Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.

Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

Neutrophils in Cancer immunotherapy: friends or foes?

Neutrophils play a Janus-faced role in the complex landscape of cancer pathogenesis and immunotherapy. As immune defense cells, neutrophils release toxic substances, including reactive oxygen species and matrix metalloproteinase 9, within the tumor microenvironment. They also modulate the expression of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand, augmenting their capacity to induce tumor cell apoptosis. Their involvement in antitumor immune regulation synergistically activates a network of immune cells, bolstering anticancer effects. Paradoxically, neutrophils can succumb to the influence of tumors, triggering signaling cascades such as JAK/STAT, which deactivate the immune system network, thereby promoting immune evasion by malignant cells. Additionally, neutrophil granular constituents, such as neutrophil elastase and vascular endothelial growth factor, intricately fuel tumor cell proliferation, metastasis, and angiogenesis. Understanding the mechanisms that guide neutrophils to collaborate with other immune cells for comprehensive tumor eradication is crucial to enhancing the efficacy of cancer therapeutics. In this review, we illuminate the underlying mechanisms governing neutrophil-mediated support or inhibition of tumor progression, with a particular focus on elucidating the internal and external factors that influence neutrophil polarization. We provide an overview of recent advances in clinical research regarding the involvement of neutrophils in cancer therapy. Moreover, the future prospects and limitations of neutrophil research are discussed, aiming to provide fresh insights for the development of innovative cancer treatment strategies targeting neutrophils.

Exopolysaccharides from the Green Microalga Strain Coelastrella sp. BGV-Isolation, Characterization, and Assessment of Anticancer Potential.

Algal metabolites have been extensively studied as potential anticancer therapeutics. Among them, polysaccharides have attracted much attention because of their beneficial biological effects and safety. In the present research, the chemical characteristics, antitumor, and proapoptotic activities of extracellular polysaccharides (EPS) isolated from a new Bulgarian strain of the green microalga Coelastrella sp. BGV were investigated. A fast and convenient method of precipitation with cold ethanol was used to isolate EPS from the culture medium. The chemical characteristics of the isolated EPS were examined by colorimetric and spectrophotometric analyses, HPSEC-RID and HPLC-UV chromatography, and FT-IR spectroscopy. The results showed that the isolated EPS sample consists of three carbohydrate fractions with different molecular weights (11.5 × 104 Da, 30.7 × 104 Da, and 72.4 × 104 Da, respectively) and contains 7.14 (w/w%) protein. HPLC-UV analysis revealed the presence of galactose and fucose. The total uronic acid content in the sample was 4.5 (w/w%). The IR-FT spectrum of EPS revealed the presence of various functional groups typical of a polysaccharide (or proteoglycan) composed primarily of neutral sugars. The anticancer potential of the obtained EPS was assessed using cell lines with cancerous and non-cancerous origins as in vitro experimental models. The results of the performed MTT assay showed that EPS reduced the viability of the cervical and mammary carcinoma cell lines HeLa and MCF-7, while the control non-cancer cell lines BALB/3T3 and HaCaT were less affected. The HeLa cell line showed the highest sensitivity to the effects of EPS and was therefore used for further studies of its anticancer potential. The ability of EPS to inhibit cancer cell migration was demonstrated by wound-healing (scratch) assay. The cell cycle FACS analysis indicated that the EPS treatment induced significant increases in the sub G1 cell population and decreases of the percentages of cells in the G1, S, and G2-M phases, compared to the control. The fluorescent microscopy studies performed using three different staining methods in combination with Annexin V-FITC flow cytometric analysis clearly demonstrate the ability of EPS to induce cancer cell death via the apoptosis pathway. Moreover, an altered pattern and intensity of the immunocytochemical staining for the apoptosis- and proliferation-related proteins p53, bcl2, and Ki67 was detected in EPS-treated HeLa cancer cells as compared to the untreated controls. The obtained results characterize the new local strain of green microalgae Coelastrella sp. BGV as a producer of EPS with selective antitumor activity and provide an opportunity for further studies of its pharmacological and biotechnological potential.

