antivirals

Question 1

anitviral drugs

Answer 1

• Most infectious disease in NA
  o 95% respiratory
• All class 4 pathogens are viruses
  o Ebola, Marburg, lassa fever, hanta virus, small pox

Question 2

class 1 pathogens

Answer 2

o No risk/limited risk
 Work on open lab bench (E.coli)
o P1 lab

Question 3

class 2

Answer 3

o Moderate risk
o Limited access to lab
o Lab coat required
o Laminar hoods
 Herpes virus
o P2 lab

Question 4

class 3

Answer 4

o Risk of death or serious illness
o Restricted access, special training required
o Surgical gowns, gloves, respirators
o All liquids/ air coming in/out is filtered
o Everything coming out is autoclaved and incinerated
 HIV , Y.Pestis, COVID
o P3 lab

Question 5

class 4

Answer 5

o Lethal, highly infectious, untreatable
o Lab accessed by air lock, special training
o Space suit worn, shower going in and out
o Low pressure in lab (leaks will be IN), airlocks
o All liquids/gases are filtered/treated in and out
 Ebola, Marburg, Lassa fever, Hanta virus, smallpox
 P4 lab

Question 6

viral structure

Answer 6

• Genetic information
  o DNA/RNA
• Surrounded by a capsid
  o Hollow container of protein
• Some viruses may have
  o Enzymes
  o Regulatory proteins
• Some viruses are enveloped
  o Capsid surrounded by a membrane
  o Remnant of the host cell membrane
  o Contains viral proteins

Question 7

self-replicating

Answer 7

• Viral proteins structurally related to host
  o Oncogenes
• Viral proteins bind to host proteins and alter its function
  o Control cellular regulatory systems
• Common elements
  o Duplication of genetic info
  o Production of viral protein
• Viral process utilize host proteins and machinery
  o Ribosome
  o Nucleic acid polymerases

Question 8

difficulties in developing antiviral drugs

Answer 8

o Difficult to target with drugs
o Proteins bind to each other very tightly
o Utilize large contact surfaces
o Need to avoid interfering with normal host cells

Question 9

cell signaling systems use protein binding

Answer 9

whereby protein a attaches to protein b via the active site and conformational change to transmit a signal into the cell
viruses do the same thing (replace protein A with a virus and protein b will change into another shape to fit the virus) a viral message will be sent into the cell instead

Question 10

blocking viral binding

Answer 10

attach a big molecule on protein B to block viral enzyme from binding and transmitting a signal. difficult to do b/c its a large surface area and big drugs are bad drugs because of bioavailability

Question 11

viral enzymes as drug targets are difficult b/c

Answer 11

o Host cells make nucleic acids
o Substrates, mechanisms, and structures are similar
o How do you block viral enzyme without blocking host enzyme?
 Limited number of targets
* Most viruses have small genomes (4 genes)
* 1-2 enzymes
o Most involve nucleic acids (host selectivity problem b/c we have similar)
o Only few antiviral drugs exist hepatitis C (cure), herpes (treat) and HIV (manage)

Question 12

problems w/antiviral drugs

Answer 12

• Selectivity
  o Kill virus w/o killing host
• Diagnosis
  o Drugs specific for each virus
  o Many viruses have similar symptoms
  o Need biochemical test
• Resistance
  o Mutation rates in viruses is high
  o Viruses quickly develop resistance to drugs (days or weeks)

Question 13

1. Absorption and penetration into cell

Answer 13

o Virus binds to host on outside of host membrane
 Capsid binds directly and is passed inside before opening
 Envelope of capsid fuses with host cell membrane releasing capsid inside cell
o Genetic info of virus injected into host
 Can inject viral protein
o This stage is poor drug targets b/c
 Binding is protein-protein binding
* SA very large
* Difficult to inhibit protein-protein binding with small molecules
 Limited success in HIV
* Fuzeon (peptide with 36 AA)
* Maraviroc (small molecule + fits into HIV protein)

Question 14

2. Capsid opens releasing contents

Answer 14

o Capsid releases genetic info into cell
o Difficult to target bc protein-protein interactions, pH changes)
o 2 successful drugs
 Influenza
* Block ion channel

Question 15

3. Synthesis of regulatory proteins

Answer 15

o Viral proteins made + take over normal cell systems
o Nucleic acid replication
o Expression of viral protein
o Suppression of host cell defenses
o Binding to host proteins
o No drug for this

Question 16

4. Synthesis of RNA or DNA

Answer 16

o Viral genome replicated using host enzymes
o Some viruses have their own enzymes for this (drug targets)
o Most anti-viral drugs target this phase
 Require unique viral enzyme
 Prevent viral nuclei acid synthesis

