Targeting ATP-binding site of WRN Helicase: Identification of novel inhibitors through pocket analysis and Molecular Dynamics-Enhanced virtual screening.

WRN helicase is a critical protein involved in maintaining genomic stability, utilizing ATP hydrolysis to dissolve DNA secondary structures. It has been identified as a promising synthetic lethal target for microsatellite instable (MSI) cancers. However, few WRN helicase inhibitors have been discovered, and their potential binding sites remain unexplored. In this study, we analyzed potential binding sites for WRN inhibitors and focused on the ATP-binding site for screening new inhibitors. Through molecular dynamics-enhanced virtual screening, we identified two compounds, h6 and h15, which effectively inhibited WRN's helicase and ATPase activity in vitro. Importantly, these compounds selectively targeted WRN's ATPase activity, setting them apart from other non-homologous proteins with ATPase activity. In comparison to the homologous protein BLM, h6 exhibits some degree of selectivity towards WRN. We also investigated the binding mode of these compounds to WRN's ATP-binding sites. These findings offer a promising strategy for discovering new WRN inhibitors and present two novel scaffolds, which might be potential for the development of MSI cancer treatment.

Constructing triazole-modified quinazoline derivatives as selective c-MYC G-quadruplex ligands and potent anticancer agents through click chemistry.

c-MYC is a hallmark of various cancers, playing a critical role in promoting tumorigenesis. The formation of G-quadruplex (G4) in the c-MYC promoter region significantly suppresses its expression. Therefore, developing small-molecule ligands to stabilize c-MYC G4 formation and subsequentially suppress c-MYC expression is an attractive topic for c-MYC-driven cancer therapy. However, achieving selective ligands for c-MYC G4 poses challenges. In this study, we developed a series of triazole-modified quinazoline (TMQ) derivatives as potential c-MYC G4 ligands and c-MYC transcription inhibitors from 4-anilinoquinazoline lead 7a using click chemistry. Importantly, the c-MYC G4 stabilizing ability and antiproliferation activity were well correlated among these new derivatives, particularly in the c-MYC highly expressed colorectal cancer cell line HCT116. Among them, compound A6 exhibited good selectivity in stabilizing c-MYC G4 and in suppressing c-MYC transcription better than 7a. This compound induced G4 formation, selectively inhibited G4-related c-MYC transcription and suppressed the progression of HCT116 cells. These findings identify a new c-MYC transcription inhibitor and provide new insights for optimizing c-MYC G4-targeting ligands.

Discovery of Novel Coumarin-quinolinium Derivatives as Pan-KRAS Translation Inhibitors by Targeting 5'-UTR RNA G-Quadruplexes.

Hyperactivated KRAS mutations fuel tumorigenesis and represent attractive targets for cancer treatment. While covalent inhibitors have shown clinical benefits against the KRASG12C mutant, advancements for non-G12C mutants remain limited, highlighting the urgent demand for pan-KRAS inhibitors. RNA G-quadruplexes (rG4s) in the 5'-untranslated region of KRAS mRNA can regulate KRAS translation, making them promising targets for pan-KRAS inhibitor development. Herein, we designed and synthesized 50 novel coumarin-quinolinium derivatives, leveraging our previously developed rG4-specific ligand, QUMA-1. Notably, several compounds exhibited potent antiproliferative activity against cancer cells as pan-KRAS translation inhibitors. Among them, 15a displayed exceptional capability in stabilizing KRAS rG4s, suppressing KRAS translation, and consequently modulating MAPK and PI3K-AKT pathways. 15a induced cell cycle arrest, prompted apoptosis in KRAS-driven cancer cells, and effectively inhibited tumor growth in a KRAS mutant xenograft model. These findings underscore the potential of 15a as a pan-KRAS translation inhibitor, offering a novel and promising approach to target various KRAS-driven cancers.

Discovery of Ingenane Diterpenoids from Euphorbia hylonoma as Antiadipogenic Agents.

