Examination I PHRM 836 Biochemistry for Pharmaceutical Sciences II September 29, 2015

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1 Examination I PHRM 836 Biochemistry for Pharmaceutical Sciences II September 29, 2015 PHRM 836 Exam I - 1 Name: Instructions 1. Check your exam to make certain that it has 9 pages including this cover page. Ask for a new copy of the exam if you are missing any pages. 2. Use a pencil for filling in the answer sheet for computerized grading. 3. Write your name on the line above (this page) AND on the answer sheet for computerized grading. Also place your student ID number on the answer sheet. Fill in the circles underneath where you write your name and student ID number. Do NOT place any OTHER identification (or signature) on your exam or your answer sheet. Also, do NOT fill in anything for section number on the answer sheet. 4. Your answers to problems 1 through 22 will be graded by computer. All computer-graded questions will be worth three points each. Put your answers for these problems on the answer sheet and fill in the appropriate circle using a pencil. It is your responsibility to be certain that the answers for these problems are correctly marked on the answer sheet at the time you hand in your completed exam. You are strongly encouraged to double check this at the time you submit the answer sheet, as no points will be given for having mistakenly marked your answer sheet. The computer grading answer sheets will not be returned to the students, so you are encouraged to record your answers to the computer-graded questions on this exam as well as on the answer sheet. However, the computerized grading of your answers to these questions will be done solely on the basis of the optically scanned answer sheet, not on what is recorded on your exam. The course policy is that there will be no re-grading of the computer answer sheets. 5. Examination problems 23 through 26 are to be answered with short answers, brief essays, or drawings. Put your answer to each of these directly on this exam, in the space provided below each of these questions. 6. After completing your exam, both the exam and the answer sheet must be submitted for grading and both must be identified only with your name. 7. This is a closed-book, closed-notes exam. Calculators and electronic devices of any kind are not allowed to be used during this exam. Any electronic device that is not in a backpack, bookbag, purse, pocket, etc, will be considered to be in use, so put away all electronic devices including cell phones and calculators. Anyone observed to be using a calculator, any book, handouts, notes, printed or handwritten material of any kind, or the exam or answer sheet of any other student will be considered to have committed an act of academic dishonesty. Students that do so will not be allowed to complete the exam and will be given a zero for this exam. Also remember that the first instance of academic dishonesty also results in a grade of "F" in this course and reporting this episode to the Pharmacy Dean and the Dean of Students office.

2 PHRM 836 Exam I - 2 MATCHING. For problems 1 to 4, a set of numbered answers is provided immediately below. For each problem, select from the list of answers the single choice that best matches the item described in the problem. Mark that answer on your answer sheet. An answer may be used more than once or not at all. [3 points each] 1. A trimer of dimers answer 4 2. Membrane transporter answer 2 3. CYP21E2 answer 3 4. Transcription factor answer 5

3 PHRM 836 Exam I - 3 MULTIPLE CHOICE. For problems 5 to 22, select from the list immediately following each question the single most correct choice to complete the statement, solve the problem, or answer the question. Mark that answer on your answer sheet. [3 points each] 5. Where molecular oxygen binds to myoglobin: between the proximal histidine residue and the heme iron in the plane of the heme the proximal histidine and Phe43 at the dimer interface to the heme iron coordinates with the distal histidine 6. Which statement is an incorrect description of protein molecules? Protein molecules range in size by more than a factor of 100. The 3-dimensional shape of protein molecules can be globular or elongated. The 3-dimensional structure of a protein is determined by crystallography. The protein structure determines what small molecule ligands will bind. Folded proteins are rigid structures, so that all side chains are locked into one structure. Many proteins fold into a stable 3-dimensional structure that has a lower free energy than the unfolded state. Some but not all proteins have a β-sheet. 7. Which statement is an accurate description of K I? The apparent K I increases for competitive inhibition. K I changes in the presence of an activating effector. K I decreases when ph increases. K I increases for competitive inhibition because the inhibitor binds only to E and not to ES. K I is considered as an equilibrium dissociation constant for an inhibitor. K I depends on total inhibitor concentration. 8. The Ig fold in an antibody has antiparallel β-sheets. has parallel β-sheets. is a 2-helix bundle. is a β-sandwich of two β-sheets. is stabilized by a disulfide link. appears in all four polypeptide chains of an antibody. Choices,, Choices, Choices,, Choices,,,

