SMALL MOLECULES DU192, DU283 AND DU325 INDUCE DIFFERENTIATION AND APOPTOSIS OF HUMAN ACUTE PROMYELOCYTIC LEUKEMIA CELLS

Acute myelogenous leukemia (AML) originates from myeloid stem cells or myeloid blasts halted in an immature state during haematopoiesis. AML represents a group of heterogeneous forms of myeloid malignancies with diverse genetic abnormalities and different stages of myeloid differentiation. The human cell line, HL-60 used in this study belongs to a sub-type of AML, namely acute promyelocytic leukemia (APL). Another pathologic condition of myeloid expansion is the ”emergency” granulo-monocytopoiesis in most of the solid malignancies in which, an army of immature myeloid cells leave the bone marrow, called monocytic and granulocytic myeloid-derived suppressor cells (MDSCs). In contrast to AML, MDSCs are not malignant cells, but promote angiogensesis and immunosuppression leading to the progression of cancer. Both in AML and in solid malignancies the differentiation of immature myeloid cells is an already established therapeutic concept. Since the differentiation of AML cells is frequently followed by apoptosis or increases the sensitivity to chemotherapy, we have screened a library of small molecules to mature the human prototype cells, HL-60. In the resazurin assay small molecules DU192, DU283 and DU325 confounded viability of HL-60 cells, half-inhibitory concentration (IC50) values were as follows: 940 nM, 210 nM and 20 nM, respectively. IC50 could not be determined for human primary fibroblasts in the applied concentration range (1.6 nM - 5 µM). Using flow cytometry we obtained ERK phosphorylation as an early response to DU325 stimulation followed by the increase of the percentage of the Bcl-xl and pAkt bright cells. The expression of the members of the AP-1 TF complex, a driver of cellular differentiation, c-Fos, JunB, c-Jun and JunD were elevated on a concentration and time dependent manner detected by qRT-PCR. As a proof of cellular differentiation the expression of haematopoietic stem cell markers CD33 and CD34 decreased. Due to maturation the size and granularity of HL-60 cells increased upon treatment. Matured myeloid cell marker CD11b elevated on the cell surface detected by flow cytometry. We confirmed that differentiation of HL-60 cells was accompanied by apoptosis. We could detect AnnexinV+/PI- early and AnnexinV+/PI+ late apoptotic populations after 24h of treatment. Caspase-3 activated gradually by time detected by the percentage of active caspase-3 positive cells by flow cytometry and the digestion of zDEVD – amino-Luciferin. Finally, as a proof of massive cell death, we have shown the appearance of the hypo-diploid apoptotic cells in the sub-G1 population and the leakage of the lactate-dehydrogenase into the supernatant. We conclude that DU molecules differentiated immature HL-60 cells, which was followed by apoptosis. We propose to further investigate the effects of DU325 on additional human AML cells obtained from clinical samples. On the other hand we plan to systematically investigate the effect of DU325 on the expansion of immature MDSCs in solid malignancies. Funding: GINOP-2.3.2-15-2016-00030 (LGP); János Bolyai Research Scholarship (GJSz, BO/00139/17/8

Enantioselective Synthesis of 8-Hydroxyquinoline Derivative, Q134 as a Hypoxic Adaptation Inducing Agent

Hypoxia is a common feature of neurodegenerative diseases, including Alzheimer’s disease that may be responsible for disease pathogenesis and progression. Therefore, the hypoxia-inducible factor (HIF)1 system, responsible for hypoxic adaptation, is a potential therapeutic target to combat these diseases by activators of cytoprotective protein induction. We have selected a candidate molecule from our cytoprotective hydroxyquinoline library and developed a novel enantioselective synthesis for the production of its enantiomers. The use of quinidine or quinine as a catalyst enabled the preparation of enantiomer-pure products. We have utilized in vitro assays to evaluate cytoprotective activity, a fluorescence-activated cell sorting (FACS) based assay measuring mitochondrial membrane potential changes, and gene and protein expression analysis. Our data showed that the enantiomers of Q134 showed potent and similar activity in all tested assays. We have concluded that the enantiomers exert their cytoprotective activity via the HIF1 system through HIF1A protein stabilization

