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High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors

A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors: Paper Summary A high throughput screening was carried out of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which resulted in multiple compound series with the potential for further development[1]. Multiple screening methods were carried out, including a Primary PFKFB3 HTS spot-test assay with an IC50 follow up, an Orthogonal IC50 assay and Enzyme Ratio Testing; these methods shall be outlined throughout this summary. Primary PFKFB3 HTS spot-test assay IC50 follow up The screening of compounds by St-Gallay et al was performed using an ADP-Glo format [2] – a dual-step process. The protein of interest (PFKFB3) was incubated with two compounds, fructose-6-phosphate (F6P) and ATP in a buffer solution, containing 5 mM MgCl2, 5 mM KPi at PH7, 200 mM KCl, 0.1 mM Triton X-100, 50 mM HEPES and 1mM dithiothreitol (DTT); thus giving the terminal concentrations of PFKFB3, ATP and F6P. (8.75 nM, 34 µM and 15 µM respectively, as outlined below in Figure 1.) Figure 1: Graphs outlining the final concentrations and rate of luminescence (RFU/RLU per minute) of ATP, F6P and PFKFB3[1] The primary run was undergone in the 1536 well format, whereby 1µL of enzyme and 1µL substrate additions were made simultaneously, to give the final volume of 2µL per well. All other assays were prepared using a very similar method, by addition of a fraction of solution containing enzyme (reactions already initiated) with an equal volume of substrate solution. After approximately 60-70 minutes of time passing, ADP-Glo reagent was introduced to the wells in order to terminate the kinase reaction, and exhaust any remaining ATP. After approximately another 40 minutes, a kinase detection reagent was added, in order to convert adenosine diphosphate (ADP) back to its original ATP form. The newly formed ATP could then be quantitated by the luciferin/luciferase reaction, where for each molecule of ADP that was produced, it was assumed that 1 molecule of ATP had regenerated. Luminescence could then be determined and used as an indicator of enzyme inhibition; it was found that molecules that did indeed inhibit PFKFB3 reduced the luminescent signal when compared to uninhibited enzyme. Both the primary screen and follow up followed the same terminal conditions, in both a 384 well (low volume plate) and 1536 well format. Both substrate and enzyme additions were added at a 1:1 ratio, although the total volume was 2µL per well for the 1536 format and 5µL per well for the 384 well format. The ADP-Glo was added stoichiometrically (1:1:2 ratio as recommended by the manufacturer) in regards to volume/ADP-Glo reagent/kinase detection reagent [1]. Bovine Serum Albumin (BSA) was considered for the determination of non-specific binding, however as it is well known that BSA has a tendency to bind drug-like molecules[1], of which many are confirmed to inhibit enzymes, so there was a real risk of removing genuine hits through false negative results. Therefore it was decided that the protein of interest (PFKFB3) was to be used, with the added benefit of the ability to construct a kinetically equivalent assay at both of the concentrations outlined above. Full IC50 (follow up) curves were produced for a specific subset of hit compounds, these were identified from the initial library of 837387 compounds; A comparson of these results with the orthogonal inhibition assay can be found in Figure 2 below. Finally those selected compounds were tested within an artifact IC50 format, whereby the detection part of the ADP-Glo assay was run again, this time without PFKFB3 and instead with an equivalent concentration of ADP/ATP – which was a substitute for what would have been produced by the aformentioned kinase reaction. Therefore if any of the selected compounds interfered with the detection of the mixture, there would be a notable decrease in signal observed; however none of compounds tested were found to cause this effect. Figure 2: A graph outlining PFKFB3 pIC50 when comparing luminescence (ADP-Glo assay) versus the Transcreener technology(orthogonal inhibition assay) [1] Orthogonal inhibition assay The compounds brought forward for an IC50 follow up using the ADP-Glo assay after the initial HTS were also tested used a Transcreener ADP Fluorescent Intensity Assay [1]. Due to the fact that this assay is based on detecting ADP, any class of compounds /enzyme that produces ADP is compatible with Transcreener technology [3]. The process of assay preparation was designed to match the conditions used in the previous ADP-Glo assay, however it was found that instead of a protein concentration of 8nM being left for 60 minutes, a concentration of 4nM being left for 120 minutes gave a linear reaction and more appropriate signal window, as outlined in Figure 3 below. Figure 3: Using ADP-Glo conditions, linearity of a reaction time course was measured with various concentrations of PFKFB3 for the orthogonal inhibition assay [1] The assay was again performed in two steps, first PFKFB3 was incubated with the compound, ATP and F6P, in the same buffer solution used prior; the only difference in final concentration was PFKFB3 at 4.3nM instead of 8.75nM, again highlighted by Figure 3, and the solutions were left for 2h instead of 1h. ADP was able to be detected due to a tracer (ADP Alexa 594) that was bound to ADP2 monoclonal antibody [1], as the tracer is displaced by ADP it is released into solution and causes a positive increase in fluorescent readout. Consequently, there is a correlation between kinase activity and an increase in fluorescence due to a rise in ADP production. Using a black, low volume 384 well plate, after 2 hours of incubation 5µL (2.5 µL of PFKFB3 and 2.5 µL of ATP/F6P) of solution was added to each well. After approximately a further 60 minutes, fluorescence readings of the plates were taken. The IC50 values were then generated from these results, and compared against the results achieved through the ADP-Glo assay, as again can be seen in Figure 2. A positive correlation was observed between the two assays in regards to compound potency values [1] suggesting a high level of accuracy in IC50 readings. Enzyme Ratio Testing A secondary enzyme test occurred in order to determine nonspecific compounds effects, thereby allowing separation of potential hits that are time-dependant or enzyme-concentration dependant. By increasing the PFKFB3 concentration 10-fold, and simultaneously decreasing the incubation time by the same amount (60 minutes decreasing to 6 minutes), it was possible to ascertain effects (both time and concentration dependant) that potentially occurred due to the compound behaviour within the enzyme assay, for example compound aggregation. [4] ERT was an excellent secondary filter that was used to specify more promising candidates from the initial hits through both HTS and the following assays. Conclusion St-Gallay et al were able to benefit from the highly accurate and selective HTS and following assays in order to determine ‘right’ compounds with positive lead-like properties [5]. By taking advantage of assays designed around ADP production and the detection of fluorescent tags, with powerful filtering and analytical techniques (Enzyme Ratio Testing, Isothermal Calorimetry and X-Ray Crystallography) the dihydropyrrolopyrimidinone series of compounds [1] were identified as a potential option in developing potential treatments for tumours – by targeting the increased glycolytic flux that occurs in tumour growth [6]. References [1] Stephen A. St-Gallay, Neil Bennett, Susan E. Critchlow. A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors, Sage Journals. SLAS Discovery. 2017, Vol. 23, 11–22. [2] Zegzouti, H., Zdanovskaia, M., Hsiao, K.. ADP-Glo: A Bioluminescent and Homogeneous ADP Monitoring Assay for Kinases. Assay Drug Dev. Technol. 2010, 7, 560–572. [3] Kleman-Leyer, K. M., Klink, T. A., Kopp, A. L.. Characterization and Optimization of a Red-Shifted Fluorescence Polarization ADP Detection Assay. Assay Drug Dev. Technol. 2009, 7, 56–67. [4] Habig, M., Blechschmidt, A., Dressler, S.. Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists. J. Biomol. Screen. 2009, 14, 679–689. [5] Teague, S. J., Davis, A. M., Leeson, P. D.. The Design of Leadlike Combinatorial Libraries. Angew. Chem. Int. Ed. 1999, 38, 3743–3748 [6] Buerkle, A., Weber, W. Imaging of Tumor Glucose Utilization with Positron Emission Tomography. Cancer Metastasis Rev. 2008, 27, 545–554.

UC Marketing Management & Marketing & Distribution Strategies Presentation

UC Marketing Management & Marketing & Distribution Strategies Presentation.

Task-1( 700+ words and in APA format including Times New Roman with font size 12 and double spaced).Reflect on the assigned readings from attached chapter 14 & 15. Identify what you thought was the most important concept(s), method(s), term(s), and/or any other thing that you felt was worthy of your understanding. Also, provide a graduate-level response to each of the following questions:Discuss how core factors, cues to quality, and interpersonal factors of a product influence your buying decisions. Discuss with supporting examples.Imagine designing a conjoint for your b-school’s café. In particular, you’re in charge of the daily pizza orders. Pizzas are tricky—while they’re a simple food, they can be created in a zillion combinations. What factors should you test in terms of your fellow students’ likely preferences? Wheat crust vs. white, thick vs. thin, plain cheese vs. sausage vs. sausage and green pepper vs. vegetarian (you get the picture). Design a conjoint that would result in identifying 2 or 3 popular slices that your café managers could order every morning. The student body knows you’re responsible—how do you make most of them happy?Task-2 – Attached a pdf file. in that topic 11 & 12(mentioned below also), Please do an PPT document with 7 or 8 slides11. Distribution Strategy 12. Promotional Strategy12.1 Marketing Mix12.2 Generic Strategies12.3 Current Market Trends
UC Marketing Management & Marketing & Distribution Strategies Presentation

Rasmussen College Importance of Constructing Confidence Intervals Questions Report

java assignment help Rasmussen College Importance of Constructing Confidence Intervals Questions Report.

I’m working on a Statistics spreadsheet and need support to help me study.

Confidence intervalsA major client of your company is interested in salary distributions of jobs in the state of Minnesota that range from $30,000 to $200,00 per year. As a business Analyst, your boss asks you to research and analyze the salary distributions. You’re given a spreadsheet that contains the following information:– A listing of the jobs by title- The salary (in dollars) for each jobYou have previosly explained some of the basic statistics to your client already, and he really liked your work. Now he wants you to analyze the confidence intervals.
Rasmussen College Importance of Constructing Confidence Intervals Questions Report

Policy Making on Federal Spending Expository Essay

It’s the responsibility of the government to acquire some resources to finance its spending. There are several sources from which the government gets finances. For instance, through taxation, fund raising by selling its goods and services, and also through borrowing from a potential donor among other sources. The main sources of centralized government revenue are the taxes from the individuals’ income, and the payroll taxes (Steuerle, 2004). Taxes play a great role in contributing to federal revenues because they are compulsory payments. Taxes are not paid in exchange of anything whether goods or services. Other sources may include, organizational income taxes, excise duties, and custom duties among others. For the last half-century, the payroll taxes have been increasing, with organizations taxes decreasing, and the individual income taxes remaining unchanged. In mid 1950s, individual income tax was the greatest source of the government revenue, followed by the payroll taxes. Starting from 1965, the payroll taxes became the major contributor of the government revenue (Steuerle, 2004). They increased rapidly due to the introduction of Medicare. The taxes that were received from Medicare, and the social security taxes led to the increment of payroll taxes from 1.6% in 1951 to 6% in early 1990s. Other sources that contributed to the increment of payroll taxes are national workers pension, and railroad retirement fund. Government spending is defined in three major ways. Firstly, the government spends its money by buying goods and services for current use by its citizens. Secondly, government spends its money to buy goods and services to be used in the future, like infrastructure (Steuerle, 2004). The third way through which government spends its money is by acquiring goods and services through its own production, and by use of its labor. The main way through which the government spends its money is by securing the future of its citizens. When the first type of spending is combined with the second type, they form gross domestic product. In the year 2010, the United States central government spent $3.6% trillion, which was an equal amount of 24% of the GDP. Out of the $3.6%, approximately $2.2% was financed from nations tax revenues, while the remaining part was borrowed, creating a deficit to be recovered by future taxpayers. The largest issue faced in federal spending is federal debt. The financial problems and recession in US economy brought about a decrease in tax revenues, and on the other hand, spending increased. In the year 2007, the national budget had a deficit of 1.2% of the gross domestic product (Schick, 2007). Get your 100% original paper on any topic done in as little as 3 hours Learn More In the year 2009, the budget deficit increased to 10%, which was the highest deficit since 1945. The current reports concerning the economy declare that, the increasing government debt may turn to be unsustainable in the long run. The greatest issue at hand is that the interest rates on the federal debts are a bit higher than the rate of income growth (Schick, 2007). This situation may lead to debt consuming the high levels of income rates, unless the debt would be settled on time. If this situation would not be considered on time, there would be a higher probability of income falling, and new debt adding to the old ones. The private sectors in turn will have an increased risk of non-payment cases. These sectors will end up with weak financial base, and reduced annual profits. Reference List Schick, A. (2007). The Federal Budget, Politics, Policies, Process. Washington, DC: Brookings Institution. Steuerle, C. E. (2004). Contemporary U.S. Tax Policy. Washington, DC: Urban Institute Press.

YouTube Search

YouTube Search. I’m stuck on a Business question and need an explanation.

YouTube search
There are some truly amazing tech clips out there that give us a glimpse of the VERY NEAR future. We know there are self-driving cars.And look at disruptive tech like Bitcoin. https://www.cbsnews.com/video/bitcoin-and-the-future-of-money/ (Links to an external site.)
Cut and paste if it doesn’t embed properly.
Example here is the google self-driving car clip
Self-Driving Car Test: Steve Mahan

Instructions:

Find (2) of the best YouTube videos on the latest coolest business technologyand post the link to share with the class.
You will post two technology videos, and then write 1-3 sentence about each of your YouTube clips
After 2-3 days from posting your YouTube videos, you most comment on 2 other students posts after you watch theirs (Students post I will share with you after 24 from getting the 2 YouTube videos from you).

YouTube Search