Targeting MutT Homolog 1 (MTH1) for Breast Cancer Suppression by Using a Novel MTH1 Inhibitor MA-24 with Tumor-Selective Toxicity.

BACKGROUND: Breast cancer is a commonly diagnosed cancer worldwide. Human MutT homolog 1 (MTH1) is found to be elevated in breast tumors and cancer cells need MTH1 for survival. Pharmacological inhibition of MTH1 may be potentially beneficial in the treatment of breast cancer. METHODS: MA-24 was screened by malachite green colorimetric assay for MTH1 inhibitors and the kinetic characteristics of MA-24 were assessed. The features of MA-24's binding with MTH1 were ascertained through molecular docking, and the cytotoxic activity of MA-24 was validated in vitro and in vivo. Target engagement assays, comet assay, and Western blot confirmed the intracellular target and mechanism of MA-24. RESULTS: MA-24 shows potent antitumor bioactivity both in vitro and in vivo. MA-24 competitively inhibited the MTH1 and further induced DNA strand breaks, leading to increased apoptosis of cancer cells depending on the upregulation of the cleaved-caspase 3-cleaved-PARP axis. In particular, MA-24 exhibited a powerful efficacy and safety in vivo (tumor growth inhibition rate: 61.8%). CONCLUSIONS: MA-24 possesses a broad spectrum of breast cancer cytotoxicity and offered valuable insights for overcoming the challenges of chemotherapy-related toxicity, which holds great potential for the further development MA-24 as an anti-cancer drug.

IDO1 Inhibition Promotes Activation of Tumor-intrinsic STAT3 Pathway and Induces Adverse Tumor-protective Effects.

Pharmacological inhibition of IDO1 exhibits great promise as a strategy in cancer therapy. However, the failure of phase III clinical trials has raised the pressing need to understand the underlying reasons for this outcome. To gain comprehensive insights into the reasons behind the clinical failure of IDO1 inhibitors, it is essential to investigate the entire tumor microenvironment rather than focusing solely on individual cells or relying on knockout techniques. In this study, we conducted single-cell RNA sequencing to determine the overall response to apo-IDO1 inhibitor administration. Interestingly, although apo-IDO1 inhibitors were found to significantly activate intratumoral immune cells (mouse colon cancer cell CT26 transplanted in BALB/C mice), such as T cells, macrophages, and NK cells, they also stimulated the infiltration of M2 macrophages. Moreover, these inhibitors prompted monocytes and macrophages to secrete elevated levels of IL-6, which in turn activated the JAK2/STAT3 signaling pathway in tumor cells. Consequently, this activation enables tumor cells to survive even in the face of heightened immune activity. These findings underscore the unforeseen adverse effects of apo-IDO1 inhibitors on tumor cells and highlight the potential of combining IL-6/JAK2/STAT3 inhibitors with apo-IDO1 inhibitors to improve their clinical efficacy.

Structure-Based Drug Design of Novel Triaminotriazine Derivatives as Orally Bioavailable IDH2R140Q Inhibitors with High Selectivity and Reduced hERG Inhibitory Activity for the Treatment of Acute Myeloid Leukemia.

Neomorphic IDH2R140Q mutation is commonly found in acute myeloid leukemia (AML), and inhibiting its activity has been validated as an effective treatment for AML. Herein, we report a series of highly potent and selective IDH2R140Q inhibitors. Among them, compound 36 was identified as the most promising inhibitor, with an IC50 value of 29 nM and more than 490-fold selectivity over wild-type IDH2. The compound significantly suppressed D2HG production (IC50 = 10 nM) and induced differentiation in TF-1/IDH2R140Q cells. Furthermore, it showed reasonable pharmacokinetic properties with high bioavailability (F = 90.3%) and an appropriate half-life (T1/2 = 6.4 h). In vivo, oral administration of compound 36 at a dose of 25 mg/kg effectively reduced D2HG levels in the tumor of TF-1/IDH2R140Q xenograft mouse model. Besides, compound 36 displayed little effect on the hERG current. These results suggest that compound 36 has the potential to be an efficacious treatment for AML.

Design, synthesis, and evaluation of 4(1H)-quinolinone and urea derivatives as KRASG12C inhibitors with potent antitumor activity against KRAS-mutant non-small cell lung cancer.

