Drug discovery of small molecules targeting the higher-order hTERT promoter G-quadruplex.

DNA G-quadruplexes (G4s) are now widely accepted as viable targets in the pursuit of anticancer therapeutics. To date, few small molecules have been identified that exhibit selectivity for G4s over alternative forms of DNA, such as the ubiquitous duplex. We posit that the lack of current ligand specificity arises for multiple reasons: G4 atomic models are often small, monomeric, single quadruplex structures with few or no druggable pockets; targeting G-tetrad faces frequently results in the enrichment of extended electron-deficient polyaromatic end-pasting scaffolds; and virtual drug discovery efforts often under-sample chemical search space. We show that by addressing these issues we can enrich for non-standard molecular templates that exhibit high selectivity towards G4s over other forms of DNA. We performed an extensive virtual screen against the higher-order hTERT core promoter G4 that we have previously characterized, targeting 12 of its unique loop and groove pockets using libraries containing 40 million drug-like compounds for each screen. Using our drug discovery funnel approach, which utilizes high-throughput fluorescence thermal shift assay (FTSA) screens, microscale thermophoresis (MST), and orthogonal biophysical methods, we have identified multiple unique G4 binding scaffolds. We subsequently used two rounds of catalogue-based SAR to increase the affinity of a disubstituted 2-aminoethyl-quinazoline that stabilizes the higher-order hTERT G-quadruplex by binding across its G4 junctional sites. We show selectivity of its binding affinity towards hTERT is virtually unaffected in the presence of near-physiological levels of duplex DNA, and that this molecule downregulates hTERT transcription in breast cancer cells.

Long promoter sequences form higher-order G-quadruplexes: an integrative structural biology study of c-Myc, k-Ras and c-Kit promoter sequences.

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34-70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.

POT1 stability and binding measured by fluorescence thermal shift assays.

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 -DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

Human POT1 unfolds G-quadruplexes by conformational selection.

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (-10.1 ± 0.3 kcal mol-1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

The hTERT core promoter forms three parallel G-quadruplexes.

The structure of the 68 nt sequence with G-quadruplex forming potential within the hTERT promoter is disputed. One model features a structure with three stacked parallel G-quadruplex units, while another features an unusual duplex hairpin structure adjoined to two stacked parallel and antiparallel quadruplexes. We report here the results of an integrated structural biology study designed to distinguish between these possibilities. As part of our study, we designed a sequence with an optimized hairpin structure and show that its biophysical and biochemical properties are inconsistent with the structure formed by the hTERT wild-type sequence. By using circular dichroism, thermal denaturation, nuclear magnetic resonance spectroscopy, analytical ultracentrifugation, small-angle X-ray scattering, molecular dynamics simulations and a DNase I cleavage assay we found that the wild type hTERT core promoter folds into a stacked, three-parallel G-quadruplex structure. The hairpin structure is inconsistent with all of our experimental data obtained with the wild-type sequence. All-atom models for both structures were constructed using molecular dynamics simulations. These models accurately predicted the experimental hydrodynamic properties measured for each structure. We found with certainty that the wild-type hTERT promoter sequence does not form a hairpin structure in solution, but rather folds into a compact stacked three-G-quadruplex conformation.

Multi-group diagnostic classification of high-dimensional data using differential scanning calorimetry plasma thermograms.

The thermoanalytical technique differential scanning calorimetry (DSC) has been applied to characterize protein denaturation patterns (thermograms) in blood plasma samples and relate these to a subject's health status. The analysis and classification of thermograms is challenging because of the high-dimensionality of the dataset. There are various methods for group classification using high-dimensional data sets; however, the impact of using high-dimensional data sets for cancer classification has been poorly understood. In the present article, we proposed a statistical approach for data reduction and a parametric method (PM) for modeling of high-dimensional data sets for two- and three- group classification using DSC and demographic data. We compared the PM to the non-parametric classification method K-nearest neighbors (KNN) and the semi-parametric classification method KNN with dynamic time warping (DTW). We evaluated the performance of these methods for multiple two-group classifications: (i) normal versus cervical cancer, (ii) normal versus lung cancer, (iii) normal versus cancer (cervical + lung), (iv) lung cancer versus cervical cancer as well as for three-group classification: normal versus cervical cancer versus lung cancer. In general, performance for two-group classification was high whereas three-group classification was more challenging, with all three methods predicting normal samples more accurately than cancer samples. Moreover, specificity of the PM method was mostly higher or the same as KNN and DTW-KNN with lower sensitivity. The performance of KNN and DTW-KNN decreased with the inclusion of demographic data, whereas similar performance was observed for the PM which could be explained by the fact that the PM uses fewer parameters as compared to KNN and DTW-KNN methods and is thus less susceptible to the risk of overfitting. More importantly the accuracy of the PM can be increased by using a greater number of quantile data points and by the inclusion of additional demographic and clinical data, providing a substantial advantage over KNN and DTW-KNN methods.

Putting a New Spin of G-Quadruplex Structure and Binding by Analytical Ultracentrifugation.

Analytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability, and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be used to characterize G4 formation and to monitor small-molecule binding.

Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.

Characterization and classification of lupus patients based on plasma thermograms.

