Inhibitory Activity Assay Research Articles

Virtual screening as a tool to discover new \u03b2-haematin inhibitors with activity against malaria parasites

Malaria remains a major public health problem. With the loss of antimalarials to resistance, the malaria burden will likely continue for decades. New antimalarial scaffolds are crucial to avoid cross-resistance. Here, we present the first structure based virtual screening using the β-haematin crystal as a target for new inhibitor scaffolds by applying a docking method. The ZINC15 database was searched for compounds with high binding affinity with the surface of the β-haematin crystal using the PyRx Virtual Screening Tool. Top-ranked compounds predicted to interact with β-haematin were submitted to a second screen applying in silico toxicity and drug-likeness predictions using Osiris DataWarrior. Fifteen compounds were purchased for experimental testing. An NP-40 mediated β-haematin inhibition assay and parasite growth inhibition activity assay were performed. The benzoxazole moiety was found to be a promising scaffold for further development, showing intraparasitic haemozoin inhibition using a cellular haem fractionation assay causing a decrease in haemozoin in a dose dependent manner with a corresponding increase in exchangeable haem. A β-haematin inhibition hit rate of 73% was found, a large enrichment over random screening, demonstrating that virtual screening can be a useful and cost-effective approach in the search for new haemozoin inhibiting antimalarials.

Inhibitory Effect of Sesamolin on Melanogenesis in B16F10 Cells Determined by In Vitro and Molecular Docking Analyses.

Melanin protects the skin against the harmful effects of ultraviolet irradiation. However, melanin overproduction can result in several aesthetic problems, including melasma, freckles, age spots and chloasma. Therefore, development of anti-melanogenic agents is important for the prevention of serious hyperpigmentation diseases. Sesamolin is a lignan compound isolated from sesame seeds with several beneficial properties, including potential for melanin inhibition. The aim of this study was to evaluate the anti-melanogenic effect of sesamolin in cell culture in vitro and the underlying mechanism of inhibition using molecular docking simulation. Melanogenesis was induced by 3-isobutyl-1-methylxanthine in B16F10 melanoma cells, and the inhibitory effects of sesamolin were evaluated using zymography, a tyrosinase inhibitory activity assay, western blotting, and real-time reverse transcription-polymerase chain reaction analysis. Docking simulations between sesamolin and tyrosinase were performed using Autodock vina. Sesamolin significantly inhibited the expression of melanogenesis-related factors tyrosinase, and tyrosinase-related proteins 1 and 2 at the mRNA and protein levels. Treatment of melanoma cells with 50 µM sesamolin demonstrated the strongest inhibition against intercellular tyrosinase and melanin synthesis without exerting cytotoxic effects. Sesamolin significantly reduced mushroom tyrosinase activity in a dose-dependent manner via a competitive inhibition mechanism. Tyrosinase docking simulations supported that sesamolin (-6.5 kcal/mol) bound to the active site of tyrosinase more strongly than the positive control (arbutin, -5.7 kcal/mol). Sesamolin could be developed as a melanogenesis inhibiting agent owing to its dual function in blocking the generation of melanogenesis-related enzymes and inhibiting the enzymatic response of tyrosinase.

Targeting Cannabinoid Receptors and Fatty Acid Amide Hydrolase: An Innovative Food-Grade Delivery System of Zingiber officinale and Acmella oleracea Extracts as Natural Adjuvant in Pain Management

