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Antiviral Drugs: Discovery and Resistance

Vincent Racaniello

vrr1@columbia.edu

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Vaccines have provided considerable success

in preventing viral disease

BUT, they have modest or often no therapeutic effect

for individuals who already are infected

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1. Compounds interfering with virus growth can adversely affectthe host cell

Side effects are common Every step in viral life cycle engages host functions 2. Many medically important viruses are dangerous, cant be

tested in model systems, or cant be propagated

Difficult or impossible to grow in the laboratory: (e.g., hepatitis B and C, papilloma viruses), Have no available animal model of human disease: (e.g., smallpox virus, HIV, measles virus). Will kill investigators who arent careful (e.g., Ebola virus, Lassa fever virus, Smallpox virus)

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Many standard pharmaceuticals can be effectiveif enzyme activity is partially blocked

Partial inhibition is not acceptable for an antiviral drug

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Think about it: if drug doesn't block virus replication completely,

bad things are inevitable:

RESISTANT VIRUSES WILL ALWAYS EMERGE!

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1. By the time the patient feels ill, the virus is no longer replicating in thepatient.

- the symptoms are due to the immune response - the antiviral drug is of no useAntiviral drugs for these viruses must be given earlyin infection or

prophylacticallyto populations at risk. - safety issues; giving drugs to healthy people is not wise

2. No broad-spectrum antiviral agents are currently available, so every virusinfection has to be identified specifically!

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The lack of rapid diagnostic reagents alone has hampered development andmarketing of antiviral drugs for many acute viral diseases

This is so despite the existence of effective therapies.

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The first modest search for antiviral drugs occurred in the early 1950's

- Chemists looked at derivatives of the sulfonamide antibiotics- Synthesis of thiosemicarbazones active againstpoxviruses.

- Natural smallpox was still a major threat after WWII.- Millions of people died from smallpox in 1967!

In the 1960's and 1970's, drug companies launched huge "blind-screening"programs to find chemicals with antiviral activity

- spurred on by successes in the treatment of bacterial infections with antibiotics

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Blind Screening:Random chemicals and natural product mixtures tested for ability to blockreplication of a variety of viruses in cell culture systems.

"Hits", compounds or mixtures that blockin vitro viral replication- purified and fractions tested in various tissue culture and animal models

for safety and efficacy.

Promising molecules, called "leads", were modified systematically bymedicinal chemists

- to reduce toxicity, increase solubility and bioavailability- to improve other pharmacokinetic properties.

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Moreover, the mechanism of how these compounds inhibited the viruswas often unknown or speculative

- For example, the mechanism of action of Symmetrel wasn't deduced

until the early 1990s

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New technology, recombinant DNA technology, and sophisticated chemistry havemade targeted discovery possible:- go directly for a particular protein or mechanismEssential viral genes can be cloned, expressed in genetically tractable organisms,

purified, and analyzed in molecular and atomic detail.

Blind-screening procedures have lost popularity among drug hunters

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Some targets for antiviral drug discovery

Peptidomimetics (HIV)

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It is not unusual for the cost to bring an antiviral drug to market to

exceed $100-200 million dollars!

Up the down staircase of drug discovery:

Significant hurdles that stand in the way of finding effective antiviral drugs

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Resistance to anyantiviral drug must be anticipated when one appliescompounds that inhibit viral growth

- viruses replicate so efficiently

- have modest to high mutation frequencies

This is a special concern during the extended therapy required forchronic infections (HIV, HBV, HCV)

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There is a bright side:

Genetic analysis of resistance provides insightinto the antiviral mechanism

Can identify new strategies to reduce or circumvent the problem.

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The masters of error-prone replication. RNA polymerases are unable to correct errors

Host repair systems do not work on RNA

The average error frequencies reported

One mis-incorporation in 104

or 105

nucleotides polymerized, More than one million times greater than the rate for a host DNA genome.

Some hotspots in RNA genomes change at rates 10-100 fold higher than theaverage high error rate (spots where polymerase pauses, slips etc)

RNA viruses

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Replication of DNA virus genomes is not as error-prone as replication of RNA virusgenomes.

Most DNA polymerases can excise and replace mis-incorporated nucleotides.

Nevertheless, the mutation rate observed for DNA virus replication tends to be

higher than that observed for the host genome during cellular DNA replication.

Error rates for replication of single-stranded DNA viral genomes are higherthan those found for double-stranded DNA genomes.