Question 17

5. Synthesis of structural proteins

Answer 17

o Utilize host ribosome
 Poor drug target
o Some viruses utilize specific enzymes for protein maturation
 Protease drugs
* HIV
* Hepatitis C

Question 18

6. Assembly of viral particles

Answer 18

o Capsid proteins self-assemble
 Nuclei acid inside
 Viral proteins outside
o Release may destroy cell
 Lytic virus (herpes/influenza)
o Cell may remain intact
 Papilloma, herpes
o Few drug targets (HIV, influenza)
* Release from host

Question 19

antiviral drugs require viral enzyme targets

Answer 19

• Enzyme should structurally be unrelated to host enzymes (selectivity)
• Most viral enzymes are involved in nucleic acid replication

Question 20

herpes

Answer 20

• Causes chronic recurrent infections
• Able to escape immune system (hides in nerve cell)

Question 21

herpes structure

Answer 21

o Genetic info is double stranded DNA
 >70 genes
 Has its own polymerases- drug target
o Components of nucleic acid
 Nucleoside (no phosphate)
 Sugar (DN/RNA)
 Base (ATCG) heterocycle (ring with heteroatoms)
* Recognized by shape and hydrogen not letter
* Draw nucleic acid
o Polymerases copy nucleic acids
 Use one strand as template to make another strand
* Nucleotides are added one at a time, matching bases

Question 22

rational drug design herpes

Answer 22

 Use knowledge of enzyme mechanism and substrate to design drug
 Selectivity issue w/nucleic acids
* Must block viral enzyme w/o blocking host enzyme

Question 23

poor selectivity= toxicity herpes

Answer 23

interference with normal cell f’n causes problems

Question 24

strategy 1 herpes add non natural base

Answer 24

add non natural base on DNA strand instead of normal ATCG
Causes disruption of nucleic acid and causes spelling error that DNA wont be read by other enzymes
requires:
 Drug is a substrate for a kinase (to be phosphorylated))
* Host
* Virus (rare)
 Drug is a substrate for viral polymerase
* Incorporated into viral nucleic acid
* Create an unreadable strand
* Drug must not be a substrate for host polymerase (side effects)

Question 25

strategy 2 herpes chain terminator

Answer 25

add isostere on 3' OH as a biological replacement o Requirements for chain termination  Drug is phosphorylated (host or virus)  Drug substrate for viral polymerase * Incorporated into viral nucleic acid * Stops replication * Viral proteins don’t get made  Drug must not be a substrate for host polymerase

Question 26

Idoxuridine

Answer 26

* Substrate for viral and host polymerase * Highly toxic * Topical use o Limits exposure to body o Eye infections of herpes * Proof of principle—antiviral drug is possible but ideally not good

Question 27

o Chain termination—selectivity is a problem

Answer 27

 Drugs have structures too similar to normal substrate * These drugs incorporated into host nucleic acid by host polymerase * Many terminators are toxic  Improve selectivity by changing sugar from 3' to 2' or opening sugar the host polymerase will not accept open sugar but viral will

Question 28

open sugar chain terminator Acyclovir (CT)

Answer 28

* Very selective for virally infected cells * Low incidence of side effects * Low severity side effects * Selectivity due to selective bioavailability * Accepted by viral polymerase not host * Topical cream in NA does not work but works in Europe * Is a prodrug add PO3 on OH end traps acyclovir in infected cells and is concentrated draw phosphorylation

Question 29

* HSV-1

Answer 29

o Cold sores and blisters on mouth nose, eye  80% infected  10-20% experience outbreaks o Virus escapes eradication by hiding in neurons  Activate by no access to immune system, stress, sunlight, epithelial cells o Lytic infection  Only in epithelial cells, no damage neurons  Viral activity 24hrs  Minimal damage  Damage caused by immune system * Overstimulated and destroys tissue * Drug administered quickly

Question 30

* HSV-2

Answer 30

o Blisters on anus and genitals o 15%-20% infected o More virulent and painful o One outbreak per year o Most damage done by immune system, administer drug quickly

Question 31

influenza structure

Answer 31

o Enveloped virus  Human membrane that virus steals when it leaves o Enveloped embedded w/2 viral proteins o Neuraminidase (viral maturation) o Hemagglutinin (viral entry)  Antibody test to identify them as they are on the outside of virus