Thirteen new Euphorbia diterpenoids, euphylonanes A-M (1-13), and eight known ones were isolated from the whole plants of Euphorbia hylonoma. Compounds 1 and 2 are two rearranged ingenanes bearing a rare 6/6/7/3-fused ring system. Compound 3 represents the first example of a 9,10-epoxy tigliane, while 4-21 are typical ingenanes varying with substituents. Structures were elucidated using a combination of spectroscopic, computational, and chemical methods. Most ingenanes exerted a significant antiadipogenic effect in 3T3-L1 adipocytes, among which 4 was the most active with an EC50 value of 0.60 ± 0.27 µM. Mechanistic study revealed that 4 inhibited the adipogenesis and lipogenesis in adipocytes via activation of the AMPK signaling pathway.

Revealing Mitochondrion-Lysosome Dynamic Interactions and pH Variations in Live Cells with a pH-Sensitive Fluorescent Probe.

Mitochondrion-lysosome interactions have garnered significant attention in recent research. Numerous studies have shown that mitochondrion-lysosome interactions, including mitochondrion-lysosome contact (MLC) and mitophagy, are involved in various biological processes and pathological conditions. Single fluorescent probes are termed a pivotal chemical tool in unraveling the intricate spatiotemporal interorganelle interplay in live cells. However, current chemical tools are insufficient to deeply understand mitochondrion-lysosome dynamic interactions and related diseases, Moreover, the rational design of mitochondrion-lysosome dual-targeting fluorescent probes is intractable. Herein, we designed and synthesized a pH-sensitive fluorescent probe called INSA, which could simultaneously light up mitochondria (red emission) and lysosomes (green emission) for their internal pH differences. Employing INSA, we successfully recorded long-term dynamic interactions between lysosomes and mitochondria. More importantly, the increasing mitochondrion-lysosome interactions in ferroptotic cells were also revealed by INSA. Further, we observed pH variations in mitochondria and lysosomes during ferroptosis for the first time. In brief, this work not only introduced a pH-sensitive fluorescent probe INSA for the disclosure of the mitochondrion-lysosome dynamic interplays but also pioneered the visualization of the organellar pH alternation in a specific disease model.

Regioselective Electrophilic Addition to Propargylic B(MIDA)s Enabled by ß-Boron Effect.

Electrophilic addition reaction to alkynes is of fundamental importance in organic chemistry, yet the regiocontrol when reacting with unsymmetrical 1,2-dialkyl substituted alkynes is often problematic. Herein, it is demonstrated that the rarely recognized ß-boron effect can confer a high level of site-selectivity in several alkyne electrophilic addition reactions. A broad range of highly functionalized and complex organoborons are thus formed under simple reaction conditions starting from propargylic MIDA (N-methyliminodiacetic acid) boronates. These products are demonstrated to be valuable building blocks in organic synthesis. In addition to the regiocontrol, this study also observes a drastic rate enhancement upon B(MIDA) substitution. Theoretical calculation reveals that the highest occupied molecular obital (HOMO) energy level of propargylic B(MIDA) is significantly raised by 0.3 eV, and the preferential electrophilic addition to the γ position is due to its higher HOMO orbital coefficient and more negative natural bond orbital (NBO) charge compared to the ß position. This study demonstrates the potential of utilizing the ß-boron effect in stereoelectronic control of chemical transformations, which can inspire further research in this area.

Design, synthesis and evaluation of 2-pyrimidinylindole derivatives as anti-obesity agents by regulating lipid metabolism.

