Intracellular Inhibitory Activity Research Articles

Nanoparticulate β-Cyclodextrin with Gallium Tetraphenylporphyrin Demonstrates in Vitro and in Vivo Antimicrobial Efficacy against Mycobacteroides abscessus and Mycobacterium avium.

The emergence of drug-resistant pathogens causes the greatest challenge for drug development research. Recently, gallium(III)-based compounds have received great attention as novel antimicrobial agents against drug-resistant pathogens. Here, we synthesized a new β-cyclodextrin Ga nanoparticle (CDGaTP) using Ga tetraphenylporphyrin (GaTP, a hemin analogue) and β-cyclodextrin. The newly synthesized nanoparticle was nontoxic and efficient at a single dose, showing sustained drug release for 15 days in vitro. CDGaTP's activity with transferrin or lactoferrin was tested, and synergism in activity was observed against nontuberculosis mycobacteria (NTM), Mycobacterium avium (M.avium), and Mycobacteroides abscessus. Human serum albumin (HSA) decreased the efficacy of both GaTP and CDGaTP in a concentration-dependent manner. The NTMs incubated with GaTP or CDGaTP significantly produced reactive oxygen species (ROS), indicating potential inhibition of antioxidant enzymes, such as catalase. The single-dose CDGaTP displayed a prolonged intracellular inhibitory activity in an in vitro macrophage infection model against both NTMs. In addition, CDGaTP, not GaTP, was effective in a murine lung M.avium infection model when delivered via intranasal administration. These results suggest that CDGaTP provides new opportunities for the development of gallium-porphyrin based antibiotics.

Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory activity over serine/arginine-rich protein kinases (SRPKs)

The serine/arginine-rich protein kinases (SRPKs) have frequently been found with altered activity in a number of cancers, suggesting they could serve as potential therapeutic targets in oncology. Here we describe the synthesis of a series of twenty-two trifluoromethyl arylamides based on the known SRPKs inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) and the evaluation of their antileukemia effects. Some derivatives presented superior cytotoxic effects against myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular, compounds 24, 30, and 36 presented IC50 values ranging between 6.0 and 35.7 μM. In addition, these three compounds were able to trigger apoptosis and autophagy, and to exhibit synergistic effects with the chemotherapeutic agent vincristine. Furthermore, compound 30 was more efficient than SRPIN340 in impairing the intracellular phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2, VEGF, and RON oncogenic isoforms. Therefore, novel compounds with increased intracellular effects against SRPK activity were obtained, contributing to medicinal chemistry efforts towards the development of new anticancer agents.

In Vitro and In Vivo Intracellular Killing Effects of Tigecycline against Clinical Nontyphoid Salmonella Isolates Using Ceftriaxone as a Comparator

Salmonella is an important, worldwide food-borne pathogen. Resistance to fluoroquinolones and cephalosporins has been increasingly reported, and new therapeutic agents are desperately needed. In this study, we evaluated the in vitro antimicrobial susceptibility of clinical nontyphoidal Salmonella isolates to tigecycline. Antibacterial activity of tigecycline, ceftriaxone, and ciprofloxacin were investigated by time-kill studies and the murine peritonitis model. The MIC₅₀/MIC₉₀ values of tigecycline, ceftriaxone, and ciprofloxacin against 76 Salmonella isolates were 0.25/0.5, 1/8, and 0.125/0.5 μg/ml, respectively. The intracellular inhibitory activity of tigecycline at 0.5 μg/ml (1 × MIC) against Salmonella isolates in human peripheral blood mononuclear cells was sustained for 24 h. In a mouse peritonitis model, tigecycline reduced the extracellular and intracellular bacterial counts from 10⁷ CFU/ml and 10⁵ CFU/ml, respectively, to an undetectable level within 96 h. The results were similar to those obtained with ceftriaxone. The survival rate of mice exposed to tigecycline after being infected by an inoculum of 1 × 10⁵ CFU was 80%, and that of mice exposed to ceftriaxone was 100%. When the inoculum was increased to 1.3 × 10⁶ CFU, the survival rate of mice treated by tigecycline was 20%, and that of mice exposed to ceftriaxone was 0% (P = 0.2). When a ceftriaxone- and ciprofloxacin-resistant but tigecycline-susceptible isolate was tested, mice treated by tigecycline had a higher survival rate than those treated by ceftriaxone (15/20 [75%] versus 6/20 [30%]; P = 0.011). Our results suggest that tigecycline is at least as effective as ceftriaxone for murine Salmonella infections and warrants further clinical investigations to delineate its potential against human Salmonella infections.

