Lecture 3 Prep for Exam 1

Question 1

What is amyloidosis?

Answer 1

Question 2

AD affects what percent of the population over age ____

Answer 2

10% of the population over the age of 65 (50% of those over 85)

Question 3

AD is a protein misfolding disease. What major protein is involved and what occurs?

Answer 3

The amyloid precursor protein (APP) is cut into fragments by Y-secretase and B-secretase…this then results in beta amyloid fragments which are crucial in the formation of senile plaques

Beta Amyloid plaques form when Aβ misfolds and aggregates into oligomers and longer polymers, the latter of which are characteristic of amyloid. Misfolded and aggregated Aβ is thought to be neurotoxic, especially in its oligomeric state.

Question 4

Why cant amyloid fibrils be dissolved by water?

Answer 4

Water cannot destroy this as once they are formed, they are dead and very hydrophobic.

Question 5

What is the most abundant protein in the brain? What is the most abundant protein in the plasma?

Answer 5

brain= transthyretin (TTR)

plasma=albumin

Question 6

What is the structure of TTR? what does it transport?

Answer 6

TTR is homo-tetrameric…it transports holo-retinol binding protein (RBP bound vitamin A)

Question 7

Describe the pathogenesis of TTR amyloidoses

Answer 7

Question 8

What occurs during the protective mutation of TTR? What does T119M do?

Answer 8

T119M adds one extra hydrogen bond between to furtherly stabilize TTR…preventing the aggregation of beta amyloid

Question 9

What mutation occurred in Senile systemic amyloidoses (SSA)?

a) V122I
b) WT
C) V30M

Answer 9

b) WT

Question 10

What mutation occurred in Familial amyloid cardiomyopathy (FAC)?

a) V122I
b) WT
C) V30M

Answer 10

a) V122I

Question 11

What mutation occurred in Familial amyloid polyneuropathy (FAP)?

a) V122I
b) WT
C) V30M

Answer 11

C) V30M

Question 12

What is Tafamidis? What is it metabolized by?

Answer 12

A potential TTR amyloidosis inhibitor…convinced that this drug stabilizes TTR which thus can prevent heart failure…it is metabolized by glucuronidation

Question 13

When you have two serine’s and two nitrogen’s what kind of interaction can you form?

a) hydrophobic
b) salt bridge
c) hydrogen bond
d) VDW’s?

Answer 13

c) hydrogen bonds!

Question 14

What is the difference between crystal structure of AG10 and Tafamidis in complex with V122I-TTR?

Answer 14

AG10 can be 100% stabilized while Tafamidis is only 50% stabilized. Beginning at the top of the structure, AG10 participates in a direct electrostatic interaction with lysine as the molecule is “longer” (look at contrast in orientation of the ketone at the top of the molecules) at t…In contrast, Tafamidis is shorter and needs water molecule to participate in bonding at the top…therefore a non-direct electrostatic bond is formed with lysine (not as strong as AG10 direct electrostatic bond)…Near the bottom of AG10 the two nitrogen groups allow for hydrogen bonding to occur with Ser117…Tafamidis is not able to participate in any bonding at the bottom

Question 15

What did the video on alpha synuclein and Parkinson’s disease highlight? What is the significance?

Answer 15

Originally, scientists believed thought that a monomer (that was unfolded) would aggregate into larger assemblies that would eventually make a “Lewy body” which was the hallmark of Parkinson’s…however the study suggests an additional step which involves the tetratamer of alpha-synuclein needing to break apart before forming dangerous clumps

if scientists, therefore, can keep alpha synuclein packages from breaking apart, they might be able to prevent Parkinson’s from progressing/developing

Question 16

What was the first recombinant human protein drug?

Answer 16

human insulin

Question 17

How does the process of recombinant DNA technology work? (molecular cloning)

Answer 17

1) have human genome
2) select gene of interest isolated from genome
3) insert gene into plasmid
4) plasmid then is inserted into bacteria population generating “clones” of the gene

aka…gene, manipulate it, let bacteria mass produce it for you

Question 18

What are advantages of protein therapeutics? What are disadvantages of protein therapeutics?

Answer 18

Question 19

What is the average size of drugs in the market?

Answer 19

about 1 dalton

Question 20

What is the MW of insulin? How many a.a.?

Answer 20

Question 21

What is the MW of Filgrastim? How many a.a.?

Answer 21

Question 22

What is the MW of IgG? How many a.a.?

Answer 22

Question 23

What are antibodies used for? What are they produced by?

Answer 23

antibodies are large proteins produced by plasma cells…they are used by the immune system to identify and neutralize foreign objects (bacteria, viruses, certain medications)

Question 24

What is the quaternary structure of an antibody?

Answer 24

2 light chains, 2 heavy chains and the top part of the antibody being the variable region in which the hands differ in all Ig yet the rest of the region is called the constant region which is the same in all Ig

Question 25

Label the heavy chain and the light chain and what type of bond connects the two…

Answer 25

Question 26

Label where the Fab fragments and the Fc fragments are…along with the antigen binding site (hypervariable region)

Answer 26

Question 27

What two things interact with FcRn?

Answer 27

the Fc portion of IgG and human serum albumin (HSA)

Question 28

How much does FcRn extend the serum half life or IgG and albumin (i.e. about 90% of serum protein content) from…

Answer 28

1 day to up to several weeks

Question 29

Majority of monoclonal antibodies are based on the structure of…

Answer 29

Question 30

Describe mAb engineering… what is chimeric? humanized? what is changed in each?