Obesity, a global pandemic posing a growing threat to human health, necessitates the development of effective and safe anti-obesity agents. Our previous studies highlighted the lipid-lowering effects of indolylquinazoline Bouchardatine and its derivatives. In this study, we employed scaffold hopping and simplification strategies to design and synthesize two new series derivatives by modifying the D ring. Extensive discussions have been conducted regarding the structure-activity relationship between lipid-lowering activity and the new compounds. These discussions have resulted in the discovery of 2-pyrimidinylindole derivatives as a promising scaffold for anti-obesity treatment. The new 2-pyrimidinylindole derivatives exhibited comparable lipid-lowering activity to the previously reported indolylquinazoline derivatives, including SYSU-3d and R17, with reduced toxicity. The most potent compound, 5a, demonstrated a larger therapeutic index, improved aqueous solubility and oral bioavailability compared to the previous lead compounds. In vivo evaluation indicated that 5a effectively reduced lipid accumulation in adipose tissue, improved glucose tolerance, and mitigated insulin resistance and liver function damage caused by a high-fat and high-cholesterol diet. Mechanism studies indicated that 5a may regulate lipid metabolism through the modulation of the PPARγ signaling pathway. Overall, our study has identified a highly active compound 5a, and provided the basis for further development of 2-pyrimidinylindole as a promising scaffold for obesity treatment.

MSN8C: A Promising Candidate for Antitumor Applications as a Novel Catalytic Inhibitor of Topoisomerase II.

MSN8C, an analog of mansonone E, has been identified as a novel catalytic inhibitor of human DNA topoisomerase II that induces tumor regression and differs from VP-16(etoposide). Treatment with MSN8C showed significant antiproliferative activity against eleven human tumor cell lines in vitro. It was particularly effective against the HL-60/MX2 cell line, which is resistant to Topo II poisons. The resistance factor (RF) of MSN8C for Topo II in HL-60/MX2 versus HL-60 was 1.7, much lower than that of traditional Topo II poisons. Furthermore, in light of its potent antitumor efficacy and low toxicity, as demonstrated in the A549 tumor xenograft model, MSN8C has been identified as a promising candidate for antitumor applications.

Divergent Fluorinations of Vinylcyclopropanes: Ring-Opening 1,5-Hydrofluorination and Ring-Retaining 1,2-Difluorination.

Organofluorine compounds have been widely used in pharmaceutical, agrochemical, and material sciences. Reported herein are divergent fluorination reactions of vinylcyclopropanes with different electrophiles, which allow the facile synthesis of homoallylic monofluorides and vicinal-difluorides through ring-opening 1,5-hydrofluorination and ring-retaining 1,2-difluorination, respectively. Both protocols feature mild conditions, simple operations, good functional group tolerance, and generally good yields. The practicality of these reactions is demonstrated by their scalability, as well as the successful conversion of the formed homoallylic monofluorides into other complex fluorinated molecules.

A rapid and highly sensitive immunosorbent assay to monitor helicases unwinding diverse nucleic acid structures.

Helicases are crucial enzymes in DNA and RNA metabolism and function by unwinding particular nucleic acid structures. However, most convenient and high-throughput helicase assays are limited to the typical duplex DNA. Herein, we developed an immunosorbent assay to monitor the Werner syndrome (WRN) helicase unwinding a wide range of DNA structures, such as a replication fork, a bubble, Holliday junction, G-quadruplex and hairpin. This assay could sensitively detect the unwinding of DNA structures with detection limits around 0.1 nM, and accurately monitor the substrate-specificity of WRN with a comparatively less time-consuming and high throughput process. Remarkably, we have established that this new assay was compatible in evaluating helicase inhibitors and revealed that the inhibitory effect was substrate-dependent, suggesting that diverse substrate structures other than duplex structures should be considered in discovering new inhibitors. Our study provided a foundational example for using this new assay as a powerful tool to study helicase functions and discover potent inhibitors.

Design and Synthesis of Bouchardatine Derivatives as a Novel AMP-Activated Protein Kinase Activator for the Treatment of Colorectal Cancer.

Metabolic reprogramming is a crucial hallmark of tumorigenesis. Modulating the reprogrammed energy metabolism is an attractive anticancer therapeutic strategy. We previously found a natural product, bouchardatine, modulated aerobic metabolism and inhibited proliferation in the colorectal cancer cell (CRC). Herein, we designed and synthesized a new series of bouchardatine derivatives to discover more potential modulators. We applied the dual-parametric high-content screening (HCS) to evaluate their AMP-activated protein kinase (AMPK) modulation and CRC proliferation inhibition effect simultaneously. And we found their antiproliferation activities were highly correlated to AMPK activation. Among them, 18a was identified with nanomole-level antiproliferation activities against several CRCs. Interestingly, the evaluation found that 18a selectively upregulated oxidative phosphorylation (OXPHOS) and inhibited proliferation by modulating energy metabolism. Additionally, this compound effectively inhibited the RKO xenograft growth along with AMPK activation. In conclusion, our study identified 18a as a promising candidate for CRC treatment and suggested a novel anti-CRC strategy by AMPK activating and OXPHOS upregulating.

Development and Characterization of Benzoselenazole Derivatives as Potent and Selective c-MYC Transcription Inhibitors.

Developing c-MYC transcription inhibitors that target the G-quadruplex has generated significant interest; however, few compounds have demonstrated specificity for c-MYC G-quadruplex and cancer cells. In this study, we designed and synthesized a series of benzoazole derivatives as potential G-quadruplex ligand-based c-MYC transcription inhibitors. Surprisingly, benzoselenazole derivatives, which are rarely reported as G-quadruplex ligands, demonstrated greater c-MYC G-quadruplex selectivity and cancer cell specificity compared to their benzothiazole and benzoxazole analogues. The most promising compound, benzoselenazole m-Se3, selectively inhibited c-MYC transcription by specifically stabilizing the c-MYC G-quadruplex. This led to selective inhibition of hepatoma cell growth and proliferation by affecting the MYC target gene network, as well as effective tumor growth inhibition in hepatoma xenografts. Collectively, our study demonstrates that m-Se3 holds significant promise as a potent and selective inhibitor of c-MYC transcription for cancer treatment. Furthermore, our findings inspire the development of novel selenium-containing heterocyclic compounds as c-MYC G-quadruplex-specific ligands and transcription inhibitors.

Discovery of the First Raptor (Regulatory-Associated Protein of mTOR) Inhibitor as a New Type of Antiadipogenic Agent.

Raptor, a regulatory-associated protein of mTOR, has been genetically proved to be an important regulator in lipogenesis. However, its druggable potential is rarely investigated, largely due to the lack of an inhibitor. In this study, the antiadipogenic screening of a daphnane diterpenoid library followed by target fishing led to the identification of a Raptor inhibitor, 1c (5/7/6 carbon ring with orthoester and chlorine functionalities). Pharmacodynamic studies verified that 1c is a potent and tolerable antiadipogenic agent in vitro and in vivo. Mechanistic studies revealed that the targeting of Raptor by 1c could block the formation of mTORC1 and then downregulate the downstream S6K1- and 4E-BP1-mediated C/EBPs/PPARγ signaling, eventually retarding adipocyte cell differentiation at the early stage. These findings suggest that Raptor can be explored as a novel therapeutic target for obesity and its related complications, and 1c as the first Raptor inhibitor may provide a new therapeutic option for these conditions.

Syntheses and evaluation of acridone derivatives as anticancer agents targeting Kras promoter i-motif structure.

Two series of novel acridone derivatives were designed and synthesized, with their anticancer activity evaluated. Most of these compounds showed potent antiproliferative activity against cancer cell lines. Among them, compound C4 with dual 1,2,3-triazol moieties exhibited the most potent activity against Hep-G2 cells with IC50 value determined to be 6.29 ± 0.93 µM. Subsequent experiments showed that C4 could bind to and destabilize Kras gene promoter i-motif structure without significant interaction with its corresponding G-quadruplex. C4 could down-regulate Kras expression in Hep-G2 cells, possibly due to its interaction with the Kras i-motif. Further cellular studies indicated that C4 could induce apoptosis of Hep-G2 cells, possibly related to its effect on mitochondrial dysfunction. These results indicated that C4 could be further developed as a promising anticancer agent.

