Binding of 1-substituted carbazolyl-3,4-dihydro-β-carbolines with DNA: Molecular dynamics simulation and MM-GBSA analysis

Molecular Mechanics-Generalized Born-Solvent Accessibility free energy calculations were used to analyse DNA binding affinity of 1-substituted carbazolyl-3,4-dihydro-β-carboline molecules. In this study, DNA structure with sequence of d(CGATCG)2 was used for simulations. 15 ns molecular dynamics simulations of the studied complexes were performed. The calculated free energy was compared with experimental antitumor activity (IC₅₀). The predicted free energies decreased with the increase of IC₅₀ values. It was shown that molecules 1–6 bind to DNA via intercalation mode, while molecules 7–9 bind through groove binding mode. Also, it was found that the vdW energy term (ΔE_vdW) and the non-polar desolvation energy (ΔG_SA) are the favorable terms for binding energy, whereas net electrostatic energies (ΔE_ele + ΔG_GB) and conformational entropy energy (TΔS) are unfavorable ones.     

Formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines in human urine

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-β-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-β-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-di-methoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-β-carboline, 6-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.     

Behavioral and biochemical evidence for serotonergic actions of tetrahydro-β-carbolines

In two groups of rats trained to discriminate LSD (0.1 mg/kg or 0.24 mg/kg) from saline, tetrahydro-β-carboline (THBC; 1–12 mg/kg as free base) and its derivative 6-methoxy-THBC (1–12 mg/kg as free base) substituted partially for LSD. The substitution of THBC for 0.1 mg/kg of LSD was analyzed further with antagonism tests in 16 animals and was attenuated by the serotonin (5-HT) antagonist BC-105 (pizotifen; 3 mg/kg) but not by the dopamine (DA) antagonist haloperidol (0.1 mg/kg). It was abolished by pre-treatment with the 5-HT synthesis inhibitor p-chlorophenylalanine (100 mg/kg/day for 3 days). In addition, THBC was found to inhibit ³H-LSD binding to homogenates of rat frontal cortex with an IC₅₀ value of 4 μM which is similar to that previously reported for other 5-HT agonists. These data indicate that THBCs exert potent 5-HT agonist actions. Since THBCs have recently been found in mammalian brain and other tissues, the present results are of interest in relation to a possible role of these substances in endogenous psychosis.     

Synthesis,antileishmanial and antitrypanosomal activities of N-substituted tetrahydro-β-carbolines

A series of N-substituted tetrahydro-β-carbolines were synthesized and screened for antileishmanial activity through an in vitro assay that involves promastigotes and axenic amastigotes of Leishmania donovani, the causative agent for visceral leishmaniasis. The thiophen-2-yl analogs 9b and 11f and naphthyl analog 11h were found to show significant activity against promastigotes with IC₅₀ values of 12.7, 9.1 and 22.1 μM, respectively. Analogs 9b and 11h were also effective against axenic amastigotes with IC₅₀ values of 62.8 and 87.6 μM, respectively. The antileishmanial activity of analogs was then tested in human macrophage cell line infected with L. donovani amastigotes and 2-naphthyl linked analog 11h was found to be effective with IC₅₀ value of 28.3 μM. Several analogs also displayed antitrypanosomal activity against Trypanosoma brucei, the causative agent for human African trypanosomiasis. Compounds 11e, 11f and 11h were more effective than others with IC₅₀ values of 1.0, 8.9 and 10.2 μM, respectively. All synthesized analogs were not cytotoxic towards mammalian cell lines including Vero (monkey kidney fibroblasts), HEPG2 (human hepatoma cells), LLC-PK₁ (pig kidney epithelial cells) and THP-1 (human macrophages).     

Optically Active Bis-β-keto Sulfonium Ylid

Particulate alcohol dehydrogenase of acetic acid bacteria that is mainly participated in vinegar fermentation was purified to homogeneous state from Gluconobacter suboxydans IFO 12528. Solubilization of enzyme from the bacterial membrane fraction by Triton X-100 and subsequent fractionation on DEAE-Sephadex A-50 and hydroxylapatite was successful in enzyme purification. A cytochrome c-like component was tightly bound to the dehydrogenase protein and existed as an enzyme-cytochrome complex. It was also confirmed that the alcohol dehydrogenase is not a cytochrome component itself. The molecular weight of the enzyme was determined to be 150,000, and gel electrophoresis showed the presence of three subunits having a molecular weight of 85,000, 49,000 and 14,400. The smallest subunit was corresponded to the cytochrome c-like component. Ethanol was oxidized in the presence of dyes in vitro but NAD or NADP were not required as hydrogen acceptor. Unlike NAD- linked alcohol dehydrogenase in yeast or liver and other primary alcohol dehydrogenases in methanol utilizing bacteria, the enzyme from the acetic acid bacteria showed its optimum pH at fairly acidic pH.     

