Halil Ibrahim Ciftci

Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents

New pyrazoline derivatives were synthesized and evaluated for their cytotoxic effects on AsPC-1 human pancreatic adenocarcinoma, U87 and U251 human glioblastoma cell lines. 1-[((5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (11) was found to be the most effective anticancer agent against AsPC-1 and U251 cell lines, with IC50 values of 16.8 µM and 11.9 µM, respectively. Tumor selectivity of compound 11 was clearly seen between Jurkat human leukemic T-cell line and human peripheral blood mononuclear cells (PBMC). Due to its promising anticancer activity, compound 11 was chosen for apoptosis/necrosis evaluation and DNA-cleavage analysis in U251 cells. Compound 11-treated U251 cells exhibited apoptotic phenotype at low concentration (1.5 µM). DNA-cleaving efficiency of this ligand was more significant than cisplatin and was clearly enhanced by Fe(II)-H2O2-ascorbic acid systems. This result pointed out the relationship between the DNA cleavage and the cell death.

A clue to unprecedented strategy to HIV eradication: “Lock-in and apoptosis”

Despite the development of antiretroviral therapy against HIV, eradication of the virus from the body, as a means to a cure, remains in progress. A “kick and kill” strategy proposes “kick” of the latent HIV to an active HIV to eventually be “killed”. Latency-reverting agents that can perform the “kick” function are under development and have shown promise. Management of the infected cells not to produce virions after the “kick” step is important to this strategy. Here we show that a newly synthesized compound, L-HIPPO, captures the HIV-1 protein Pr55Gag and intercepts its function to translocate the virus from the cytoplasm to the plasma membrane leading to virion budding. The infecting virus thus “locked-in” subsequently induces apoptosis of the host cells. This “lock-in and apoptosis” approach performed by our novel compound in HIV-infected cells provides a means to bridge the gap between the “kick” and “kill” steps of this eradication strategy. By building upon previous progress in latency reverting agents, our compound appears to provide a promising step toward the goal of HIV eradication from the body.

Novel metal chelating molecules with anticancer activity. Striking effect of the imidazole substitution of the histidine–pyridine–histidine system

Previously we have reported a metal chelating histidine-pyridine-histidine system possessing a trityl group on the histidine imidazole, namely HPH-2Trt, which induces apoptosis in human pancreatic adenocarcinoma AsPC-1 cells. Herein the influence of the imidazole substitution of HPH-2Trt was examined. Five related compounds, HPH-1Trt, HPH-2Bzl, HPH-1Bzl, HPH-2Me, and HPH-1Me were newly synthesized and screened for their activity against AsPC-1 and brain tumor cells U87 and U251. HPH-1Trt and HPH-2Trt were highly active among the tested HPH compounds. In vitro DNA cleavage assay showed both HPH-1Trt and HPH-2Trt completely disintegrate pUC19 DNA. The introduction of trityl group decisively potentiated the activity.

Mutational analysis of HIV-2 Vpx shows that proline residue 109 in the poly-proline motif regulates degradation of SAMHD1.

In this study, we performed a mutational analysis to determine whether the mechanism by which HIV‐2 Vpx confers the capacity for infectivity and viral replication in macrophages is solely dependent on its ability to degrade the host antiviral factor SAMHD1. Contrary to expectations, we demonstrated that P109 in the C‐terminal poly‐proline motif of HIV‐2 Vpx has two unique roles: to facilitate the specific degradation of SAMHD1 in macrophages, and to facilitate multimerization of Vpx, therefore preventing SAMHD1 degradation in the presence of high levels of Vpx.

Zinc-binding site of human immunodeficiency virus 2 Vpx prevents instability and dysfunction of the protein

Human immunodeficiency virus 2 Vpx coordinates zinc through residues H39, H82, C87 and C89. We reported previously that H39, H82 and C87 mutants maintain Vpx activity to facilitate the degradation of SAMHD1. Herein, the expression of Vpx mutants in cells was examined in detail. We demonstrated that the zinc-binding site stabilizes the protein to keep its function in virus growth when low levels of Vpx are expressed. At higher levels of expression, Vpx aggregation could occur, and zinc binding would suppress such aggregation. Among the amino acids involved in zinc coordination, H39 plays the most critical role. In summary, zinc binding appears to mitigate flexibility of the three-helix fold of Vpx, thereby preventing dysfunction.HIV-2 Vpx coordinates zinc through residues H39, H82, C87 and C89. We reported previously that H39, H82 and C87 mutants maintain Vpx activity to facilitate the degradation of SAMHD1. Herein, the expression of Vpx mutants in cells was examined in detail. We demonstrated that the zinc-binding site stabilizes the protein to keep its function in virus growth when low levels of Vpx are expressed. At higher levels of expression, Vpx aggregation can occur, and zinc binding would suppress such aggregation. Among the amino acids involved in zinc coordination, H39 plays the most critical role. In summary, zinc binding appears to mitigate flexibility of the three-helix fold of Vpx, thereby preventing dysfunction.

Design, Synthesis, and Biological Evaluation of Novel 1,3,4-Thiadiazole Derivatives as Potential Antitumor Agents against Chronic Myelogenous Leukemia: Striking Effect of Nitrothiazole Moiety

In an attempt to develop potent antitumor agents, new 1,3,4-thiadiazole derivatives were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the K562 chronic myelogenous leukemia cell line that expresses the Bcr-Abl tyrosine kinase. N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (2) inhibited the Abl protein kinase with an IC50 value of 7.4 µM and showed selective activity against the Bcr-Abl positive K562 cell line. Furthermore, a Bcr-Abl-compound 2 molecular modelling simulation highlighted the anchoring role of the nitrothiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. These results provide promising starting points for further development of novel kinase inhibitors.

Introduction of H2C2-type zinc-binding residues into HIV-2 Vpr increases its expression level

Human immunodeficiency virus type 2 has two structurally similar proteins, Vpx and Vpr. Vpx degrades the host anti‐viral protein SAMHD1 and is expressed at high levels, while Vpr is responsible for cell cycle arrest and is expressed at much lower levels. We constructed a Vpr mutant with a high level of expression by replacing the amino acids HHCR/HHCH with a putative H2C2‐type zinc‐binding site that is carried by Vpx. Our finding suggests that during the evolution of Vpr and Vpx, zinc‐binding likely became a mechanism for regulating their expression levels.

The First Pentacyclic Triterpenoid Gypsogenin Derivative Exhibiting Anti-ABL1 Kinase and Anti-chronic Myelogenous Leukemia Activities

The discovery of the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL)-targeted drug imatinib conceptually changed the treatment of chronic myelogenous leukemia (CML). However, some CML patients show drug resistance to imatinib. To address this issue, some artificial heterocyclic compounds have been identified as BCR-ABL inhibitors. Here we examined whether plant-derived pentacyclic triterpenoid gypsogenin and/or their derivatives show inhibitory activity against BCR-ABL. Among the three derivatives, benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) was found to be the most effective anticancer agent on the CML cell line K562, with an IC50 value of 9.3 µM. In contrast, the IC50 against normal peripheral blood mononuclear cells was 276.0 µM, showing better selectivity than imatinib. Compound 1c had in vitro inhibitory activity against Abelson kinase 1 (ABL1) (IC50=8.7 µM), the kinase component of BCR-ABL. In addition, compound 1c showed a different inhibitory profile against eight kinases compared with imatinib. The interaction between ATP binding site of ABL and 1c was examined by molecular docking study, and the binding mode was different from imatinib and newer generation inhibitors. Furthermore, 1c suppressed signaling downstream of BCR-ABL. This study suggests the possibility that plant extracts may be a source for CML treatment and offer a strategy to overcome drug resistance to known BCR-ABL inhibitors.

A Dithiol Compound Binds to the Zinc Finger Protein TRAF6 and Suppresses its Ubiquitination

Despite various inhibitors targeting the zinc center(s) of enzymes, drugs that target zinc fingers have not been examined in detail. We previously developed a dithiol compound named SN‐1 that has an inhibitory effect on the function of zinc finger transcription factors, but its mechanism of action has not yet been elucidated. To establish a general principle for new drugs, the details of the action of SN‐1 against a zinc finger protein were examined. As a zinc‐finger‐containing protein, we focused on TRAF6, which is related to cancer and inflammation. Binding of SN‐1 to TRAF6 and its effect on TRAF6 ubiquitination were examined in vitro, and the binding mode was calculated by computational methodology. Furthermore, ubiquitination of TRAF6 and downstream signaling was examined by cell‐based experiments. The results show that SN‐1 binds to TRAF6, inhibiting its auto‐ubiquitination and downstream NF‐κB signaling. Docking studies indicate that SN‐1 binds directly to the first zinc finger of TRAF6. This binding disrupts the neighboring structure, that is, the RING finger domain, to suppress the ubiquitin ligase activity of TRAF6. Taken together, this study provides a platform for developing new small molecules that target zinc finger proteins.