Fluorescent Quinolinium Derivative as Novel Mitochondria Probe and Function Modulator by Targeting Mitochondrial RNA.

Mitochondria have a crucial role in regulating energy metabolism and their dysfunction has been linked to tumorigenesis. Cancer diagnosis and intervention have a great interest in the development of new agents that target biomolecules within mitochondria. However, monitoring and modulating mitochondria RNA (mtRNA), an essential component in mitochondria, in cells is challenging due to limited functional research and the absence of targeting agents. In this study, we designed and synthesized a fluorescent quinolinium derivative, QUCO-1, which actively lit up with mtRNA in both normal and cancer cells in vitro. Additionally, we evaluated the function of QUCO-1 as an mtRNA ligand and found that it effectively induced severe mitochondrial dysfunction and OXPHOS inhibition in RKO colorectal cancer cells. Treatment with QUCO-1 resulted in apoptosis, cell cycle blockage at the G2/M phase, and the effective inhibition of cell proliferation. Our findings suggest that QUCO-1 has great potential as a promising probe and therapeutic agent for mtRNA, with the potential for treating colorectal cancer.

Discovery of Clinically Used Octenidine as NRAS Repressor That Effectively Inhibits NRAS-Mutant Melanoma.

Mutations in NRAS promote tumorigenesis and drug resistance. As this protein is often considered an undruggable target, it is urgent to develop novel strategies to suppress NRAS for anticancer therapy. Recent reports indicated that a G-quadruplex (G4) structure formed in the untranslated region of NRAS mRNA can downregulate NRAS translation, suggesting a potential NRAS suppression strategy. Here, we developed a novel cell-based method for large-scale screening of NRAS G4 ligand using the G-quadruplex-triggered fluorogenic hybridization probe and successfully identified the clinically used agent Octenidine as a potent NRAS repressor. This compound suppressed NRAS translation, blocked the MAPK and PI3K-AKT signaling, and caused concomitant cell cycle arrest, apoptosis, and autophagy. It exhibited better antiproliferation effects over clinical antimelanoma agents and could inhibit the growth of NRAS-mutant melanoma in a xenograft mouse model. Our results suggest that Octenidine may be a prominent anti-NRAS-mutant melanoma agent and represent a new NRAS-mutant melanoma therapy option.

Regulation of c-Kit gene transcription selectively by bisacridine derivative through promoter dual i-motif structures.

BACKGROUND: c-Kit protein is a signal transduction protein involved in multiple signal pathways, which play an important role in a variety of cellular events such as cell proliferation, apoptosis and differentiation. Special DNA secondary structures on the promoter of c-Kit gene, including G-quadruplex and i-motif structures, could act as "molecular switch" for gene transcriptional regulation, which are potentially important target for development of new anti-cancer drugs. METHODS: We screened and evaluated the effect of compounds on c-Kit through several experiments, including SPR, FRET, CD, MST, NMR, dual-luciferase reporter assay, Western blot, qPCR, immunofluorescence, MTT assay, colony formation, cell scrape, cell apoptosis, cell cycle analysis, and transwell assay. RESULTS: After extensive screening, we found that bisacridine derivative B05 had selective binding and stabilization to dual i-motif structures on c-Kit gene promoter, which could down-regulate c-Kit gene transcription and translation, resulting in inhibition of cell proliferation and metastasis. B05 exhibited potent anti-tumor activity on HGC-27 cells, and strongly suppressed tumor growth in HGC-27 xenograft mice model. CONCLUSIONS: B05 could interact with c-Kit promoter dual i-motif structures with excellent selectivity, which make it possible for selective regulation of gene transcription and translation. B05 could be further developed for selective anti-cancer agent targeting c-Kit promoter i-motifs. GENERAL SIGNIFICANCE: i-Motifs on different proto-oncogene promoters are diversified, and especially binding of dual i-motifs on the same promoter simultaneously could significantly down-regulate gene transcription with decreased dosage, and therefore increasing the selectivity. This new strategy shed bight light on development of selective DNA-targeting ligands.

