Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X.

Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.

In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells.

BACKGROUND: Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be "undrugable" because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription. METHODS: This study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt's lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents. RESULTS: GQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin. CONCLUSION: These results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

The Consequences of Overlapping G-Quadruplexes and i-Motifs in the Platelet-Derived Growth Factor Receptor ß Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression.

The platelet-derived growth factor receptor ß (PDGFR-ß) 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-ß 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-ß NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-ß pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-ß 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-ß 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-ß 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-ß 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-ß i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-ß promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-ß-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.

The quinone methide aurin is a heat shock response inducer that causes proteotoxic stress and Noxa-dependent apoptosis in malignant melanoma cells.

Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters.

Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.

The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure.

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.

Novel Brain-Penetrant, Small-Molecule Tubulin Destabilizers for the Treatment of Glioblastoma.

Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide (TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (Cmax of 7807 ng/mL (20 µM), Tmax at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC50s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC50 values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.

Gaining insights into the small molecule targeting of the G-quadruplex in the c-MYC promoter using NMR and an allele-specific transcriptional assay.

G-quadruplexes (four-stranded DNA secondary structures) are showing promise as new targets for anticancer therapies. Specifically, G-quadruplexes in the proximal promoter region of regulatory genes have the potential to act as silencer elements and thereby turn off transcription. Thus, compounds that are capable of binding to and stabilizing G-quadruplexes would be of great benefit. In this chapter we describe two recent studies from our labs. In the first case, we use NMR to elucidate the structure of a 2:1 complex between a small molecule and the G-quadruplex in the c-MYC promoter. In the second case, we use an allele-specific transcription assay to demonstrate that the effect of a G-quadruplex-interactive compound is mediated directly through the G-quadruplex. Finally, we use this information to propose models for the interaction of various small molecules with the c-MYC G-quadruplex.

Synthesis and biological activity of aminophthalazines and aminopyridazines as novel inhibitors of PGE2 production in cells.

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE(2) reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R(1)=H, R(2)=p-CH(3)O) exhibited the most potent activity in cells (EC(50)=0.02 µM) and minimal inhibition of COX-2 activity (3% at 5 µM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.

Demonstration that drug-targeted down-regulation of MYC in non-Hodgkins lymphoma is directly mediated through the promoter G-quadruplex.

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.

Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events.

The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but also this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.

Identification of a novel class of anti-inflammatory compounds with anti-tumor activity in colorectal and lung cancers.

Chronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E(2) (PGE(2)) is one of the major players. PGE(2) synthases (PGES) are the enzymes downstream of the cyclooxygenases (COXs) in the PGE(2) biosynthesis pathway. Microsomal prostaglandin E(2) synthase 1 (mPGES-1) is inducible by pro-inflammatory stimuli and constitutively expressed in a variety of cancers. The potential role for this enzyme in tumorigenesis has been reported and mPGES-1 represents a novel therapeutic target for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we screened the ChemBridge library and identified 13 compounds as potential hits. These compounds were tested for their ability to bind directly to the enzyme using surface plasmon resonance spectroscopy and to decrease cytokine-stimulated PGE(2) production in various cancer cell lines. We demonstrate that the compound PGE0001 (ChemBridge ID number 5654455) binds to human mPGES-1 recombinant protein with good affinity (K(D) = 21.3 ± 7.8 µM). PGE0001 reduces IL-1ß-induced PGE(2) release in human HCA-7 colon and A549 lung cancer cell lines with EC(50) in the sub-micromolar range. Although PGE0001 may have alternative targets based on the results from in vitro assays, it shows promising effects in vivo. PGE0001 exhibits significant anti-tumor activity in SW837 rectum and A549 lung cancer xenografts in SCID mice. Single injection i.p. of PGE0001 at 100 mg/kg decreases serum PGE(2) levels in mice within 5 h. In summary, our data suggest that the identified compound PGE0001 exerts anti-tumor activity via the inhibition of the PGE(2) synthesis pathway.

Synthesis and biological activity of 2-aminothiazoles as novel inhibitors of PGE2 production in cells.

