Problem

The purpose of this experiment is to test potential cAMP dependent protein kinase inhibitors using computational methods and gel electrophoresis essays in the search for new viable anti-cancer drugs.

Review of Literature

Protein kinases are enzymes that phosphorylate a protein-specific substrate by transferring a phosphate from adenosine triphosphate (ATP) to the substrate. ATP is an important part of cellular activity because it stores and transports energy within the cell. Protein kinases are classified according to the amino acid residue that they phosphorylate. They are important because they regulate cell-signal pathways and are involved in signal cascade mechanisms. In signal cascade mechanisms, phosphorylation is used to regulate activities within the cell. These activities include the regulation of glycogen synthesis and breakdown, fatty acid synthesis, the oxidation of pyruvate to acteyl-CoA, mobilization of triacylglycerols, and the regulation of glycolysis and gluconeogenesis. A change in the level of phosphorylation can have significant effects on cellular activity, including irregular cellular growth, regulation, and differentiation (Balis, 2002). Because protein kinases regulate this activity, aberrations in kinase activity can lead to diseases such as cancer (Cohen, 2002). Since the realization of their role in diseases, protein kinases have become appealing targets for drug discovery.

The idea that a protein can be “switched” off by antagonist was introduced in 1905 (Drews, 2000). An antagonist inhibits biological effects after binding to a ligand site, whereas an agonist induces a biological effect similar to the natural ligand. In modern pharmacology, this idea has translated into protein kinase inhibitors. An inhibitor can prevent the activity of one of the 500 molecular targets (Drews, 2000). By inhibiting kinase activity when a protein is over-expressed, the signal cascade mechanism can be repaired and the disease can be cured (Noble, 2004). If a viable protein kinase inhibitor can antagonize protein kinase action, over-expression can theoretically be fixed.

Small molecules are small pieces of amino acids that can be used as inhibitors because of their binding ability. If a small molecule binds to a protein kinase, it can prevent phosphorylation and consequentially protein activity (Jorgensen, 2004). Drug discovery, i.e. the discovery of viable small molecules for inhibition, involves finding which small molecules will bind to the protein kinase and inhibit activity. A novel technique for the discovery of viable small molecules involves virtual screening in combination with a kinase activity assay (Jorgensen, 2004).

Virtual screening is used to determine which small molecules theoretically have the potential to bind to a given protein. Databases of thousands of small molecules are used to evaluate the potential of the small molecules they contain (Gray, 2000). To discern which small molecules will bind to a protein kinase, the library of compounds is screened against a panel of proteins and binding affinity for each compound is determined (Drews, 2000). The potential of a ligand for binding can be assessed by considering kinase structure, charge distribution, energy minimization through potential aromatic pi-stacking, reduction of steric repulsion, and dynamic simulations (Teague, 2003). By determining the structure of the small molecule and the protein, and by considering their potential steric and electrostatic interaction, it is possible to predict whether a ligand will bind in an assay (Teague, 2003). “Guided docking” is the term used for this approach because it uses chemical information to guide the orientation of the small molecule into the binding site (Fradera, 2004).

Generally, the ligand has potential to become a drug if it binds at certain sites and inhibits activity (Shah, 2004). Site targets for the small molecule can be the ATP binding site, substrate sites, or allosteric sites. Most small molecules are used to inhibit kinase function by binding to the ATP binding site, between the two lobes of the kinase lobe (Noble, 2004). However, binding ligands to the substrate binding site is becoming a prevalent approach. If a small molecule binds to the substrate binding site, then the substrate cannot bind, phosphorylation cannot occur, and activity is inhibited.

After the identification of possible inhibitors, each candidate is tested with the protein in a gel electrophoresis assay to determine actual capabilities. The assay tests the potential of the small molecule with by a fluorescence test (Humphries, 2004). The basic components are purified kinase, substrate, and ATP, and small molecule. A florescent label on peptide directly assesses phosphorylation (Humphries, 2004). The florescent label is important during the primary screening, which identifies compounds with inhibitor potential. After primary screening, a cell-based assay is used to test the small molecules activity with actual cells. It can confirm the activity of agents tested in target-based screening assays and can assess the agent’s pharmacologic effects (Balis, 2002). Selectivity is an important indicator of pharmacological efficiency. The small molecule should be specific to the ligand site and not produce secondary effects due to binding with another ligand site.

Kinase inhibitors are being developed for cancer (Gibbs, 2000) in addition to auto-immune disorders, metabolic disorders, inflammatory diseases, and neuro-generative diseases. Because of their potential for such a wide variety of diseases, protein kinases are important considerations for modern biochemistry. Their activity can be inhibited with small molecules, which can be used as drugs to stem the loss of life. A growing interest in protein kinase inhibitors has recently culminated in the approval of the first drugs for clinical use (Shah, 2004).

My research is important because it tests inhibitors for cAMP dependent protein kinases A (PKA A) and assesses the potential. Cyclic AMP dependent protein kinases are important factors in cellular regulation. They are dependent on cyclic AMP for the transfer of gamma phosphate from ATP to either a serine or threonine substrate. Using the techniques previously discussed, the potential for inhibition is assessed by first predicting ligand potential, and then testing actual binding ability on an assay. This research is very important for new drug discovery.

Procedure

A. Virtual Screening and Isolation of Compounds

1. Construct a homology model of the desired receptor. With this structure, “drugable” sites, i.e. those sites with potential to be sites of ligand binding, can be found by looking at the cavities in the structure.
2. Choose a site in the protein where the ligand binding will be assessed.
3. Select small molecules from a library of compounds or from drugs designed to optimize pharmacological properties of similar drugs. This should be based on drug-like properties, such as low toxicity, absorption, and metabolic properties.
4. Using the protein model, target the ligand in the specific binding site.
5. Assess efficiency of ligand by considering hydrogen bonding and charge distribution.
6. Select the molecules that appear to have the most potential as an inhibitor.