Background: Anti-inflammatory and analgesic properties are displayed by several natural products, in particular Acmella oleracea and Zingiber officinale. Literature data suggests that both botanical extracts can be useful as inflammation/pain modulators, so technological efforts have been made to associate them together in a unique delivery new food-grade formulation, Mitidol, with the purpose of improving their beneficial effects. Objective: The aim of the present work was to assess if Mitidol and its active ingredients are able to modulate the endocannabinoid 2 receptor system, one of the major systems responsible for positive effects of Cannabis treatment. Cannabinoid 2 receptor represents an attractive pharmacological target in obtaining an anti-inflammatory/analgesic effect with low central nervous system side-effects. Methods: The new food-grade Phytosome, together with all the botanical ingredients and active compounds, were tested in a cell-based assay in human recombinant Cannabinoid 2 Receptor cells, in order to evaluate a possible agonist effect on those receptors, and in a Fatty Acid Amide Hydrolase Inhibition Activity assay, to evaluate potential inhibition of that hydrolase, which is responsible for degradation of the endogenous cannabinoid anandamide. Results: Zingiber officinale showed potent activity in Fatty Acid Amide Hydrolase Inhibition Activity assay, being about six fold more effective than the flavonoid Kaempferol. Acmella oleracea proved to be highly active in Fatty Acid Amide Hydrolase inhibition and Cannabinoid 2 activation. Conclusion: Our data suggest a strong rationale for the use of Mitidol as a natural adjuvant in pain management.

Inhibition of Heme Polymerization Invitro Assay Of Extract of Sirih Leaf (Piper betle Linn.) and Sun Flower Leaves (Helianthus annuus L.)

Malaria is a disease that occurs in tropical countries like Indonesia. The incidence of malaria in the world is still quite high and the occurrence of cases of Plasmodium resistance to antimalarial drugs and the widespread of resistance have prompted researchers to look for new antimalarial drugs, especially from natural materials. Betel leaf (Piper betle Linn.) And sunflower leaf (Helianthus annuus L.) have long been used by the people of Indonesia as an antimalarial drug. The purpose of this study was to determine antimalarial activity through inhibition of heme polymerization and determine secondary metabolite compounds by phytochemical screening from betel leaves and sunflower leaves. The heme polymerization inhibition activity assay was carried out by the in-vitro method using a microplate reader at 415 nm and 630 nm wavelengths. IC50 values of betel leaf extract and sunflower leaf were 178.67 μg/ml and 160.10 μg/ml, respectively. Phytochemical screening results from betel leaf showed the presence of flavonoids, polyphenols, tannins, quinones, saponins, and monoterpenoids-sesquiterpenoids, while sunflower leaves contain alkaloids, polyphenols, flavonoids, steroids and monoterpenoids-sesquiterpenoids.Keywords: Piper betle Linn., Helianthus annuus L., Malaria, Heme Polymerization

Bioactive constituents from Manilkara obovata (Sabine & G.Don) J.H.Hemsl

The phytochemical investigation of the methanolic extracts of roots, stem bark, leaves and twigs of Manilkara obovata has led to the isolation of one new friedelane triterpene, lacefriedelic acid or 3β,23-dihydroxy D:A-friedooleanan-28-oic acid (1) and one new prenylated xanthone, lacexanthone or 4,7-dihydroxy-2,3,3,9,9-pentamethyl-2,2-dihydrofurano[2,3-a]pyrano[2,3-i]xanthen-13(9H)-one (2) alongside twenty-four known compounds. Compounds 1-11 are reported here for the first time from the genus Manilkara. The structures of all compounds were determined by spectroscopic analyses and X-ray crystallography. The methanolic extracts of twigs and leaves showed anti-oxidant activity of 93.2 and 91.1%, respectively, at 100 µg/mL when measured by DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), while the twig extract displayed 86.3% at 100 µg/mL against the urease inhibition assay. Some isolated compounds (1-4, 15 and 20) showed significant to moderate anti-oxidant activity and urease inhibition assay. It is estimated that significantly active anti-oxidants and urease inhibitors metabolized by the plant may find future application in food industry.

