General Aspects of Pharmacology

General Aspects of Pharmacology

Efficacy homework

 You are performing a set of in vitro studies to evaluate a library of novel compounds that your chemistry group has designed to target the μ-opioid receptor (MOR), the central nervous system receptor that is activated by endogenous endorphins and exogenously administered morphine. New agonists of this receptor may be useful as analgesics; new antagonists may be useful to treat opioid addiction and overdose…

You’ve received some CHO cells that express high levels of MOR. You can grow these cells easily and use them for functional studies. MOR is a Gαi-coupled GPCR, meaning that receptor activation reduces the activity of the enzyme adenylyl cyclase, in turn reducing the concentration of intracellular cAMP. It is known that MOR has some intrinsic signaling.

Before you delve into the new compounds, you’ll need to characterize your assay system with some control compounds. Your assay is designed to measure cAMP levels in the cells using a kit from Promega called cAMP-Glo, which works as follows:

The cAMP-Glo™ Assay is a homogeneous, bioluminescent and high-throughput assay for measuring cAMP levels in cells. The cAMP-Glo™ Assay monitors cAMP production in cells in response to the effects of test compounds on G protein-coupled receptors (GPCRs). GPCRs that couple with adenylate cyclase will increase (Gαs-coupled) or decrease (Gαicoupled) intracellular cyclic AMP (cAMP). The assay is based on the principle that cAMP stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction.

The cAMP-Glo™ Assay can be performed in 96-, 384or 1536-well plates. The cells are induced with a test compound for an appropriate period of time to modulate cAMP levels. After induction, cells are lysed to release cAMP, then the cAMP detection solution, which contains protein kinase A, is added. The Kinase-Glo® Reagent is then added to terminate the PKA reaction and detect the remaining ATP via a luciferase reaction. Plates are read using a microplate-reading luminometer. Luminescence can be correlated to the cAMP concentrations by using a cAMP standard curve. The half-life for the luminescent signal is greater than 4 hours. This extended signal half-life eliminates the need for luminometers with reagent injectors and allows batch-mode processing of multiple plates. https://www.promega.com/products/drug-discovery/gpcr-assays/camp_gloassay/ In simpler terms: as intracellular [cAMP] increases, light emitting from the cells decreases, and vice versa.

Following the assay instructions, you produced the following data for four control compounds. In columns 2, 3, 5, & 7 you are using only the single compounds at the doses in column 1. In columns 4, 6, & 8, you are varying the dose of the listed drug but keeping the dose of morphine steady.

Compound: Morphine alone (agonist) [drug] (M) 0 1.00E-11 3.00E-11 1.00E-10 3.00E-10 1.00E-09 3.00E-09 1.00E-08 3.00E-08 1.00E-07 3.00E-07 1.00E-06 3.00E-06 1.00E-05 3.00E-05 1.00E-04 luminescence 550 558 574 631 786 1268 2272 3926 5204 5914 6159 6250 6277 6286 6289 6290 Naloxone alone (neutral antagonist) luminescence 550 550 550 550 550 550 550 550 550 550 550 550 550 550 550 550 Naloxone + 10 μM morphine BNTX alone BNTX + 10 μM morphine (inverse agonist) luminescence 6290 6285 6276 6243 6150 5848 5142 3681 2190 1165 771 618 573 557 552 551 luminescence 550 550 549 547 542 523 477 373 251 154 114 98 93 92 91 91 Buprenorphine alone Buprenorphine + 10 μM morphine (partial agonist) luminescence 6290 6286 6278 6251 6176 5925 5311 3906 2247 946 405 188 124 101 94 92 luminescence 550 551 553 559 576 636 791 1202 1819 2448 2762 2897 2939 2954 2959 2960 luminescence 6290 6289 6286 6278 6253 6171 5957 5390 4538 3669 3236 3048 2990 2970 2964 2962

Determine a rational way to normalize your data. Then, plot these normalized data on a single graph and answer the following questions:

1. What are the Emax & EC50/IC50 values for morphine, naloxone, BNTX, and buprenorphine?

2. Briefly but clearly explain (i.e., in 1-2 sentences for each column above) why each of these seven data sets has their characteristic curves. Your responses should demonstrate that you 1) understand the assay technique and 2) understand the consequences of combining mixed-efficacy compounds in the manner done here.

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