Novel quinoxaline analogs as telomeric G-quadruplex ligands exert antitumor effects related to enhanced immunomodulation.

G-quadruplexes (G4s) are commonly formed in the G-rich strand of telomeric DNA. Ligands targeting telomeric G4 induce DNA damage and telomere dysfunction, which makes them potential antitumor drugs. New telomeric G4 ligands with drug-likeness are still needed to be exploited, especially with their antitumor mechanisms thoroughly discussed. In this study, a novel series of quinoxaline analogs were rationally designed and synthesized. Among them, R1 was the most promising ligand for its cytotoxic effects on tumor cells and stabilizing ability with telomeric G4. Cellular assays illustrated that R1 stabilized G4 and induced R-loop accumulation in the telomeric regions, subsequently triggering DNA damage responses, cell cycle arrest in G2/M phase, apoptosis and antiproliferation. Moreover, R1 evoked immunogenic cell death (ICD) in tumor cells, which promoted the maturation of bone marrow derived dendritic cells (BMDCs). In breast cancer mouse model, R1 exhibited a significant decrease in tumor burden through the immunomodulatory effects, including the increase of CD4+ and CD8+ T cells in tumors and cytokine levels in sera. Our research provides a new idea that targeting telomeric G4 induces DNA damage responses, causing antitumor effects both in vitro and in vivo, partially due to the enhancement of immunomodulation.

Development of a fluorescent ligand that can illuminate nuclear G-quadruplexes and modulate oncogene expression.

In this study, we developed a promising dual-function fluorescent ligand termed KS-1 by a slight structural modification on a reported carbazole-based scaffold. KS-1 was then found to mainly bind and illuminate the nuclear DNA G-quadruplexes (G4s) in a sandwich-like interacting mode, and also effectively modulate the oncogene expression through a G4-mediated manner. Furthermore, KS-1 was proved to inhibit cancer cell growth either in 2D monolayer cells or 3D multicellular tumor spheroids. To be noted, this ligand could overcome the shortcomings of other reported dual-function ligands that appeared to accumulate in the lysosomes or mitochondria, and may be used as a theranostic agent in the future.

Discovery of a triphenylamine-based ligand that targets mitochondrial DNA G-quadruplexes and activates the cGAS-STING immunomodulatory pathway.

Stabilization of G-quadruplex (G4) structures in mitochondria leads to the damage of mitochondrial DNA (mtDNA), making mtDNA G4s a promising target in the field of cancer therapy in recent years. Damaged mtDNA released into the cytosol can stimulate cytosolic DNA-sensing pathways, and cGAS-STING pathway is a typical one with potent immunostimulatory effects. A few small molecule ligands of mtDNA G4s are identified with antitumor efficacy, but little is known about their results and mechanisms on immunomodulation. In this study, we engineered a series of triphenylamine-based analogues targeting mtDNA G4s, and A6 was determined as the most promising compound. Cellular studies indicated that A6 caused severe mtDNA damage. Then, damaged mtDNA stimulated cGAS-STING pathway, resulting in the following cytokine production of tumor cells and the maturation of DCs. In vivo experiments certified that A6 exerted suppressive influences on tumor growth and metastasis in 4T1 cell-bearing mice by the regulation of TME, including the remodeling of macrophages and the activation of T cells. To our knowledge, it is the first time to report a ligand targeting mtDNA G4s to activate the cGAS-STING immunomodulatory pathway, providing a novel strategy for the future development of mtDNA G4-based antitumor agents.

Combination Therapy with a TLR7 Agonist and a BRD4 Inhibitor Suppresses Tumor Growth via Enhanced Immunomodulation.