Evaluation of in vitro biological activities of three Hypericum species (H. calycinum, H. confertum, and H. perforatum) from Turkey

Hypericum species have been used since ancient times for medicinal purposes due to their important pharmacological properties. As the most abundant species of the genus Hypericum, Hypericum perforatum is unquestionably one of the best-known and most consumed medicinal plants in the world. The aim of this current study is to evaluate antioxidant, anticholinesterase, and antiurease activities of the methanol extracts of flowering aerial parts of Hypericum calycinum L. Hypericum confertum Choisy, and Hypericum perforatum L. from Turkey. In this study, total phenolic and total flavonoid contents were determined as pyrocatechol and quercetin equivalents, respectively. Antioxidant activities of the species were carried out by using DPPH free radical scavenging, ABTS cation radical scavenging, metal chelate, and CUPRAC activity methods.Among the species that were investigated, it was determined that H. calycinum had the highest total phenolic content (141.12±2.40 μg PEs/mg), whereas H. confertum had the highest total flavonoid content (70.61±0.51 μg QEs/mg). The biochemical results in this study showed that all three species exerted strong antioxidant activity. H. calycinum appeared the most active in all antioxidant activity assays which is mainly due to its rich phenolic content. Additionally, H. calycinum showed the highest acetylcholinesterase inhibitory activity, while H. perforatum had the best inhibition towards butyrylcholinesterase. According to the urease inhibitory activity assay results, only H. calycinum showed low antiurease activity, whereas the other species did not show any activity against urease. The obtained results suggest that the studied three Hypericum species, especially H. calycinum could be promising sources for the treatment of several diseases because of their antioxidant and anticholinesterase properties.

In vitro antineurodegenerative activity and in silico predictions of blood-brain barrier penetration of Helichrysum plicatum flower extract

This study aimed to assess the antineurodegenerative and antioxidant activity of Helichrysum plicatum flower extract, as well as to identify extract ingredients with acceptable pharmacokinetic parameters such as gastrointestinal absorption, blood-brain barrier permeation, and P-glycoprotein-mediated effusion for optimal therapeutic brain exposure. Antioxidant activity was evaluated by ABTS, FRAP, and b-carotene bleaching assays, while antineurodegenerative activity was tested using acetylcholinesterase (AChE) and tyrosinase (TYR) inhibitory activity assays. In the ABTS test, the dry extract at the highest applied concentration (500 µg/mL) showed better or similar antioxidant activity compared to the standards. In the b-carotene assay, all applied concentrations of the extract showed significantly higher activity than vitamin C. No concentration-dependent activity was observed in the AChE assay, while in the TYR assay the lowest extract concentration (100 µg/mL) showed the highest percentage of inhibition (27.92 %). Pharmacokinetic parameters of compounds were predicted by in silico SwissADME online tool in accordance by the rules of drug-likeness. According to the pharmacokinetic properties, we concluded that pentoxymethoxylated flavones may represent CNS drug candidates for further studies.

Temperature dependent studies on nutritional, total polyphenols, flavonoids content and antioxidant activities of Aframomum citratum (C.Pereira) K.Schum and Tetrapleura tetraptera (Schum. & Thonn.) Taub. fruits