B. Analysis of Proteins by PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

1. Add reaction mixture to sample buffer and put 3nm into each well of SDS-Page.
2. Separate samples by SDS-PAGE under non-reducing conditions.
3. Stain gel for visualization (Humphries et al. 2004).
4. Use same technique to purify mutant strain of protein.

C. Assay

1. Prepare .65% Agarose gel and set up gel electrophoresis.
2. Make cyclic AMP dependent protein kinase reaction mix by mixing ATP solution, Biotinylated Kemptide Solution, cAMP Solution, cAMP dependent protein kinase Reaction Buffer, adenosine triphosphate (ATP) working Solution, deionized water, and test substance.
3. Pipette into vials 1 through 7 and 9 through 15.
4. Make cAMP dependent protein kinase control mix by following above materials, excluding Biotinylated Kemptide and small molecule. Pipette into vials 8 and 16, which are the controls.
5. Pipette 20 µL of cAMP dependent protein kinase reaction mix into vials 1 though 16. Initiate reaction by adding cAMP dependent protein kinase sample and centrifuge. Incubate for 30 seconds and end reaction by adding stop solution.
   6. Fill wells 1 through 16 with corresponding tube solutions and run gel electrophoresis for 25 to 30 minutes. Remove gel from buffer solution.
6. Repeat procedure with mutant protein.
7. Repeat both assays (wild type and mutant) to verify results.

D. Analysis

1. 1. Analyze protein kinase activity using a non-radioactive system based on the fluorescent tagged protein.
   The experiment tested whether different small molecules had the potential to bind in place of the substrate and disallow the production of the phosphorylated product. The presence of a second band in each lane indicates the presence of the product. If there is a strong second band, the product was produced because the small molecule did not bind and the substrate was phosphorylated. Therefore, the respective small molecules are not potential inhibitors of kinase activity.

The absence of a second band indicates that no product was produced. This is because the substrate was not phosphorylated. This may be because the small molecule binded to each protein kinase in the sample and prevented any kinase activity. However, it is possible that clumping occurred because the small molecule was defective. If the small molecule stuck together, then it is difficult to determine whether or not it actually bound to the protein kinase. It is possible that the small molecule just became large enough to stick to any site on the kinase. Because of the ambiguity involved in the complete absence of a second band, the procedure should be repeated to verify these results. Also, it is difficult to determine whether these are good inhibitors.

However, the wells with weak second bands are potential targets. The presence of a weak second band proposes that some phosphorylation occurred, but also a lot of protein kinase activity was inhibited by the ligand. Further cell-based assays could analyze the actions of these compounds on the protein.

The second part of this experiment tested the effects of the small molecules on a mutated protein. The mutant protein was constructed with a mutation to disallow the binding of the small molecules but still allow the binding of the substrate. Therefore, this test should have resulted in the presence of second bands because the ligand could not inhibit action. This was meant to prove that the small molecule actually bound to the protein kinase in the wild-type assay. However, the results are the same as the previous wild-type test, raising questions about the efficiency of the small molecules tested. The lack of a second band may mean that the protein had been denatured before the experiment and did not function properly. It is also possible that one or more small molecules have a structure that allows it to block the kinase active site and so are potential inhibitors. There is ambiguity involved in the results because they are not definitively explainable. Therefore, it is difficult to determine the potential of these molecules. The assay should be run again to confirm these results.Acknowledgment

We wish to express our thanks and gratitude to Dr. Ahmed El Sawi Shoukry for suggesting the project, and to him and Dr. Mahmoud El Marsafy for their teaching, guidance, and help, and for donating a lot of their valuable time for our project.

Results

A. Wild Type

Lanes 1, 2, 6, 9, and 15 are empty. No product was made because phosphorylation did not occur.

Lanes 5,7,11,12,14 have weak bands and indicate the presence of some product. Therefore, a reduced amount of phosphorylation occurred.

Lanes 3, 4, 10, and 13 have strong bands indicating a large amount of product. This indicates that normal amounts of phosphorylation occurred.

Lanes 8 and 16 are the controls. They indicate that phosphorylation occurred by the presence of a second band. No small molecule inhibitor was used in the reaction. This was to show that with ATP, protein kinase, and substrate, phosphorylation occurs and product is produced.

B. Mutant

There is no apparent change in the presence or lack thereof of the second band.

Fig. 1 – 0.65% Agarose Gel Results for wild type assay of protein kinase activity	Fig. 2 – 0.65% Agarose Gel Results for mutant strain of protein kinase

Conclusions

This research is one step in the search for new drug discovery. The results did not clearly demonstrate any potential inhibitors, but they were useful in that they provided initial results for the selected small molecules. Some of the results are difficult to result because of the various explanations. The strong bands show that the small molecules are not likely inhibitor, but weak bands are possible inhibitors.

Computational methods such as virtual screening have become increasingly important in drug discovery. Continuing research will provide insights into the best available inhibitors for a wide range of protein kinases, including cAMP-dependent kinases. In addition to testing the available compounds, research should focus on mechanisms of protein kinase resistance to ligands, including issues like Single Nucleotide Polymorphisms (SNPs). Drug discovery based on ligand docking to a protein kinase is an important consideration for modern biochemistry. This is an area in which full potential will only be reached with continued research.

Acknowledgement

Thank you to Adrian Saldanha and the Scripps Research Institute.

Works Cited

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