Transcript Lecture 30 - University of Maryland, College Park

Lecture 27
Pharmacology
Flint et al., Chapter 19, pp. 725 –
757.
The paradox of antiviral drugs
• Although we know more about the
molecular biology of most viruses than
about any other pathogen, we have very
few drugs in our antiviral arsenal.
• One of the reasons is that viruses utilize
so many host functions, that to poison the
virus is also to poison the host.
• Need to target virus-specific mechanisms
The pathway to drug discovery
Fig. 19.1
1) Identify the medical need, 2) identify the mechanism, 3) devise a screen,
4) use a “screen” to identify a “lead compound” that affects the mechanism,
5) chemically alter it to increase activity, 6) then take the drug candidate
through physiological and clinical characterization.
Drug Discovery
1. Identify mechanism: use Mol. Biol and Genetics to
identify mutants that can’t propagate virus.
2. Screen for compounds.
1. Mechanism based, e.g. inhibits HIV-1 protease
2. Cell based, e.g. inhibits virus production in cells
3. High Throughput screens. Look through hundreds of
thousands of candidate compounds quickly and
accurately.
4. Compound sources
1. Libraries of natural compounds
2. Computer based “Designer” compounds – structure based
design
3. Genomics – use microarray technology to identify host
mRNAs/proteins that are affected by virus
infection…identify new antiviral gene products or toxic
responses to drug candidates.
5. Combinatoral chemistry
Mechanism-based screens
Example: a Fluorescence based assay to monitor HIV-1
protease activity
Fig. 19.14
Drug
Cell-based screens
Fig. 19.15
Combinatoral chemistry
Fig. 19.16
•Can provide all
possible combinations
of a basic set of
modular chemical
components.
•Often coupled to
beads or other solid
supports.
•Allows active
compounds to be
traced, purified and
quickly identified.
•Thousands of
compounds can be
made and screened in
days.
Structure based drug design
Squanivir
(Roche)
Indinavir
(Merck)
Protease inhibitors that were designed by modeling chemical structures on the
computer to fit inside of the catalytically active site of HIV-1 protease using the
HIV-1 protease X-ray crystal structure.
The long road from discovery to market
Fig. 19.16
•Initial screen of hundreds of
thousands of compounds
•Some have the desired effects
•Of these, only a few aren’t totally
toxic to cells
•Fewer have the desired effect in
animals but many of these are
toxic in this context.
•This pattern is repeated in
humans.
•Finally, after some 5 – 10 years,
one compound is approved for
human use.
•The cost of such an enterprise typically ranges from $100M -- $500M.
•Cost has significant impact on what kind of entity can do this kind of work (large
corporations) and also on what diseases will be targeted for market (diseases of
rich Americans).
Classes of antiviral compounds
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Viral adsorption inhibitors
Viral-cell fusion inhibitors
Viral DNA polymerase inhibitors
Reverse transcriptase inhibitors
Protease inhibitors
Neuraminidase inhibitors
IMP dehydrogenase inhibitors
S-adenosylhomocysteine hydrolase inhibitors
Nucleoside/nucleotide analogs
• Look like substrates for DNA replication
• Incorporation into DNA  highly mutagenic
• Take advantage of the fact that viral DNA
polymerases and RT’s are not as discriminating
as cellular DNA pol.
• Still,
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relatively toxic to humans,
can cause mutations,
teratogenic,
potentially carcinogenic
Examples of
nucleoside analogs
Acyclovir.
• Specific, non-toxic
• Highly effective against Herpesviruses and to some extent Varicella
• Acyclic sugar group can be phosphorylated for incorporation into nucleic
acids (3’ – 5’ link), but lack of 3’ OH prevents the next nucleotide from being
incorporated
Chain termination
Acyclovir is a Prodrug: precursor to the active antiviral compound. Must first be
phosphorylated by HSV thymidine kinase before it can be activated.
Looks like a 5’ –OH group (i.e. acceptor)
Nonnucleoside RT inhibitors
• Rather than binding in the active site of HIV RT, they
inactivate by binding elsewhere.
• Problem: mutations in any of 7 orange (right) residues
confers drug resistance.
• Still useful in combination with other drugs.
Protease inhibitors
•The natural substrate
for HIV-1 Pro is 7 amino
acids long.
•Ro 31-8959 mimics
this structure
•Fits into the HIV-1 Pro
active site and doesn’t
leave, inactivating the
enzyme.
•An example of an
inhibitory suicide
substrate.
Amantadine
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Also known as Symmetrel (DuPont)
3 ringed symmetric amine
Specifically blocks the influenza A M2 protein ion channel.
Very effective against influenza A.
Must be given within 24 – 48 hrs of infection: typically in to susceptible patients in
anticipation of infection, e.g. nursing homes.
Must be adminstered for 10 days
Neural side effects led to the discovery that it can alleviate some symptoms of
Parkinson’s disease. Today, Amantadine is primarily prescribed for nervous system
disease.
A methylated derivative called Rimantadine cannot cross the blood brain barrier  this is
the primary drug used for Influenza A.
New drug targets
• Inhibitors of virus entry and uncoating
– e.g. Target HIV Env protein with a drug that occupies its CD4 or
CCR5 binding sites.
• Viral Proteases
– Known inhibitors of HIV Pro. Many other viral proteases, e.g.
from CMV, Hepatitis C NS 3 protein.
• Virus-specific nucleic acid synthesis and processing
– E.g. target RDRP’s from RNA viruses, viral proteins involved in
cap snatching, synthesis of primers, recognition of RNA
secondary structure, viral helicases.
• Regulatory proteins
– Viruses encode numerous proteins that regulate the switches
between early, intermediate, and late stages of gene expression.
Drug resistance
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Viruses have high rates of replication
High rates of mutation
 Accelerated rates of evolution.
Mutants that are more “fit” in the presence of
drug will be selected for.
•  Drug resistant varients.
– e.g. AZT resistant HIV-1 RT
– Protease inhibitor resistant HIV-1 Pro
– Acyclovir resistant Herpesvirus Thymidine Kinase
doesn’t recognize acyclovir as a substrate.