Description. Hard shelled fruits, Tetrapleura tetraptera and Aframomum citratum nonwood forest products are used as spices and for many medicinal recipes. Mostly used together, they undergo heat treatment before being milled when cooking Mbongo Tchobi, a black spicy stew from central and coastal regions in Cameroon. Objectives. Evaluate the impact of roasting these spices on the physicochemical properties (dry matter, ash, raw protein, total sugar, raw fibre, pH, aw, total energy, total fat), phytochemical content (total polyphenol, total flavonoids) and the antioxidant activities (total antioxidant activity, FRAP, DPPH and lipid peroxidation inhibition assays) of their aqueous extracts in a home food consumption context. Method. Tetrapleura tetraptera and Aframomum citratum fruits from Ngola locality in Yokadouma (east region in Cameroon) locality were collected, sorted, cleaned and dried (72 h, 45 °C) in an oven. Cut into small pieces, a part was heated using roasting parameters deduced from preliminary tests (140 °C/10 min; 180 °C/20 min and 150 °C/12 min respectively for T. tetraptera, A. citratum and a formulation 95/5 of the two spices). The heated and unheated spices were ground and an infusion was prepared from boiling water in the proportion of 1/5 (g·ml-1) for both types of spices. Physicochemical and phytochemical characteristics (dry matter, total ash, fat, sugar, crude fibre and energy contents, pH, aw, total flavonoids and polyphenols) as well as the antioxidant activities of the aqueous extracts of the two spices and a mix of the two spices were evaluated before and after the heat treatment. Results. Heat treatment significantly (p < 0.05) reduces fat and total sugars contents but improves raw fiber contents for all spices. Phenolic content is more important for heated spices and percentage differences before and after heat treatment are between 8.0 and 23.2% for both spices. Extracts obtained with heated spices possess the best antioxidant potential. Strong significant positive correlations have been established between the phenolic compounds and the reductive antioxidant activities and negative correlations with the antiradical antioxidant activities as they are expressed as IC50 (mg·ml-1). Conclusions. In most of the features studied, the heated formulation (95/5) possesses the highest characteristics studied. The choice of heating these spices under the roasting parameters identified should be guided by the desire of improving their antioxidant potential because of the nutritional losses observed after heat treatment.

Antioxidant, antimicrobial and phytotoxic activities of Rhaponticum acaule DC. essential oil

The in-vitro antioxidant activity of Rhaponticum acaule essential oil (RaEO) was evaluated using β-carotene/linoleic acid bleaching, chelating activity, and lipid peroxidation inhibition (TBARS) assays. The antimicrobial activity of RaEO was assessed by disc diffusion and microdilution methods against 8 bacteria and 4 yeast. Finally, the allelopathic activity of RaEO on the seed germination and the shoot and root elongation of lettuce (Lactuca sativa L.) seedlings were investigated. According to our results, the RaEO exhibited significant antioxidant activity, similar to those of standards (BHT and ascorbic acid) with IC50 values of 0.042 and 0.045 mg/mL obtained by β-carotene bleaching and TBARS assays, respectively. On the other hand, despite its interesting ferrous chelating activity, RaEO possesses moderate IC50 value (0.35 mg/mL) as compared with that of EDTA (0.015 mg/mL). RaEO exhibited a strong antimicrobial activity against all the tested microorganisms, with IZ, MIC and MBC values being in the range of 7.67 ± 0.58 to 13.33 ± 0.58 mm, 1.25 to 5.00 and 5.00 to 10.00 mg/mL, respectively. The results reveled also that RaEO inhibited the shoot and root growth of Lactuca sativa L. seedlings. Our data suggested that the RaEO had pharmaceutical benefits and could be used as a potential natural herbicide resource.

Anti-oxidant and anti-diabetes activities of agarwood extracts from Gyrinops versteegii (Gilg.) Domke and their cytotoxicity

Gyrinops versteegii is an endemic plant in Lombok, West Nusa Tenggara, which produce agarwood. In vitro anti-oxidant and anti-diabetes activities from several extracts of agarwood produced by G. versteegii were investigated. The 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging activity assay was used to determine the anti-oxidant activity, whereas the α-glucosidase inhibitory activity assay was conducted to determine the anti-diabetes activity. The toxicity test using brine shrimp lethality test (BSLT) and phytochemical analysis were also conducted in this experiment. As the results, the acetone extracts of infected wood of G. versteegii had the highest anti-oxidant activity with the IC50 value of 65.62 μg/mL, whereas the acetone extracts of agarwood had the α-glucosidase inhibitory activity with the IC50 value of 53.46 μg/mL. The toxicity test to the both samples shows the high toxicity with the LC50 values of 1.98 and 15.44 μg/mL, respectively. The high anti-oxidant and anti-diabetes activities in both of the acetone extracts of infected wood and agarwood may be caused by their high content of total phenolic (12.75 and 9.17%, respectively) and total flavonoid (19.96 and 14.34%, respectively). The anti-diabetes activity found in agarwood extracts produced by G. versteegii from Lombok is firstly reported.