OBJECTIVE: Plasma thermograms (thermal stability profiles of blood plasma) are being utilized as a new diagnostic approach for clinical assessment. In this study, we investigated the ability of plasma thermograms to classify systemic lupus erythematosus (SLE) patients versus non SLE controls using a sample of 300 SLE and 300 control subjects from the Lupus Family Registry and Repository. Additionally, we evaluated the heterogeneity of thermograms along age, sex, ethnicity, concurrent health conditions and SLE diagnostic criteria. METHODS: Thermograms were visualized graphically for important differences between covariates and summarized using various measures. A modified linear discriminant analysis was used to segregate SLE versus control subjects on the basis of the thermograms. Classification accuracy was measured based on multiple training/test splits of the data and compared to classification based on SLE serological markers. RESULTS: Median sensitivity, specificity, and overall accuracy based on classification using plasma thermograms was 86%, 83%, and 84% compared to 78%, 95%, and 86% based on a combination of five antibody tests. Combining thermogram and serology information together improved sensitivity from 78% to 86% and overall accuracy from 86% to 89% relative to serology alone. Predictive accuracy of thermograms for distinguishing SLE and osteoarthritis / rheumatoid arthritis patients was comparable. Both gender and anemia significantly interacted with disease status for plasma thermograms (p<0.001), with greater separation between SLE and control thermograms for females relative to males and for patients with anemia relative to patients without anemia. CONCLUSION: Plasma thermograms constitute an additional biomarker which may help improve diagnosis of SLE patients, particularly when coupled with standard diagnostic testing. Differences in thermograms according to patient sex, ethnicity, clinical and environmental factors are important considerations for application of thermograms in a clinical setting.

Clinical application of plasma thermograms. Utility, practical approaches and considerations.

Differential scanning calorimetry (DSC) studies of blood plasma are part of an emerging area of the clinical application of DSC to biofluid analysis. DSC analysis of plasma from healthy individuals and patients with various diseases has revealed changes in the thermal profiles of the major plasma proteins associated with the clinical status of the patient. The sensitivity of DSC to the concentration of proteins, their interactions with other proteins or ligands, or their covalent modification underlies the potential utility of DSC analysis. A growing body of literature has demonstrated the versatility and performance of clinical DSC analysis across a range of biofluids and in a number of disease settings. The principles, practice and challenges of DSC analysis of plasma are described in this article.

Activation of the proapoptotic Bcl-2 protein Bax by a small molecule induces tumor cell apoptosis.

The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer.

Structural analysis of the DNA target site and its interaction with Mbp1.

The solution structure of a 14 base-pair non-self complementary DNA duplex containing the consensus-binding site of the yeast transcription factor Mbp1 has been determined by NMR using a combination of scalar coupling analysis, time-dependent NOEs, residual dipolar couplings and 13C-edited NMR spectroscopy of a duplex prepared with one strand uniformly labeled with 13C-nucleotides. As expected, the free DNA duplex is within the B-family of structures, and within experimental limits is straight. However, there are clear local structural variations associated with the consensus CGCG element in the binding sequence that are important for sequence recognition. In the complex, the DNA bends around the protein, which also undergoes some conformational rearrangement in the C-terminal region. Structural constraints derived from paramagnetic perturbation experiments with spin-labeled DNA, chemical shift perturbation experiments of the DNA, previous cross-saturation, chemical shift perturbation experiments on the protein, information from mutational analysis, and electrostatics calculations have been used to produce a detailed docked structure using the known solution conformation of the free protein and other spectroscopic information about the Mbp1:DNA complex. A Monte Carlo-based docking procedure with restrained MD in a fully solvated system subjected to available experimental constraints produced models that account for the available structural data, and can rationalize the extensive thermodynamic data about the Mbp1:DNA complex. The protein:DNA interface is closely packed and is associated with a small number of specific contacts. The structure shows an extensive positively charged surface that accounts for the high polyelectrolyte contribution to binding.

Thermodynamics and specificity of the Mbp1-DNA interaction.

The DNA binding domain of the yeast transcription factor Mbp1 is a winged helix-turn-helix structure, with an extended DNA binding site involving C-terminal "tail" residues. The thermodynamics of the interaction of the DNA binding domain with its target DNA sequence have been determined using fluorescence anisotropy and calorimetry. The dissociation constant was determined as a function of pH and ionic strength in assessing the relative importance of specific and nonspecific ionic interactions. Mutational analysis of the residues in the binding site was used to determine their contributions to binding. The three tail histidine residues and His 63 in the recognition helix accounted for most of the pH dependence of the DNA binding. The tail histidine residues, along with two previously identified lysine residues, account for a major part of the polyelectrolyte contribution to binding and for the nonspecific affinity of Mbp1 for DNA. Gln67 was shown to be a very important residue, which interacts in the minor groove of the target DNA. Systematic mutations of the DNA consensus binding sites showed that the CGCG core contributes most to recognition. Isothermal titration calorimetry revealed a strong temperature-dependent enthalpy change, with a Delta Cp of -1.3kJ mol(-1) K(-1), consistent with a specific binding mode and burial of surface area. Parsing the free energy contributions demonstrates that polyelectrolyte effects account for half of the total free energy at the physiological pH and salt concentration. We present a model for the origin of the sequence specificity and overall affinity of the protein that accounts for the observed thermodynamics.