Antitumor activities against breast cancers by an afucosylated anti-HER2 monoclonal antibody H2 Mab-77-mG2a -f.

Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2 Mab-77 (mouse IgG1 , kappa). This was then altered to produce H2 Mab-77-mG2a -f, an afucosylated mouse IgG2a . In the present work, we examined the reactivity of H2 Mab-77-mG2a -f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressing breast cancer cell line, was identified by H2 Mab-77-mG2a -f with a strong binding affinity (a dissociation constant [KD ]: 5.0 × 10-9 M). H2 Mab-77-mG2a -f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H2 Mab-77-mG2a -f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2 Mab-77-mG2a -f. Additionally, in the BT-474-bearing tumor xenograft model, H2 Mab-77-mG2a -f substantially suppressed tumor development when compared with the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2 Mab-77-mG2a -f. These findings point to the possibility of H2 Mab-77-mG2a -f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

Selective killing of castration-resistant prostate cancer cells by formycin A via the ATF4-CHOP axis.

Prostate cancer is initially androgen-dependent but often relapses to an androgen-independent state called castration-resistant prostate cancer (CRPC). Currently approved therapies have limited efficacy against CRPC, highlighting the need for novel therapeutic strategies. To address this need, we conducted a drug screen in our previously established aggressive CRPC cell model. We found that formycin A induced cell death in CRPC model cells but not in parental prostate cancer cells. In addition, formycin A upregulated death receptor 5 through the induction of endoplasmic reticulum stress, activating the "extrinsic" apoptosis pathway in CRPC model cells. Moreover, formycin A showed in vivo antitumor efficacy against CRPC xenografts in castrated nude mice. Thus, our findings highlight the potential of formycin A as a CRPC therapeutic.

A monoclonal antibody recognizing CD98 on human embryonic stem cells shows anti-tumor activity in hepatocellular carcinoma xenografts.

CD98, also known as SLC3A2, is a multifunctional cell surface molecule consisting of amino acid transporters. CD98 is ubiquitously expressed in many types of tissues, but expressed at higher levels in cancerous tissues than in normal tissues. CD98 is also upregulated in most hepatocellular carcinoma (HCC) patients; however, the function of CD98 in HCC cells has been little studied. In this study, we generated a panel of monoclonal antibodies (MAbs) against surface proteins on human embryonic stem cells (hESCs). NPB15, one of the MAbs, bound to hESCs and various cancer cells, including HCC cells and non-small cell lung carcinoma (NSCLC) cells, but not to peripheral blood mononuclear cells (PBMCs) and primary hepatocytes. Immunoprecipitation and mass spectrometry identified the target antigen of NPB15 as CD98. CD98 depletion decreased cell proliferation, clonogenic survival, and migration and induced apoptosis in HCC cells. In addition, CD98 depletion decreased the expression of cancer stem cell (CSC) markers in HCC cells. In tumorsphere cultures, the expression of CD98 interacting with NPB15 was significantly increased, as were known CSC markers. After cell sorting by NPB15, cells with high expression of CD98 (CD98-high) showed higher clonogenic survival than cells with low expression of CD98 (CD98-low) in HCC cells, suggesting CD98 as a potential CSC marker on HCC cells. The chimeric version of NPB15 was able to induce antibody-dependent cellular cytotoxicity (ADCC) against HCC cells in vitro. NPB15 injection showed antitumor activity in an HCC xenograft mouse model. The results suggest that NPB15 may be developed as a therapeutic antibody for HCC patients.

Boosting antitumor efficacy using docetaxel-loaded nanoplatforms: from cancer therapy to regenerative medicine approaches.