Lipase inhibitory activity assay for fermented milk

The lipase inhibitory activity method described here was developed to identify potential anti-obesity properties in milk fermented with different strains of lactic acid bacteria via inhibition of pancreatic lipase and a subsequent decrease in fat digestion and absorption in the gut. The method is based on the hydrolysis of 4-nitrophenyl octanoate by pancreatic lipase and the subsequent release of p-nitrophenol, a coloured product whose absorbance can be measured at 412 nm. Inhibition of lipase leads to a decrease in the amount of p-nitrophenol released and a subsequent reduction in the absorbance with respect to a 100% activity control. The assay was developed by adapting various methods previously described in published literature and includes modifications that are key to adapt the existing protocols to fermented milk samples, in particular the pH issues encountered when analysing acidic samples:•A two buffer system is introduced to allow optimal pH control after addition of fermented milk samples with pH values between 3.5 and 6.5.•A post-clarification filtration step is added for samples where turbidity remains after addition of the clarifying reagent for dairy products.•An absorbance correction factor is calculated and applied to the samples to account for the reduction in the absorbance of p-nitrophenol caused by milk.

Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi.

Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species ( Pacheco et al., 2018 ) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions.

Evaluation of the antioxidant and anti-arthritic potential of Zingiber officinale Rosc. by in vitro and in silico analysis

Rheumatoid Arthritis (RA) is an autoimmune disorder that leads to joint and bone destruction. The available anti-inflammatory chemo drugs are associated with adverse side-effects providing only temporary relief. Herbal medicine can act as an alternative source for RA treatment with improved efficacy, lesser toxicity and side effects. An attempt was made to evaluate the antioxidant and anti-arthritic potential of ginger (Zingiber officinale Rosc.). Crude ginger extracts were prepared with different organic solvents using soxhlet approach. The extracts were assessed for their anti-oxidant activity using DPPH, ABTS and nitric oxide radical inhibition assays and anti-arthritic activity through membrane stabilization assay, anti-proteinase activity and protein denaturation inhibition assays. The highest scavenging potential was exhibited by Z. officinale methanolic extract (ZOME) as assessed by DPPH assay (86.26 ± 0.97%), ABTS assay (91.04 ± 0.96%) and nitric oxide assay (86.72 ± 1.51%). Similarly, the ZOME showed 84.72 ± 1.38% of inhibition in membrane stabilization assay, 82.72 ± 1.48% in anti-proteinase activity and 81.68 ± 1.66% in protein denaturation inhibition capacity. GC-MS analysis of ZOME showed 30 different phytoconstituents. The major ginger bioactive compounds were virtually docked with novel RA targets using PyRx and the pharmacokinetic properties like ADMET properties were evaluated. Among all, 8-Gingerol showed the highest covalent interaction along with suitable binding affinity to the RA targets of COX-II (−7.2), TNF-α (−4.7), MCSF (−5.0), IL-1β (−5.7) and MMP9 (−6.7) kcal/mol. 8-Gingerol can be a better drug candidate that must be further investigated for mechanistic studies to verify its therapeutic potential in treating RA.

Study on the active polyphenol constituents in differently colored Rubus Chingii Hu and the structure-activity relationship of the main ellagitannins and ellagic acid

Color is considered an index to evaluate the quality of the unripe fruit of Rubus chingii Hu in China. This study compared the contents of the active polyphenolic constituents in unripe differently colored fruits. The data showed that there were significant differences between the yellow fruit (YF) and green fruit (GF) in the composition of ellagitannins (ETs) and ellagic acid (EA). Then, the structure-activity relationship of the main ellagitannins (lambertianin A, sanguiin H-6, casuarictin) and ellagic acid in unripe fruits were investigated by the antioxidant activities assay, non-enzymatic glycation inhibitory activity assay and glucose uptake assay in L6 cells. The results showed that the number of hydroxyl groups and galloyl configuration at the glucose C-1 significantly affected the activity of ellagitannins. However, ellagic acid, which had fewer hydroxyl groups, exhibited the highest bioactive properties. Moreover, the fluorescence quenching analysis and molecular docking results demonstrated that ellagitannin was a concentration-dependent antiglycation compound. This study suggested that EA and ETs had different action mode and the content of ETs was an important factor influencing the unripe fruits of Rubus chingii Hu.

