Drug Design_L1

Question 1

the structure of an antibody(Ab)

Answer 1

Ab is the immunoglobin folds produced by the immune cells in specificity to the antigen for neutralisation, for example, the antigen can be spike proteins embedded in viral surface membrane, an Ab consists of heavy chains and light chains.

Question 2

the function of an antibody(Ab)

Answer 2

(1) Abs have very high affinity and specific binding
(2) Non-toxic
(3) Typical affinity range of antibodies
(4) Enzymes stabilise TS
(5) Extracted enzymes cannot be used for treatments becuse of the possibility of immune reponse triggered and degradation in blood stream

Question 3

Ab production, role and association in the immune response

Answer 3

(1) humoral response system: the Ab production is blood/serum based. The antigen molecule is recognised by the B-cell surface bound receptor, the B-cell is activated and induces the plasma cell antibody production and memory cell generation targeting the specific antigen in the long term.
(2) cell-mediated(cellular) response system: cellular contact delivers the immune signal to the other immune cells. Pathogen interacts with the T-cell at the receptor, activated T-cell triggers the macrophage action by cytokines to digest the pathogen down, meanwhile, crosstalking the B-cells by cytokines to promote the memory cell formation. Moreover, activated T cells differentiate into the cytotoxic T lymphocytes

Question 4

Ab production, role and association in the immune response

Answer 4

(1) humoral response system: the Ab production is blood/serum based. The antigen molecule is recognised by the B-cell surface bound receptor, the B-cell is activated and receives the cytokines from T-cells to differentiate into plasma cell for antibody production. Memory cells can be generated both from the T-cells and T-cells.
(2) cell-mediated(cellular) response system: cellular contact delivers the immune signal to the other immune cells. Pathogen interacts with the T-cell at the receptor, activated T-cell triggers the macrophage action by cytokines to digest the pathogen down, meanwhile, crosstalking the B-cells by cytokines to promote the memory cell formation. Moreover, activated T cells differentiate into the cytotoxic T lymphocytes

Question 5

What is complementary determining region(CDR)?

Answer 5

CDR is a hypervariable domain yielded from heavy and light chain folds, 3 domains at each chain will be resulted. Due to the presence of heavy and light chains, the CDR is tightly fixed by hydrogen bonds and allowed to extend conformationally. CDR binds to the antigen in high specificty, for example, sugar moiety in the glycoprotein on the surface of the virus/pathogen.

Question 6

pros and cons of clinically designed Abs?

Answer 6

pros:

(1) target specifically to the tissue
(2) selective activation of non-toxic prodrug over the toxic ones
(3) allow more complex chemistry

cons:

(1) some Abs are chemically impossible to be manufactured
(2) the haptens fail to activate the antibodies which catalyse the desired reaction
(3) haptens clsoely resemble the structure of the end-product and therefore act as inhibitor of the reaction
(4) the catalytic efficiency depends on solvent-exposed binding site

Question 7

difference between the antigen and hapten?

Answer 7

(1) antigen: the foreign molecule evoking the immune response either alone or after forming a complex with a larger molecule (as a protein) and that is capable of binding with a product (as an antibody or T cell) of the immune response
(2) hapten: a small molecule that reacts specifically with an antibody but is incapable of stimulating antibody production except in combination with an associated protein molecule

Question 8

mechanism of Ab catalysing the reaction under body temperature?

Answer 8

Ab stabilises the TS state by lowering the energy level to reduce the energy barrier, however, if the Ab binds to the substrate with higher affinity, the TS intermediate formation is more favourable

Question 9

cocaine degradation mechanism aided by the enzyme?

Answer 9

two tyrosine residues stabilise the signly positively charged amine group on the coanine ring, hydroxyl group from the water attacks on the ester bond to bring an electron to the oxygen atom in double bond. The intermediate is therefore formed: the double bond is turned to single bond with a negative charge on the oxygen, the negatively charged chemical group is stabilised by the tyrosine residues nearby. The excessive electron on the oxygen orbital jumps to the neighboring bonds, the tetrahedral intermediate is split up into the ecgonine methyl ester and benzoic acid respecitively, the original ester bond is broken down to the carboxylic acid and hydoxyl group

Question 10

cocaine addiction mechanism?