Selective inhibition of 17β-hydroxysteroid dehydrogenase type 1 (17βHSD1) reduces estrogen responsive cell growth of T47-D breast cancer cells

The most potent estrogen estradiol (E2) plays a pivotal role in the initiation and progression of estrogen dependent diseases. 17β-Hydroxysteroid dehydrogenase type 1 (17βHSD1) catalyses the NADPH-dependent E2-formation from estrone (E1). It is often overexpressed in breast cancer and endometriosis. For this reason, inhibition of 17βHSD1 is a promising strategy for the treatment of these diseases. In the present paper, we investigate the estrogen responsive cell growth of T47-D breast cancer cells, the intracellular inhibitory activity of non-steroidal 17βHSD1-inhibitors and their effects on estrogen dependent cell growth in vitro. At equal concentrations the estrogens E1 and E2 induced the same extent of growth stimulation indicating fast intracellular conversion of E1 into E2. Application of inhibitors selectively prevented stimulation of proliferation evoked by E1-treatment whereas E2-mediated stimulation was not affected. Furthermore, intracellular E2-formation from E1 was significantly inhibited with IC 50-values in the nanomolar range. In conclusion, our findings strongly support suitability of non-steroidal 17βHSD1-inhibitors for the treatment of estrogen dependent diseases.

Synthesis and anti-hepatitis B virus evaluation of novel ethyl 6-hydroxyquinoline-3-carboxylates in vitro

A series of non-nucleoside ethyl 6-hydroxyquinoline-3-carboxylate derivatives were prepared and evaluated in HepG2.2.15 cells. Most compounds inhibited the expression of viral antigens HBsAg or HBeAg at low concentration. Six compounds, 9f 3, 12b 6, 12f 6, 13b 2, 13b 6, and 13f 6, displayed excellent intracellular inhibitory activity and selectivity towards the replication of HBV DNA. Of these six initial hits, compound 13b 6 was the most active.

Synthesis and HCV inhibitory properties of 9-deaza- and 7,9-dideaza-7-oxa-2′- C-methyladenosine

As a part of an ongoing medicinal chemistry effort to identify inhibitors of the Hepatitis C Virus RNA replication, we report here the synthesis and biological evaluation of 9-deaza- and 7,9-dideaza-7-oxa-2′- C-methyladenosine. The parent 2′- C-methyladenosine shows excellent intracellular inhibitory activity but poor pharmacokinetic profile. Replacement of the nucleoside-defining 9-N of 2′- C-methyladenosine with a carbon atom was designed to yield metabolically more stable C-nucleosides. Modifications at position 7 were designed to exploit the importance of the hydrogen bond accepting properties of this heteroatom in modulating the adenosine deaminase (ADA) mediated 6-N deamination. 7-Oxa-7,9-dideaza-2′- C-methyladenosine was found to be a moderately active inhibitor of intracellular HCV RNA replication, whereas 9-deaza- 2′- C-methyladenosine showed only weak activity despite excellent overlap of both of the synthesized target compounds with 2′- C-methyladenosine’s three dimensional structure. Position 7 of the nucleobase proved to be an effective handle for modulating ADA-mediated degradation, with the rate of degradation correlating with the hydrogen-bonding properties at this position.