The intersection of nanotechnology and pharmacology has revolutionized the delivery and efficacy of chemotherapeutic agents, notably docetaxel, a key drug in cancer treatment. Traditionally limited by poor solubility and significant side effects, docetaxel's therapeutic potential has been significantly enhanced through its incorporation into nanoplatforms, such as nanofibers and nanoparticles. This advancement offers targeted delivery, controlled release, and improved bioavailability, dramatically reducing systemic toxicity and enhancing patient outcomes. Nanofibers provide a versatile scaffold for the controlled release of docetaxel, utilizing techniques like electrospinning to tailor drug release profiles. Nanoparticles, on the other hand, enable precise drug delivery to tumor cells, minimizing damage to healthy tissues through sophisticated encapsulation methods such as nanoprecipitation and emulsion. These nanotechnologies not only improve the pharmacokinetic properties of docetaxel but also open new avenues in regenerative medicine by facilitating targeted therapy and cellular regeneration. This narrative review highlights the transformative impact of docetaxel-loaded nanoplatforms in oncology and beyond, showcasing the potential of nanotechnology to overcome the limitations of traditional chemotherapy and pave the way for future innovations in drug delivery and regenerative therapies. Through these advancements, nanotechnology promises a new era of precision medicine, enhancing the efficacy of cancer treatments while minimizing adverse effects.

Silver ciprofloxacin (CIPAG): a multitargeted metallodrug in the development of breast cancer therapy.

The anti-proliferative activity of the known metalloantibiotic {[Ag(CIPH)2]NO3∙0.75MeOH∙1.2H2O} (CIPAG) (CIPH = ciprofloxacin) against the human breast adenocarcinoma cancer cells MCF-7 (hormone dependent (HD)) and MDA-MB-231 (hormone independent (HI)) is evaluated. The in vitro toxicity and genotoxicity of the metalloantibiotic were estimated toward fetal lung fibroblast (MRC-5) cells. The molecular mechanism of the CIPAG activity against MCF-7 cells was clarified by the (i) cell morphology, (ii) cell cycle arrest, (iii) mitochondrial membrane permeabilization, and (iv) by the assessment of the possible differential effect of CIPAG on estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) transcriptional activation, applying luciferase reporter gene assay. Moreover, the ex vivo mechanism of CIPAG was clarified by its binding affinity toward calf thymus (CT-DNA).

The Effect of Lipid Composition on the Liposomal Delivery of Camptothecin Developed by Active Click Loading.

In the present study, we investigated the role of lipid composition of camptothecin (CPT)-loaded liposomes (CPT-Lips) to adjust their residence time, drug distribution, and therefore the toxicities and antitumor activity. The CPT was loaded into liposomes using a click drug loading method, which utilized liposomes preloaded with GSH and then exposed to CPT-maleimide. The method produced CPT-Lips with a high encapsulation efficiency (>95%) and sustained drug release. It is shown that the residence times of CPT-Lips in the body were highly dependent on lipid compositions with an order of non-PEGylated liposomes of unsaturated lipids < non-PEGylated liposomes of saturated lipids < PEGylated liposomes of saturated lipids. Interestingly, the fast clearance of CPT-Lips resulted in significantly decreased toxicities but did not cause a significant decrease in their in vivo antitumor activity. These results suggested that the lipid composition could effectively adjust the residence time of CPT-Lips in the body and further optimize their therapeutic index, which would guide the development of a liposomal formulation of CPT.

Synthesis and Biological Evaluation of Methoxypolyethylene-Glycol-Substituted Abiraterone Derivatives as Potential Antiprostate Cancer Agents.

Globally, prostate cancer is the most commonly diagnosed tumor and a cause of death in older men. Abiraterone, an orally administered irreversible CYP17 inhibitor, is employed to treat prostate cancer. However, abiraterone has several clinical limitations, such as poor water solubility, low dissolution rate, low bioavailability, and toxic side effects in the liver and kidney. Therefore, there is a need to identify high-efficiency and low-toxicity water-soluble abiraterone derivatives. In this work, we aimed to design and synthesize a series of abiraterone derivatives by methoxypoly(ethylene glycol) (mPEG) modification. Their antitumor activities and toxicology were analyzed in vitro and in vivo. The most potent compound, 2e, retained the principle of action on the CYP17 enzyme target and significantly improved the abiraterone water solubility, cell permeability, and blood safety. No significant abnormalities were observed in toxicology. mPEG-modification significantly improved abiraterone's antitumor activity and efficiency while reducing the associated toxic effects. The finding will provide a theoretical basis for future clinical application of mPEG-modified abiraterone.