Environmentally-safe scCO2P. pinaster branches extracts: Composition and properties

Considering the vast array of compounds that plants manufacture that help protect them against insects’ attacks, plant raw materials are a potential source of future insecticides/larvicides. In this work, volatiles from maritime pine (Pinus pinaster) branches, a pruning residue, were extracted by supercritical carbon dioxide (scCO2), and also by hydrodistillation for comparison purposes. The volatiles naturally emitted from the pine branches were identified as well. Experimental design results showed higher extraction yields (up to 6%), when scCO2 density and temperature increased, while the inverse occurred for total phenolic contents (1.2–3.3% GAE, gallic acid equivalents). Emitted volatiles from the branches were mainly α-pinene, β-pinene, β-myrcene, limonene, and β-caryophyllene. In general, the volatile profiles of the scCO2 and hydrodistillation extracts were quite similar, revealing abietadiene, the precursor of abietic acid, as the main compound. Oxidation inhibitions of ∼42 – 59% were obtained for the scCO2 extracts in the β-carotene assay. The acetylcholinesterase inhibitory activity assay revealed the potential of these extracts to have insecticidal/larvicidal activity, with IC50 values of 2.8–10.7 mg/mL. Overall, these extracts may be applied in environmentally-safe insect/larvae control strategies, in formulations that benefit from the presence of antioxidants.

In vitro Antioxidant and Anti-obesity Activities of Freeze-dried Canarium sp., Averrhoa bilimbi L. and Malus domestica

BACKGROUND: The number of obesity cases is still increasing worldwide and has reached an epidemic scale. Plants are known to have a protection role in the development of obesity, however their antioxidant and anti-obesity activities have not widely known. This study was conducted to assess the in vitro antioxidant and anti-obesity activities of different types of freeze-dried fruits cultivated in Indonesia, especially Canarium sp., Averrhoa bilimbi L. and Malus domestica.METHODS: Total phenolic content of freeze-dried fruits was identified by Folin-Ciocalteu method, while the total flavonoid content was measured by aluminium chloride colorimetric assay. To assessed the antioxidant activity, 2,2-diphenyl 1-picrylhydrazyl (DPPH) scavenging activity assay and 2,2’-azino-bis (3-ethylbenzothiazoline6-sulphonic acid) (ABTS) reducing activity assay were performed. The α-amylase and lipase inhibitory activity assay were performed to assess the anti-obesity activity. For comparison, hydroxycitric acid (HCA) compound was also assessed with DPPH, ABTS, α-amylase and lipase assays.RESULTS: A. bilimbi had the highest total phenol content (6.35 µg GAE/mg), meanwhile M. domestica had the highest total flavonoid content (2.06 µg QE/mg). A. bilimbi also showed the highest antioxidant activity both in DPPH and ABTS assay, with inhibitory concentration (IC50)=279.99 µg/mL and 631.78 µg/mL, respectively. The freeze-dried M. domestica had the highest anti-α-amylase activity (IC50=258.85 µg/mL), while Canarium sp. had the highest anti-lipase activity (IC50=118.66 µg/mL).CONCLUSION: Freeze-dried fruits demonstrate in vitro benefits toward obesity. A. bilimbi has potent antioxidant activity and is beneficial against obesity-related adverse health effect by relieving oxidative stress. M. domestica and Canarium sp. hamper the fat accumulation by reducing the carbohydrate absorption and dietary lipid.KEYWORDS: antioxidant, anti-obesity, Canarium sp., Averrhoa bilimbi L., Malus domestica, hydroxycitric acid

