Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action

Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.

Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature.  Black = 30 min preincubation Grey = No preincubation.

The first small-molecule kinase inhibitor approved as a cancer therapeutic, imatinib mesylate (Gleevec® treatment), has been amazingly successful. However, a thorough understanding of its molecular mechanism of action (MMOA) was not truly obtained until more than ten years after the molecule had been identified.

Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.

Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.

One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). This disease, which puts approximately 70 million people in 36 African countries at risk, and has approximately 30,000 active cases, is currently treated by five medicines that must be administered by IV or IM injection. Three of these medicines are only used for late-stage infection, after the parasite has crossed the blood brain barrier. Although two additional drugs are in development, the need to develop an inexpensive, orally administered treatment is obvious.

Recently Swinney et al. published a paper in which they describe a 4-point method for assessing MMOA for candidate therapeutic compounds for HAT. In their paper they describe a method to characterize the critical parameters of time-dependence and competition of the GSK3ß protein kinase from Trypanosoma brucei. Using this method they assess the effect of compounds on kinase activity at a single concentration of the compound (inhibitor) both with and without a preincubation to determine the effect of time dependence on kinase activity. Then, they look at the effect of compound (inhibitor) concentration to determine the nature of concentration. Loss of activity of the kinase at higher concentrations could indicate competitive binding for a target site.  For their method kinase activity was measured using the ADP-Glo® universal kinase assay which measures the ADP formed from a kinase reaction.

They applied their 4-point method of assessing MMOA to the kinase inhibitors tideglusib and GW8510. The authors were able to identify tidelusib, as a time-dependent inhibitor that exhibits ATP competitive initial binding but transitions to ATP non-competitive binding with time. By contrast, GW8510 showed competitve inhibition and it’s inhibition of the kinase was not time-dependent (see figure).

The authors point out that their 4-point method can be used at any point during the drug research and discovery process, and can be used with any enzyme target, regardless of the number of substrates simply by using two concentrations of the substrate of interest.

 

 

Gleevec is a registered trademark of Novartis Pharmaceuticals.

 

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Michele Arduengo

Social Media Manager at Promega Corporation
Michele earned her B.A. in biology at Wesleyan College in Macon, GA, and her PhD through the BCDB Program at Emory University in Atlanta, GA. Michele manages the Promega Connections blog. She enjoys leisure reading, writing creative nonfiction and knitting, and the occasional cross-country skiing jaunt.

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