A newly identified secretory phospholipase A2 group XIIA homolog (LcPLA2XIIA) in Larimichthys crocea exhibits antimicrobial and antitumor activities.

The phospholipase A2 (PLA2) superfamily has attracted increasing attention in recent years due to the multiple physiological and pathological functions exerted by its members. Up to date, the knowledge about the biological role of PLA2XIIA subfamily members remains limited. In this study, a new member of PLA2XIIA subfamily, LcPLA2XIIA, was characterized in large yellow croaker. Different from most members of the PLA2 superfamily with positive charge, LcPLA2XIIA encodes an anionic protein, which is similar to other members of PLA2XIIA subfamily. LcPLA2XIIA is highly expressed in the intestine, and afterwards, it is up-regulated after with Pseudomonas plecoglossicida or Staphylococcus aureus. LcPLA2XIIA exhibits strong inhibitory activity against these two bacteria. The results indicate that LcPLA2XIIA plays an important role in the antimicrobial immune responses of large yellow croaker. LcPLA2XIIA displays strong binding activity to all the tested bacteria. It specifically interacts with LTA, a unique component on the surface of Gram-positive bacteria. It also significantly promotes bacterial agglutination in the presence of Ca2+. These findings reveal that the binding and agglutinating abilities of LcPLA2XIIA to bacteria contribute greatly to its antibacterial activity. In addition, LcPLA2XIIA significantly inhibits the proliferation of infectious hematopoietic necrosis virus instead of recombinant human adenovirus type 5. It also suppresses the growth of human colorectal adenocarcinoma cells by inducing apoptosis, but it has no obvious inhibitory effect on the growth of epithelioma papulosum cyprinid cells. This study provides new insights into the antibacterial activity, and the mechanism of LcPLA2XIIA in large yellow croaker, and antiviral and antitumor functions of PLA2XIIA subfamily members.

Preparation of valine-curcumin conjugate and its in vitro antibacterial and antitumor activity and in vivo biological effects on American eels (Anguilla rostrata).

Curcumin (Cur) exhibits diverse natural pharmacological activities, despite its limited water solubility (hydrophobicity) and low bioavailability. In this investigation, a valine-curcumin conjugate (Val-Cur) was synthesized through amino acid side chain modification, and its solubility increased to 1.78 mg/mL. In vitro experimental findings demonstrated that the antibacterial activity of Val-Cur against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus was significantly superior to that of Cur. The inhibition rate of Val-Cur against HepG2 (human hepatocellular carcinoma) cells was higher than that of Cur at low concentrations (below 25 µmol/L), although the IC50 value of Val-Cur did not differ significantly from that of Cur. In vivo biological effects of Val-Cur were assessed by adding it into the feed (150 mg/kg) of American eels (Anguilla rostrata). Val-Cur significantly improved the growth performance (↑weight gain rate, ↑specific growth rate, and ↓feed conversion rate) and activities of intestinal digestive enzymes (amylase and lipase) and antioxidant enzymes (superoxide dismutase) in American eels. Additionally, Val-Cur significantly improved serum biochemical indices (↑high-density lipoprotein cholesterol, ↓low-density lipoprotein cholesterol, ↓aspartate and alanine aminotransferases). Furthermore, Val-Cur increased intestinal microbial diversity, reduced the abundance of potentially pathogenic bacteria (Spiroplasma, Clostridium, and Pseudomonas), and elevated the abundance of beneficial digestion-promoting bacteria (Romboutsia, Phyllobacterium, Romboutsia sedimentorum, and Clostridium butyricum) conducive to glucose metabolism (P < 0.05). To the best of our knowledge, this study is the first to explore water-soluble curcumin in aquaculture, and the findings will lay the groundwork for the potential application of water-soluble curcumin in the field of aquaculture.