Nucleosides. II. Synthesis and Properties of 3,4-Diaryl-4,5-dihydro-1-(β-D-ribofuranosyl)-1,2,4-triazole-5-thiones

Abstract

3,4-Diaryl-4,5-dihydro-1,2,4-triazole-5-thiones (1a-c) were silylated to give compounds (2a-c) which were condensed with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (3) in the presence of trimethylsilyl trifluoromethane sulfonate to afford the corresponding nucleosides 4a-c. Treatment of 4a-c with sodium methoxide in methanol at room temperature afforded the debenzoylated nucleosides 5a-c. The reaction of 5a with acetone in the presence of p-toluenesulfonic acid gave the 2′, 3′-isopropylidene derivative (6a). Phosphorylation of 6a with phosphoryl chloride and triethylphosphate followed by treatment with barium hydroxide afforded barium 3,4-diphenyl-4,5-dihydro(β-D-ribofuranosyl)-1,2,4-triazole-5-thione-5′- monophosphate, which gave after lyophilization the free acid (7a)     

Synthesis and biological activity of N-aroyl-tetrahydro-γ-carbolines

Research on dual inhibitors of both 5-LOX and COXs gained interest due to the overexpressions of these enzymes during the malignant state of the evolution of prostate cancer. In order to take part in this research, new N-aroyl-tetrahydro-γ-carbolines issued from the modification of Indomethacin have been synthesised. As for the NSAIDs, the compounds have been tested for their activity against COX₁, COX₂ plus against 5-LOX and against the proliferation of malignant prostate cancer. Interesting cytotoxic activities and selectivities of some tetrahydro-γ-carboline derivatives have been obtained.     

Actions of amino-β-carbolines on induction of sister-chromatid exchanges

Synthetic 3-aminoharman and 3-aminonorharman (amino-β-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-β-carbolines are ranked between 2-amino-α-carboline and 2-amino-6-methyl-9a-aza-δ-carboline (Glu-P-2) and much lower than 3-amino-γ-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-β-carboline, harman and norharman had very weak, if any, SCE-inducer activity.Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.     

Synthesis and Cytotoxic Activity of Novel 5-Substituted-1-(β-L-Arabinofuranosyl) Pyrimidine Nucleosides

A series of new 5-halogeno-1-(ß-L-arabinofuranosyl)uracils and their cytosine analogues were synthesized by halogenation of ara-L-uridine and ara-L-cytidine, respectively. The 5-(2-thienyl) and 5-halogenothienyl derivatives of both series were also prepared in excellent yields by Stille coupling followed by halogenation. All of these syntheses were based on benzoyl-protected derivatives. In vitro cytotoxicity experiments carried out using L1210 mouse leukemia cells showed that 5-(2-thienyl)-ara-L-uridine was the most potent compound of the new compounds; the majority of the analogues were not effective up to 200 μM concentrations.     

6-chloro-3-β-d-erythrofuranosyl-l-phenyl- and -l-p-tolylpyrazolo-[3,4-b]quinoxaline

The C-nucleoside analogs 6-chloro-3-β-d-erythrofuranosyl-l-phenylpyrazolo-[3,4-b]quinoxaline (5) and 3-β-d-erythrofuranosy]-l-p-tolylpyrazolo[3,4-b]quinoxaline (10) were prepared by dehydration of the polyhydroxyalkyl chain of 6(7)-chlorolo-phenyl-3-(d-arabino-tetritol-l-yl)-pyrazolo(3,4-b]quinoxaline and 3-(d-arbino-tetritol-l-yl)-l-p-tolylpyrazolo[3,4-b]quinoxaline, respectively. The structure and anomeric configuration of 5 and 10 were determined by high-resolution, n.m.r. spectroscopy. The mass spectra and biological activities of some of these compounds are discussed.     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

Discovery of tetrahydro-β-carbolines as inhibitors of the mitotic kinesin KSP

Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the synthesis and biological evaluation of a novel series of tetrahydro-β-carboline analogs based on the structure of the known KSP inhibitor HR22C16. Preferred compounds 11b, 12a and 19b were identified as potent inhibitors in a KSP ATPase assay with good anti-proliferative activity in A549 cells.     

Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16⁺ NK cells, delineated a unique dual-function cytotoxic-CD107a⁺/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56⁺ NK cells, α4β7⁺ and α4β7⁻ NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.Human peripheral blood contains two primary subsets of natural killer (NK) cells—a major CD16⁺ CD56^dim subset and a minor CD16⁻ CD56^bright subset. We have defined subsets of rhesus macaque (Macaca mulatta) NK cells and found that, similarly, macaque peripheral blood is dominated by a CD16⁺ CD56⁻ subset but contains two minor CD16⁻ CD56⁺ and CD16⁻ CD56⁻ subpopulations (34). As in humans, macaque CD16⁻ CD56⁺ NK cells are the primary producers of gamma interferon (IFN-γ) and express cell surface markers such as CCR7 and CD62L that mediate homing to lymph nodes, whereas CD16⁺ CD56⁻ NK cells do not express CCR7 or CD62L and primarily mediate cytolytic functions (7, 12, 30, 34). In both humans and macaques, the distribution of NK subsets in peripheral blood is distinct from that observed in lymph nodes and mucosal tissues, where NK cells are primarily CD56⁺ (9, 12, 30, 35).NK cells are important for the control of multiple viral infections, and increasing evidence suggests that NK cells play a significant role in controlling human immunodeficiency virus (HIV) infection (3, 5, 13, 14, 19, 21, 22, 24, 33), as well as simian immunodeficiency virus (SIV) infection of rhesus macaques and sooty mangabeys (6, 16, 26). HIV and SIV primarily replicate in the gut mucosa (18), and although we and others have demonstrated the presence of NK cells in the gastrointestinal tracts of both humans and rhesus macaques (8, 9, 25, 30), the nature of these NK cells is still poorly understood. Interestingly, the integrin α4β7, which directs lymphocyte trafficking to the gut (4), has been shown to be expressed on peripheral blood NK cells in humans and rhesus macaques (11, 27). This finding suggests that the gut mucosa is a site of active NK cell trafficking.Despite the importance of gut-associated lymphoid tissue in HIV/SIV pathogenesis, little is known about the effects of infection on NK cell homing to these tissues. In order to address this deficit, a total of 47 Indian rhesus macaques were studied, 27 of which were SIV naïve and 20 infected with either SIVmac239 (5) or SIVmac251 (15) for more than 150 days (mean duration of infection, 293 days). All animals were housed at the New England Primate Research Center and maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (23a).PBMC isolation and polychromatic flow cytometry staining were carried out using protocols described previously for our laboratory (29, 31); the antibodies used are listed in Table ​Table1.1. NK cells were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34), and CD16 and CD56 expression were used to delineate three primary subsets: CD56⁻ CD16⁺ (CD16⁺), the dominant subset; CD56⁺ CD16⁻ (CD56⁺); and CD56⁻ CD16⁻ (double negative [DN]) (Fig. ​(Fig.11 A). The results of polychromatic flow cytometry analyses demonstrated that α4β7 was expressed at the highest levels on CD16⁺ NK cells and that, while expression on this subset was not altered during SIV infection, α4β7 was significantly upregulated on both CD56⁺ and DN NK cells in SIV-infected animals (Fig. 1B and C). Interestingly, CCR7, which is expressed only on the CD56⁺ and DN NK cell subsets in macaques (30, 34), was concomitantly downregulated on these subsets of NK cells during chronic SIV infection (Fig. ​(Fig.1B).1B). The relationship between the two markers delineated a dichotomous expression pattern between naïve and SIV-infected macaques (Fig. ​(Fig.1D).1D). This dramatic shift in CD56⁺ and DN NK cell trafficking repertoires is likely indicative of increased homing of these NK subsets to the gut coupled to decreased homing to lymph nodes. Also, as shown in Fig. ​Fig.1E,1E, the absolute numbers of both CD16⁺ and DN NK cells increased during chronic SIV infection, resulting in increased absolute numbers of gut-homing α4β7⁺ cells in both subsets. Interestingly, while the absolute numbers of all CD56⁺ NK cells tended to decrease during chronic SIV infection, the absolute numbers of the α4β7⁺ CD56⁺ NK cell subset increased slightly (Fig. ​(Fig.1E,1E, middle panel), further suggesting that multiple subsets of α4β7⁺ NK cells increase during chronic SIV infection.Open in a separate windowFIG. 1.Comparison of α4β7 expression on NK cell subsets in naïve and SIV-infected macaques. (A) Macaque NK cell subsets were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34) and then further delineated into CD56⁺, CD16⁺, and DN subsets. (B) Representative flow cytometry plots of α4β7 and CCR7 expression on NK cell subsets in naïve and SIV-infected macaques. (C) Percentages of α4β7⁺ cells above the background level were compared between naïve and SIV-infected macaques for CD56⁺, CD16⁺, and DN NK subsets. (D) Relationships between α4β7 and CCR7 expression on CD56⁺ and DN NK cells in naïve and SIV-infected macaques. (E) Absolute numbers of total circulating NK cells were determined by using a bead-based flow cytometric assay as described previously (29, 30), and α4β7⁺ NK cell subset counts were extrapolated using these data combined with NK cell frequency data determined by polychromatic flow cytometry (panel A). Horizontal bars indicate median values for 20 to 27 animals. Student''s t tests were used to compare naive and SIV-infected animal groups; P values of >0.05 are considered statistically significant.