KRASG12C is the most prevalent KRAS mutation in non-small cell lung cancer (NSCLC) and has emerged as a promising therapeutic target. Herein, two series of novel 4(1H)-quinolinone and urea compounds were designed based on the reported KRASG12C inhibitor SH-9. Many compounds showed significantly growth inhibitory activity against human NSCLC cells with KRASG12C mutation in cell viability assays. Compound 20a exhibited an IC50 value of 0.5 µM in KRASG12C-mutant NCI-H358 cells with 21-fold selectivity over KRASWT NCI-H2228 cells. LC-MS analysis indicated that compounds 14c, 14h and 20a covalently bound to KRASG12C rather than KRASWT. Moreover, these compounds could remarkably trap KRASG12C in its inactive state by blocking SOS1-mediated GDP/GTP exchange. Furthermore, treatment of NCI-H358 but not NCI-H2228 cells with 20a dose-dependently reduced the phosphorylation of KRAS downstream effectors ERK and AKT. Importantly, 20a significantly inhibited tumor growth in NCI-H358 xenograft models by suppressing KRASG12C signalling. These results indicate that 20a is a promising candidate worthy of further investigation.

Combination of apatinib with apo-IDO1 inhibitor for the treatment of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer in the world. Recently, many clinical studies have demonstrated the therapeutic potential of immune checkpoint therapy combined with inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) in colon cancer. Compound B37, identified in our previous experiment, is an apo-form indoleamine-2,3-dioxygenase 1 inhibitor (apo-IDO1 inhibitor), which has been shown to significantly suppress tumor growth combined with an anti-PD1 antibody. We speculated whether this apo-IDO inhibitor (B37) combined with a VEGFR2 inhibitor (apatinib) would further improve its anti-tumor activity. Therefore, a syngeneic mouse colon cancer model (mouse colon cancer cell line CT26) was established to investigate the anti-tumor activity of B37 combined with apatinib. As expected, the combination of B37 and apatinib (VEGFR2 inhibitor) improved the therapeutic effect compared with apo-IDO1 inhibitor and apatinib monotherapy, as shown by the reduced growth of transplanted tumors, weakened proliferation, and increased apoptosis of cancer cells. Specifically, there was a 24.8% reduction in tumor volume using apatinib and 31.3% reduction using B37. The combination-treated group showed remarkable inhibition of tumor growth (52.2%). For tumor weight, there was a 29.2% reduction in the apatinib-treated group and 35.0% reduction in the B37-treated group. The combination-treated group showed a 56.3% reduction. Moreover, the combination therapy reprogrammed the immune microenvironment by increasing infiltration of CD4+ and CD8+ T cells, decreasing the ratio of regulatory T cells, and promoting the killing ability of T cells manifested by elevated expression of IFN-γ and granzyme B in the combination-treated group. Our study indicates that the combination of apo-IDO1 inhibitor and apatinib is a promising strategy for CRC therapy.

Apo-Form Selective Inhibition of IDO for Tumor Immunotherapy.

The pharmacological inhibition of IDO1 is considered an effective therapeutic approach for cancer treatment. However, the inadequate response of existing holo-IDO1 inhibitors and unclear biomarkers available in clinical practice limit the possibility of developing efficacious IDO1 inhibitors. In the current study, we aimed to elucidate the activity and mechanism of a potent 1H-pyrrole-2-carboxylic acid derivative (B37) targeting apo-IDO1 and to determine its role in tumor therapy. By competing with heme for binding to apo-IDO1, B37 potently inhibited IDO1 activity, with an IC50 of 22 pM assessed using a HeLa cell-based assay. The x-ray cocrystal structure of the inhibitor-enzyme complex showed that the B37-human IDO1 complex has strong hydrophobic interactions, which enhances its binding affinity, determined using isothermal titration calorimetry. Stronger noncovalent interactions, including π stacking and hydrogen bonds formed between B37 and apo-human IDO1, underlay the enthalpy-driven force for B37 for binding to the enzyme. These binding properties endowed B37 with potent antitumor efficacy, which was confirmed in a mouse colon cancer CT26 syngeneic model in BALB/c mice and in an azoxymethane/dextran sulfate sodium-induced colon carcinogenesis model in C57BL/6 mice by activating the host immune system. Moreover, the combination of B37 and anti-PD1 Ab synergistically inhibited tumor growth. These results suggested that B37 may serve as a unique candidate for apo-IDO1 inhibition-mediated tumor immunotherapy.

