Reference no: EM133207411 , Length: 5 pages.
Assignment: General Aspects of Pharmacology- Radioligand Binding
You are performing a set of binding studies to evaluate a library of novel compounds that your chemistry group has designed to target the 5-HT1A receptor, a serotonin receptor. You hope to create new agonists of this receptor-these could potentially be used to treat anxiety and depression. Your first step, however, if screening the compound library...
You've received some HEK293 cells that have been genetically modified to overexpress 5-HT1A. You can grow these cells easily and harvest their cell membranes for binding studies using standard techniques.
You've decided to use the radioligand [3H]8-OH-DPAT for radioligand competition studies. First, you must characterize the radioligand in your system. The following are the data you collected from a hot saturation binding experiment:
|
[[3H]8-OH-DPAT] (M)
|
Total CPM
|
Non-specific CPM
|
|
1.00E-11
|
252.35
|
225.03
|
|
1.00E-10
|
471.44
|
225.29
|
|
3.00E-10
|
831.28
|
225.87
|
|
1.00E-09
|
1465.47
|
227.90
|
|
2.00E-09
|
1825.11
|
230.80
|
|
3.00E-09
|
1997.48
|
233.70
|
|
4.00E-09
|
2099.39
|
236.60
|
|
5.00E-09
|
2167.21
|
239.50
|
|
6.00E-09
|
2215.97
|
242.40
|
|
7.00E-09
|
2252.98
|
245.30
|
|
8.00E-09
|
2282.25
|
248.20
|
|
9.00E-09
|
2306.15
|
251.10
|
|
1.00E-08
|
2326.16
|
254.00
|
|
3.00E-08
|
2493.11
|
312.00
|
|
1.00E-07
|
2737.00
|
515.00
|
|
3.00E-07
|
3328.97
|
1095.00
|
|
1.00E-06
|
5363.19
|
3125.00
|
For total binding, each test vial contained 20 μg of cell membrane homogenate + the listed concentration of radioligand in a Tris buffer at pH = 7.4. For non-specific binding, each test vial contained 20 μg of cell membrane homogenate + the listed concentration of radioligand + 100 μM of pindolol (a high-affinity 5-HT1A receptor blocker) in a Tris buffer at pH = 7.4.
All samples were allowed to incubate at room temperature for 90 minutes (sufficient time to allow equilibrium binding). Samples were then filtered through Whatman GF/B glass fiber filters, which will readily bind and trap the cell membranes but not free small molecules (like radioligands). Those filters were placed in scintillation fluid and radioactivity was counted in a scintillation counter. The counts per minute (CPM) values reported above indicate the amount of radioactivity present on each filter.
Determine specific binding for each sample. Determine the Bmax(measured in CPMs) of this experiment. Plot a single graph that shows CPMs vs. [radioligand] for total binding, nonspecific binding, and specific binding. Normalize your specific binding data and plot a semilog graph that shows ? vs. [radioligand]. Determine the Kd(in nM) of [3H]8-OH-DPAT at 5-HT1A under these experimental conditions.
Now that you've characterized your radioligand, you can use it to screen your compound library. The following data are from 10 library compounds in a radioligand competition assay. For each library compound, test vials contained 10 ug of cell membrane homogenate + the listed concentration of test compound + 5.0 nM [3H]8-OH-DPATall in a tris buffer at pH=7.4. Each individual compound was tested with control total and nonspecific vials: total vials had 10 ug of cell membrane homogenate + 5.0 nM [3H]8-OH-DPAT; non-specific vialshad 10 ug of cell membrane homogenate + 5.0 nM [3H]8-OH-DPAT + 100 μM of pindolol.
