, and that simple thermodynamic analysis does not directly provide interpretable thermodynamic parameters.

A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/AKT signaling

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.