Introduction: Cancer treatment is complicated by the fact that cancer is not a single disease. Initially, doctors and scientists thought that all growing cells were equal and one drug would cure all cancers. Today we recognize that there are many routes that can lead to a cancerous cell, and different biochemical pathways require different medical responses. The genes that produce cancers are, in most cases, misregulated versions of important cellular genes. Some genes are turned off when they should be on, whereas others are on at inappropriate times or locations. This makes treatment difficult because drugs and treatments that alter the expression of these cancer genes or affect the proteins they encode must be able to distinguish cancer cells from healthy cells; otherwise, these treatments are likely to destroy healthy cells along with cancerous ones. To further complicate treatment, cancers mutate over time to produce additional genomic abnormalities that can make them even harder to treat.

In this assignment we’re going to examine kinases, which are a class of regulatory proteins that are commonly misregulated in cancers. Broadly speaking, kinases are enzymes that catalyze the transfer of a phosphoryl group from ATP to the side chain of an amino acid. This phosphoryl transfer, called phosphorylation, changes the properties of the amino acid, leading to changes in the properties/function of the protein that contains it.

Phosphorylation often results in a series of cascading chemical reactions that triggers a change in gene expression within the cell, allowing for the regulation of important cellular events like growth and division. Kinases are specific in which amino acid side chains they phosphorylate, and regulation of kinase activity ensures that these modifications are only made at the appropriate time and location. Many types of cancers involve mutations in kinases, causing phosphorylation to happen at inappropriate times and leading to uncontrolled cell growth and division. We are going to examine one such kinase, EGFR, and see how its misregulation can be treated using the drug gefitinib.

1. (10 points) As discussed in the introduction, protein kinases are a class of enzymes that catalyze the

transfer of a phosphoryl (-PO32-) group from ATP onto the side chain of an amino acid. Usually an alcohol group in the side chain of tyrosine, serine, or threonine is the recipient of the phosphoryl group. Shown below is the arrow pushing mechanism for the phosphoryl transfer reaction that is catalyzed by the kinase enzyme. The tyrosine substrate is the amino acid side chain that will receive the phosphoryl group (it may be part of the kinase or part of a different protein). The other two amino acid side chains shown in the diagram (Lys and Asp) are part of the kinase’s active site.

a. (6 points) In the space below, draw the structure of the product of the reaction mechanism shown above. You do not need to draw the structure of ribose and adenine in your answer.

b. (4 points) Which atoms acted as nucleophiles in the reaction above? Which atoms acted as electrophiles? Briefly explain how you know.

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2. (16 points) The human genome contains an enormous number of kinases, with about 1.7% of human genes encoding kinase proteins. These kinase genes are evolutionarily related, and because of this all kinases have a similar core structure, called a kinase domain, that is responsible for catalyzing the phosphorylation reaction. Although they have similar core structures, the different kinase proteins in the human genome differ in how they’re regulated and in what specific substrates they bind and phosphorylate.
Open the file “Kinase (Take Home Exam).pse”, which you can download from Canvas. Click on the scene titled “Kinase_Surface”. The structure shown is a specific kinase called EGFR (epidermal growth factor receptor), whose misregulation causes numerous types of cancers. Only a portion of EGFR is shown in this structure (just the kinase domain), and it is shown using a cartoon/surface representation. The ATP molecule is also shown bound to the kinase. The short segment in cyan is a portion of the polypeptide chain from the substrate of this kinase.
a. (4 points) In the “Kinase_Surface” scene the kinase domain is shown using two colors, light green and dark green. These two colors are used to differentiate the two lobes of the kinase domain, the n-lobe and c-lobe. The n-lobe is named as such because it is closer to the N-terminus of the kinase, whereas the c-lobe is closer to the C-terminus of the kinase. Which color, light green or dark green, is used to label the n-lobe in this structure? Briefly explain how you can tell.
b. (3 points) Briefly describe where ATP binds in the structure. Reference the n-lobe and/or c-lobe in your description.
c. (3 points) Briefly describe where the substrate polypeptide binds in the structure. Reference the n-lobe and/or c-lobe in your description.
d. (3 points) What is the sequence of the substrate polypeptide that is shown? List all amino acids in the sequence and label the N- and C-termini.

e. (3 points) How many amino acids are in the c-lobe of the EGFR structure shown in the “Kinase_Surface” scene? Use the sequence bar to answer this question (visible at the top of the structure display window in PyMOL). If the sequence bar is not visible, you can enable it by clicking “display” from the menu toolbar and then selecting “sequence”.

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3. (10 points) Click on the scene titled “ATP_binding”. This structure zooms in on the binding site where ATP binds to the kinase.
a. (4 points) The adenine base in ATP forms two hydrogen bonds using the same acceptor and donor that are used to form base pairs in DNA. Which amino acids (give their names) are forming these H-bonds to the adenine base? What part of the amino acid is forming the H-bond?
b. (6 points) Draw a structure showing the hydrogen bonds you described in the previous question.
Label the donor and acceptor for each hydrogen bond in your drawing.