Answer 30

Question 31

Describe the transgenic mouse platform that is used to generate fully human monoclonal antibodies…

Answer 31

Question 32

Describe the phage display platform that is used to generate fully human monoclonal antibodies…

Answer 32

Question 33

What are the limitations of antibody therapeutics?

Answer 33

not orally available, poor penetration across blood-brain barrier…LARGE SIZE-DOES NOT GET TO TUMORS WELL***

Question 34

How can you make a cancer antibody more powerful? Specifically to cancer cell and not to normal cell?

Answer 34

an antibody-drug conjugate (ADC) goes to the cell that has the antigen on it (targeted drug therapy) and sparing the normal cell

Overall…you take a toxic drug molecule and attach it to an antibody to deliver the cytotoxic molecule of interest to the cancer cell of interest sparing the normal cells…these are called antibody drug conjugates

Question 35

If you gave Mertansine (microtubule inhibitor for HER2-positive metastatic breast cancer) by itself, it a patient…would it be toxic? How would you get around this?

Answer 35

Mertansine alone is toxic as it will kill everything it will come in contact with. However, if you attach it to a HER2 monoclonal antibody such as Trastuzum, it will float in the body and find an antigen and the antibody will latch onto it and go inside (via endocytosis) and release Mertansine (making this new complex altogether no longer toxic to other normal cells)

Question 36

What kind of linker would you use for linking the Mertansine (microtubule inhibitor for HER2 metastatic breast cancer) and Trastuzum (HER2 monoclonal antibody)?

Answer 36

Since you want something to get inside the cells it could be lysosome enzymes, or mainly disulfide bonds as disulfide bonds will be chopped immediately by glutathione after having entered inside the cell

Question 37

What are the three ways we can modify mAb fragments?

Answer 37

Antibody drug conjugate (ADCs), Fab fragments and Fc fusion protein

Question 38

Explain Fc fusion protein

Answer 38

You take the protein of interest and attach it to the Fc (alike taking the legs of the antibody) to increase its half-life…you then replace the Fab with the protein of interest to then use Fc only increasing its half life

Question 39

You have a patient that has rheumatoid arthritis… what type of treatment/method would you give the patient (ADC or Fc fusion protein)…how would this work for getting around the receptor signaling?

Answer 39

Fc fusion protein such as Etanercept, as the drug’s half-life becomes longer (due to the replacement of the Fab with the Tumor necrosis factors (TNF) receptor and you don’t have to give it as often to the patient since you increased the half life)

It will sequester all the TNF alpha (using the decoy receptor which is the Fc fusion protein) in its solvent form (not attached to the cell unlike the actual TNF alpha receptor that is) outside of the cell and nothing will thus happen for activation as binding does not occur with the actual TNF alpha receptors as the TNF alphas bounded to these decoy receptors (the Fc fusion protein)

Question 40

Does the Fc or Fab define the longer half life of the protein?

Answer 40

Fc

Question 41

How would a Fab fragment work if being used as therapy for rheumatoid arthritis?

Answer 41

You would take solely the Fab fragment and you need to increase its half life (since no Fc) and so you bind it to polyethylene glycol (PEG) and then the fragment will now bind to TNF alpha for a long period of time

Question 42

How do you increase the half life of a Fab fragment that binds to TNF-alpha?

Answer 42

You bind it to PEG (polyethylene glycol)!!!

Question 43

What are the 4 ways to increase serum half-life of proteins?

Answer 43

Question 44

What are the 3 ways to increase serum half-life of peptides?

Answer 44

Question 45

What question does reversible binding to transthyretin answer?

Answer 45

Is there a way to make a peptide still hydrophilic?…what he showed was he can maintain the polarity of the conjugate and we increase the half life and we maintain the binding affinity

Question 46

You want one of these components to be either 1 nanomolar and the other 100 nanomolar…which one will be 100 nM-the target receptor or TTR?

Answer 46

100 nanomolar would be the better receptor because lower binding affinity is better

Question 47

What protein does the spike of the COVID-19 virus bind to on the human cell?

Answer 47

the angiotensin-converting enzyme 2 (ACE2)

Question 48

What is the difference between ACE2 and ACE?

Answer 48

ACE2 lowers blood pressure and is relevant in that it is the entry point for the covid virus into our human cells… while ACE increases blood pressure and is a drug target for ACE inhibitor/anti-hypertensive agents…

Question 49

When are the antibodies (IgG’s) created after infection of COVID-19? How do they function?

Answer 49

They usually begin to accumulate/are made 7-14 days after infection…they work by binding to the spikes of the COVID-19 to not allow for them to try to bind to ACE2 and allow access into the human cell…

Question 50

How does the spike protein bind to the ACE2?

Answer 50

Question 51

What is the structure of the spiked protein? Primary, secondary, tertiary, etc.

Answer 51

What terminus right? We have 3 identical subsunits…this is a homotriamer

Question 52

How can we prevent the spike from binding (in regards to covid19)?

Answer 52

You bring an antibody that will be able to bind to the spike protein, and then since the antibody is very large, it will prevent the binding…but antibodies are very expensive…thus, they took nanobodies (which have a short half life but are similar to antibodies) and put them in a nebulizer (this nanobody form is very stable/can store at room temperature)