Antiviral Activity of Dammarane Saponins Against Herpes Simplex Virus I

AbstractTwo dammarane saponins, ginsenoside Rb1 (GRb1) and chikusetsusaponin III (Chi III), were investigated for antiviral activity against herpes simplex virus type I using an in vitro plaque elimination assay. Chi III prevented plaque formation at 50 μM, while GRb1 had no effect at 100 μM. Chi III was further examined for effects on virus yield and for cytotoxicity. The ID50 value for virus yield reduction was 29 μM, and cell growth was inhibited by 50% at 66 μM, indicating that Chi III exhibited marginal antiviral selectivity with a therapeutic index of about 2-fold. Although Chi III exhibited an in vitro virucidal effect with an ID50 value of 62 μM, drug treatment for 24 h reduced production of the cell-associated virus as well as the virus present in the medium, indicating Chi III has intracellular inhibitory activity. Intracellular levels of viral proteins were marginally affected at five hours post-infection, indicating Chi III has a minor impact on the early events of infection.

Clostridium butyricum neurotoxin: partial amino acid sequence of major proteolytic fragments and intracellular activity in PC12 cells

The ∼ 150 kDa single-chain neurotoxin produced by Clostridium butyricum, reported to be similar to C. botulinum neurotoxin serotype E ( Giménez and Sugiyama, Infect. Immun. 54, 926–929, 1988), was probed with trypsin and endoproteinase Lys-C. The two proteases cleaved the butyricum neurotoxin between residues Arg 421-Lys 422 and Lys 418-Gly 419, respectively. Cleavage at this region, highly susceptible to proteolysis, generated ∼ 50 kDa light and ∼ 100 kDa heavy chains, whose identities were established by amino acid sequence determination. In the ∼ 150 kDa dichain neurotoxin these two chains remained linked by an interchain disulfide bond. The endoproteinase Lys-C also cleaved the heavy chain between residues Lys 594-Ile 595, yielding a ∼ 73 kDa fragment. A total of 77 amino acid residues was identified by Edman degradation of the major fragments generated by proteolysis. By analogy with other botulinum neurotoxin serotypes, the light chain contains the intracellular inhibitory domain and the heavy chain the receptor-binding and channel-forming domains. Butyricum neurotoxin, whether in single-chain or dichain form, inhibited ∼ 70% of the Ca 2+ stimulated [ 3H]norepinephrine release from permeabilized PC12 cells. Reduction with 5 mM dithiothreitol enhanced the intracellular inhibitory activity of dichain neurotoxin about 30-fold and increased the extent of inhibition to about 80%, while the activity of single-chain neurotoxin was slightly affected. Increase in the intracellular inhibitory activity of butyricum neurotoxin following mild treatment with trypsin (i.e. conversion of the single-chain protein to the dichain form) was virtually complete within 6 min.

Calcium-dependent release of norepinephrine from permeabilized PC12 cells is inhibited by ∼48 and ∼112 kDa fragments of botulinum neurotoxin type E

Permeabilized PC12 cells exhibit a Ca 2+-stimulated norepinephrine secretory pathway which is sensitive to botulinum neurotoxin serotypes A, B and E [Lomneth R., Martin T. F. J. and DasGupta B. R. (1991) J. Neurochem. 57: 1413–1421]. Two novel amino terminal fragments of the 150 kDa neurotoxin serotype E (∼ 112 and 48 kDa), produced by digestion with pepsin, were tested in permeabilized PC12 cells. The intracellular inhibitory activity of the ∼ 112 kDa amino terminal fragment, like that of the 150 kDa neurotoxin, was progressively enhanced after trypsinization and dithiothreitol reduction. The ∼ 50 kDa C-terminal half of the heavy chain therefore does not contribute to the enhancement of inhibitory activity. The ∼ 48 kDa amino terminal light chain-like fragment completely inhibited release of norepinephrine, with an IC 50 = 500pM (more potent than the light chain isolated after digestion with trypsin) not requiring reduction with dithiothreitol. These results clarify the molecular basis of activation of neurotoxin by trypsin and dithiothreitol.