RATIONAL DRUG DESIGN

TEACHER NOTES
There are three separate tasks here that students can work on. Each task should be completed within approximately 40 minutes with 10 minutes using the instructional Power Point to give students background on the particular protein of choice. The Power Point presentation also covers aspects of protein structure and function. f this has already been covered in class you can skip ahead to the slide covering the protein you are interested in. Students can explore drugs designed to target the acti it! o" the "ollo#ing proteins$ the en!yme amylase " developing a diet pill the flu en!yme #euraminidase " developing $elen!a% an antiviral drug for the flu The &cetylcholine receptor channel " blocking sensory nerve impulses

Designing a Diet Pill

1. #ame some foods that have a high content of starch. $ice% potato% cereals% etc '. (hoose from the list of words below to fill in the blanks for the following statements) The en!yme amylase breaks down starch into disaccharide molecules called %ALTOSE The en!yme maltase breaks down maltose into the monosaccharide GLUCOSE &mylase is produced by the salivary glands and the &ANCREAS The monosaccharide glucose is re*uired by our bodies to provide ENERG' Open the Cn3D file named amylase +. ,ow many alpha helices are there in this polypeptide- . 4. ,ow many beta sheets are there in this polypeptide- Tell them to use the se(uence align)ent ie#er to count these. (ount the number of brown amino acid se*uence blocks / '. 0. 1hat parts of the en!yme appear to be making up) 2a3 the entrance to the active site- These are the random coils or random loops that are cradling the drug 2b3 the active site- The actual active site is made up of a ring of beta sheets 2the cofactor chloride ion is also found in the active site3 4. (harged ions are often re*uired to assist an en!yme to do its 5ob. These ions are cofactors. 1hich cofactor seems to be involved in the functioning of amylase 2is in the active site3- (l* 6. 7ugar units often form a ring shape. ,ow many sugar units are there in the drug molecule 2the larger molecule seen here3- 4 .. 1hat organism was this en!yme found in% and what part of the organism- ,uman pancreas 8. The large carbohydrate in this molecule is an inhibitor molecule. t stops the en!yme from breaking down starch. 9ooking at the location of the inhibitor% how might it be exerting its effect- t is

blocking the entrance to the active site so the substrate starch can no longer get into the active site 2a little like rolling a rock in front of a tunnel3 10. Explain how the drug shown interacting with amylase would help someone to lose weight. 1hen we eat foods high in starch% such as bread% amylase breaks down the starch into maltose. This is in turn broken down by the en!yme maltase into glucose. :lucose is used to provide cells with energy in respiration. f we have more glucose than the cells need% it is stored as glycogen in the liver and as fat in adipose tissue. f you stop amylase from working% then starch will pass through the body without being digested. &s a result there will be less glucose available so less will be stored as fat. 11. ;o you think the drug being designed to inhibit the action of amylase would block the active site of this en!yme reversibly or irreversibly- Explain your answer. 1ould be best if it is reversible so that some starch is broken down. 1e don<t want to permanently block amylase or we could end up not having enough glucose for cell metabolism.

Saving Lives: Designing a Flu Drug

t is really important that you ensure your students have made the connection between the # protein on the virus coat and the # protein they view in (n+;. =ften they will think they are looking at the whole virus when they open up # in (n+;. $emind them that # acts like a pair of scissors% cutting the links between the virus and the host cell membrane.

This is what your # will look like when you view it in (n+;. The drug sitting in the active site is highlighted in yellow

f they move the molecule around so the drug is at the top% they can see that it looks like its being cradled in a cup>like indentation. The important part 2the active site3 or the blades of the scissors is shown here being blocked by the drug. f they think of the active site like the scissor blades we can use an analogy. f you put a rock between scissor blades% can they still cut- #o. f we place a rock 2the drug3 in the active site of #% can it still cut- #o. 7o the virus cannot escape the host cell which means that it won<t spread. 1hat they need to think about is that if we want to block the active site% the drug we design must interact more strongly with one or more of the amino acids in the active site that the normal substrate does. n this way the drug will competitively bind with the active site rather than the substrate% sialic acid.

+N, the ?scissors<

used by the flu virus to leave host cells

This shows the drug nestled in the active site pocket. To get this view select ?molecule< from colouring shortcuts dropdown menu.

'

Open the Cn3D file named Neuraminidase 1'. (omplete the following statement by circling the correct response. #euraminidase protein is composed of random coils and alpha helices @ -eta sheets. 1+. 9ocate a disulfide bond found in #euraminidase. ;ouble click on the two amino acids making up this bond. Aind out the one letter code for the amino acids in this bond and then use the table below to find out the name of the amino acid. ( / (ysteine

.* letter code G A L % 1 2 3 4 E S

Na)e o" a)io acid :lycine &lanine 9eucine Cethionine Phenylalanine Tryptophan 9ysine :lutamine :lutamic &cid 7erine

/*letter code :ly &la 9eu Cet Phe Trp 9ys :ln :lu 7er

.* letter code & 0 I C ' H R N D T

Na)e o" a)io acid Proline Baline soleucine (ysteine Tyrosine ,istidine &rginine &sparagine &spartic &cid Threonine

/*letter code Pro Bal le (ys Tyr ,is &rg &sn &sp Thr

14. ,ow many sugar groups are bound to #euraminidase 2do not include the drug molecule in this3- + 10. The normal substrate that # acts on is called sialic acid. t enters the active site of # and is ?stressed< so bonds break cutting the virus free of the host cell so it can go off to infect more host cells. The drug you can see on your screen is $elen!a. 7ialic acid and $elen!a are shown below. (ircle any differences you can see on the $elen!a drug molecule. These differences make it bind to the active site of # more strongly.