JQ-1 is a typical BRD4 inhibitor with the ability to directly fight tumor cells and evoke antitumor immunity via reducing the expression of PD-L1. However, problems arise with the development of JQ-1 in clinical trials, such as marked lymphoid and hematopoietic toxicity, leading to the investigation of combination therapy. SZU-101 is a TLR7 agonist designed and synthesized by our group with potent immunostimulatory activity. Therefore, we hypothesized that combination therapy of SZU-101 and JQ-1 would target innate immunity and adaptive immunity simultaneously, to achieve a better antitumor efficacy than monotherapy. In this study, the repressive effects of the combination administration on tumor growth and metastasis were demonstrated in both murine breast cancer and melanoma models. In 4T1 tumor-bearing mice, i.t. treatment with SZU-101 in combination with i.p. treatment with JQ-1 suppressed the growth of tumors at both injected and uninjected sites. Combination therapy increased M1/M2 ratio in TAMs, decreased PD-L1 expression and promoted the recruitment of activated CD8+ T cells in the TME. In summary, the improved therapeutic efficacy of the novel combination therapy appears to be feasible for the treatment of a diversity of cancers.

Discovery of a far-red carbazole-benzoindolium fluorescent ligand that selectively targets mitochondrial DNA and suppresses breast cancer growth.

G-quadruplex (G4) formation was considered to be more prevalent in the mitochondrial DNA (mtDNA) of cancer cells compared with normal cells. Stabilization of these G4s may induce mtDNA instability and cause mitochondrial dysfunction and subsequent cell death, which may be treated as a new strategy for cancer treatment. However, few ligands were developed to target mtG4s, leaving a huge room to improve. In this study, we designed and synthesized a series of carbazole-based ligands, among which, BKN-1 was identified as the most promising mitochondrial targeting fluorescent ligand with far-red emission. Then, we demonstrated that BKN-1 may robustly interact with mtG4s via a variety of biophysical, biological experiments. Subsequently, we proved that BKN-1 may cause mtDNA loss, disrupt mitochondrial integrity, decrease ATP level and trigger unbalanced ROS, thereby leading to apoptosis and autophagy. Finally, we verified that BKN-1 had good anti-tumor activity in both cellular and in vivo models. Altogether, this study provided a dual-function ligand that may not only track the formation of mtG4s but also induce mitochondrial dysfunction, which may be developed into an applicable chemical tool for investigating the structure and function of mtG4s, and moreover, an effective therapeutic agent for cancer interference.

Novel phenanthrene imidazoles as telomeric G-quadruplex ligands trigger potent immunogenic cell death in triple-negative breast cancer.

Immunogenic cell death (ICD) is a typical type of regulated cell demise, and ICD inducers stimulate the immune responses against dead-cell antigens and exert specific antitumor effects. G-quadruplex (G4) binders targeting the telomeres lead to DNA damage response (DDR) and the potential of harnessing the immune system for cancer therapy. However, the immunostimulatory effects of G4 ligands in cancer cells are still seldomly determined. In this study, we rationally designed and synthesized a series of novel phenanthrene imidazoles targeting telomeric G4. Among them, PI-2 was identified as the most promising ligand with high cytotoxicity, cellular uptake efficiency and G4-interacting ability. Cellular studies indicated that PI-2 inhibited the proliferation and migration of both human and mouse triple-negative breast cancer (TNBC) cells. PI-2 triggered the occurrence of DDR and ICD, where the related pathways were further decided. In vivo experiments displayed that PI-2-treated dying cells could be an effective vaccination to reduce tumor burden and promote the infiltration of CD8+ and CD4+ T cells to the tumor microenvironment (TME). To our knowledge, it is the first time to report a DDR-targeted G4 ligand with ICD-inducing ability in immunocompetent animals, which may provide new insights for the development of promising G4-based immunochemotherapeutic agents.

Non-fused imidazole-biphenyl analogs repress triple-negative breast cancer growth by mainly stabilizing the c-MYC G-quadruplex via a multi-site binding mode.