Answer 10

Cocaine binds to the dopamine re-uptaker at the post-synaptic neurone membrane to inhibit the dopamine internalisation and degradation, the concentration of dopamine accumulates in the synaptic cleft and continuously stimulates the neurone activation.

Question 11

how to tackle with the cocaine degradation problem by using catalytic Ab?

Answer 11

Ab mimics the structure of the intermediate by replacing the carbon atom by phosphorous which is not able to initiate a nucleophilic attack from water molecule to stabilise the compound, the best analogous form can be selected out from the candidates. The analogue bound in the Ab is so stable that the binding site is unavailable to cocaine.

Question 12

What should be taken into consideration when designing the effective Ab?

Answer 12

peptide linkers which are more prone to modification: linker length, tether site and linker type are the three major factors. Linker length should not be too long or too short, overlengthed linker is more likely to be digested and underlengthed linker is not capable oof reaching the destination. The tether site determines the position of the hapten binding to the carrier protein. To successfully trigger the immune response, the hapten is conjugated with the carrier protein which is larger in size by linker. The carrier protein could be BSA or ovalbumin.

The number of haptens attached to the carrier protein should be selected as well, depending on the identidity of the carrier. The unstable monophosphonate product was coupled to carrier protein to yield TSA 1. The analog:carrier coupling ratio was 6:1 for bovine serum albumin (BSA) and 15:1 for ovalbumin based on the incorporation of radiolabel into protein. Theoretically, the likelihood of heptan contacts increases as the number goes up.

Question 13

the commercial Ab mass production approach?

Answer 13

Mice are injected with the antigen triggering the production of the antibodies, spleen is extracted from the mice bodies, spleen cells of interest are cultured on the plate. Myeloma cells are introduced to the mice cells for cell fusion. Hybrid cells are produced are isolated in individual clones. The activity of the clones are assessed in terms of catalytic efficiency.
However, because of the organ extraction stage, the production amount is limited.

Question 14

essential parameters for testing the hapten quality?

Answer 14

(1) the catalytic efficiency
(2) the toxicity of the drug
(3) mice survival rate after being treated with the drug

Question 15

What will be behaviour of the hapten in the catalytic site?

Answer 15

Hapten molecule resembles the docking position of the intermediate at the active site

Question 16

What is prodrug and where do most reactions take place?

Answer 16

Prodrug is the designed drug structure which is inactive under normal circumstances and only active when encountering specific enzymes. The metabolic reaction mostly happens in the liver. Prodrug should be fed according to its side effect, does and clearance. Below listed some conversion between the prodrug and active drug:
Prodrug				Active Drug	
Protonsil				Sulfanilamide	
Levodopa				Dopamine	
Talampicillin			Ampicillin		
Cortisone				Hydrocortisone	
Dipivefrin				Adrenaline

Question 17

What routes are involved in creating the theraputic Abs?

Answer 17

murines —-> chimeric —-> humanised —-> human
Chimeric consist of variable regions (VLand VH) derived from a mouse and constant regions derived from human. ~65% human
Humanized therapeutic mAbs are predominantly derived from a human source except for the CDRs, which are murine. >90% human

Question 18

What is butyrylcholinesterase (BChE) and what mechanism it conducts cleavage of butyrylcholine?

Answer 18

BChE hydrolyses butyrylcholine
It can also hydrolyse toxic compounds that contain an ester
Human esterases are slow to degrade cocaine
> 11 isoforms found in liver, brain, heart

two main side chains: the histidine and serine surrounding the butyrylcholine, histidine initiates the nucleophilic attack as a Bronsted-Lowry base to the serine which transfers the electron from the ring structure to the doubly bonded oxygen atom in the ester bond, tetrahedral intermediate is formed from the electron transfer and the serine is covalently attached to the molecule at this stage. The negative charge on the oxygen is stabilised by the NH groups. The electron is retrieved to histidine so that the bond is broken and the choline is released, followed by the incoming of a water molecule attacked by the histidine. The hydroxyl group dissociated from the water attacks onto the remainder group which covalently bonded to the serine, serine is returned to the physiological condition and benzonic acid is released