14. Dsing the se*uence alignment viewer% place your cursor over the amino acids that are now yellow and read their location in the protein chain. $ecord their location in the table below. 1hen you have done this use the amino acid table on page 4 to find out the name of each amino acid.

Location and na)e o" highlighted a)ino acids$ Na)e o" a)ino acid .*letter Location Na)e o" a)ino acid code 1. &rginine r +6 0. :lutamic acid '. &spartic acid d 60 4. :lutamic acid +. &rginine r 61 6. &rginine 4. &rginine r 144 .. &rginine

.*letter code e e r r

Location 146 184 '1' '80

Chec5 !our ans#er$ Eou should have 0 arginines 2r3% two glutamic acids 2e3 and one aspartic acid 2d3. There are also three non>charged amino acids in the active site that also bind with the drug. They are Tryptophan 2w3% soleucine 2i3 and Tyrosine 2y3. The uncharged molecules are grey.
16. 1hat is the function of # for the flu virus# functions to cut the flu virus away from the host cell so the virus can spread to infect more cells 1.. Explain how the drug shown interacting with # stops the flu virus from spreading and infecting new host cells. The drug blocks the active site so the flu virus gets stuck to the host cell and can<t move off to infect more of your cells. 18. 1ould you design a drug to inhibit the action of # reversibly or irreversibly- Explain your answer. deally the drug will irreversibly bind to the active site so # can never work. The virus is forever stuck to the host cell and the host cell covered in virus would be engulfed by a macrophage. '0. The region of # that cuts the flu virus away from receptors on the host cell surface is known as the Factive siteG. The genetic code of the influen!a virus mutates rapidly to help influen!a escape our immune system. ,owever% the regions of the genome that encode the Factive siteG are highly conserved. ;iscuss specificity of the active site to describe why this might be- f the shape of the active site changes then the action of the protein will no longer work. n this case% the # active site would no longer bind with its substrate% sialic acid% so the virus would never escape host cells to infect new cells. This mutation would not be passed on to subse*uence viruses as they would never make it to a new host cell to replicate.

Controlling Chronic Pain: Venoms for Drugs

=PE# &cetycholine 2neurotransmitter3 bound to the shapes at the #ote the difference between (9=7E; ion channel. This causes a shape areas indicated bychange the arrows. The ion channel in the protein so the pore through the receptor on the right is closed% the one on left is membrane open s. The one on right will not generate a nerve open. impulse so the pain message is not passed on.

Open the Cn3D file named Ion channel and neurotransmitter '1. ,ow many acetylcholine molecules are found binding with this molecule 2this chemical causes the ion channel to open3- 1 ''. ,ow many parts make up this ion channel- 2look for the different colours3 0 20 polypeptides are found making up the *uaternary structure3 '+. $otate the molecule so you have an aerial view. ;raw ' *uick sketches showing what it looks like from the side and from above. ndicate on each where transport of sodium ions would occur.

6inding site

'4.

6inding site

ndicate on each sketch above where the acetylcholine binding site is located. '0. 1ould you expect negatively charged ions to be attracted to or repelled away from this ring of negatively charged amino acids- Explain your answer. 7hould be repelled as like charges repel.

'4. =utside the cell there is a soup of positive and negative ions and other chemicals. Explain how the structure of this ion channel would stop negative ions from moving through the pore and entering the cell. This ring of negatively charged amino acids will attract positive ions like sodium and calcium " the ones that set of a nerve impulse. The negatively charged ones will be repelled leaving the pore entrance free for positive ions to move through. '6. ,ow many disulfide bridges does each of these alpha>conotoxins have- 1 '.. Huickly sketch each protein from the side showing the effect that binding of alpha>conotoxin exerts 2#I@ &lpha>conotoxin changes the shape of the receptor protein so the pore in the ion channel won<t open. =ur structure only shows the receptor% not the pore going through the cell membrane3. 7ee the first diagrams after the heading ?controlling chronic pain) venoms for drugs< 3iller conotoxins$ 1e now know that the 5iller drugs have a loop of + amino acids and then 0 amino acids separated by a disulfide bond 2+@03. These drugs target nicotinic &cethylcholine $eceptors in muscles so the heart and diaphragm are affected adversely. Therapeutic conotoxins$ &lpha conotoxins that have 4 amino acids and 6 amino acids separated by a disulfide bond 2 4@63% act on neuronal nicotinic &cetylchoine $eceptors found in the sensory nerves. These drugs do not kill. 9ook at the se*uence for an conotoxin shown below) eccnpacgrhyscx

;isulfide bonds form between two cysteine amino acids 2c>c3. 1e need to count the number of amino acids between these c<s. n the example above we have + and then 0 / 7 /89 conotoxin3. t<s a killerJ '8. 1rite down the se*uences of alpha>conotoxin & and alpha>conotoxin I below and determine the name you would use for each 2i.e. :8: conotoxin3 &lpha>conotoxin &) xccnpacgpkyscx +@0 conotoxin &lpha>conotoxin I) gccslppcaannpdycx 4@6 conotoxin

+0. 1hich of these two conotoxins would you choose to develop as a drug for blocking peripheral nerve pain- Explain. &lpha conotoxin I as it is not a killer but blocks sensory nerve pain.