Question 32

influenza life cycle

Answer 32

o Virus life cycle start with binding  Virus hemagglutinin binds to host glycoproteins containing sialic acidsugar o Endosome forms o Virus envelope and cell membrane fuse o Contents spill into cell o Viral RNA and proteins are replicated * Capsid self-assemble with RNA * Capsid form inside cell complete RNA * Viral envelope proteins accumulate on cell membrane o Hemagglutinin o Neuraaminidase * Virus particles bud from the cell * Virus particles still contain host proteins o Host proteins may contain sialic acid (sugar)  If sialic acid remains on the virus, hemagglutinin form other virus particles will stick o This will cause virus particles to clump together  To prevent this, neuraminidase removes sialic acid from host proteins on the virus envelop  Virus particles don’t stick together o Immature virus stick to each other o Neuraminidase removed the sialic acids DRAW

Question 33

Neuraminidase

Answer 33

* Essential enzyme o Highly conserved—other viral proteins variable * Cuts bond between sialic acid and other sugars in virus o Mechanism of hydrolysis o Enzymes bind to transition state (rational drug design= mechanism, enzyme for trans, design drug that binds trans to enzyme similar to the trans) o Trans state vs trans state mimic

Question 34

Xray crystallography

Answer 34

* Crystallize molecule * Bombard crystal with x ray beam o Wavelength similar to bond lengths * Xray are scattered by interaction with atoms in crystals o Angle determined by WL and bond distance o Bragg equation * Crystal generates large scale order o All molecules arranged in a fixed lattice o Magnifies the affect ( all molecules diffract the same way) = diffraction pattern * Computer calculates the e density map o Molecular formula known o Fix molecular structure into the EDM o Technique important for accelerator design  Designed to incorporate beam lines

Question 35

tamiflu

Answer 35

* Very expensive * Limited supply * Given in first 24-48hrs * Diagnosis problem (flu vs cold; symptoms are the same) o Only works for influenza (20% colds) * Reduces disease by 1 day * Benefit/risk analysis indicates clinical use is questionable * Toxicity issues * Patent expired in 2017 o Generic companies cannot replicate b/c they cant replicate raw material (why cant they?)

Question 36

hepatitis characteristics

Answer 36

o Single stranded RNA o Small genome o Protein is cut into smaller proteins  First few released by host proteases  Remaining is cut by viral proteases o Virus has an RNA dependant RNA polymerase  Copies viral RNA o Virus proteins spontaneously assemble around viral RNA o Transported in blood to liver as a complex with lipopolysaccharides o Virus is replicated inside membrane of ER o Viral enzymes  RNA dependant RNA polymerase (makes viral RNA copies)  Protease (cuts viral polypeptide into small proteins)

Question 37

viral enzymes HC

Answer 37

 RNA dependant RNA polymerase (makes viral RNA copies)  Protease (cuts viral polypeptide into small proteins)

Question 38

inhibiton of viral polymerase HC

Answer 38

 Sofobuvir * Prodrug * Active drug is a chain terminator

Question 39

inhibiton of viral enzyme formation HC

Answer 39

 Large protein binds to a small protein to form functional enzyme  Ledipasvir * Sticks to small protein * Prevents protein association and formation of viral enzyme * Doesn’t follow Lipinski  Harvoni * Combination of ledipasvir and sofobuvir * Capable of curing HCV infection 8-24 weeks

Question 40

AIDS

Answer 40

* Patients suffered from T-cell depletion o Key components of immune system * Victims dies from opportunistic infections o Normally ctl by a healthy immune system

Question 41

HIV

Answer 41

o Agent causes AIDS o Spread  Unsafe sexual practice  Intravenous drug use  Congenital infection of newborns  Fluid transfers o Targets CD4 cells of immune system o Virus may have spread from chimpanzees

Question 42

Retrovirus HIV

Answer 42

 Viral genome is carried by single stranded RNA  Viral enzyme—reverse transcriptase: copies ssRNA into dsDNA (what infects the person)  RT destroys RNA template as it is copied * RT uses the single stranded DNA to prepare double stranded viral DNA

Question 43

Replication HIV

Answer 43

 Virus binds CD4 protein on outside of cells  Virus envelope fuses w/membrane and injects capsid inside  Capsid opens, RT copies viral RNA into dsDNA  Viral integrase enzyme inserts viral DNA into host chromosome—infection permanent  Viral genome activatesviral RNA is made, single viral protein is made  Viral protease cuts itself out of viral protein and cuts the viral protein into smaller viral proteins  Virus particles self-assemble around viral RNA  Virus buds from the cell taking membrane piece with it (envelope)

Question 44

HIV Drug targets

Answer 44

RT (Chain terminator), protease, GP41 (Changes shape) not good--intergrase, RNAase H, GP120

Question 45

Acute phase HIV (6-10 WKS)

Answer 45

o Flu like symptoms o High viral titters o Phase ends when immune system reduce viral load (get infection under ctrl)

Question 46

Chronic Phase (8-12)