TABLE 1.

Antibodies used in polychromatic flow cytometry analyses

The discovery of tetrahydro-β-carbolines as inhibitors of the kinesin Eg5

A series of tetrahydro-β-carbolines were identified by HTS as inhibitors of the kinesin Eg5. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of removing potential metabolic liabilities and improving cellular potency.     

Cytotoxic activity,apoptosis induction and structure–activity relationship of 8-OR-2-aryl-3,4-dihydroisoquinolin-2-ium salts as promising anticancer agents

As our continuing research, a series of 2-aryl-8-OR-3,4-dihydroisoquinolin-2-ium bromides were evaluated for cytotoxic activity on cancer cells and apoptosis induction in the present study. SAR was derived also. Among them, 23 compounds showed the higher cytotoxicity on MKN-45 cells with IC₅₀ values of 1.99–11.3 μM than a standard anticancer drug cis-platinum (IC₅₀ = 11.4 μM) or their natural model compound chelerythrine (IC₅₀ = 12.7 μM); 16 compounds possessed the medium to high activity on NB4 cells with IC₅₀ values of 1.67–4.62 μM. SAR analysis showed that both substitution patterns of the N-aromatic ring and the type of 8-OR significantly impact the activity. AO/EB staining and flow cytometry analysis with Annexin V/PI double staining showed that the compounds were able to induce apoptosis in a concentration-dependent manner. The results above suggested that the title compounds are a class of promising compounds for the development of new anti-cancer drugs.     

Cytotoxic and α-Glucosidase Inhibitory Xanthones from Garcinia mckeaniana Leaves and Molecular Docking Study

A new racemic xanthone, garmckeanin A ( 1 ), and eight known analogs 2 – 9 were isolated from the ethyl acetate (AcOEt) extract of the Vietnamese Garcinia mckeaniana leaves. Their structures were determined by MS and NMR spectral analyses and compared with the literature. The AcOEt extract showed good cytotoxicity against cancer cell lines KB, Lu, Hep-G2 and MCF7, with IC₅₀ values of 5.40–8.76 μg/mL, and it also possessed α-glucosidase inhibitory activity, with an IC₅₀ value of 9.17 μg/mL. Garmckeanin A ( 1 ) exhibited inhibition of all cancer cell lines, with an IC₅₀ value of 7.3–0.9 μM. Allanxanthone C ( 5 ) successfully controlled KB growth, with an IC₅₀ value of 0.54 μM, higher than that of the positive control, ellipticine (IC₅₀ 1.22 μM). Norathyriol ( 8 ) was a promising α-glucosidase inhibitor, with an IC₅₀ value of 0.07 μM, much higher than that of the positive control, acarbose (IC₅₀ 161.0 μM). The interactions of the potential α-glucosidase inhibitors with the C- and N-terminal domains of human intestinal α-glucosidase were also investigated by molecular docking study. The results indicated that bannaxanthone D ( 2 ), garcinone E ( 4 ), bannaxanthone E ( 6 ), and norathyriol ( 8 ) exhibit higher binding affinity to the C-terminal than to the N-terminal domain through essential residues in the active sites. In particular, compound 8 could be assumed to be the most potent mixed inhibitor.     

Synthesis of novel β-carbolines with efficient DNA-binding capacity and potent cytotoxicity

A series of water-soluble β-carbolines, bearing a flexible amino side chain, was prepared and evaluated in vitro against a panel of human tumor cell lines. The N⁹-arylated alkyl substituted β-carbolines represented the most interesting cytotoxic activities, and compound 7b was found to be the most potent antitumor agent with IC₅₀ values lower than 10 μM against eight human tumor cell lines. The results confirmed that the N⁹-arylated alkyl substituents of β-carboline nucleus played an important role in the modulation of the cytotoxic potencies. In addition, these compounds were found to exhibit significant DNA-binding affinity.     

Substituted tetrahydro-β-carbolines as potential agents for the treatment of human papillomavirus infection

The identification and optimization of a series of substituted tetrahydro-β-carbolines with potent activity against human papillomavirus is described. Structure–activity studies focused on the substitution pattern and chirality of the β-carboline ring system are discussed. Optimization of these parameters led to compounds with antiviral activities in the low nanomolar range.