Construction of a TICT-AIE-Integrated Unimolecular Platform for Imaging Lipid Droplet-Mitochondrion Interactions in Live Cells and In Vivo.

Inter-organelle interactions play a vital role in diverse biological processes. Thus, chemical tools are highly desirable for understanding the spatiotemporal dynamic interplay among organelles in live cells and in vivo. However, designing such tools is still a great challenge due to the lack of universal design strategies. To break this bottleneck, herein, a novel unimolecular platform integrating the twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) dual mechanisms was proposed. As a proof of concept, two organelles, lipid droplets (LDs) and mitochondria, were selected as models. Also, the first TICT-AIE integration molecule, BETA-1, was designed for simultaneous and dual-color imaging of LDs and mitochondria. BETA-1 can simultaneously target LDs and mitochondria due to its lipophilicity and cationic structure and emit cyan fluorescence in LDs and red fluorescence in mitochondria. Using BETA-1, for the first time, we obtained long-term tracking of dynamic LD-mitochondrion interactions and identified several impressive types of dynamic interactions between these two organelles. More importantly, the increase in LD-mitochondrion interactions during ferroptosis was revealed with BETA-1, suggesting that intervening in the LD-mitochondrion interactions may modulate this cell death. BETA-1 was also successfully applied for in vivo imaging of LD-mitochondrion interactions in C. elegans. This study not only provides an effective tool for uncovering LD-mitochondrion interactions and deciphering related biological processes but also sheds light on the design of new probes with an integrated TICT-AIE mechanism for imaging of inter-organelle interactions.

Design, synthesis and evaluation of N3-substituted quinazolinone derivatives as potential Bloom's Syndrome protein (BLM) helicase inhibitor for sensitization treatment of colorectal cancer.

The homologous recombination repair (HRR) pathway is critical for repairing double-strand breaks (DSB). Inhibition of the HRR pathway is usually considered a promising strategy for anticancer therapy. The Bloom's Syndrome Protein (BLM), a DNA helicase, is essential for promoting the HRR pathway. Previously, we discovered quinazolinone derivative 9h as a potential BLM inhibitor, which suppressed the proliferation of colorectal cancer (CRC) cell HCT116. Herein, a new series of quinazolinone derivatives with N3-substitution was designed and synthesized to improve the anticancer activity and explore the structure-activity relationship (SAR). After evaluating their BLM inhibitory activity, the SAR was discussed, leading to identifying compound 21 as a promising BLM inhibitor. 21 exhibited the potent BLM-dependent cytotoxicity against the CRC cells but weak against normal cells. Further evaluation revealed that 21 could disrupt the HRR level while inhibiting BLM located on the DSB site and trigger DNA damage in the CRC cells. This compound effectively suppressed the proliferation and invasion of CRC cells, along with cell cycle arrest and apoptosis. Consequently, 21 might be a promising candidate for treating CRC, and the BLM might be a new potential therapeutic target for CRC.

Euphylonoids A and B, Two Highly Modified Jatrophane Diterpenoids with Potent Lipid-Lowering Activity from Euphorbia hylonoma.

Euphylonoids A (1) and B (2), two highly modified jatrophane diterpenoids, were isolated from Euphorbia hylonoma. 1 represents a new 9(10→18)-abeo-8,12-cyclojatrophane skeleton containing a cage-like 3,8-dioxatricyclo[5.1.2.04,9]decane core, while 2 is a 14(13→20)-abeo-8,12-cyclojatrophane featuring an unusual 17-oxatetracyclo[12.2.1.01,5.09,13]heptadecane framework. Their structural elucidation was completed by spectroscopic, chemical, computational, and single-crystal X-ray diffraction means. 2 significantly inhibited early adipogenesis in 3T3-L1 adipocytes via activating AMP-activated protein kinase signaling.