Ultrafast chiral peptides purification via surface plasmon enhanced spin selectivity.

By D-arginine and L-arginine chiral peptides induced spin selectivity and Au NPs enhanced spin polarization, chiral peptides purification has been effectively simplified and the purification performance has raised from a mixture system. The angular momentums of light are operated by the polarizer and wave plates. Au NPs decorated ZnO nanorods electrodes are utilized to modulate the polarization of spintronic. Seed growth methods are for synthesizing spherical Au NPs. UV light reduction methods are for urchin-liked Au NPs. Au NPs are decorated on ZnO nanorods electrodes for rising photon to electron conversion efficiency and enhancing spin polarization rates by surface plasmon effect. From our results, photon to the electron conversion efficiency of ZnO nanorods electrodes has effectively enhanced by urchin-liked Au NPs decorating. Ultrahigh localized plasmon conversion efficiency as high as 60% was also obtained. Besides, density functional theory (DFT) calculations simulated the force on spintronic. Since the D-arginine and L-arginine are on Au substrate, DFT results demonstrate different angular momentum and spin polarization coupling. Along with urchin-liked Au NPs rising chiral induced spin polarization by surface plasmon resonance, the sensitivity of chiral arginine has been raised around 5000% from bare ZnO nanorods electrodes. The purification and separation time of a specific chiral arginine only needs 5 min.

Design, Synthesis, and Evaluation of o-(Biphenyl-3-ylmethoxy)nitrophenyl Derivatives as PD-1/PD-L1 Inhibitors with Potent Anticancer Efficacy In Vivo.

Two series of novel o-(biphenyl-3-ylmethoxy)nitrophenyl compounds (A1-31 and B1-17) were designed as programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) inhibitors. All compounds showed significant inhibitory activity with IC50 values ranging from 2.7 to 87.4 nM except compound A17, and compound B2 displayed the best activity. Further experiments showed that B2 bound to the PD-L1 protein without obvious toxicity in Lewis lung carcinoma (LLC) cells. Furthermore, B2 significantly promoted interferon-gamma secretion in a dose-dependent manner in vitro and in vivo. Especially, B2 exhibited potent in vivo anticancer efficacy in an LLC-bearing allograft mouse model at a low dose of 5 mg/kg, which was more active than BMS-1018 (tumor growth inhibition rate: 48.5% vs 17.8%). A panel of immunohistochemistry and flow cytometry assays demonstrated that B2 effectively counteracted PD-1-induced immunosuppression in the tumor microenvironment, thereby triggering antitumor immunity. These results indicate that B2 is a promising PD-1/PD-L1 inhibitor worthy of further development.

Molecular modeling studies to discover novel mIDH2 inhibitors with high selectivity for the primary and secondary mutants.

Mutant isocitrate dehydrogenase 2 (mIDH2) is an emerging target for the treatment of cancer. AG-221 is the first mIDH2 inhibitor approved by the FDA for acute myeloid leukemia treatment, but its acquired resistance has recently been observed, necessitating the development of new inhibitor. In this study, a multi-step virtual screening protocol was employed for the analysis of a large database of compounds to identify potential mIDH2 inhibitors. To this end, we firstly utilized molecular dynamics (MD) simulations and binding free energy calculations to elucidate the key factors affecting ligand binding and drug resistance. Based on these findings, the receptor-ligand interaction-based pharmacophore (IBP) model and hierarchical docking-based virtual screening were sequentially carried out to assess 212,736 compounds from the Specs database. The resulting hits were finally ranked by PAINS filter and ADME prediction and the top compounds were obtained. Among them, six molecules were identified as mIDH2 putative inhibitors with high selectivity by interacting with the capping residue Asp312. Furthermore, subsequent docking and MD experiments demonstrated that compound V2 might have potential inhibitory activity against the AG-221-resistant mutants, thereby making it a promising lead for the development of novel mIDH2 inhibitors.

Discovery of secondary sulphonamides as IDO1 inhibitors with potent antitumour effects in vivo.

