Danzhou Yang - US grants

DESCRIPTION (Applicant's Description): The research objective of this five- year grant application is to explore the molecular level details of the DNA and topoisornerase I interactions of the camptothecin family of anticancer drugs. The research will take place at University of Kentucky, Department of Chemistry and College of Pharmacy. Camptothecin is an experimental anticancer agent renowned for its novel mechanism of action, the inhibition of DNA- processing enzyme topoisomerase I. Two camptothecins (TPT and CPT-11) have recently gained the U.S. Food and Drug Administration's approval for clinical use in 1996 and 1998. The project involves the implementation of a variety of state-of-the-art analytical and biophysical methods including high field nuclear magnetic resonance spectroscopy (NMR), computer molecular modeling, high pressure liquid chromatography, high sensitivity differential scanning c a l o rimetry and isothermal titration calorimetry, photon correlation spectroscopy, laser-induced one- and two-photon fluorescence spectroscopy, and Fourier transform mass spectrometry. This variety of highly complementary biophysical methods provides a powerful approach for obtaining a detailed, biophysical view of the target interactions of the camptothecins. The aim of the project is to understand the molecular level details of the interactions of clinically relevant water-soluble as well as lipophilic camptothecins with dsDNA and genomic DNA. Molecular level details of cleavable complexes formed between dsDNA, topoisomerase I and camptothecin drugs will be sought. Also to be studied is the structural basis of drug binding in dsDNA and cleavable c o m plexes. Mechanistic information and structure-function correlations concerning the inhibition of topoisomerase I function by camptothecins will be pursued. This research project is intended to define the academic research program of Dr. Yang who is intent on achieving a faculty position in a first- rate research-oriented College of Pharmacy or Medicine in the United States.

This proposal requests a triple-resonance 600 MHz Nuclear Magnetic Resonance (NMR) spectrometer, which will be located in the College of Pharmacy at the University of Arizona. Common features of the user grouip are (1) NIH supported research programs with a significant NMR component and (2) immediate need for high-field NMR instrumentation not available in the proximity of the College of Pharmacy (all users are located in the College of Pharmacy). Most major users have just relocated to the University of Arizona, and access to a high-field NMR spectrometer is critical to the maximum productivity and success of these projects.

[unreadable] DESCRIPTION (provided by applicant): The four oncogenes, c-MYC, bcl-2, VEGF, and HIF-1a, all contain polyG/polyC tracts in their promoter regions critical for transcriptional activation. The occurrence of DNA G-quadruplex secondary structures has been demonstrated in the promoter regions of these four oncogenes and has been shown to be a transcriptional modulator. While the DNA G-quadruplexes are promising new drug targets, the evaluation of their potential as cancer therapeutic targets depends on the understanding of biologically relevant G-quadruplex structures. Our preliminary studies have allowed us to identify the predominant forms of G-quadruplexes in the promoter regions of c-MYC, bcl-2, VEGF, and HIF-1a, which appear to represent three different basic G-quadruplex structures with VEGF/HIF-1a G-quadruplexes being the same basic type. The different molecular structures of the promoter G-quadruplexes make these structures attractive targets for pathway-specific drug design. In this proposal we intend to define the specific molecular structure of each G-quadruplex and its drug-complex(es). The structural information obtained will be correlated with the biological data to understand the effective gene modulation. Insight into the structures of the promoter G-quadruplexes and their drug complexes will provide an important basis for structure-based rational drug design. We will test our hypothesis that each promoter G-quadruplex can be specifically targeted by different small molecule drug compounds. In addition to the principle that selectivity can be achieved by interactions with different G-quadruplex core structures, we expect that selectivity can also be achieved by interactions within the external loops and capping structures in which binding pockets are generated. Proof of principle will be important in this regard. We will use a combination of NMR, CD, molecular modeling, and microcalorimetry data in concert with appropriate mutant promoter elements. Our primary approach, high field NMR spectroscopy, represents a major tool for structure determination of biologically relevant G-quadruplexes, due to the difficulty of crystallization of such structures. Our ultimate objective is to use a structure-based approach to rationally design small molecule G-quadruplex-interactive compounds that specifically target the G-quadruplex structure unique to each promoter and modulate gene transcription. Specifically, we plan to 1) determine the molecular structure of the biologically relevant G-quadruplex formed in the promoter region of c-MYC and its drug interactions; 2) determine the molecular structure of the biologically relevant G-quadruplex formed in the promoter region of bcl-2 and its drug interactions; and 3) determine the molecular structure of the biologically relevant G-quadruplex formed in the promoter region of VEGF/HIF-1a and its drug interactions. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Overexpression of PDGFR-beta is an important factor in pancreatic cancer as well as other inflammatory diseases, including arteriosclerosis and therefore is a highly significant molecular target. The long-range goal of this proposal is to develop small molecule therapeutics that will specifically suppress PDGFR-beta gene expression. Our strategy takes advantage of the recent insight into the importance of G-quadruplexes in transcriptional silencing of a number of genes, including c-Myc. Preliminary data reported in this proposal demonstrate that a cluster of four overlapping G-quadruplexes are key elements in the control of PDGFR-beta transcription. We have also demonstrated that known G-quadruplex-interactive compounds have differential effects on PDGFR-beta gene expression dependent on the selectivity for the constituent G-quadruplexes. Transcriptionally induced superhelicity has been demonstrated to be important in the conversion of duplex DNA to G-quadruplex in promoter region. Thus our hypothesis to be tested is that the negative superhelicity induced by transcription provides a real-time feedback mechanism into the NHE in the PDGFR-beta promoter to modulate both the firing rate (cruise control) and activation or silencing (on/off switch) of PDGFR-beta transcription. The specific aims are: (1) To determine the biological function of the 54-end, mid-54, mid-34, and 34-end G-quadruplex-forming sequences by deletion and mutational analysis of the PDGFR-beta promoter element and then determine the effect of supercoiling on the pattern of G-quadruplex formation in the PDGFR-beta promoter. (2) To determine by NMR the folding patterns and structures of the G-quadruplexes in the PDGFR-beta promoter. (3) To identify specific proteins and small molecules that bind differentially to the constituent G-quadruplexes found in the PDGFR-beta promoter. For specific aim 1, we will construct supercoiled plasmids containing PDGFR-beta promoter inserts and a luciferase reporter system. For specific aim 2, high-field NMR will be used to determine the structures of the constituent G-quadruplexes located in the PDGFR-beta promoter element. For specific aim 3, we will use affinity chromatography and small molecule screening methods to identify proteins and drug-like molecules that bind to the individual G-quadruplexes. The biological effects of these entities will be determined in specific aim 1.

DESCRIPTION (provided by applicant): Bcl-2 is a membrane protein that functions as an inhibitor of cell apoptosis. Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. We have found that the human bcl-2 gene contains a GC-rich proximal promoter region that can form two stable intramolecular G-quadruplex DNA secondary structures using overlapping guanine-rich DNA sequences. This GC-rich region contains a binding site of the WT1 protein which has been shown to be a negative regulator of the bcl-2 gene expression. We have recently developed a screening assay of small molecule compounds that can selectively bind the bcl-2 promoter G-quadruplex structures. Intriguingly, these compounds have been shown to upregulate the bcl-2 gene transcription. The hypothesis to be tested is that stabilization of the bcl-2 promoter G-quadruplex secondary structure(s) with small molecules upregulates bcl-2 gene transcription by inhibiting the binding of the negative regulator WT1 protein. A G-quadruplex DNA secondary structure has been demonstrated to be a transcriptional silencer element in the proximal promoter region of the human c-Myc gene and is amenable to small molecule drug targeting. The G-quadruplexes formed in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-Myc promoter in that their formation could serve to modulate gene transcription. However, the complexity of the G-quadruplex structures in the bcl-2 promoter is higher than is the case for the c-Myc promoter. The presence of two interchangeable G-quadruplexes overlapping in the region of the G-rich strand is likely to be important for the precise regulation of bcl-2 gene transcription, as each G-quadruplex may bind to different proteins leading to different gene modulation, in a manner analogous to the genetic switch in the bacteriophage lambda controlled by the interactive Cro and Repressor proteins, whose operator regions (ORs) overlap with each other's promoter regions and thereby inhibit each other's transcription. In this proposal we aim to determine the biological roles and molecular structures for the bcl-2 promoter G-quadruplex structures. Our primary approach, high-field NMR spectroscopy, represents a major tool for determination of DNA secondary structures under physiological conditions, due to the difficulty of crystallization of such structures. Our long-term goal is to use structure-based rational design to develop small molecule compounds that specifically target the bcl-2 promoter G-quadruplex structures and effectively modulate bcl-2 gene expression. Specifically, we plan to 1) determine the functional significance of the two interchangeable bcl-2 promoter G-quadruplexes in bcl-2 gene regulation and how binding of G-quadruplex-stabilizing compounds affects the regulation of the bcl-2 gene; and 2) to determine the structures of the two interchangeable bcl-2 promoter G-quadruplexes and their complexes with G-quadruplex-stabilizing compounds. PUBLIC HEALTH RELEVANCE: Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. The proposed research represents a novel new strategy for modulating bcl-2 gene expression by small molecule drugs.

DESCRIPTION (provided by applicant): Modulating c-MYC Transcription by G-quadruplex-interactive Small Molecules DNA G-quadruplex secondary structures have recently been found to form in proximal promoter regions as transcriptional regulators, and are considered as a new class of molecular targets for anticancer drugs. Specifically, c-MYC, one of the most commonly deregulated genes in human cancers, has a DNA G-quadruplex motif in the promoter Nuclease Hypersensitive Element (NHE) III1 which regulates 80-95% of its total transcription. The DNA G-quadruplex formed in the c-MYC NHE III1 has been shown to be a transcriptional silencer element; compounds that bind to and stabilize the G-quadruplex conformation can reduce c-MYC expression and are anti- tumorigenic. We have recently discovered that the NM23-H2 protein unfolds the c-MYC promoter G-quadruplex to activate gene transcription. However, although the c-MYC promoter G-quadruplex is the first and most extensively studied system, little is known about its molecular interactions with small molecules and proteins. The hypothesis to be tested is that the physiological functions of c-MYC G-quadruplex-interactive compounds are mediated through not only the G-quadruplex but also the G-quadruplex-interactive protein. We have identified an Ellipticine analog as our lead compound for further optimization to target the c-MYC promoter G- quadruplex. Ellipticine has good drug-like properties and has been shown to selectively bind the c-MYC G-quadruplex. We will use NMR to understand the molecular interactions with the c- MYC G-quadruplex and ITC to characterize the thermodynamic contributions of drug binding (Aim 1). Based on this information, we will rationally design and synthesize new Ellipticine analogs with various substituents at C9, N2, N6, and C3 positions (Aim 2). We will use biochemical, biophysical, and biological assays to examine the effects of the Ellipticines on inhibiting NM23-H2 binding and unfolding of the c-MYC G-quadruplex and their effectiveness in c-MYC transcriptional suppression. A combination of structural and biological studies will allow us to understand the specific G-quadruplex interactions of Ellipticine that lead to inhibition of the NM23-H2 protein and suppression of c-MYC transcription. The overall objectives of this research are to establish the structure-activity relationship and underlying molecular mechanism of Ellipticines for c-MYC suppression and to design/synthesize new analogs for further drug development. The specific aims are: 1) To determine structural and thermodynamic details of molecular interactions of Ellipticines and related molecules with the c-MYC G- quadruplex. 2) To design and synthesize new C9-, N2-, N6-, and C3-substituted Ellipticine analogs and to study structure-activity relationship (SAR) of Ellipticines targeting the c-MYC G- quadruplex. 3) To determine how Ellipticine analogs modulate NM23-H2 binding and unfolding of the c-MYC G-quadruplex, and how this correlates with c-MYC transcriptional suppression.