Interleukin-33 and liver natural killer cells: A novel perspective on antitumor activity in liver fibrosis.

AIM: Liver fibrosis, heralding the potential progression to cirrhosis and hepatocellular carcinoma (HCC), compromises patient survival and augments post-hepatectomy recurrence. This study examined the detrimental effects of liver fibrosis on the antitumor functions of liver natural killer (NK) cells and the interleukin-33 (IL-33) signaling pathway. METHODS: Our investigation, anchored in both human physiologies using living and deceased donor livers and the carbon tetrachloride (CCl4)-induced mouse fibrosis model, aimed to show a troubling interface between liver fibrosis and weakened hepatic immunity. RESULTS: The Fibrosis-4 (FIB-4) index emerged as a salient, non-invasive prognostic marker, and its elevation correlated with reduced survival and heightened recurrence after HCC surgery even after propensity matching (n = 385). We established a strong correlation between liver fibrosis and liver NK cell dysfunction by developing a method for extracting liver NK cells from the liver graft perfusate. Furthermore, liver fibrosis ostensibly disrupted chemokines and promoted IL-33 expression, impeding liver NK cell antitumor activities, as evidenced in mouse models. Intriguingly, our results implicated IL-33 in diminishing the antitumor responses of NK cells. This interrelation, consistent across both mouse and human studies, coincides with clinical data suggesting that liver fibrosis predisposes patients to an increased risk of HCC recurrence. CONCLUSION: Our study revealed a critical relationship between liver fibrosis and compromised tumor immunity, emphasizing the potential interference of IL-33 with NK cell function. These insights advocate for advanced immunostimulatory therapies targeting cytokines, such as IL-33, aiming to bolster the hepatic immune response against HCC in the context of liver fibrosis.

Synthesis and evaluation of anticancer activity of new 4,5,6,7-tetrabromo-1H-benzimidazole derivatives.

An efficient method for the synthesis of new 4,5,6,7-tetrabromo-1H-benzimidazole derivatives has been developed. New ketones were obtained by N-alkylation of TBBi or 2-Me-TBBi with various phenacyl halides and then reduced to the corresponding alcohols. All compounds were obtained with satisfactory yields in the range of 40-91 %. The synthesized compounds appeared a weak CK2 and PIM-1 inhibitors but exhibit an interesting cytotoxic activity against cancer cell lines, i.e. MCF-7, PC-3, CCRF-CEM, K-562. 1-Phenyl-2-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)ethanone 3aA exhibits the highest cytotoxic activity with IC50 value of 5.30 µM for MCF-7 and 6.80 µM for CCRF-CEM. Moreover, this compound shows the highest selectivity against both MCF-7 and CCRF-CEM with SI selectivity coefficients (against MRC-5 and Vero cells) equal 5.45 and 4.30 for MCF-7 and 4.25 and 3.35 for CCRF-CEM, respectively. Furthermore, it was shown that compound 3aA exhibits very good pro-apoptotic properties, through induction of the mitochondrial apoptotic pathway in CCRF-CEM cells. These results correlate with data showing the effect of 3aA on intracellular level of CK2α protein and CK2-mediated phosphorylation of Ser529 in NF-κBp65. Study of the effect of compound 3aA on mRNA levels of CK2α and CK2α' showed no significant differences in gene expression levels in control CCRF-CEM and cells treated with 3aA, indicating 3aA action at the protein level.

Design, synthesis and antitumor activities of phthalazinone derivatives as PARP-1 inhibitors and PARP-1/HDAC-1 inhibitors.