Indoleamine 2,3-dioxygenase 1 (IDO1) as a key rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism plays an important role in tumour immune escape. Herein, a variety of secondary sulphonamides were synthesised and evaluated in the HeLa cell-based IDO1/kynurenine assay, leading to the identification of new IDO1 inhibitors. Among them, compounds 5d, 5l and 8g exhibited the strongest inhibitory effect with significantly improved activity over the hit compound BS-1. The in vitro results showed that these compounds could restore the T cell proliferation and inhibit the differentiation of naïve CD4+ T cell into highly immunosuppressive FoxP3+ regulatory T (Treg) cell without affecting the viability of HeLa cells and the expression of IDO1 protein. Importantly, the pharmacodynamic assay showed that compound 5d possessed potent antitumour effect in both CT26 and B16F1 tumours bearing immunocompetent mice but not in immunodeficient mice. Functionally, subsequent experiments demonstrated that compound 5d could effectively inhibit tumour cell proliferation, induce apoptosis, up-regulate the expression of IFN-γ and granzyme B, and suppress FoxP3+ Treg cell differentiation, thereby activate the immune system. Thus, compound 5d could be a potential and efficacious agent for further evaluation.

Effective Virtual Screening Strategy toward heme-containing proteins: Identification of novel IDO1 inhibitors.

Developing small molecules occupying the heme-binding site using computational approaches remains a challenging task because it is difficult to characterize heme-ligand interaction in heme-containing protein. Indoleamine 2,3-dioxygenase 1 (IDO1) is an intracellular heme-containing dioxygenase which is associated with the immunosuppressive effects in cancer. With IDO1 as an example, herein we report a combined virtual screening (VS) strategy including high-specificity heme-binding group (HmBG)-based pharmacophore screening and cascade molecular docking to identify novel IDO1 inhibitors. A total of four hit compounds were obtained and showed proper binding with the heme iron coordinating site. Further structural optimization led to a promising compound S18-3, which exerted potent anti-tumor efficacy in BALB/c mice bearing established CT26 tumors by activating the host's immune system. These results suggest that S18-3 merits further study to assess its potential for the intervention of cancer. Furthermore, our study also unveils a novel in silico-based strategy for identifying potential regulators for hemeproteins within short timeframe.

Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour­targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni­NTA column and a G­50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high­performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec­1, respectively. The anti­hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF­7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.

Novel Ligustrazine-Based Analogs of Piperlongumine Potently Suppress Proliferation and Metastasis of Colorectal Cancer Cells in Vitro and in Vivo.

Piperlongumine 1 increases reactive oxygen species (ROS) levels and preferably induces cancer cell apoptosis by triggering different pathways. However, the poor solubility of 1 limits its intensive investigation and clinical application. Ligustrazine possesses a water-soluble pyrazine skeleton and can inhibit proliferation and metastasis of cancer cells. We synthesized compound 3 by replacement of the trimethoxyphenyl of 1 with ligustrazine moiety and further introduced 2-Cl, -Br, and -I to 3 for synthesis of 4-6, respectively. Compound 4 possessed 14-fold greater aqueous solubility than 1 and increased ROS levels in colorectal cancer HCT-116 cells. Additionally, 4 preferably inhibited proliferation, migration, invasion, and heteroadhesion of HCT-116 cells. Treatment with 4 suppressed tumor growth and lung metastasis in vivo and prolonged the survival of tumor-bearing mice. Furthermore, 4 mitigated TGF-ß1-induced epithelial-mesenchymal transition and Wnt/ß-catenin activation by inhibiting the Akt and GSK-3ß phosphorylation in HCT-116 cells. Collectively, 4 displayed significant antiproliferation and antimetastasis activities, superior to 1.

Transcriptome analysis to assess the cholestatic hepatotoxicity induced by Polygoni Multiflori Radix: Up-regulation of key enzymes of cholesterol and bile acid biosynthesis.