4. (18 points) Click on the “EGFR_Sticks” scene. Pick an amino acid whose one letter abbreviation is the first letter of your last name (if the first letter of your last name is not an amino acid abbreviation, please proceed to the next letter). Find an example of this amino acid in the structure of EGFR and measure its phi and psi dihedral angles.
a. (6 points) What are the phi and psi angles that you measured?
b. (8 points) Attach a saved image from PyMOL that shows the measured dihedral angles. Be sure that the side chain is visible in the image.
c. (4 points) Shown below is the Ramachandran plot that we showed in lecture. Circle the approximate location on the Ramachandran plot that corresponds to the dihedral angles you measured.

5. (10 points) Click on the “Gefitinib” scene to show the structure of the drug gefitinib (magenta) bound to EGFR (cyan). Gefitinib is an incredibly effective drug that is used to treat certain lung and breast cancers in which inappropriate activation of EGFR leads to uncontrolled cell growth. The skeletal structure of Gefitinib is shown below.
[PyMOL Hint: If you have you have trouble zooming in and out, right click on the black space and select “zoom (vis)” to correct the problem and make zooming smoother.]
a. (3 points) Briefly describe where gefitinib binds to the structure of EGFR. Reference the n-lobe and c-lobe in your description and compare this location to where ATP and/or the polypeptide substrate bind.
b. (3 points) Based on your answer to the previous question, explain how gefitinib prevents EGFR from catalyzing the phosphorylation reaction.
c. (4 points) Click on the scene “Gefitinib_H-bonding”. How do the H-bonds formed between gefitinib and EGFR compare to those formed between ATP and EGFR (as seen in question 3)?
Briefly describe any similarities/differences.

6. (18 points) Click on the scenes “Gefitinib_sticks” and “Gefitinib_spheres” to see a surface
representation showing how gefitinib binds to EGFR.
a. (6 points) Shown below is a variant of gefitinib that binds poorly to EGFR. In terms of changes in entropy that take place upon binding of the drug to EGFR, explain why the molecule below binds poorly to EGFR compared to gefitinib.
b. (6 points) Shown below is a variant of gefitinib that binds poorly to EGFR. Use the structure in PyMOL to explain why this molecule would bind poorly to EGFR compared to gefitinib.
c. (6 points) Shown below are three variants of gefitinib in which a methyl (-CH3) group has been added at different locations on the molecule. In which variant would you expect the methyl group to interfere the least with binding to EGFR (i.e., which variant would you expect to bind best to EGFR)? Use the PyMOL structure to support your answer and explain why the other two structures would bind more poorly. [Hint: use the “Gefitinib_spheres” to answer this question.]

7. (6 points) Like all living organisms, cancer cells evolve in response to selective pressure. Although gefitinib is initially very effective at treating EGFR-dependent cancers, disease progression nearly always occurs within 10-12 months of treatment. Through random mutation, some cancer cells will acquire mutations that allow the cell to grow in the presence of gefitinib. Cancer cells with these mutations grow and divide, leading to a population of cancer cells that are not affected by drug treatment.
The most commonly observed of these mutations is the replacement of threonine #790 with methionine (“T790M”). Click on scene “EGFR_T790M_Gefitinib” to view a structure in which EGFR (red) carries this mutation, rendering it immune to gefitinib. The structure of gefitinib (magenta) has been artificially inserted into the PyMOL structure based on alignment with other EGFR structures.
The structure of the mutated methionine side chain is also shown using a stick representation.
a. (3 points) Use the structure to explain why gefitinib fails to bind to the T790M version of EGFR.
Focus specifically on methionine #790.
b. (3 points) Cancers that are resistant to gefitinib are generally treated using other molecules that
are similar to gefitinib but that still bind to the mutated EGFR protein. Imagine that you are a scientist at a pharmaceutical company and are part of a team that is designing a new drug to bind to the T790M mutant of EGFR. Use the PyMOL structure to propose a change to the structure of gefitinib that might allow it to bind to EGFR T790M.

8. (12 points) Lecture 37 featured a class discussion of the ethics of pharmaceutical marketing. During this discussion we evaluated whether pharmaceutical marketing should be permitted. In order to make this decision we first determined who the stakeholders were, which ethical concerns were involved, and how each stakeholder benefited or was harmed by pharmaceutical marketing.
a. (4 points) Write a short paragraph in which you formulate an argument for why pharmaceutical marketing is a good thing. Your response must address the relevant stakeholders and identify the specific ethical issues that are involved.

b. (4 points) Write a short paragraph in which you formulate an argument for why pharmaceutical marketing is a bad thing. Your response must address the relevant stakeholders and identify the specific ethical issues that are involved.
c. (4 points) Write a short paragraph in which you describe how best to resolve the ethical
dilemma presented here. Be specific in your response (e.g., what amount of marketing should be permitted? In what contexts? What type of rules/regulation should be applied to drug marketing?). Defend your proposal by specifically referencing the ethical issues involved in your decision.