DNA viruses

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Many antiviral compounds are nucleoside

and nucleotide analogs

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acyclovir

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Why are mutants resistant?

Two reasons:1. cannot phosphorylate the pro-drug

- mutations are in the viral thymidine kinase (TK) gene

2. unable to incorporate the phosphorylated drug into DNA

- mutations in the viral DNA polymerase gene- cant bind the acyclovir-nucleotide

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TK minus, acyclovir -resistant mutants can be devastating in AIDS patients, - can cause disseminated disease (invade large body organs) - are often resistant to other nucleoside analogs that require viral TK activity (cross-resistance)

These infections can be treated with Foscarnet, a DNA polymerase inhibitor - this drug has side-effect issues

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Figure 19.23

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Influenza virus neuraminidase inhibitors

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Zanamivir Oseltamivir

Inhibitors were design to mimic the natural ligand, sialic acid

The closer the inhibitor is to the natural compound, the less likely the target can mutateto avoid binding drug, while still maintaining viable function

No resistant influenza virus mutants have been obtained from immunocompetentpatients

Resistant virus mutants have been isolated in cell culture, but dont grow well

Resistance likely to appear with increased usage

Influenza virus neuraminidase inhibitors

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The attack on this devastating virus by academics and industry has beenunprecedented. 25 years of intense effort.

The entire pharmaceutical industry, the biotech industry, and many

academic centers mobilized to focus on finding a way to block the

replication of HIV and deal with the pathogenesis of AIDS.

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Current

Current

New

New

Nothing yet

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Acyclovir is phosphorylated by a viral kinase, but AZT is phosphorylated to theactive form by cellular enzymes.

Like Acyclovir, AZT acts as a chain terminator if incorporated into DNA because

the 3' OH of adenosine is replaced by an azido (N3) group.

Although phosphorylated AZT is NOT a good substrate for most cellularpolymerases, it is incorporated into the DNA copy of the HIV genome by HIVreverse transcriptase.

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AZT can be given orally and is absorbed rapidly,-but also is degraded rapidly by liver enzymes- effective half-life in a patient is only about 1 hour.

Patients must be given drug 2 or 3 times daily to maintain an effective

antiviral concentration.

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Mutants resistant to AZ arose immediately after AZT was licensed

These mutants have single amino acid changes at one of four sites in RT

RT enzymes with these amino acid changes do not bind phosphorylated AZT

New nucleoside analogs subsequently developed: Didanosine (ddI), Zalcitabine

(ddC), Stavudine (d4T), Lamivudine (3TC)

This lead to combination therapy, the use of two antiviral drugs to combatresistance

However, mutants resistant to two drugs still arose in less than a year

Resistance to nucleoside analogs

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Structure of HIV-1 RT highlighting the polymerase active site and NNRTI binding pocket.

nevirapine delavirdine efavirenz

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Resistance to NNRTIs

Resistant mutants are selected rapidly

Amino acid substitutions in any of seven residues that line binding sites on

enzyme confers resistance

Cannot be used alone for treatment of AIDS

Now used largely in combination therapy

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HIV antiviral drugs that target viral protease

HIV protease is absolutely required for

production of mature, infectious virions

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The active site is large enough to accommodate seven amino acids

The first inhibitor "leads" were peptide mimics of the natural cleavage sites(peptidomimetics)

- inhibitors of other proteases such as renin, a protease involved in hypertension,provided the firstleads for protease inhibitors.

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Figure 19.17

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Combination therapy:Based on the concept that the probability of resistance to a

combination of drugs is the product of their individual resistances

Two RT inhibitors and a protease inhibitor

Also known as HAART: highly active antiretroviral therapy

Used to require many costly pills; this summer a new, single pill

(size of a vitamin tablet) with THREE inhibitors was approved

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Table 19.11

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Raltegravir (Merck)

Licensed October 2007

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HIV fusion inhibitors

Fuzeon, licensed March 2003

36 amino acid syntheticpeptide: Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu- Ile-Glu-Glu-Ser-Gln-Asn-

Gln-Gln-Glu- Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu- Leu-Asp-

Lys-Trp-Ala-Ser-Leu-Trp-Asn- Trp-Phe-NH2

Binds to gp41 subunit of viralspike glycoprotein, blockstransition into fusion activeconformation

Peptide is expensive toproduce ($25,000/yr), must beinjected

Resistant mutants emerge,have amino acid changes inpeptide binding site on gp41

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