A Novel Combination Regimen of BET and FLT3 Inhibition for FLT3-ITD Acute Myeloid Leukemia

Background: Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors such as quizartinib and gilteritinib improve overall survival in relapsed patients, but their efficacy is limited and most such patients die of the disease. This is because even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as c-Myc and Bcl2, but these drugs thus far have shown only limited single-agent clinical potential. PLX51107 is a novel BET inhibitor designed to inhibit BET activity in intermittent daily fashion to allow for greater tolerability. We investigated whether the addition of PLX51107 to potent FLT3 inhibition with quizartinib could overcome the protective effect of the bone marrow stroma through inhibition of c-Myc expression. Methods: We developed a plasma inhibitory activity assay for c-Myc (c-Myc PIA) to assess in vivo efficacy of the PLX51107 in patients and to identify c-Myc-inhibitory doses of the drug. We tested PLX51107 alone and in combination with quizartinib in murine models of AML and against primary FLT3-ITD AML cells co-cultured with human bone marrow stroma. In addition, we analyzed gene expression patterns in the treatment models to explore the basis of any observed synergistic cytotoxic effect. Results: In a murine xenograft model of AML using MV4-11 cells, PLX51107 alone induced suppression of tumor growth in association with a 90% decrease in c-Myc gene expression. The combination of PLX51107 and quizartinib induced complete tumor regression in 5 out of 7 animals after 14 days of treatment (Figure 1). Animals treated with a 5-day course of quizartinib alone displayed tumor regression persisting until day 26 of treatment, while the addition of PLX51107 resulted in tumor regression until day 39. In patients treated with a dose of 120 mg/day of PLX51107, the c-Myc PIA demonstrated a robust suppression of c-Myc expression for roughly 6 hours, returning to baseline between 7 and 9 hours post-treatment (Figure 2). The mean plasma concentration to achieve this inhibition was 3.3 uM, which, accounting for the difference in protein drug binding between plasma and culture medium with 10% FBS, corresponded to a concentration of 250 nM PLX51107 in culture medium. With this same concentration and schedule (i.e., at concentrations and exposure times equivalent to what human patients would experience taking 120 mg daily of PLX51107 and 60 mg daily of quizartinib), the combination induced synergistic cytotoxicity in a series of 10 different FLT3-ITD AML patient blast samples co-cultured with bone marrow stroma (Figure 3). C-Myc RNA and protein were directly suppressed in these primary samples, and ingenuity pathway analysis of RNA expression confirmed that c-Myc associated genes displayed the highest level of down-regulation. Conclusions: These studies suggest that combination therapy with approximately 120 mg/day PLX51107 and 60 mg/day quizartinib will be a more effective therapy for relapsed FLT3-ITD AML than 60 mg/day of quizartinib alone. The combination of FLT3 inhibition and BET inhibition may represent an attractive therapeutic option for FLT3-ITD AML. Disclosures Hizukuri: Daiichi Sankyo Co, Ltd: Employment. Severson:Plexxikon Inc.: Employment. Powell:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Ma:Plexxikon Inc: Employment. Narahara:Daiichi Sankyo Co, Ltd: Employment. Sumi:Daiichi Sankyo, Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levis:FUJIFILM: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Menarini: Consultancy, Honoraria.

Design, Synthesis and Preliminary Biological Evaluation of Benzylsulfone Coumarin Derivatives as Anti-Cancer Agents.

