The unique activity of saponin: Induction of cytotoxicity in HTLV-1 infected cells.

Infection with the retrovirus human T-cell leukemia virus type 1 (HTLV-1) sometimes causes diseases that are difficult to cure. To find anti-HTLV-1 natural compounds, we opted to screen using the HTLV-1-infected T-cell line, MT-2. Based on our results, an extract of the pulp/seeds of Akebia quinata Decaisne fruit killed MT-2 cells but did not affect the Jurkat cell line that was not infected with virus. To determine the active ingredients, seven saponins with one-six sugar moieties were isolated from A. quinata seeds, and their activities against the two cell lines were examined. Both cell lines were killed in a similar manner by Akebia saponins A and B. Further, Akebia saponins D, E, PK and G did not exhibit cytotoxicity. Akebia saponin C had a similar activity to the extract found in the screening. This compound was found to enhance Gag aggregation, induce the abnormal cleavage of Gag, suppress virion release, and preferentially kill HTLV-1 infected cells; however, their relationship remains elusive. Our findings may lead to the development of new therapies for infectious diseases based on the removal of whole-virus-infected cells.

New insight into the bioactivity of substituted benzimidazole derivatives: Repurposing from anti-HIV activity to cell migration inhibition targeting hnRNP M.

Drug repurposing is a distinguished approach for drug development that saves a great deal of time and money. Based on our previous successful repurposing of a compound BMMP from anti-HIV-1 therapy to anti-cancer metastatic activity, we adopted the same techniques for repurposing benzimidazole derivatives considering MM-1 as a lead compound. An extensive structure-activity relationship (SAR) study afforded three promising compounds, MM-1d, MM-1h, and MM-1j, which inhibited cell migration in a similar fashion to BMMP. These compounds suppressed CD44 mRNA expression, whereas only MM-1h further suppressed mRNA expression of the epithelial-mesenchymal transition (EMT) marker zeb 1. Using benzimidazole instead of methyl pyrimidine as in BMMP resulted in better affinity for heterogeneous nuclear ribonucleoprotein (hnRNP) M protein and higher anti-cell migration activity. In conclusion, our study identified new agents that surpass the affinity of BMMP for hnRNP M and have anti-EMT activity, which makes them worthy of future attention and optimization.

A New 1,2-Naphthoquinone Derivative with Anti-lung Cancer Activity.

1,2-Naphthoquinone (2-NQ) is a nucleophile acceptor that non-selectively makes covalent bonds with cysteine residues in various cellular proteins, and is also found in diesel exhaust, an air pollutant. This molecule has rarely been considered as a pharmacophore of bioactive compounds, in contrast to 1,4-naphthoquinone. We herein designed and synthesized a compound named N-(7,8-dioxo-7,8-dihydronaphthalen-1-yl)-2-methoxybenzamide (MBNQ), in which 2-NQ was hybridized with the nuclear factor-κB (NF-κB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) as a nucleophile acceptor. Although 50 µM MBNQ did not inhibit NF-κB signaling, 10 µM MBNQ induced cell death in the lung cancer cell line A549, which was insensitive to 2-NQ (10 µM). In contrast, MBNQ was less toxic in normal lung cells than 2-NQ. A mechanistic study showed that MBNQ mainly induced apoptosis, presumably via the activation of p38 mitogen-activated protein kinase (MAPK). Collectively, the present results demonstrate that the introduction of an appropriate substituent into 2-NQ constitutes a new biologically active entity, which will lead to the development of 2-NQ-based drugs.

Discovery of anti-cell migration activity of an anti-HIV heterocyclic compound by identification of its binding protein hnRNP M.