Polygoni Multiflori Radix (PMR) has been commonly used as a tonic in China for centuries. However, PMR-associated hepatotoxicity is becoming a safety issue. Cholestasis often occurs in PMR-induced hepatotoxicity in clinical medicine, but the exact mechanism is not completely understood. An RNA-Seq method was employed, in the present study, to explore the molecular mechanism of cholestatic liver injury induced by PMR, characterized by the hepatic transcriptional response in rats exposed to 1 and 20 g/kg PMR for 90 days. Pathological changes seen in rat livers exposed to PMR included increased bile ducts in portal areas and biliary epithelial cell hyperplasia, which were accompanied by the elevation of serum biochemistries. Dose-dependent increases in the expression of 14 transcripts encoding enzymes involved in the cholesterol biosynthetic pathway were identified. Furthermore, cholesterol 7-alpha hydroxylase (Cyp7a1), a rate-limiting enzyme in the synthesis of bile acids (BAs) from cholesterol, was found to be upregulated by PMR treatment. Protein analysis by western blot suggested that expression of 3-hydroxy-3-methylglutaryl CoA reductase (Hmgcr) and Cyp7a1 were increased in a dose-dependent manner. Collectively, the present study demonstrates that PMR upregulates key enzymes for biosynthesis of cholesterol and BA, which poses the risk of cholestatic liver injury. SIGNIFICANCE: To the best of our knowledge, this is the first transcriptome analysis to highlight the main molecular changes occurring in rats chronic exposed to PMR. We have identified 39 specific differentially expressed genes (DEGs) that were present in various comparisons. A total of 14 of these altered gene transcripts were associated with cholesterol biosynthesis. Another factor of great importance in our opinion seemed to be the enhancement of bile acid (BA) biosynthesis, which were closely linked to cholesterol biosynthesis or metabolism. Our findings suggested that the disturbance on balance of BA formation and elimination might lead to a BA overload in hepatocytes, thereby resulting in liver injury.

Firefly-like Water Splitting Cells Based on FRET Phenomena with Ultrahigh Performance over 12.

A firefly-like chemiluminescence reaction was utilized in a ZrO2 nanoparticle matrix of water splitting cells, where the chlorophyll of Lantana camara was used as the major photosensitizer to excite electrons to the conduction band of ZrO2. The fluorescence resonance energy transfer (FRET) was induced by rubrene, a firefly-like chemiluminescence molecule, and Lantana camara chlorophyll combined with 9,10-diphenylanthracene. The ZrO2 nanoparticle film coated by the chlorophyll of Lantana camara and 9,10-diphenylanthracene under chemiluminescence irradiation in 1 M KHCO3 water solution demonstrated the highest photocurrent density (88.1 A/m2) and the highest water splitting efficiency (12.77%).

Novel Derivative of Bardoxolone Methyl Improves Safety for the Treatment of Diabetic Nephropathy.

Currently, no effective and safe medicines are available to treat diabetic nephropathy (DN). Bardoxolone methyl (CDDO-Me) has displayed promising anti-DN activity as well as serious side effects in clinical trials, probably because the highly reactive α-cyano-α,ß-unsaturated ketone (CUK) in ring A of CDDO-Me can covalently bind to thiol functionalities in many biomacromolecules. In this study, we designed and synthesized a γ-glutamyl transpeptidase (GGT)-based and CUK-modified derivative of CDDO-Me (2) to address this issue. 2 can be specifically cleaved by GGT, which is highly expressed in the kidney, to liberate CDDO-Me in situ. It should be noted that 2 exhibited anti-DN efficacy comparable to that of CDDO-Me with much less toxicity in cells and db/db mice, suggesting that its safety is better than CDDO-Me. Our findings not only reveal the therapeutic potential of 2 but also provide a strategy to optimize other synthetic molecules or natural products bearing a pharmacophore like CUK to achieve safer pharmaceutical drugs.

Discovery of imidazoleisoindole derivatives as potent IDO1 inhibitors: Design, synthesis, biological evaluation and computational studies.

Indoleamine-2,3-dioxygenase-1 (IDO1) is an attractive target for cancer immunotherapy. Herein, a series of novel imidazoleisoindole derivatives were prepared and evaluated for their ability to inhibit IDO1. Among these, derivative 11r was the most active compound with nanomolar potency in the Hela cell-based assay, while showed negligible cellular toxicity. UV-visible spectra study demonstrated that compounds 11p and 11r bound to IDO1 and coordinated with the heme iron. Furthermore, they could significantly promote T cell proliferation, increase IFN-γ production, and reduce the numbers of Foxp3+ regulatory T cells. Finally, induced fit docking (IFD) and quantum mechanics/molecular mechanics (QM/MM) calculation were performed to understand the interactions of these compounds to IDO1 protein, which provided a comprehensive guide for further structural modification and optimization.