In this work, a series of benzylsulfone coumarin derivatives 5a–5o were synthesized and characterized. Kinase inhibitory activity assay indicated that most of the compounds showed considerable activity against PI3K. Anti-tumor activity studies of the active compounds were also carried out in vitro on the Hela, HepG2, H1299, HCT-116, and MCF-7 tumor cell lines by MTS assay. The structure–activity relationships (SARs) of these compounds were analyzed in detail. Compound 5h exhibited the most potent activities against the mentioned cell lines with IC50 values ranging from 18.12 to 32.60 μM, followed by 5m with IC50 values of 29.30–42.14 μM. Furthermore, 5h and 5m clearly retarded the migration of Hela cells in vitro. Next, an in silico molecular docking study was conducted to evaluate the binding models of 5h and 5m towards PI3Kα and PI3Kβ. Collectively, the above findings suggested that compounds 5h and 5m might be promising PI3K inhibitors deserving further investigation for cancer treatment.

A Phase II Study of Midostaurin and 5-Azacitidine for Untreated Elderly and Unfit Patients With FLT3 Wild-type Acute Myelogenous Leukemia

BackgroundMidostaurin, a multikinase inhibitor, is approved for treatment of FLT3-mutant acute myeloid leukemia (AML). A phase I study established that midostaurin 75 mg orally twice daily for 14 days with standard dose azacitidine was safe and tolerable in elderly patients with AML. Herein, we report the phase II expansion cohort of previously untreated elderly or unfit patients with AML. Patients and MethodsPrimary objectives were to further describe the toxicity profile and determine the response rate in untreated patients with AML. Patients received midostaurin 75 mg orally twice daily on days 8 to 21 in combination with intravenous azacitidine at 75 mg/m2 on days 1 to 7. Plasma inhibitory activity assay for FLT3 was performed pretreatment and on day 8 and day 15 of each cycle. ResultsTwenty-six patients (median age, 74 years; range, 59-85 years) with FLT3 wild-type AML were accrued. Patients received a median of 2 cycles of therapy (range, 1-10 cycles). Seven (29%) of 24 evaluable patients achieved a clinical response (4 complete response; 1 complete response with incomplete count recovery; and 2 partial response). The median overall survival was 244 days (95% confidence interval, 203-467 days). Hematologic, infectious, and gastrointestinal toxicities were comparable to similar studies. Peripheral blood FLT3 wild-type phosphorylation declined to 8% to 55% of pretreatment by day 15 of cycle 1 (7 patients) and declined with subsequent cycles (< 10% baseline) in 2 patients who were analyzed after cycle 3. ConclusionMultiple cycles of azacitidine and midostaurin were not well-tolerated, but persistent inhibition of FLT3 wild-type phosphorylation suggest intermittent dosing of midostaurin should be considered in future low-intensity regimens for FLT3-mutant AML.

Elastase, Hyaluronidase and Tyrosinase inhibitor activities antiaging of Curcuma mangga Val. extract and its fractions

Curcuma mangga Val. has bioactive compounds such as curcumin. Curcumin is a natural antioxidant and known as antiaging agent. Curcumin can stimulate the synthesis of collagen type I, inhibit melanogenesis and help to maintain correct skin hydration. This show C.mangga Val. has potential as antioxidant and antiaging agents. This research aims to find out the antiaging activities of C. mangga ethanolic extracts (CMEE) and its fractions. In this study, the antiaging activity was performed by measuring inhibition of elastase, hyaluronidase, and tyrosinase inhibitory activity assay. Ascorbic acid using as a control. In elastase inhibitory activity, ascorbic acid has the highest activity (15.53 µg/mL) but ethyl acetate fraction of C. Mangga (EACM) also has high activity (26.34 µg/mL) compared to other fractions and CMEE. In hyaluronidase inhibitory activity, ascorbic acid has the highest activity (27.67 µg/mL) but CMEE also has high activity (80.04 µg/mL) compared to other fractions, while in tyrosinase inhibitory activity EACM has the highest value (40.34 µg/mL) compared to ascorbic acid (65.99 µg/mL). In summary, C. mangga extracts have anti-hyaluronidase, while EACM also has good antiaging properties through anti-elastase and antityrosinase activity.