Answer 46

o Asymptomatic  Fully infectious  Viral replication is high 10^9)  Immune system destroys viral particles  Few viruses escape to continue infection  CD4 cells in IS slowly depleted * Helps; no symptoms b/c IS taking care, but leakage causes virus to remain  Phase ends when IS runs out of CD4 cells

Question 47

AIDS phase (2-4)

Answer 47

o Secondary infections o Person fully infectious o Viral replication is very high o High viral titers o Immune system is completely destroyed o Secondary infectious cannot be cleared by immune system o Person dies b/c of secondary infection

Question 48

AZT

Answer 48

* Failed ad a cancer drug * Toxic * Tested against HIV * Gained FDA approval after truncated clinical trial o Terminated b/c of ethical grounds

Question 49

AZT failed b/c

Answer 49

o Short period of antiviral activity o Resistant virus appears in few weeks and drug no longer works  RT is error prone * Enzyme has 2 domains (functions o Polymerase o RNASE H o Enzyme destroys template RNA as it copied (RNAse H)  As RT makes DNA, * No error checking o Virus in the body is different b/c of mistake * Sloppy replication  Very high mutation rate * 1 althernation/genome * Every virus particle is different

Question 50

3TC racemate ACT

Answer 50

* Natural enantiomer highly toxic o Substrate host polymerase * Unnatural enantiomer low toxicity o Not recognized by host enzymes * Pseudoenantiomers have diff toxicities o Natural highly toxic  Substrate for host kinase and polymerase o Unnatural low toxicity  Substrate for host kinase  Nucleotide not recognized by host polymerase

Question 51

Patent for 3TC

Answer 51

* Drug is unnatural enantiomer by Bernard Belleau * Patent o Described as racemate o Isomers can be separated but did not say how o Patent pure enantiomers and did not say how to make them o Clinical trials  Drug low toxicity  Resistance appeared quickly in drug  Effective if combined with AZT  Approved  1 billion/yr 2000

Question 52

Liotta

Answer 52

 Noticed patent did not describe how to make enantiomers of 3TC  Discovered a method to make pure enantiomers  Patent both synthesis  Holds right to the company’s drug  Negotiated licence agreement with OG’S  Also noticed company missed key compound * Fluorinated analogue was common change when non-natural base anticancer drugs were developed * Patented FTC

Question 53

HIV protease

Answer 53

Essential enzyme Cleavage of long viral peptide into smaller viral proteins Aspartyl protease Active site has 2 conserved aspartic acids know mechanism!!!!!!!!!!!!!!!!!!!!!!!!!!!

Question 54

Approved Protease Inhibitors

Answer 54

hard to manufacture on large scale because of complexity i.e chiral centers, large molecules, poor bioavailbility (rule of 5), high dose required, impossible manufact economically

Question 55

cost of drugs is small

Answer 55

Most of the cost of drugs is not the active ingredient Marketing Research Infrastructure Until AIDS, no drug program had ever been cancelled because the drug itself was too hard to manufacture

Question 56

indinavir

Answer 56

Utilized brand-new catalytic methods to construct stereocentres

Question 57

HIV resistance

Answer 57

HIV replicates rapidly 10 billion new viral particles every day Replication sloppy--RT no error checking, each virus particle is diff so resistance is fast n drug cant keep up The genome of each virus is different Resistant mutants generated every day Anti-HIV agents select resistant strains unless all viral proliferation is blocked No single anti-HIV agent is powerful enough to do this

Question 58

Combination Therapy

Answer 58

If one drug fails, second picks up slack Third drug provides insurance Virus resistance more difficult

Question 59

HAART

Answer 59

Highly Active Antiretroviral Therapy Combination of 3 drugs Compliance 16 or more HIV pills a day Complex regimen with/without food, empty stomach, without other drugs etc… side effects: rash, nightmares, lipodistrophy (forever lose fat in cheeks) Compliance problems produce resistance b/c they would stop taking it at the specific time

Question 60

Patient compliance problem

Answer 60

HAART therapy initially very successful Improved quality of life Side effects severe, dosing inconvenient Appearance of resistance and increased spread Patients skipped doses Regression to high risk behaviours Second generation drugs designed to be more “drug like” Reduced side effects Convenient dosing

Question 61

Patient compliance solution

Answer 61

Current therapies based on “clean” drugs Pseudoenantiomer Nucleosides 3TC, FTC Few side effects Good patient compliance

Question 62

HIV is a manageable infection in developed world

Answer 62

Lifespan now more than 20 years Disease cannot be cured Mild side effects Can manage the progression of the illness

Question 63

Current Therapies HIV

Answer 63

Pseudoenantiomer nucleosides 3TC FTC (Liotta) Clean drugs Integrase inhibitors Second generation Protease inhibitors Improved properties