One compound sometimes shows two biological functions, becoming important aspect of recent drug discovery. This study began with an attempt to confirm the previously reported molecular mechanism of the anti-human immunodeficiency virus (HIV) heterocyclic compound BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], i.e., induction of abnormal uncoating of the viral core at the post-entry step. Our mechanistic study gave results consistent with this mechanism. We further attempted to find out the molecular target of BMMP by a pulldown approach using previously synthesized biotinylated BMMP (Biotin-BMMP) and successfully identified heterogenous nuclear ribonucleoprotein M (hnRNP M) as a BMMP-binding protein. This protein was found not to be accountable for the anti-HIV activity of BMMP. As hnRNP M has been reported to promote cancer metastasis, we tested this mechanism and found that BMMP suppressed migration of the human lung carcinoma cell line A549 stimulated with transforming growth factor-ß (TGF-ß). Mechanistic study showed that BMMP suppressed the expression of CD44 mRNA via the regulation of hnRNP M. Furthermore, six new derivatives of BMMP were synthesized, and the patterns of their activities against HIV-1 and cell migration were not uniform, suggesting that the anti-HIV mechanism and the anti-cell migration mechanism of BMMP are independent. Taken together, the anti-cell migration activity of the anti-HIV heterocyclic compound BMMP was newly discovered by identification of its binding protein hnRNP M using a chemical biology approach.

The stability of HIV-2 Vpx and Vpr proteins is regulated by the presence or absence of zinc-binding sites and poly-proline motifs with distinct roles.

The Vpx and Vpr proteins of human immunodeficiency virus type 2 (HIV-2) are important for virus replication. Although these proteins are homologous, Vpx is expressed at much higher levels than Vpr. Previous studies demonstrated that this difference results from the presence of an HHCC zinc-binding site in Vpx that is absent in Vpr. Vpx has another unique region, a poly-proline motif (PPM) of seven consecutive prolines at the C-terminus. Using PPM point mutants of Vpx, this study demonstrated that these seven consecutive prolines are critical for suppressing proteasome degradation of Vpx in the absence of Gag. Both the PPM and the zinc-binding site stabilize Vpx but do so via different mechanisms. PPM and zinc-binding site mutants overexpressed in Escherichia coli aggregated readily, indicating that these motifs normally prevent exposure of the hydrophobic region outside the structure. Furthermore, introduction of the zinc-binding site and the PPM into Vpr increased the level of Vpr expression so that it was as high as that of Vpx. Intriguingly, HIV-2 has evolved to express Vpx at high levels and Vpr at low levels based on the presence and absence of these two motifs with distinct roles.

Structure activity study of S-trityl-cysteamine dimethylaminopyridine derivatives as SIRT2 inhibitors: Improvement of SIRT2 binding and inhibition.

Sirtuin proteins are a highly conserved class of nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases. The pleiotropic human isoform 2 of Sirtuins (SIRT2) has been engaged in the pathogenesis of cancer in a plethora of reports around the globe. Thus, SIRT2 modulation is deemed as a promising approach for pharmaceutical intervention. Previously, we reported S-Trityl-l-Cysteine (STLC)-ornamented dimethylaminopyridine chemical entity named STC4 with a significant SIRT2 inhibitory capacity; this was separate from the conventional application of STLC scaffold as a kinesin-5 inhibitor. An interactive molecular docking study of SIRT2 and STC4 showed interaction between Asn168 of SIRT2 and the methyl ester of STC4, that appears to hinder STC4 to reach the selective pocket of the protein unlike strong SIRT2 inhibitor SirReal2. To improve its activity, herein, we utilized S-trityl cysteamine pharmacophore lacking the methyl ester. Nine compounds were synthesized and assayed affording three biopertinent SIRT2 inhibitors, and two of them, STCY1 and STCY6 showed higher inhibitory activity than STC4. These compounds have pronounced anti-proliferative activities against different cancer cell lines. A molecular docking study was executed to shed light on the supposed binding mode of the lead compound, STCY1, into the selective pocket of SIRT2 by interaction of the nitrogen of pyridine ring of the compound and Ala135 of the protein. The outcome of the study exposes that the active compounds are effective intermediates to construct more potent biological agents.

HSP70 induction by bleomycin metal core analogs.