This Letter presents the synthesis and biological evaluation of a collection of 2-aminothiazoles as a novel class of compounds with the capability to reduce the production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 36 analogs were synthesized and assayed for PGE(2) reduction, and those with potent cellular activity were counter screened for inhibitory activity against COX-2 in a cell free assay. In general, analogs bearing a 4-phenoxyphenyl substituent in the R(2) position were highly active in cells while maintaining negligible COX-2 inhibition. Specifically, compound 5l (R(1)=Me, R(2)=4-OPh-Ph, R(3)=CH(OH)Me) exhibited the most potent cellular PGE(2) reducing activity of the entire series (EC(50)=90 nM) with an IC(50) value for COX-2 inhibition of >5 µM in vitro. Furthermore, the anti-tumor activity of analog 1a was analyzed in xenograft mouse models exhibiting promising anti-cancer activity.

Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1.

G-quadruplexes (G4s) are non-canonical nucleic acid structures that regulate key biological processes, from transcription to genome replication both in humans and viruses. The herpes simplex virus-1 (HSV-1) genome is prone to form G4s that, along with proteins, regulate its viral cycle. General G4 ligands have been shown to hamper the viral cycle, pointing to viral G4s as original antiviral targets. Because cellular G4s are also normally present in infected cells, the quest for improved anti-HSV-1 G4 ligands is still open. Here, we evaluated a series of new quindoline-derivatives which showed high binding to and stabilization of the viral G4s. They displayed nanomolar-range anti-HSV-1 activity paralleled by negligible cytotoxicity in human cells, thus proving remarkable selectivity. The best-in-class compound inhibited the viral life cycle at the early times post infection up to the step of viral genome replication. In infected human cells, it reduced expression of ICP4, the main viral transcription factor, by stabilizing the G4s embedded in ICP4 promoter. Quindoline-derivatives thus emerge as a new class of G4 ligands with potent dual anti HSV-1 activity.

In Silico Targeting of the Long Noncoding RNA MALAT1.

RNA targeting has gained traction over the past decade. It has become clear that dysregulation of RNA can be linked to many diseases, leading to a need for new scaffolds recognizing RNA specifically. Long noncoding RNAs are emerging as key controllers of gene expression and potential therapeutic targets. However, traditional targeting methods have overwhelmingly been focused on proteins. In this study, we used a protein computational tool and found several possible targetable pockets in a structurally characterized long noncoding RNA, MALAT1. Screening against those identified pockets revealed several hit compounds. We tested the binding of those compounds to MALAT1 RNA and tRNA as a negative control, using SPR. While several compounds were nonspecific binders, others were able to recognize MALAT1 specifically. One of them, MTC07, has an apparent affinity of 400.2 ± 14.4 µM. Although it has weak affinity, MTC07 is the first compound targeting MALAT1 originating from in silico docking.

Selective targeting of NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in rodents.

The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified >200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics­without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.

Application of a novel [3+2] cycloaddition reaction to prepare substituted imidazoles and their use in the design of potent DFG-out allosteric B-Raf inhibitors.

B-Raf protein kinase, which is a key signaling molecule in the RAS-RAF-MEK-ERK signaling pathway, plays an important role in many cancers. The B-Raf V600E mutation represents the most frequent oncogenic kinase mutation known and is responsible for increased kinase activity in approximately 7% of all human cancers, establishing B-Raf as an important therapeutic target for inhibition. Through the use of an iterative program that utilized a chemocentric approach and a rational structure based design, we have developed novel, potent, and specific DFG-out allosteric inhibitors of B-Raf kinase. Here, we present efficient and versatile chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile.

Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene.

A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the vascular endothelial growth factor (VEGF) gene is essential for transcriptional activation. The guanine-rich (G-rich) and cytosine-rich (C-rich) strands on this tract are shown to form specific secondary structures, characterized as G-quadruplexes and i-motifs, respectively. Mutational analysis of the G-rich strand combined with dimethyl sulfate (DMS) footprinting, a polymerase stop assay, and circular dichroism (CD) spectroscopy revealed that the G-quadruplex containing a 1:4:1 double-chain reversal loop is the most thermodynamically stable conformation that this strand readily adopts. These studies provide strong evidence that the size of loop regions plays a critical role in determining the most favored folding pattern of a G-quadruplex. The secondary structure formed on the complementary C-rich strand was also determined by mutational analysis combined with Br(2) footprinting and CD spectroscopy. Our results reveal that at a pH of 5.9 this strand is able to form an intramolecular i-motif structure that involves six C-C(+) base pairs and a 2:3:2 loop configuration. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form on the G- and C-rich strands, respectively, of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition from B-DNA to non-B-DNA conformations.

Erratum: Nucleolin represses transcription of the androgen receptor gene through a G-quadruplex.

[This corrects the article DOI: 10.18632/oncotarget.27589.].