Sunday, 9 December 2007

Virus Prevention and Control Part II

Continuing on antiviral drugs...

While viruses have fewer targets for drugs due to their smaller number of gene products, there are still many targets for potential drugs. Processes such as viral attachment, absorption, fusion, replication, transcription, translation, assmebly, anf virion release are a few such targets.

Entry inhibitors: One particularly effective drug target is that of virus entry into the cell. If the virus cannot enter a cell, then it canont cause an infection. One example is TAK799, a drug that blocks the HIV gp120 protein from attaching to the CCR5 co-receptor on T-cells. This prevents viral fusion, effectively blocking the entry of the virus. Other examples are Heparin and dextran sulfate, which are effective against many viruses, and Amantadine, which is used against influenza (specifically, it blocks the action of the M2 ion pump, thereby preventing the pH change in the endosome, resulting in the virion being trapped).

Replication inhibitors: Inhibiting viral replication is a favoured target of antiviral drugs. Most of the replication inhibitors are analogues of nucleosides, and are also chain terminators. They can be acyclic GTP analogues, acyclic nucleoside phosphonates, dideoxynucleotides, and pyrophosphate analogues. There are some non-nucleoside inhibitors as well. A well known example is that of Lamivudine which is given for Hepatitis B and HIV. Also known as 3TC, it has a sulphur at the 3' carbon instead of a hydroxyl group. This means that the growing DNA chain cannot be extended and elongation is terminated. It is only incorporated by reverse transcriptase, and not by cellular replication machinery, giving it specificity for infected cells.

Usully the mechanism by which replication inhibitors work is complex. First the drug is activated by the viral thymidine kinase enzyme, and then is selectively use by the viral replication machinery. This means that specificity is dependand upon two factors: that cellular kinases do not recognize it as a substrate, and that the cellular replication machinery does not use it as a valid nucleoside. Strains which are resistant, then, can have mutations in two different aspects.

Non-nucleoside analogue inhibitors work by some other method. They may affect the binding of a nucleoside to the polymerase or affect protein oligmerization.

Protease inhibitors: Many viruses require proteases to cleave their gene products from polyproteins (such as HCV, or the HIV protease). By inhibiting these, the assembly of the virus is also inhibited.

Neuraminidase inhibitors: In influenza, the virion is assembled at the cellular membrane and is attached to the cell surface by sialic acid. Neuaminidase cleaves the sialic acid, freeing the virion. Drugs which inhibit this chemical thus prevent virus release.

Viral Stability Inhibitors: Viruses with lipid envelopes are sensitive to detergents. Using detergents to break up the envelope will render the virus avirulent. Nonoxynol-9 is one such reagent. It is commonly used in spermacides to prevent HIV and HSV infections. Another is ST-246 which is used against poxviruses.

Antiviral Drug Resistance
Resistance to antiviral drugs is as big of a problem as that of bacteria to antibiotics. For example, before the introduction of HAART (higly active antiretroviral therapy), the drug Foscornet was used to treat HCMV which caused retinitis in 30% of HIV patients. Unfortunately, HCMV strains quickly became resistant to the drug and it can no longer be used.

How can viruses become resistant to drugs?
Polymerase fidelity: DNA Polymerase has a proofreading ability which allows it to remove incorrect nucleotides and nucleoside analogues. This means that viruses that use DNA Polymerase may be albe to avoid incorporating the drug into its growing DNA chain. RNA polymerase, however, lacks proofreading ability, meaning RNA viruses have a much higer error rate, and consequently, mutation rate, than DNA viruses. This could be the cause behind the aparent restriction in genome size of RNA viruses.

Also, no polymerase is 100% efficent at proofreading; if a mistake is made, it could potentially lead to a mutation that confers resistance to a drug.

Quasispecies: Viruses do not reproduce clonally, so each virus may be different. They are subject to evolutionary selection pressures so many different forms of a virus are produced; a drug is then not effective against all of them. An example is the resistance of Cidofovir in orthopoxvirus.

How can we determine if resistance will be a problem?
1) Pass the virus through a cell culture, increasing the concentration of a drug
2) Select the strains which gain resistance
3) Sequence the polymerase gene (or other gene target, depending on the drug) and determine where the mutations lie
4) make recombinate strains that contain the mutations
5) check for resistace in cell cultures and in mice
The last three steps are known as "marker rescue".

How can resistance be prevented?
Use drugs more cleverly! Using two or more antiviral drugs which have independent mechanisms will reduce the chances of a strain becoming resistant. A "drug vacation" may also work by reducing the selective pressure on strains to become resistant (though this has the added risk of allowing virus titres to increase).

No comments: