Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III 1 region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III 1 region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.The MYC proto-oncogene is a key component of normal cell growth and differentiation, with roles in a multitude of cellular processes. Normally, this gene is subject to tight transcriptional regulation; however, aberrant MYC expression is a common feature in an estimated 80% of all human malignancies (1-3); it is estimated that one-seventh of cancer deaths in the United States are associated with alterations in the MYC gene or its expression (4). Deregulation can arise through a variety of mechanisms (5-13), but most often MYC is activated through alterations in cell signaling that lead to increased transcription (14).Deregulated MYC can lead to transformation (15), often as an early step in the process of multistage cancer development, and one on which all other mutations are based (16,17). Cancer cells appear to be addicted to a deregulated MYC (18), which can be the "Achilles heel", offering the potential for a therapeutic window (19,20). The ability to selectively and potently down-regulate MYC would have considerable potential for both efficacy and safety in a variety of tumor types.There are several upstream elements within the MYC promoter that can potentially undergo strand separation to form either single-stranded or other non-B-DNA structures (21), which play a critical role in transcriptional control of MYC: the distant Far Upstream Element acts as a cruise control element, Z-DNA found both in the far upstream and the promoter regions, and a GC-rich region within the proximal promoter that acts as an on/off switch (22-28). This near upstream core promoter region, which is responsible for the initiation of 80 -90% of MYC transcription (29), contains the GC-rich nuclease hypersensitive element (NHE) 2 III 1 to which doublestranded (Sp1) and single-stranded (CNBP and hnRNP k) transcriptional factors bind. Within the MYC gene's NHE III 1 , a 31-base pair element consisting of five repeats of the sequence (C/T)C(C/T)TCCCCA serves as the "on/off switch" for MYC trans...

The platelet-derived growth factor receptor β (PDGFR-β) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-β NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-β pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-β is the 3′-end G-quadruplex, which has a GGA sequence at the 3′-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-β NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-β NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-β promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3′-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-β i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-β promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-β-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.

Overexpression of PDGFR-β has been associated with cancers, vascular and fibrotic disorders. PDGFR-β has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in GC-rich nuclease hypersensitivity element (NHE) of the human PDGFR-β gene promoter has been found to inhibit PDGFR-β transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-β promoter. Instead of using the four continuous runs with three or more guanines, this G-quadruplex adopt a novel folding with a broken G-strand, to form a primarily parallel-stranded intramolecular structure with three 1-nt double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-β promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1-nt loop. Together with recent progress on G-quadruplexes formed in gene promoter sequences, the 1-nt-looped GiNGj motif may be evolutionarily selected to serve as a stable foundation for the promoter G-quadruplexes to build upon. The novel folding of the PDGFR-β promoter G-quadruplex may represent an attractive target for small molecule drugs that specifically target this secondary structure and modulate PDGFR-β gene expression.

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Understanding the mode of action (MOA) of many natural products can be puzzling with mechanistic clues that seem to lack a common thread. One such puzzle lies in the evaluation of the antitumor properties of the natural product withaferin A (WFA). A variety of seemingly unrelated pathways have been identified to explain its activity, suggesting a lack of selectivity. We now show that WFA acts as an inhibitor of the chaperone, p97, both in vitro and in cell models in addition to inhibiting the proteasome in vitro. Through medicinal chemistry, we have refined the activity of WFA toward p97 and away from the proteasome. Subsequent studies indicated that these WFA analogs retained p97 activity and cytostatic activity in cell models, suggesting that the modes of action reported for WFA could be connected by proteostasis modulation. Through this endeavor, we highlight how the parallel integration of medicinal chemistry with chemical biology offers a potent solution to one of natures’ intriguing molecular puzzles.