4 PHRM 836 Exam I If you were to design a monoclonal antibody to develop into a drug, what part of the antibody molecule would be necessary for you to modify? the β-sandwich region one of the constant domains in one of the two heavy chains the two disulfide bonds the constant domains of the light chains the six hypervariable loops 10. The phrase that does not accurately describe the binding of a substrate to an enzyme. as the affinity for binding substrate increases, the catalytic rate also increases generally involves interactions with only a small part of the enzyme structure can involve hydrogen bonds can induce a change in the structure at a dimer interface is energetically favorable because the substrate size matches the size of the binding site can involve a salt bridge can induce a change in the enzyme structure can be driven by hydrophobic interactions none of the above (all choices accurately describe substrate binding) 11. The best choice that correctly describes the Bohr effect on the oxygen-binding curve of hemoglobin. The Bohr effect shifts the curve to the right because of a loss in cooperativity of binding O 2. Hemoglobin binds O 2 with greater affinity at lower ph because of the production of CO 2 in metabolizing tissues. A salt bridge at the α1β2 interface is lost when O 2 binds. The Bohr effect and an increase in BPG levels change the oxygen-binding curve similarly because H + and BPG bind hemoglobin in the same site. There is a ph dependence of CO 2 binding to heme to carry CO 2 from metabolizing tissues to the lungs. The Bohr effect is a shift in this curve to higher po 2 at ph 7.2 relative to ph 7.6. choices, choices, choices, choices,, 12. The phrase that correctly describes the catalytic triad of serine proteases. The triad comprises three residues essential for catalyzing the oxidation of a peptide bond. The two serine proteases chymotrypsin and tissue plasminogen activator have the catalytic Ser, His, Asp but these residues are positioned in different orientations in the catalytic site. One mechanism to regulate a serine protease is to block access to the catalytic triad by a serpin. The catalytic triad could be a cause for ph dependence of the catalytic activity of serine proteases. Almost all serine proteases have a catalytic triad comprised of Ser, His, and Asp. The triad is optimally oriented in 3 dimensions between S1 and S2 to promote catalysis. Choices and Choices and Choices, and All of the above

5 PHRM 836 Exam I Transcription factors are hetero-oligomeric complexes comprising up to 10 protein chains, DNA and RNA. have a variety of structural motifs to recognize DNA via nonspecific and specific interactions. are proteins with at least one, and sometimes multiple zinc-finger motifs. cleave DNA in a sequence specific manner. are DNA-binding proteins regulated by a variety of mechanisms, one of which is zymogen cleavage. have a total length of less than 50 residues. 14. A function of serine proteases is to degrade amino acids to protealyze itself to activate IgG antibodies to hydrolyze ATP to catalyze the hydrolysis of a peptide bond to facilitate digestion choices and choices and choices, and choices and 15. Sickle-cell hemoglobin, HbS, is a mutation of Glu 6 to Val, which causes anemia and promotes the deoxygenated form of hemoglobin and the formation of long stiff fibers in red blood cells. You want to develop a drug to treat sickle cell disease using the strategy of designing a molecule that alters the oxygen-binding curve to improve hemoglobin oxygen delivery. How would you alter this curve? Find a compound that lowers the maximum percent saturation. Find a compound to shift the curve to the left. Find a compound to shift the curve to the right. Find a compound that changes the curve from sigmoidal to hyperbolic. All of the above are viable. This strategy won t work. 16. Rosuvastatin is known to lower cholesterol and to competitively inhibit HMG-coA reductase. Rosuvastatin is competitive with HMG-coA because the apparent affinity of Rosuvastatin is increased in the presence of HMG-coA. the presence of HMG-coA does not affect the binding of Rosuvastatin to HMG-coA reductase. the Rosuvastatin binding site overlaps only part of the binding site of HMG-coA. Rosuvastatin does not bind to an allosteric site. choice and choice and all of the above none of the above

6 17. The cytochrome P450s PHRM 836 Exam I - 6 contain iron. are membrane proteins. that is named CYP3A4 has greater sequence identity with CYP3A7 than with CYP2A4. bind O 2. absorb at 450 nm. add oxygen to a variety of substrates. choices,, and choices,,, and choices,,, and all of the above 18. ATP-binding cassette transporters have both a membrane-spanning domain that recognizes ATP and a cytoplasmic domain that binds substrate. are all P-glycoproteins. are secondary active transporters. are phosphorylated upon hydrolysis of ATP. have a nucleotide binding domain that has a distinct sequence known as the ATP cassette. include the multidrug resistance family of ABC transporters, which cause the genetic disease cystic fibrosis. 19. The phrase that does NOT describe why membrane transporters are relevant to drug development. Membrane transporters are major determinants in pharmacokinetic, safety and efficacy profiles of drugs. Transporters are important for activating cytochrome P450s. Transporters play a role in drug-drug interactions. Numerous transporters are clinically important in drug adsorption. A drug can inhibit the transport function of certain transporters. Drugs are substrates of transporters. None of the above (all choices are accurate descriptions) 20. All DNA-binding proteins bind DNA. oligomerize. induce structural changes in DNA. bind specifically to DNA. interact with the DNA phosphate backbone. exhibit cooperative binding to DNA. bind the minor groove. interact with RNA polymerase. choices and choices and