Induction of heat shock protein 70 (HSP70) is known to be effective against various diseases. We are interested in HSP70 induction capability of an antitumor antibiotic bleomycin which produces oxidative stress by iron chelate formation and oxygen activation in a cell. The HSP70 induction activity of bleomycin and its six metal core analogs was examined, and a compound HPH-1Trt of 10 µM was found to induce this protein in a pheochromocytoma cell line and some T cell and monocytic cell lines. Its mechanism is increase of HSP70 mRNA, but higher concentration of this compound showed toxicity. Two new derivatives were then synthesized, and one of them named DHPH-1Trt was shown to have less toxicity and higher HSP70 induction activity. This study would lead to a clue for new HSP70 inducer clinically used in near future.

Interruption of Vif/Elongin C interaction: In silico and experimental elucidation of the underlying molecular mechanism of benzimidazole-based APOBEC3G stabilizers.

In 2014, two novel and promising benzimidazole-based APOBEC3G stabilizers MM-1 and MM-2 (MMs) were uncovered with an elusive mechanism of action. Vif-APOBEC3G axis has been recognized as a novel therapeutic target for anti HIV-1 drug development. The unexplored mechanism of MMs hindered their further development into lead compounds. To recognize their underlying mechanism we adopted an exhaustive in silico workflow by which we tested their ability to interrupt Vif complex network formation. The preliminary outcome guided us to a high likelihood of MMs interaction within Elongin C binding site, which in turn, perturbs Vif/Elongin C binding and ultimately undermines Vif action. To validate our estimation, we synthesized only MM-1 as a model to complement our study by in vitro assay for a real-time understanding. An immunoprecipitation experiment confirmed the capacity of MM-1 to interrupt Vif/Elongin C interaction. This is an integral study that lies at the interface between theoretical and experimental approaches showing the potential of molecular modelling to address issues related to drug development.

Minimum structural requirements for inhibitors of the zinc finger protein TRAF6.

Zinc fingers have rarely been regarded as drug targets. On the contrary, the zinc-binding site of enzymes has often been considered a target of inhibitors. We previously developed a dithiol compound called SN-1 that binds to the zinc finger protein tumor necrosis factor receptor-associated factor 6 (TRAF6) and suppresses downstream nuclear factor-κB (NF-κB) signaling. To determine the minimal structure requirements of TRAF6 inhibitors based on SN-1, NF-κB inhibitory activity and cytotoxicity of its derivatives including new compounds were examined. SN-2, an oxidative type of prodrug of SN-1 with 2-nitrophenylthio groups via disulfide, has the minimum structure for an inhibitor of TRAF6, as seen with cellular experiments. The importance of two side chains with a thiol group was shown with molecular modelling. This study may lead to development of selective TRAF6 inhibitors in the near future.

New SIRT2 inhibitors: Histidine-based bleomycin spin-off.

Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently synthesized. In this paper, we developed inhibitors of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of Sirtuin protein (SIRT2), based on HPH-1Trt/HPH-2Trt, and aimed to generate new anti-cancer drugs. HPH-1Trt and HPH-2Trt had in vitro anti-SIRT2 inhibitory activity with 50% inhibitory concentration (IC50) values of 5.5 and 8.8 µM, respectively. A structural portion of HPH-1Trt/HPH-2Trt, a tritylhistidine derivative TH-1, had stronger activity (IC50 = 1.7 µM), and thus, fourteen derivatives of TH-1 were synthesized. Among them, TH-3 had the strongest activity (IC50 = 1.3 µM). Selective binding of TH-3 in the pocket of SIRT2 protein was confirmed with a molecular docking study. Furthermore, TH-3 strongly lowered viability of the breast cancer cell line MCF7 with an IC50 of 0.71 µM. A structure-activity relationship study using cell lines suggested that the mechanism of TH-3 to suppress MCF7 cells involves not only SIRT2 inhibition, but also another function. This compound may be a new candidate anti-cancer drug.

Anti-cancer activity of the cell membrane-permeable phytic acid prodrug.