As oncogene c-MYC is abnormally expressed during TNBC pathogenesis, stabilizing its promoter G-quadruplex (G4), which may thus inhibit c-MYC expression and promote DNA damage, may be a potential anti-TNBC strategy. However, large quantities of potential G4-forming sites exist in the human genome, which represents a potential drug selectivity problem. In order to achieve better recognition for c-MYC G4, we herein presented a new approach of designing small-molecule ligands by linking tandem aromatic rings with the c-MYC G4 selective binding motifs. Thus, a series of non-fused, conformation-tunable imidazole-biphenyl analogs were designed and synthesized. Among them, the optimal ligand appeared more effective on stabilizing c-MYC G4 than other types of G4s possibly through an adaptive, multi-site binding mode involved of end-stacking, groove-binding and loop-interacting. Then, the optimal ligand exerted good inhibitory activity on c-MYC expression and induced remarkable DNA damage, leading to the occurrence of G2/M phase arrest, apoptosis and autophagy. Furthermore, the optimal ligand exhibited potent antitumor effects in a TNBC xenograft tumor model. To sum up, this work offers new insights for the development of selective c-MYC G4 ligands against TNBC.

New triazole-attached quinoxalines selectively recognize the telomeric multimeric G-quadruplexes and inhibit breast cancer cell growth.

The telomeric 3'-overhang had potential to form into higher-order structures termed multimeric G-quadruplexes (G4s), which may mainly exist in telomeres, representing an attractive drug target for development of anticancer agents with few side effects. However, only a few molecules that selectively bind to multimeric G4s have been found by random screening, which means a lot of room for improvement. In this study, we raised a feasible strategy to design small-molecule ligands with possible selectivity to multimeric G4s, and then synthesized a focused library of multi-aryl compounds by attaching triazole rings to the quinoxaline skeleton. Among them, QTR-3 was identified as the most promising selective ligand that may bind at the G4-G4 interface, which accordingly stabilized multimeric G4s and induced DNA damage in telomeric region, thereby leading to cell cycle arrest and apoptosis. Notably, QTR-3 showed more significant inhibition on breast cancer cells against normal mammary cells.

Discovery of a fluorescent, long chain-bridged bispurine that selectively targets the c-MYC G-quadruplex.

G-quadruplexes (G4s) are special nucleic acid structures which are involved in the regulation of some key biological events like transcription and translation, which are now treated as promising therapeutic targets for cancers. Stabilizing the promoter G4 by small-molecule ligands can suppress the c-MYC oncogene transcription, thus inhibiting cancer cell proliferation. So far, targeting the very structure, a number of ligands have been reported. However, most of them showed unsatisfactory specificity to the c-MYC G4 over other G4s, resulting in uncertain side effects. In this contribution, we discovered a new class of bispurines bridged with flexible hydrocarbon chains, which presented somewhat selectivity to the c-MYC G4 possibly by adaptive binding, which then showed clear inhibition on the c-MYC expression rather than other G4-driven oncogenes. Moreover, these novel molecules had the potential to fluorescently label G4s. We believed that this study may shed light on the discovery of new functional small molecules targeting a specific G4 structure.

New Dibenzoquinoxalines Inhibit Triple-Negative Breast Cancer Growth by Dual Targeting of Topoisomerase 1 and the c-MYC G-Quadruplex.

As c-MYC is one of the central players in triple-negative breast cancer (TNBC) oncogenesis, inhibiting c-MYC expression would be an effective anticancer strategy. Transcription-induced negative supercoiling is crucial in the regulation of c-MYC transcription, which facilitates the formation of a G4 structure in NHE III1 that can silence the transcription. However, topoisomerase 1 (Topo1) can dissipate this negative supercoiling, leading to continuous activation of c-MYC transcription. Thus, dual ligands targeting both Topo1 and c-MYC G4 appear to be significant in cancer therapy. In this study, a series of new dibenzoquinoxaline derivatives were designed, synthesized, and evaluated for both Topo1 and c-MYC inhibition. Among them, 5 was identified as the most promising dual ligand, which could effectively inhibit Topo1 activity and strongly stabilize c-MYC G4, thereby inhibiting cancer cell growth. Accordingly, this work suggests that this dual-targeting strategy may be effective in cancer therapy.

Drug-like biimidazole derivatives dually target c-MYC/BCL-2 G-quadruplexes and inhibit acute myeloid leukemia.

Chemotherapy is the main approach for treating acute myeloid leukemia (AML). However, this therapy can cause severe side effects as well as drug resistance, hence calling for new therapeutic strategies. As c-MYC and BCL-2 are often overexpressed in AML, and synergism between c-MYC and BCL-2 promotes tumorigenesis, therefore, dual targeting of c-MYC/BCL-2 promoter G-quadruplexes (G4s) and then inhibiting the targeted gene expression would be a potential strategy in ALM treatment. In this work, in the search of dual ligands, we performed a screening assay with an in-house, imidazole-based compound library. Consequently, two drug-like biimidazole derivatives were identified as selective c-MYC/BCL-2 G4 binders, of which, BIM-2 was selected as the candidate for inhibiting AML cell growth. Then, BIM-2 was demonstrated to downregulate both c-MYC and BCL-2 expression, and thereby cause cell cycle arrest at G0/G1 phase and apoptosis in AML cells. Furthermore, the possible end-stacking binding modes between BIM-2 and c-MYC/BCL-2 G4s were revealed by NMR and molecular docking studies. Accordingly, this study provides a new class of drug-like dual-selective c-MYC/BCL-2 G4 ligands for the potential treatment of AML.

A drug-like imidazole-benzothiazole conjugate inhibits malignant melanoma by stabilizing the c-MYC G-quadruplex.

Aberrant expression of c-MYC oncogene is significantly associated with the occurrence and development of malignant melanoma. Suppression of the c-MYC transcriptional activity accordingly provides a new idea for treating melanoma. Notably, stabilizing the G-quadruplex (G4) structure in the promoter is proved to be effective in downregulating c-MYC transcription. In this work, we developed a drug-like imidazole-benzothiazole conjugate called IZTZ-1, which was confirmed to preferentially stabilize the promoter G4 and thus lower c-MYC expression. Intracellular assays revealed that IZTZ-1 induced cell cycle arrest, apoptosis, thereby inhibiting cell proliferation. Furthermore, IZTZ-1 was demonstrated to effectively inhibit tumor growth in a melanoma mouse model. Consequently, IZTZ-1 showed good potential in the treatment of melanoma. This study provides an alternative strategy to treat melanoma by targeting the c-MYC G4.

Oxaliplatin-Based Platinum(IV) Prodrug Bearing Toll-like Receptor 7 Agonist for Enhanced Immunochemotherapy.

A combination of platinum drugs with immunotherapy has shown promising anticancer effects, especially in the drug resistance cancer model. Herein, a new type of immunochemotherapeutic was designed by tethering the toll-like receptor 7 (TLR7) agonist on the axial position of oxaliplatin-based platinum(IV) prodrug. The prodrug simultaneously induced immunogenic cell death of 4T1 cancer cells to initiate an immune response and activate dendritic cells (DCs) to secrete proinflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12, to further enhance the adaptive immunity. The prodrug exhibited better in vivo anticancer effects than oxaliplatin in the 4T1 allograft mouse model, a later stage breast cancer model, which showed poor response to traditional chemotherapy. Mechanism studies revealed that enhanced activation of cytotoxic T cells within tumor contribute to the high in vivo anticancer efficiency of the prodrug. Moreover, the prodrug displayed much lower cytotoxicity to DCs compared with oxaliplatin, indicating its safety to normal cells. These results highlight the potential of the conjugation of TLR7 agonist with oxaliplatin-based Pt(IV) prodrug as an effective anticancer agent to overcome the toxic side effects and drug resistance of traditional platinum chemotherapy.

Dimeric aryl-substituted imidazoles may inhibit ALT cancer by targeting the multimeric G-quadruplex in telomere.