7 PHRM 836 Exam I The choice that is NOT a reason that the function of cytochrome P450s is critically important to the pharmaceutical industry: CYP3A4 metabolizes over 50% of the approved drugs. CYP2E1 is induced by alcohol. A component of grapefruit inhibits a cytochrome P450. Acetaminophen is metabolized to a toxic compound by CYP2E1. CYP3A4 is induced by herbal medicines. A drug interaction between acetaminophen and alcohol is harmful when acetaminophen is taken in large doses. P450s oxygenate endogenous compounds in steroid synthesis 22. Patients with gout show an overproduction of uric acid, a product of purine degradation. The enzyme PRPP synthetase is one cause for this disease because the K M of PRPP synthetase to bind substrate is increased. PRPP synthetase catalyzes the last step in purine degradation, and the product of this step is uric acid. the inhibiting effector of PRPP synthetase does not function properly in patients with gout. the activating effector of PRPP synthetase does not function properly in patients with gout. PRPP synthetase is an allosteric enzyme that is activated by uric acid. the V max of PRPP synthetase is normal in patients with gout. ESSAY PROBLEMS. Write your answers to problems 23 to 26 in the space immediately below each problem. 23. [5 points] The catalytic activity of subtilisn, a serine protease, was measured for two peptide substrates as shown in the table below. Give an explanation, including a possible structural basis for the higher activity to hydrolyze peptide A. peptide P 2 P 1 P 1 P 2 relative activity A VFDA 1,000 B VFAA 1 Subtilisn cleaves between F-D for peptide A and between F-A for peptide B. Given that these are the only sequence differences of A and B, the higher activity for A indicates that the S 1 site on subtilisn has higher affinity for D than A and likely has basic residues (R or K) in this region.

8 PHRM 836 Exam I [5 points] The Hill plot below shows the saturation, Y, of ligand A binding to an oligomeric protein with cooperativity and ligand A binding to a monomeric protein without cooperativity. Indicate on the plot which line corresponds to the cooperative binding and non-cooperative binding. Explain your answer in a phrase or short sentence. The line with circles shows cooperative binding because the slope is not equal to 1.0. [It is greater than 1 and thus positive cooperativity.] The triangles has slope of 1 and therefore indicates non-cooperative binding. 25. [15 points] A preclinical profile of boilercoxib, a new orally active NSAID, was reported (Riendeau, et al 2001 JPET 296:558). These researchers were attempting to develop a new selective inhibitor of COX-2 that would be an improvement over simexcoxib or ibuprofen. Data from a sensitive enzyme assay are shown in the table below. compound K I COX-1 (µm) K I COX-2 (µm) selectivity ratio K I COX1/COX2 selectivity ratio K I COX2/COX1 Boilercoxib ~100 (106) OR ~0.01 (0.0095) Simexcoxib ~7 (7.7) ~0.1 (0.13) Ibuprofen a. Define a selectivity ratio in terms of K I values for COX-2 and COX-1. Insert a value for this ratio in the table above. Either Selectivity ratio = K I COX-1 / K I COX-2 (should be > 1.0 for COX-2 selectivity) Or Selectivity ratio = K I COX-2 / K I COX-1 (should be < 1.0 for COX-2 selectivity) b. Are any of the three compounds selective for COX-2 and did these investigators accomplish their goal? Boilercoxib and Simexcoxib are selective for COX-2 but Ibuprofen is not. The goal was accomplished given that the ratio of K I COX1/COX2 is much greater for Etoricoxib than either Celecoxib or Ibuprofen. c. Several known COX-2 selective inhibitors were discussed in class. What is the structural basis for their selectivity? Etoricoxib and Celecoxib are selective for COX-2 because: COX-2 has more open space occupied by inhibitors OR steric clash between COX-1 residues and inhibitors OR V523 allows inhibitors to bind COX-2 but I523 in COX-1 does not.

26. [9 points] A ribbon drawing of an antibody is shown below. PHRM 836 Exam I - 9 a. Circle one of the antigen binding sites. b. Draw a square around an Fab fragment. c.

9 26. [9 points] A ribbon drawing of an antibody is shown below. PHRM 836 Exam I - 9 a. Circle one of the antigen binding sites. b. Draw a square around an Fab fragment. c. Explain in 1-2 sentences why antibodies recognize different antigens with specificity and high affinity. The residues in 6 hypervariable loops of each antigen binding site are varied during an immune response. The large number of possible combinations of residues is able to match the antigen in size and shape and chemical nature to confer specificity and high affinity.

Examination I PHRM 836 Biochemistry for Pharmaceutical Sciences II September 29, 2015

Examination I PHRM 836 Biochemistry for Pharmaceutical Sciences II September 29, 2015 PHRM 836 Exam I - 1 Name: Instructions 1. Check your exam to make certain that it has 9 pages including this cover