Phytic acid (IP6) is an ingredient in cereals and legumes, and limited amounts of this compound are considered to enter the cell and exert anti-cancer effects. These effects have been seen by studying cells treated with around 1-5 mM IP6. However, such a large amount of IP6 chelates metals and changes the pH in cell culture medium. To overcome this problem, we synthesized a prodrug of IP6 (Pro-IP6) and elucidated generation of IP6 from Pro-IP6 in cells. Cellular experiments using Pro-IP6 demonstrated selective anti-cancer effects including apoptosis and inhibition of Akt activation. Furthermore, an in vivo study using mice with adult T-cell leukemia also showed that Pro-IP6 reduced the size of the cancer. Taken together, Pro-IP6 is a useful biological tool and may lead to development of new anti-cancer drugs.

Serial Femtosecond X-Ray Diffraction of HIV-1 Gag MA-IP6 Microcrystals at Ambient Temperature.

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cell's plasma membrane. High-resolution structures of the MA domain determined using cryo X-ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first X-ray free-electron laser (XFEL) study of the HIV-1 MA domain's interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using an XFEL to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of the MA-IP6 complex both diffracted to beyond 3.5 Å resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and will provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.

Antiproliferative S-Trityl-l-Cysteine -Derived Compounds as SIRT2 Inhibitors: Repurposing and Solubility Enhancement.

S-trityl-l-cysteine (STLC) is a well-recognized lead compound known for its anticancer activity owing to its potent inhibitory effect on human mitotic kinesin Eg5. STLC contains two free terminal amino and carboxyl groups that play pivotal roles in binding to the Eg5 pocket. On the other hand, such a zwitterion structure complicates the clinical development of STLC because of the solubility issues. Masking either of these radicals reduces or abolishes STLC activity against Eg5. We recently identified and characterized a new class of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of sirtuin protein (SIRT2) inhibitors that can be utilized as cytotoxic agents based on an S-trityl-l-histidine scaffold. Herein, we propose new STLC-derived compounds that possess pronounced SIRT2 inhibition effects. These derivatives contain modified amino and carboxyl groups, which conferred STLC with SIRT2 bioactivity, representing an explicit repurposing approach. Compounds STC4 and STC11 exhibited half maximal inhibitory concentration values of 10.8 ± 1.9 and 9.5 ± 1.2 µM, respectively, against SIRT2. Additionally, introduction of the derivatizations in this study addressed the solubility limitations of free STLC, presumably due to interruption of the zwitterion structure. Therefore, we could obtain drug-like STLC derivatives that work by a new mechanism of action. The new derivatives were designed, synthesized, and their structure was confirmed using different spectroscopic approaches. In vitro and cellular bioassays with various cancer cell lines and in silico molecular docking and solubility calculations of the synthesized compounds demonstrated that they warrant attention for further refinement of their bioactivity.

Antileukemic Activity of Twig Components of Caucasian Beech in Turkey.

Despite the development of a range of anti-cancer agents, cancer diagnoses are still increasing in number, remaining a leading cause of death. Anticancer drug treatment is particularly important for leukemia. We screened Turkish plants and found the unique antileukemic activity of twig components in Turkish Caucasian beech, selectively inducing apoptosis in leukemia cells. This effect is unique among some kinds of beeches, presumably related to oxidative stress. This study would lead to effective use of discarded material, i.e., twig of beech, and a new anti-leukemic drug based on large tree.

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.

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.

A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group.

Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity.

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.

Synthesis of the biotinylated anti-HIV compound BMMP and the target identification study.

BMMP [2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine], an inhibitor of HIV-1 replication, was linked to biotin to study the interaction with the presumed target, HIV-1 Pr55(Gag) or CA, by means of surface plasmon resonance. The synthesized Biotin-BMMP inhibited HIV-1 replication to a similar extent as BMMP alone, but did not interact with Pr55(Gag) or CA, suggesting that BMMP exerts its activity by a different mechanism.

Zinc-mediated binding of a low-molecular-weight stabilizer of the host anti-viral factor apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination.