32 - influenza

Question 1

what is influenza, describe its mutation rate, who is at risk

Answer 1

• Seasonal respiratory infection
  
  • fall and winter
• High mutation rate
  
  • Virus changes yearly
    
    • why they ask you to get flu vaccine every year
  • Virus infects humans and animals
    
    • when it transmits from animals to humans there are a lot of problems that arise
• Most forms not dangerous
  
  • Elderly and very young children are exceptions
    
    • damaged immune systems are the issues

Question 2

how many influenza pandemics have there been and when? how many people die on average from it each year.

Answer 2

• 1918 20,000,000 deaths
• 1957 1,000,000 deaths
• 1968 700,000 deaths
• average 250,000-500,000 deaths per year

Question 3

what are the three sub types of influenza. rank them from most dangerous to least dangerous

Answer 3

• Type A
  
  • Most dangerous
  • Pigs, horses, seals, whales, birds, humans
  • Lots of genetic variation
• Type B
  
  • Only humans
  • Little genetic variation
  • not the most concerning one
• Type C
  
  • Does not cause serious disease

Question 4

describe the virus structure of influenza. what is in the envelope? and what two proteins are found embedded in the envelope?

Answer 4

• Enveloped virus
  
  • piece of human membrane that the virus steals when it exits from the cell
• Envelope embedded with two viral proteins (located on outside)
  
  • Hemagglutinin (viral entry)
    
    • important for entrance
  • Neuraminidase (viral maturation)
    
    • important for exit

Question 5

what are viruses classified by? and how is this done

Answer 5

• Classified by serotype
  
  • antibody test on the flu viruses to see which antibody reacts (because the proteins are on the outside of the virus they are easy to detect using lab tests)
  • Antibody reaction to the envelope proteins
    
    • Hemagglutinin (H) - 16 types
    • Neuraminidase (N) - 9 types

Question 6

what are the most common types of hemagglutinins and neuraminidases found in human flus

Answer 6

N1 or N2

H1, H2, or H3

Question 7

what does the influenza life cycle start with?

Answer 7

binding

Question 8

describe the binding of the viral hemagglutinin to the host glycoproteins. what are glycoproteins and what is sialic acid?

Answer 8

• Virus hemagglutinin binds to host glycoproteins containing sialic acid
• glycoprotiein is a protein that is glycocylated = certain residues attached to the protein are connected to sugar strcutures (have HBA and HBD which help the proteins recognize different structures)
• sialic acid is a special type of sugar, it is the target for influenza

Question 9

how do viral proteins induce the endosome formation

Answer 9

• human proteins that the virus sticks to and viral proteins that stick to the human proteins. as the viral proteins stick to the human proteins, this causes the human proteins to wrap around the virus. this will eventually form an endosome.
• once the endosome is in the cell, there are pH changes within the endosome that cause it to open up, releasing the virus capsid into the cell = starts the cycle of the virus

Question 10

what happens when the viral capsid opens up in the cell? what two things are replicated?

Answer 10

when the capsid opens up, it releases viral RNA and it begins to get replicated. the RNA is then used by the ribosomes to start manufacturing proteins. get accumulation of viral proteins and viral RNA within the cell

Question 11

how do capsids self-assemble with RNA? what accumulates on the cell membrane

Answer 11

• Capsids form inside cell complete with RNA
  
  • form on the inside of the cell
  • capsids are attracted to the viral proteins on the outside of the cell. there is an interaction that happens between the viral membrane proteins and the proteins in the capsid that cause the final assembly of the virus particle
• makes little miniature packages of virus (capsid)
• Viral envelope proteins accumulate on cell membrane
  
  • Hemagglutinin
  • neuraminidase

Question 12

how do virus particles bud from the cell

Answer 12

on the inside of the membrane, you have bits of the viral protein that span the entire membrane, that have parts on the inside and the outisde. the parts of the viral proteins that are on the inside have specialized parts that are able to recognize the outside of the capsid. the shape of the viral protein has a shape that is complimentary to the shape of a viral protein on the capsid - they recognize each other by virtue of their shape and H-bonds. the capsid ends up getting attached to the membrane and it wraps the membrane around itself by the attractions between the viral protein. this results in a newly formed viral particle being released from the cell

Question 13

what is a technical problem that arises with virus particles and how can this be prevented

Answer 13

• technical problem for the virus is that it is using human membrane and it still may contain human derived proteins.
• If sialic acid remains on the virus, hemagglutinin from other virus particles will stick to the virus. will form big balls of virus all glued together = results in virus not able to infect other cells
• To prevent this, neuraminidase removes sialic acid from host proteins on the virus envelope = cleans off the outside of the virus envelope

Question 14

what does neuraminidase do and what is meant by calling it highly conserved?

Answer 14

Highly conserved – means it doesn’t change its structure

its job is to remove sialic acid. neuraminidase just breaks the bond between the two chairs and what you get is a virus particle that is free to go and infect

Question 15

what is the key step in the hydrolysis mechanism and how is this a target for rational drug design?

Answer 15

• key step is the stuff on the right (clipping off the sugar)
  
  • design a drug that is designed to bind to the enzyme that carries out the reaction of cutting the sugar.
    
    • rational drug design

Question 16

what do you want your influenza drug to resemble and why

Answer 16

because enzyme binds the tightest to the transition state, you want to make a drug that closely resembles the transition state of the reaction. this will give the drug the best chance of blocking the enzyme

Question 17

what is a transition state mimic and how does its structure differ from the normal transition state structure

Answer 17

transition state mimic will fit very well into the pocket of the active site on the enzyme, and the enzyme will bind to it tightly. the pi bond was just moved over in the ring.

Question 18

what is the purpose of x-ray crystallography and how does it work

Answer 18

• Bombard crystal with coherent X-ray beam (all X-rays in the same direction)
  
  • Wavelength similar to bond lengths in the crystal
    
    • Approx. 1 - 3 Angstroms
    • because wavelengths have a length that is similar to the C-C bonds in the crystal, the x-ray beams become defracted
• X-rays are scattered by interaction with the atoms in the crystal
  
  • Angle determined by wavelength and bond distance
  • nλ = 2dsinθ (Bragg equation)
  • use a calculator to solve this

Question 19

how does the crystal structure generate

Answer 19

• Large-scale order
• All molecules arranged in a fixed lattice
  
  • stacked in exactly the same way = all the beams diffract in the same way
• Magnifies the effect (all molecules diffract the same way)
  
  • by measuring the angle = calculate 3D structure

Question 20

what is the result of x-ray crystallography

Answer 20

the individuals dots on the right represent the diffraction pattern. by measuring the distance between the dots, you can recreate the angle and the distance between the atoms in the structure and generate a map of what the molecule looks like

Question 21

how does the computer calculate the electron density map

Answer 21

• hollow structure that shows you where all of the atoms are in the protein
  
  • use a computer to fit the atoms into the hollow parts = 3D structure showing location of all the atoms in the molecule
• Molecular formula usually known or approximately known
• Fit the molecular structure into the ED

Question 22

what is the significance of particle movement in accelerator design

Answer 22

• particles accelerate around in a circle
• each time they change directions they give off X-rays on a tangent to where they are turning. you get x-ray beams that are all coherent

Question 23

why do pharmaceutical companies want to sponsor accelerator design?

Answer 23

• pharmaceutical companies want to sponsor accelerator design because they want to incorporate beam lines as a part of the operation.

Question 24

how does a beam line work

Answer 24

you put your crystal sample in the middle of the beam line and you get a very high quality beam of xrays to get a very high quality 3D xray crystal structure.

Question 25

describe the binding site that the first influenza drug went inside and the interactions that took place. what did scientists notice in terms of empty space and what did this prompt them to do

Answer 25

• there is a salt bridge on the right between the CO2- and the NH2+’s
• van der walls pocket on the left
• noticed that OH was the only “empty” region in the active site, except for some glutamic acids on the bottom. made them wonder if they could redesign the inhibitor to take advantage of the empty space on the glutamic acids
• came up with the idea of making a filler structure to fill the empty space. attached something positively charged to the end of the filler structure and that will cause electrostatic interactions between the filler molecule and the CO2- at the bottom

Question 26

what is zanamivir, what was its antiviral effect, was it orally bioavailable, how was it administered, and what is meant by proof of principle

Answer 26

• Mild antiviral effect
  
  • worked in animals
• Not orally bioavailable
  
  • acid sensitive
  • Rule of 5
• Drug was administered as a nasal spray
• Proof of principle
  
  • probed that they could lower amount of influenza by inhibiting neuraminidase, and using rational drug design to make a transition state look alike

Question 27

describe the process of changing the transition state into the final drug

Answer 27

look at the lecture for this

Question 28

what is the time-line for tamiflu

Answer 28

• 1966 – high throughput screening gives no hits
  
  • shelved until 1969 where they found the mechanism of the enzyme
• 1969 – rational drug design starts
• 1974 – proof-of-principle (lab)
• 1983 – X-ray structure
• 1993 – Zanamivir
  
  • proof-of-principle (humans)
• 1997 – Tamiflu first synthesized
• 1999 – Tamiflu enters market

Question 29

why is tamiflu a very poor drug

Answer 29

• Very expensive
• Limited supply (manufacture)
  
  • made by a semi-synthesis and the first part is hard to get access to
• Must be given in first 24 – 48 hours
  
  • Diagnosis problem
    
    • have to know you have the flu before you even have symptoms
  • Only works for influenza
    
    • 20 % of colds
• Reduces course of disease by approx 1 day
  
  • instead of 10-14 days you only have it for 9-13

Question 30

what does the benefit/risk analysis indicate about tamiflu

Answer 30

• Benefit/risk analysis indicates clinical use is questionable
  
  • FDA is looking into it again

Question 31

when did tamiflu sales expane

Answer 31

• In 2008
  
  • 400,000 treatments per year
  • Approx $800 million in sales
• After 2009 averaged $3 billion/year
  
  • huge demand because of the fear surrounding influenza

Question 32

when did the tamiflu patent expire and what effect did this have on its price?

Answer 32

• Patent expired in 2017
  
  • Little if any effect on prices (limited supply)
  • generic companies didn’t lower the price