|
Compound #
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
|
TOTAL
|
2320
|
2233
|
2408
|
2295
|
2174
|
2302
|
2262
|
2236
|
2373
|
2412
|
|
Non-specific
|
276
|
262
|
271
|
288
|
254
|
302
|
294
|
266
|
298
|
293
|
|
[Compound] (M)
|
|
|
|
|
|
|
|
|
|
|
|
1.00E-12
|
2320.0
|
2232.8
|
2406.9
|
2295.0
|
2174.0
|
2299.9
|
2261.4
|
2236.0
|
2373.0
|
2412.0
|
|
3.00E-12
|
2319.9
|
2232.5
|
2404.6
|
2295.0
|
2174.0
|
2295.6
|
2260.3
|
2236.0
|
2373.0
|
2412.0
|
|
1.00E-11
|
2319.7
|
2231.2
|
2396.8
|
2294.9
|
2173.9
|
2280.7
|
2256.4
|
2236.0
|
2373.0
|
2411.9
|
|
3.00E-11
|
2319.0
|
2227.6
|
2374.8
|
2294.8
|
2173.6
|
2239.5
|
2245.3
|
2236.0
|
2372.9
|
2411.6
|
|
1.00E-10
|
2316.5
|
2215.2
|
2301.2
|
2294.4
|
2172.7
|
2107.8
|
2207.3
|
2235.9
|
2372.8
|
2410.8
|
|
3.00E-10
|
2309.7
|
2180.7
|
2116.6
|
2293.3
|
2170.2
|
1814.2
|
2106.6
|
2235.7
|
2372.3
|
2408.5
|
|
1.00E-09
|
2285.9
|
2068.8
|
1671.1
|
2289.4
|
2161.3
|
1265.7
|
1824.7
|
2235.1
|
2370.7
|
2400.3
|
|
3.00E-09
|
2221.1
|
1810.6
|
1099.6
|
2278.4
|
2136.4
|
775.3
|
1353.7
|
2233.3
|
2366.0
|
2377.3
|
|
1.00E-08
|
2023.8
|
1294.4
|
612.2
|
2240.8
|
2054.0
|
472.2
|
804.2
|
2227.1
|
2349.9
|
2300.5
|
|
3.00E-08
|
1631.0
|
790.8
|
398.3
|
2140.6
|
1854.0
|
362.1
|
499.6
|
2209.5
|
2305.3
|
2109.3
|
|
1.00E-07
|
1034.5
|
457.3
|
310.8
|
1858.7
|
1406.0
|
320.4
|
360.6
|
2150.3
|
2163.4
|
1655.2
|
|
3.00E-07
|
611.9
|
331.7
|
284.4
|
1382.7
|
894.0
|
308.2
|
316.7
|
1999.6
|
1849.9
|
1087.6
|
|
1.00E-06
|
389.9
|
283.4
|
275.1
|
819.3
|
504.4
|
303.9
|
300.9
|
1620.4
|
1275.1
|
616.2
|
|
3.00E-06
|
315.4
|
269.2
|
272.4
|
503.0
|
345.4
|
302.6
|
296.3
|
1099.5
|
772.7
|
412.9
|
|
1.00E-05
|
288.0
|
264.2
|
271.4
|
357.7
|
282.4
|
302.2
|
294.7
|
621.2
|
467.6
|
330.5
|
|
3.00E-05
|
280.0
|
262.7
|
271.1
|
311.8
|
263.6
|
302.1
|
294.2
|
400.6
|
357.8
|
305.6
|
|
1.00E-04
|
277.2
|
262.2
|
271.0
|
295.2
|
256.9
|
302.0
|
294.1
|
308.4
|
316.3
|
296.8
|
Normalize the data in each column, using the total and nonspecific values.Plot a single semilog graph that shows ? vs. [drug] for all 10 test compounds. Determine the IC50 and Kivalues of each library compound at 5-HT1A under these experimental conditions. If you wanted to test the 3 highest affinity compounds in further tests, which 3 would you choose?
Write a brief report that explains how you organized & analyzed these data and calculated the required parameters. Show representative calculations, where appropriate, to demonstrate how you determined the requested values. There are no page requirements, but please use standard fonts/margins.
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