In recent years, poly(ADP-ribose)polymerase-1 (PARP-1) and histone deacetylase (HDAC) have emerged as significant targets in tumor therapy, garnering widespread attention. In this study, we designed and synthesized two novel phthalazinone PARP-1 inhibitors and dual PARP-1/HDAC-1 inhibitors, named DLC-1-46 containing dithiocarboxylate fragments and DLC-47-63 containing hydroxamic acid fragments, and evaluated their inhibitory activities on enzymes and cells. Among the PARP-1 inhibitors, most compounds exhibited high inhibitory activity against the PARP-1 enzyme, with DLC-1-6 being particularly notable, showing IC50 values <0.2 nM. Notably, DLC-1 demonstrated significant anti-proliferative activity, with IC50 values for inhibiting the proliferation of MDA-MB-436, MDA-MB-231, and MCF-7 cells reaching 0.08, 26.39, and 1.01 µM, respectively. Further investigation revealed that DLC-1 arrested MDA-MB-231 cells in the G1 phase and induced apoptosis in a dose-dependent manner. Among the designed dual PARP-1/HDAC-1 inhibitors, several compounds exhibited potent dual-target inhibitory activity, with DLC-49 displaying IC50 values of 0.53 nM and 17 nM for PARP-1 and HDAC-1, respectively. DLC-50 demonstrated the most potent anti-proliferative activity, with IC50 values for inhibiting the proliferation of MDA-MB-436, MDA-MB-231, and MCF-7 cells at 0.30, 2.70, and 2.41 µM, respectively. Cell cycle arrest and apoptosis assays indicated that DLC-50 arrested the cell cycle in the G2 phase and induced apoptosis in HCT-116 cells. Our findings present a novel avenue for further exploration of PARP-1 inhibitors and dual PARP-1/HDAC-1 inhibitors.

Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies.

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

Design, synthesis, and evaluation of antitumor activity of Mobocertinib derivatives, a third-generation EGFR inhibitor.

Mobocertinib, as a structural analog of the third generation TKI Osimertinib, can selectively act on the EGFRex20 mutation. We have structurally modified Mobocertinib to obtain new EGFR inhibitors. In this paper, we chose Mobocertinib as a lead compound for structural modification to investigate the effect of Mobocertinib derivatives on EGFRT790M mutation. We designed and synthesized 63 Mobocertinib derivatives by structural modification using the structural similarity strategy and the bioelectronic isoarrangement principle. Then, we evaluated the in vitro antitumor activity of the 63 Mobocertinib derivatives and found that the IC50 of compound H-13 against EGFRL858R/T790M mutated H1975 cells was 3.91 µM, and in further kinase activity evaluation, the IC50 of H-13 against EGFRL858R/T790M kinase was 395.2 nM. In addition, the preferred compound H-13 was able to promote apoptosis of H1975 tumor cells and block the proliferation of H1975 cells in the G0/G1 phase; meanwhile, it was able to significantly inhibit the migratory ability of H1975 tumor cells and inhibit the growth of H1975 cells in a time-concentration-dependent manner. In the in vivo anti-tumor activity study, the preferred compound H-13 had no obvious toxicity to normal mice, and the tumor inhibition effect on H1975 cell-loaded nude mice was close to that of Mobocertinib. Finally, molecular dynamics simulations showed that the binding energy between compound H-13 and 3IKA protein was calculated to be -162.417 ± 14.559 kJ/mol. In summary, the preferred compound H-13 can be a potential third-generation EGFR inhibitor.

Identification of new 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids as p38α MAPK inhibitors: Design, synthesis, antitumor evaluation, molecular docking and in silico studies.