1,3-Dipolar Cycloaddition of Isatin-Derived Azomethine Ylides with 2H‐Azirines: Stereoselective Synthesis of 1,3- Diazaspiro[bicyclo[3.1.0]hexane]oxindoles

A regio- and diastereoselective 1,3-dipolar cycloaddition of 2H-azirines with azomethine ylides generated in situ from isatins and α-amino acids has been elaborated, affording an unprecedented aziridine-fused spiro[imidazolidine-4,3′-oxindole] framework. This one-pot three-component reaction tolerates a wide range of substrates and enables the construction of highly diverse 1,3-diazaspiro[bicyclo[3.1.0]hexane]oxindoles in isolated yields up to 81% under mild conditions

Synthesis and Cytoprotective Characterization of 8- Hydroxyquinoline Betti Products

The 8-hydroxyquinoline pharmacophore scaffold has been shown to possess a range of activities as metal chelation, enzyme inhibition, cytotoxicity, and cytoprotection. Based on our previous findings we set out to optimize the scaffold for cytoprotective activity for its potential application in central nervous system related diseases. A 48-membered Betti-library was constructed by the utilization of formic acid mediated industrial-compatible coupling with sets of aromatic primary amines such as anilines, oxazoles, pyridines, and pyrimidines, with (hetero)aromatic aldehydes and 8-hydroxiquinoline derivatives. After column chromatography and re-crystallization, the corresponding analogues were obtained in yields of 13–90%. The synthesized analogs were optimized with the utilization of a cytoprotection assay with chemically induced oxidative stress, and the most active compounds were further tested in orthogonal assays, a real time cell viability method, a fluorescence-activated cell sorting (FACS)-based assay measuring mitochondrial membrane potential changes, and gene expression analysis. The best candidates showed potent, nanomolar activity in all test systems and support the need for future studies in animal models of central nervous system (CNS) disorders

Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations

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Polyunsaturated fatty acids synergize with lipid droplet binding thalidomide analogs to induce oxidative stress in cancer cells

<p>Abstract</p> <p>Background</p> <p>Cytoplasmic lipid-droplets are common inclusions of eukaryotic cells. Lipid-droplet binding thalidomide analogs (2,6-dialkylphenyl-4/5-amino-substituted-5,6,7-trifluorophthalimides) with potent anticancer activities were synthesized.</p> <p>Results</p> <p>Cytotoxicity was detected in different cell lines including melanoma, leukemia, hepatocellular carcinoma, glioblastoma at micromolar concentrations. The synthesized analogs are non-toxic to adult animals up to 1 g/kg but are teratogenic to zebrafish embryos at micromolar concentrations with defects in the developing muscle. Treatment of tumor cells resulted in calcium release from the endoplasmic reticulum (ER), induction of reactive oxygen species (ROS), ER stress and cell death. Antioxidants could partially, while an intracellular calcium chelator almost completely diminish ROS production. Exogenous docosahexaenoic acid or eicosapentaenoic acid induced calcium release and ROS generation, and synergized with the analogs <it>in vitro</it>, while oleic acid had no such an effect. Gene expression analysis confirmed the induction of ER stress-mediated apoptosis pathway components, such as GADD153, ATF3, Luman/CREB3 and the ER-associated degradation-related HERPUD1 genes. Tumor suppressors, P53, LATS2 and ING3 were also up-regulated in various cell lines after drug treatment. Amino-phthalimides down-regulated the expression of CCL2, which is implicated in tumor metastasis and angiogenesis.</p> <p>Conclusions</p> <p>Because of the anticancer, anti-angiogenic action and the wide range of applicability of the immunomodulatory drugs, including thalidomide analogs, lipid droplet-binding members of this family could represent a new class of agents by affecting ER-membrane integrity and perturbations of ER homeostasis.</p

Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2-b]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xlbright and pAktbright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38bright cells followed apoptosis (IC50: 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo

Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options. RESULTS: In this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size. CONCLUSION: These results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids