Anti-viral Chemotherapy

Guest · **posting in usmle forum about Microbiology**

Anti-bacterial drugs such as the penicillin antibiotics have proved very successful since they act against a bacterial structure, the cell wall, that is not present in eukaryotic cells. In contrast, most anti-viral agents have proved of little use therapeutically since the virus uses host-cell metabolic reactions and thus, for the most part, anti-viral agents will also be anti-cell agents. Thus, the alternative approach of stimulating the host's immune responses using vaccines has been most often pursued. Nevertheless, there are activities (i.e. enzymes) that are virus-encoded and therefore offer potential virus-specific targets. This is particularly the case with the viruses that have large genomes and code for their own replicative enzymes. Even so, unfortunately, many apparent anti-virals that are effective in vitro are ineffective in vivo.

A successful anti-viral will:

(i) interfere with a virus-specific function (either because the function is unique to the virus or the similar host function is much less susceptible to the drug)

or

(ii) interfere with a cellular function so that the virus cannot replicate. To be specific, the anti-viral must only kill virus-infected cells. This could be done by restricting drug activation to virus-infected cells.

An ideal drug will be:

Water-soluble
Stable in the blood stream
Easily taken up by cells

An ideal drug should NOT be:

Toxic
Carcinogenic
Allergenic
Mutagenic
Teratogenic

Toxicity of an anti-viral drug may be acceptable if there is no alternative: such as, for example, in symptomatic rabies or hemorrhagic fever

Obviously, a good drug must show much more toxicity to the virus than the host cell. We measure selectivity by the therapeutic index of the drug

Therapeutic index (T.I.): Minimum dose that is toxic to cell
Minimum dose that is toxic to virus

Effective drugs have a T.I. of 100-1000.

Just as with anti-bacterials, we must find a virus Achilles heel. This could be an enzyme that is unique to the virus so that the drug is not toxic to the host cell.

Among the other enzymes are: proteases, mRNA capping enzymes, neuraminidases, ribonucleases, kinases and uncoating enzymes.

The very first licensed anti-viral drug was idoxuridine (1963), a pyrimidine analog that acts against viral DNA synthesis. It is still used topically for epithelial herpetic keratitis but has largely been replaced because other drugs are less toxic. It is toxic because it lacks specificity, i.e. the drug inhibits the host DNA polymerase as well as the viral enzyme.

One of the better anti-viral drugs that we have dates from 1983: Acyclovir (acycloguanosine) which is a purine analog. It inhibits herpes DNA replication. It is also a nucleoside analog but, in contrast to idoxuridine, is highly specific and does not exhibit severe toxic side effects...

POSSIBLE PHASES OF LIFE CYCLE ON WHICH ANTI-VIRAL ATTACK MIGHT BE LAUNCHED

1) Attachment to cell surface, perhaps competition with a specific viral receptor
2) Uptake into intracellular vesicles (endosomes)
3) Uncoating of virus (loss of protein coat, fusion of lipid membrane with endosome/lysosome). Note: the endosome/lysosome compartment is acidic and inhibition of acidification of this compartment might be a good target
4) Transcription of genome to new RNA or DNA (polymerases are the target)
5) mRNA transcription
6) mRNA processing (poly A, methylation, capping, splicing)
7) Translation to protein
Cool

Post-translational modification of proteins (glycosylation, phosphorylation, fatty acylation, proteolysis). Some of these are essential for functional, infective viral progeny.
9) Assembly of the components into the whole virus

BINDING TO RECEPTOR OR UPTAKE INTO INTRACELLULAR VESICLES

There, were until recently, no good drugs that stop this for any virus (but see influenza sialidase inhibitor below). We could use a peptide that mimics the receptor such as soluble CD4 protein. This would bind HIV gp120 and stop it binding to the receptor on the cell surface. However, there is a stability problem. The soluble protein is rapidly broken down and cleared from the circulation, i.e. an efficacious concentration is not achieved for a useful period. Attempts have been made to stabilize proteins but little success has been achieved. There have been attempts to couple soluble CD4 to toxins to kill infected cells. Again with little success.

RFI-641 (biphenyl triazine) inhibits fusion of the membrane of respiratory syncytial virus (RSV) with the cell membrane. It seems to alter the conformation of the fusion (F) protein of the virus and is active in vivo in several animal models. It is active against RSV A and B strains. The drug is much better than ribovirin (the only routinely used drug in treating RSV infections) and seems to be RSV-specific. The drug has now been abandoned for routine use because of toxicity problems and delivery problems. It cannot be taken orally and so is delivered as an aerosol but elderly patients would likely find such a mode of delivery problematical. It may be of use in infants and derivatives may be less toxic

UNCOATING

Uncoating of the virus (i.e. the loss of the lipid envelope in membrane-containing viruses or the loss of nucleocapsid proteins in non-enveloped viruses) often occurs in low pH endosome or lysosomes, as the result of a pH-dependent fusogen. Note: Some viruses fuse with the plasma membrane (non-pH dependent); this is the case with herpes viruses and HIV.

Arildone, WIN compounds inhibit uncoating of picornaviruses which do not have a lipid membrane. The drug inserts into a canyon in VPI protein of virus and blocks ion transport.

Pleconaril This acts like a WIN compound in that it fits into a hydrophobic pocket in the nucleocapsid and interrupts the replication of the virus by stopping the shedding of nucleocapsid proteins from the RNA. This orally taken compound is broadly active against a variety of entero- and rhinoviruses (picornaviruses)

Contrary to promising earlier reports, the antiviral drug pleconaril does not appear to reduce the duration of the common cold any more than placebo, according to results of a preliminary study. In a separate study, the drug was ineffective in managing viral meningitis in children.

