Virtual screening and biophysical studies lead to HSP90 inhibitors

Heat shock protein 90 (HSP90) is a molecular chaperone that plays important functional roles in cells. The chaperone activity of HSP90 is regulated by the hydrolysis of ATP at the protein’s N-terminal domain. HSP90, in particular the N-terminal domain, is a current inhibition target for therapeutic treatments of cancers. This paper describes an application of virtual screening, thermal shift assaying and protein NMR spectroscopy leading to the discovery of HSP90 inhibitors that contain the resorcinol structure. The resorcinol scaffold can be found in a class of HSP90 inhibitors that are currently undergoing clinical trials. The proved success of the resorcinol moiety in HSP90 inhibitors validates this combined virtual screen and biophysical technique approach, which may be applied for future inhibitor discovery work for HSP90 as well as other targets.     

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C₁₈, C₂₀, and C₂₂ fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Inhibition of carbohydrate and lipid digestive enzymes activities by Zygophyllum album extracts: effect on blood and pancreas inflammatory biomarkers in alloxan-induced diabetic rats

Zygophyllum album has been used as herbal medicine in Southern Tunisia to treat several diseases such as diabetes mellitus. This study is aimed to reveal the mechanisms underlying the antihyperglycemic potential, the anti-inflammatory and the protective hematological proprieties of this plant in diabetic rats. The inhibition of the α-amylase activity by different solvent-extract fractions of Z. album was tested in vitro. The fraction endowed with the powerful inhibitory activity against α-amylase was administered to surviving diabetic rats for 30 days. Data from in vitro indicated that each extract from the medicinal plant showed moderate inhibition of α-amylase enzyme except the ethyl acetate extract which was ineffective. The powerful inhibition was achieved by ethanol extract of Z. album (EZA) with an IC50 of 43.48 μg/ml as compared to acarbose (Acar) with an IC50 of 14.88 μg/ml. In vivo, the results showed that EZA decreased the α-amylase levels in serum, pancreas and intestine of diabetic rats by 40 %, 45 % and 46 %, respectively, associated with considerably reduction in blood glucose rate by 61 %. Moreover, the EZA helped to protect the structure and function of the β-cells. Interestingly, EZA had a potent anti-inflammatory effect which is manifested by decreases in CRP and TNF-α levels. Overall, a notable reduction in lipase activity both in serum and small intestine of treated diabetic rats resulted in the improvement of serum and liver lipids profile. Z. album showed a prominent antidiabetic effect via inhibition of carbohydrate and lipid digestive enzymes and ameliorated the inflammation and the disturbance of hematological biomarkers in diabetes.     

Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations

The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0–11.0 and 65–70 °C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 °C.

The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 °C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 °C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.     

Purification and biochemical characterization of a detergent stable α-amylase from Pseudomonas stutzeri AS22

This study reports the purification and biochemical characterization of a novel maltotetraose-forming-α-amylase from Pseudomonas stutzeri AS22, designated PSA. The P. stutzeri α-amylase (PSA) was purified from the culture supernatant to homogeneity by Sepharose mono Q anion exchange chromatography, ultrafiltration and Sephadex G-100 gel filtration, with a 37.32-fold increase in specific activity, and 31% recovery. PSA showed a molecular weight of approximately 57 kDa by SDS-PAGE. The N-terminal amino acid sequence of the first 7 amino acids was DQAGKSP. This enzyme exhibited maximum activity at pH 8.0 and 55°C, performed stably over a broad range of pH 5.0 ≈ 12.0, but rapidly lost activity above 50°C. Both potato starch and Ca²⁺ ions have a protective effect on the thermal stability of PSA. The enzyme activity was inhibited by Hg²⁺, Mn²⁺, Cd²⁺, Cu²⁺, and Co²⁺, and enhanced by Ba²⁺. PSA belonged to the EDTA-sensitive α-amylase. The purified enzyme showed high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), and oxidizing agents, such as sodium per borate and H₂O₂. In addition, PSA showed excellent compatibility with a wide range of commercial solid and liquid detergents at 30°C, suggesting potential application in the detergent industry. Maltotetraose was the specific end product obtained after hydrolysis of starch by the enzyme for an extended period of time, and was not further degraded.     

Effects of microwave (2.45 GHz) irradiation on some biological characters of Salmonella typhimurium

The present study was carried out to evaluate the effects of sub-lethal doses of microwave radiation on some biological characteristics in Salmonella typhimurium. The aim was to show the relationship between this treatment and the development of radiotolerance in this pathogen because there is a need for more information on physiological responses of pathogens to sub-lethal doses of microwave radiation. So, the bacterial strain was treated with a dose of 3600 J (40-s exposure with power P = 90 W) to cause cellular damage. The results have shown that the exposure of bacteria to microwaves resulted in a significant inhibition of cellular growth. This treatment has notably increased the effectiveness of the most tested antibiotics by the amelioration or the appearance of sensitivity in exposed bacteria. Gas chromatography (GC) analysis was performed to demonstrate the modification of the fatty acids (FA) composition. Results obtained have shown that this treatment had a significant effect on the FA content with an increase of unsaturated FA percentage. The acquisition of sensitivity to the sodium deoxycholate and the significant increase in the amount of extracellular proteins in exposed bacteria has confirmed the weakening of the bacterial membrane by microwaves. This study represents one of the few demonstrating the modifications on the bacterial membrane as a cellular response to survive the non-ionising radiation stress.     

Varied tolerance to NaCl salinity is related to biochemical changes in two contrasting lettuce genotypes

Salt stress perturbs a multitude of physiological processes such as photosynthesis and growth. To understand the biochemical changes associated with physiological and cellular adaptations to salinity, two lettuce varieties (Verte and Romaine) were grown in a hydroponics culture system supplemented with 0, 100 or 200 mM NaCl. Verte displayed better growth under 100 mM NaCl compared to Romaine, but both genotypes registered relatively similar reductions in growth under 200 mM NaCl treatment. Both varieties showed differences in net photosynthetic activity in the absence of salt and 8 days after salt treatment. These differences diminished subsequently under prolonged salt stress (14 days). Verte showed enhanced leaf proline and restricted total cations especially Na⁺, lesser malondialdehyde (MDA) formation and lignification in the roots under 100 mM NaCl salinity. Membrane damage estimated by electrolyte leakage increased with elevated salt concentrations in roots of both varieties, but Verte had significantly lower electrolyte leakage relative to Romaine under 100 mM NaCl. Moreover, Verte also accumulated greater levels of carotenoids under increasing NaCl concentrations compared to Romaine. Taken together, these findings suggest that the greater tolerance of Verte to 100 mM NaCl is related to the more restricted accumulation of total cations and toxic Na⁺ in the roots and enhanced levels of antioxidative metabolites in root and leaf tissue.     

Enhanced activity of hyperthermostable Pyrococcus horikoshii endoglucanase in superbase ionic liquids

Biotechnology Letters - Ionic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the...