PROJECT SUMMARY Nucleolin recognition of MYC promoter G-quadruplex and its role in MYC regulation by MycG4-ligands G-quadruplexes (G4s) are non-canonical DNA secondary structures. c-Myc, one of the central driver oncogenes in human cancers, has a DNA G4 motif in its proximal promoter region that functions as a transcription silencer. However, little is known about how the c-Myc promoter G4 (MycG4) is regulated. Nucleolin is identified as the major c-Myc G4 binding protein and shows a remarkably higher binding affinity for MycG4 over its known cellular substrate NRE RNA. Nucleolin directly binds to the MycG4 promoter region in vivo and overexpression of nucleolin represses the activity of the c-Myc promoter. We hypothesize that nucleolin recognizes the c-Myc promoter G4 and that MycG4-targeted small molecules regulate c-Myc gene expression through interactions with the nucleolin-MycG4 complex. In this proposal, we will study how nucleolin recognizes MycG4 by determining the molecular structure of the nucleolin-MycG4 complex. We will also study how small molecules interact with the nucleolin-MycG4 complex and how the interactions affect c-Myc transcription. The c-Myc promoter G4 is an attractive anticancer drug target. Our preliminary data show that the nucleolin-MycG4 complex is clearly involved in the c-Myc regulation by MycG4-ligands. The proposed research represents the first structural and functional study of the nucleolin-MycG4 complex. A structural level understanding of the nucleolin- MycG4 complex and its interactions with small molecules will provide important information for MycG4 function and MycG4-targeted drug development. The proposed research will use a combination of high-field NMR spectroscopy, X-ray crystallography, and other biophysical, biochemical, and cellular approaches. We have assembled a strong collaboration team. The specific aims are: 1) To determine the structure of nucleolin in complex with the c-Myc promoter G-quadruplex. Nucleolin is a multi-domain protein containing four tandem RNA- binding domains (RBDs). We hypothesize that nucleolin uses all four RBDs to wrap around MycG4 and recognize the G4 external loops to achieve a high-affinity binding. This structure would be the first to show how the unusual DNA G4 structure is recognized by a modular protein, likely through multi-valent interactions. 2) To investigate how small molecules interact with the nucleolin-MycG4 complex and their effects on c-Myc gene regulation. Our preliminary data shows that only MycG4-ligands that stabilize the nucleolin-MycG4 complex can lower c-Myc levels. We will use a combination of in vitro and cell-based assays to identify compounds that can stabilize the nucleolin-MycG4 complex and downregulate c-Myc. We will determine the specificity for the c-Myc gene and determine major cellular response for the top compounds. For the MycG4-ligands that form the stable ternary complexes with nucleolin-MycG4, we will also determine the structural basis of the nucleolin-MycG4 stabilization.

PROJECT SUMMARY Targeting MYC promoter G-quadruplex for MYC inhibition by Indenoisoquinolines G-quadruplex (G4) DNA is a globular DNA secondary structure and considered as a new class of molecular targets for anticancer drugs. MYC, one of the most commonly deregulated genes in human cancers, has a DNA G4 motif in its promoter that functions as a transcriptional silencer. Compounds that bind to and stabilize the G-quadruplex formed in the MYC promoter have been shown to significantly lower MYC levels in cancer cells. Thus, the MYC promoter G-quadruplex (MycG4) represents a novel target for MYC inhibition by small molecules. However, little is known about how MycG4 is regulated by proteins and development of MycG4- targeting drugs has been focused solely on G4 DNA. Whereas drug-DNA interactions may be insufficient for MYC inhibition, the effective mechanism of drug action could involve protein-DNA interactions, which is analogous to topoisomerase inhibitors. Very recently, we have discovered that indenoisoquinolines, a clinically tested scaffold with excellent drug-like properties, are strong MycG4 binders and potent MYC inhibitors. We have also discovered that the DDX5 helicase actively unfolds MycG4 and is critically involved in MYC gene transcriptional activation. These results provide new and critical insights to effectively downregulate MYC transcription by targeting MycG4 and its interactions with DDX5. Our central hypothesis is that indenoisoquinolines effectively suppress MYC transcription by binding to the MYC promoter G-quadruplex and disrupting DDX5-MycG4 interactions. The overall objective is to determine the molecular mechanism of effective MYC inhibition by indenoisoquinolines, establish the structure?activity relationships (SAR), and discover lead indenoisoquinolines for preclinical testing. The long-term research goal is to develop potent indenoisoquinoline MYC inhibitors as new anticancer drugs. The specific aims are: 1) Structural characterization of the MycG4-indenoisoquinoline complexes. 2) Establishing a compound library to determine indenoisoquinolines that bind MycG4 and inhibit MYC. 3) Determining the effect of MycG4-interactive indenoisoquinolines on DDX5 unfolding of the MYC promoter G4 and how this correlates with MYC suppression. 4) Designing and synthesizing optimized indenoisoquinolines for MYC suppression using structure-based rational approach; establishing SAR for MycG4-binding and inhibition of DDX5 unfolding. The expected outcome of this work is a determination of the SAR of indenoisoquinolines for MycG4-targeting, demonstration of the effective MYC suppression by inhibiting DDX5-MycG4 interaction, and discovery of lead compounds for future preclinical testing. The results will have an important positive impact because they lay the groundwork to develop new indenoisoquinoline anticancer drugs with MYC-targeted activity.