In 10-15% of cancers, telomere maintenance is provided by a telomerase-independent mechanism known as alternative lengthening of telomere (ALT), making telomerase inhibitors ineffective on these cancers. Ligands that stabilize telomeric G-quadruplex (G4) are considered to be able to inhibit either the ALT process or disrupt the T-loop structure, which would be promising therapeutic agents for ALT cancers. Notably, the 3'-terminal overhang of telomeric DNA might fold into multimeric G4 containing consecutive G4 subunits, which offers an attractive target for selective ligands considering large numbers of G4s widespread in the genome. In this study, a dimeric aryl-substituted imidazole (DIZ-3) was developed as a selective multimeric G4 ligand based on a G4-ligand-dimerizing strategy. Biophysical experiments revealed that DIZ-3 intercalated into the G4-G4 interface, stabilizing the higher-order structure. Furthermore, this ligand was demonstrated to induce cell cycle arrest and apoptosis, and thus inhibited cell proliferation in an ALT cancer cell line. Cancer cells were more sensitive to DIZ-3, relative to normal cells. Notably, DIZ-3 had little effect on the transcription of several G4-dependent oncogenes. This study provides a nice example for discovering dimeric agents to potentially treat ALT cancers via targeting telomeric multimeric G4.

New substituted quinoxalines inhibit triple-negative breast cancer by specifically downregulating the c-MYC transcription.

Conventional chemotherapy remains the primary treatment option for triple-negative breast cancer (TNBC). However, the current chemotherapeutic drugs have limited effects on TNBC, and often lead to serious side effects as well as drug resistance. Thus, more effective therapeutic options are sorely needed. As c-MYC oncogene is highly expressed during TNBC pathogenesis, inhibiting c-MYC expression would be an alternative anti-TNBC strategy. In this study, we designed and synthesized a serial of quinoxaline analogs that target c-MYC promoter G-quadruplex (G4), which is believed to be a repressor of c-MYC transcription. Among them, a difluoro-substituted quinoxaline QN-1 was identified as the most promising G4-stabilizing ligand with high selectivity to c-MYC G4 over other G4s, which is distinguished from many other reported ligands. Intracellular studies indicated that QN-1 induced cell cycle arrest and apoptosis, repressed metastasis and inhibited TNBC cell growth, primarily due to the downregulation of c-MYC transcription by a G4-dependent mechanism. Notably, inhibition by QN-1 was significantly greater for c-MYC than other G4-driven genes. Cancer cells with c-MYC overexpression were more sensitive to QN-1, relative to normal cells. Furthermore, QN-1 effectively suppressed tumor growth in a TNBC mouse model. Accordingly, this work provides an alternative strategy for treating TNBC.

Development of a Smart Fluorescent Sensor That Specifically Recognizes the c-MYC G-Quadruplex.

The specific sensing of an exact G-quadruplex structure by small molecules has never been reported. A fluorescent sensor based on the photoinduced electron transfer (PeT) mechanism provides possibilities for such specific, one-to-one recognition, indicated by fluorescence. We have rationally developed a PeT fluorescent sensor IZFL-2 by linking triarylimidazole and fluorescein moieties. IZFL-2 is a distinctive, smart sensor whose fluorescence is tunable by its molecular conformations. We then applied IZFL-2 to sensing G-quadruplexes and found that it could exactly distinguish the wild-type c-MYC G-quadruplex from other types of G-quadruplexes, as shown by the activation of its fluorescence. To understand this behavior, we performed various experiments, including fluorescence assays, absorption assays, and multiscale molecular dynamics simulations, to thoroughly investigate the optimal binding mode of IZFL-2 in the c-MYC G-quadruplex. Then, the corresponding HOMO-LUMO of IZFL-2 was analyzed, and the results demonstrated that the PeT process of IZFL-2 is suppressed only in the wild-type c-MYC G-quadruplex via specific loop interactions, which restores its fluorescence. To our knowledge, this smart molecule provides the first example of and new insights into the development of sensors specific for a particular G-quadruplex structure by utilizing intramolecular PeT-controlled fluorescence switching.

Natural Aromatic Compounds as Scaffolds to Develop Selective G-Quadruplex Ligands: From Previously Reported Berberine Derivatives to New Palmatine Analogues.

In this paper, the selective interactions of synthetic derivatives of two natural compounds, berberine and palmatine, with DNA G-quadruplex structures were reported. In particular, the previous works on this subject concerning berberine were further presented and discussed, whereas the results concerning palmatine are presented here for the first time. In detail, these palmatine derivatives were developed by inserting seven different small peptide basic chains, giving several new compounds that have never been reported before. The preliminary studies of the interactions of these compounds with various G-quadruplex-forming sequences were carried out by means of various structural and biochemical techniques, which showed that the presence of suitable side chains is very useful for improving the interaction of the ligands with G-quadruplex structures. Thus, these new palmatine derivatives might act as potential anticancer drugs.

Discovery of a New Four-Leaf Clover-Like Ligand as a Potent c-MYC Transcription Inhibitor Specifically Targeting the Promoter G-Quadruplex.

Downregulating transcription of the oncogene c-MYC is a feasible strategy for cancer therapy. Stabilization of the G-quadruplex structure present in the c-MYC promoter can suppress c-MYC transcription. Thus, far, several ligands targeting this structure have been developed. However, most have shown no selectivity for the c-MYC G-quadruplex over other G-quadruplexes, leading to uncertain side effects. In this study, through structural modification of aryl-substituted imidazole/carbazole conjugates, a brand-new, four-leaf clover-like ligand called IZCZ-3 was found to preferentially bind and stabilize the c-MYC G-quadruplex. Further intracellular studies indicated that IZCZ-3 provoked cell cycle arrest and apoptosis and thus inhibited cell growth, primarily by blocking c-MYC transcription through specific targeting of the promoter G-quadruplex structure. Notably, IZCZ-3 effectively suppressed tumor growth in a mouse xenograft model. Accordingly, this work provides an encouraging example of a selective small molecule that can target one particular G-quadruplex structure, and the selective ligand might serve as an excellent anticancer agent.

Discovery of Novel 11-Triazole Substituted Benzofuro[3,2-b]quinolone Derivatives as c-myc G-Quadruplex Specific Stabilizers via Click Chemistry.

The specificity of nucleic acids' binders is crucial for developing this kind of drug, especially for novel G-quadruplexes' binders. Quindoline derivatives have been developed as G-quadruplex stabilizers with good interactive activities. In order to improve the selectivity and binding affinity of quindoline derivatives as c-myc G-quadruplex binding ligands, novel triazole containing benzofuroquinoline derivatives (T-BFQs) were designed and synthesized by using the 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks. The selectivity toward c-myc G-quadruplex DNA of these novel T-BFQs was significantly improved, together with an obvious increase on binding affinity. Further cellular and in vivo experiments indicated that the T-BFQs showed inhibitory activity on tumor cells' proliferation, presumably through the down-regulation of transcription of c-myc gene. Our findings broadened the modification strategies of specific G-quadruplex stabilizers.

Specific targeting of telomeric multimeric G-quadruplexes by a new triaryl-substituted imidazole.

IZNP-1: Multiple G-quadruplex units in the 3΄-terminal overhang of human telomeric DNA can associate and form multimeric structures. The specific targeting of such distinctive higher-order G-quadruplexes might be a promising strategy for developing selective anticancer agents with fewer side effects. However, thus far, only a few molecules were found to selectively bind to telomeric multimeric G-quadruplexes, and their effects on cancer cells were unknown. In this study, a new triaryl-substituted imidazole derivative called was synthesized and found to specifically bind to and strongly stabilize telomeric multimeric G-quadruplexes through intercalating into the pocket between the two quadruplex units. The pocket size might affect the binding behavior of . Further cellular studies indicated that could provoke cell cycle arrest, apoptosis and senescence in Siha cancer cells, mainly because of telomeric DNA damage and telomere dysfunction induced by the interactions of with telomeric G-quadruplexes. Notably, had no effect on the transcriptional levels of several common oncogenes that have the potential to form monomeric G-quadruplex structures in their promoter regions. Such behavior differed from that of traditional telomeric G-quadruplex ligands. Accordingly, this work provides new insights for the development of selective anticancer drugs targeting telomeric multimeric G-quadruplexes.