In pursuit of discovering novel scaffolds that demonstrate potential inhibitory activity against p38α MAPK and possess strong antitumor effects, we herein report the design and synthesis of new series of 17 final target 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids (4-20). Chemical characterization of the compounds was performed using FT-IR, NMR, elemental analyses and mass spectra of some representative examples. With many compounds showing potential inhibitory activity against p38α MAPK, two derivatives, 8 and 9, demonstrated the highest activity (>70 % inhibition) among the series. Derivative 9 displayed IC50 value nearly 2.5 folds more potent than 8. As anticipated, they both showed explicit interactions inside the kinase active site with the key binding amino acid residues. Screening both compounds for cytotoxic effects, they exhibited strong antitumor activities against lung (A549), breast (MCF-7 and MDA MB-231), colon (HCT-116) and liver (Hep-G2) cancers more potent than reference 5-FU. Their noticeable strong antitumor activity pointed out to the possibility of an augmented DNA binding mechanism of antitumor action besides their kinase inhibition. Both 8 and 9 exhibited strong ctDNA damaging effects in nanomolar range. Further mechanistic antitumor studies revealed ability of compounds 8 and 9 to arrest cell cycle in MCF-7 cells at S phase, while in HCT-116 treated cells at G0-G1 and G2/M phases. They also displayed apoptotic induction effects in both MCF-7 and HCT-116 with total cell deaths more than control untreated cells in reference to 5-FU. Finally, the compounds were tested for their anti-migratory potential utilizing wound healing assay. They induced a significant decrease in wound closure percentage after 24 h treatment in the examined cancer cells when compared to untreated control MCF-7 and HCT-116 cells better than 5-FU. In silico computation of physicochemical parameters revealed the drug-like properties of 8 and 9 with no violation to Lipinski's rule of five as well as their tolerable ADMET parameters, thus suggesting their utilization as potential future drug leads amenable for further optimization and development.

A novel FAK-degrading PROTAC molecule exhibited both anti-tumor activities and efficient MDR reversal effects.

FAK (focal adhesion kinase) is widely involved in cancer growth and drug resistance development. Thus, FAK inhibition has emerged as an effective strategy for tumor treatment both as a monotherapy or in combination with other treatments. But the current FAK inhibitors mainly concentrate on its kinase activity, overlooking the potential significance of FAK scaffold proteins. In this study we employed the PROTAC technology, and designed a novel PROTAC molecule F2 targeting FAK based on the FAK inhibitor IN10018. F2 exhibited potent inhibitory activities against 4T1, MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells with IC50 values of 0.73, 1.09, 5.84 and 3.05 µM, respectively. On the other hand, F2 also remarkably reversed the multidrug resistance (MDR) in HCT8/T, A549/T and MCF-7/ADR cells. Both the effects of F2 were stronger than the FAK inhibitor IN10018. To our knowledge, F2 was the first reported FAK-targeted PROTAC molecule exhibiting reversing effects on chemotherapeutic drug resistance, and its highest reversal fold could reach 158 times. The anti-tumor and MDR-reversing effects of F2 might be based on its inhibition on AKT (protein kinase B, PKB) and ERK (extracellular signal-regulated kinase) signaling pathways, as well as its impact on EMT (epithelial-mesenchymal transition). Furthermore, we found that F2 could reduce the protein level of P-gp in HCT8/T cells, thereby contributing to reverse drug resistance from another perspective. Our results will boost confidence in future research focusing on targeting FAK and encourage further investigation of PROTAC with potent in vivo effects.

Synthesis of novel 4-substituted isatin Schiff base derivatives as potential autophagy inducers and evaluation of their antitumour activity.

Induction of autophagic death in cancer cells is one of the promising strategies for the development of anti-cancer therapeutics. In the present study, we designed and synthesized a series of isatin Schiff base derivatives containing thioether structures. After discovering the highly active target compound H13 (IC50 = 4.83 µM) based on in vitro antiproliferation, we also found it had a high safety against normal cells HEK293 with CC50 of 69.01 µM, indicating a sufficient therapeutic window. In addition, to provide reference for subsequent studies, a model was successfully constructed by Sybyl software. Preliminary mechanistic studies suggested that H13-induced apoptosis may be closely related to ROS accumulation and mitochondrial dysfunction. Subsequent studies revealed that H13 inhibited cell proliferation by inducing cellular autophagy mainly through blocking signal of the PI3K/AKT/mTOR pathway. Altogether, these results suggested that H13 was potentially valuable as a lead compound.