Even so, ViroPharma, the manufacturer, says it plans to begin 2 new trials of the drug. The company notes that although some of the preliminary results with pleconaril were disappointing, it did produce some satisfactory results. For example, in patients who were not taking other cold medications at the time, pleconaril reduced the average duration of symptoms from 9 days to 6.75 days, according to the company. ViroPharma also says it intends to confer with the FDA regarding the possibility of conducting another trial of pleconaril in the treatment of viral meningitis, this time in an adult population.

Amantadine, Rimantadine are lysosomotropic. They were originally thought to stop acidification of the endocytic vesicles but it is now thought that they may act on maturation of virus in trans-Golgi network which is also acidic . This is because cells that exhibit drug resistance seem to have a mutation in a Golgi function. These drugs act on maturation of influenza HA glycoprotein so that progeny virus is poorly infective.

These drugs good for oral prophylaxis against influenza A (but not influenza B). They are a good alternative to vaccine in immunocompromised patients and the elderly. Other than this, they are not used much in western countries. Prophylactic rimantadine has been used a lot in countries of the former USSR. Both of these drugs are licenced for use in US.

Acyclovir is phosphorylated first by a viral kinase to acycloGMP and then by cellular kinases to acycloGDP and acyclo GTP

NUCLEIC ACID SYNTHESIS

The best anti-viral drugs that we have are of this type.

They are selective because:

the virus may use its own enzyme to activate drug
and/or
the viral polymerases may be much more sensitive to the drug than the corresponding host enzymes
Thymidine kinase is an enzyme encoded by some viruses and used in the synthesis of their DNA. This enzyme can activate certain drugs so that there is selectivity. This is because the virus activates the drug to a toxic form while the uninfected cell does not

Three phosphates are added to thymidine. The first is added by the viral enzyme and the remainder by cellular enzymes.

The thymidine kinase of herpes simplex (and other) viruses allows the virus to grow in cells that do not have a high concentration of phosphorylated nucleic acid precursors. These are usually cells that are not replicating their genome (e.g. nerve cells). Resting cells do, however, have unphosphorylated nucleosides. By bringing in its own kinase, the virus can grow in non-dividing cells by phosphorylating the cells' nucleosides.

The name of the enzyme is a bit of a misnomer since it can work on other nucleosides than thymidine (thymidine happens to be the best substrate) i.e. the enzyme is non-specific as to substrate. This is in contrast to the host cell thymidine kinase which is very specific to thymidine. This lack of specificity of the viral enzyme allows it also to work on nucleoside-analog drugs and phosphorylate them. The host enzyme, because of its greater specificity, is much less good at this (and often does not phosphorylate the drug at all).

The fact that the viral enzyme is quite good at phosphorylating the drug has another advantage. We can administer the nucleoside-analog in a non-phosphorylated form. This is useful as it is difficult to get phosphorylated drug into the cell because plasma membranes are poorly permeable to phosphorylated compounds in the absence of a specific transport protein.

Thus the need for activation restricts use of drug to viruses with their own thymidine kinase or that cause cell to overproduce the endogenous enzyme (which may, if we are lucky, activate the drug to a lesser degree).

To recapitulate, the great use of these drugs results from the facts that:

i) they are only activated by the virus-infected cell
and
ii) the activated form of the drug is rendered even more specific as a result of the viral DNA polymerase being more sensitive to the drug than the host enzyme.

Most nucleic acid synthesis inhibiting drugs are nucleoside analogs with altered sugar, base or both.

Acyclovir (acycloguanosine) is the best example of such a drug. It is phosphorylated specifically by herpes simplex virus thymidine kinase to an active form and then it blocks DNA synthesis by inhibiting the polymerase competitively (binds to the active site of the enzyme)

DNA POLYMERASE INHIBITORS

(1) Sugar modifications

Acyclovir/Acycloguanosine. (Zovirax). As noted above, this drug is very selective and one of the better anti-virals. It is non-toxic to uninfected cells (except some renal dysfunction) because it is not activated by uninfected cells. It is a poor substrate for the cell thymidine kinase. Moreover, the viral polymerase of herpes simplex virus is 10 times more sensitive than cellular DNA polymerase. This drug is a competitive inhibitor - it competes with dGTP. When it gets incorporated into DNA, it acts as a chain terminator. HSV-1, HSV-2 and VZV are susceptible to acyclovir.

Chain termination

This drug also inhibits Epstein-Barr virus and Cytomegalovirus which do not have their own thymidine kinase. In this case, the specificity results from their DNA polymerase being very sensitive to the small amounts of drug that are activated by the host cell enzyme.

Acyclovir is effective against herpes simplex keratitis, latent HSV, fever blisters (H. labialis), genital herpes. Acyclovir-resistant mutants are a problem after long term use and have been shown to result from changes in the kinase or polymerase gene.

Ganciclovir (Cytovine). This drug is very similar to acyclovir, it just has an extra -OH. This makes it active against cytomegalovirus (CMV) which does not encode a thymidine kinase. The reason that it is active against CMV is that it is a good substrate of host cell thymidine kinase. Selectivity is achieved because the viral polymerase has 30 times greater affinity for gancyclovir than the host enzyme. But it is still toxic and it is difficult to achieve therapeutic dose orally. Use mostly for CMV retinitis in AIDS patients.

Adenosine arabinoside. (Ara-A) This has severe side effects and is only used in potentially lethal disease. It is also easily deaminated in the bloodstream to a less effective form, ara-hypoxanthine

Azidothymidine. (AZT, Retrovir, Zidovudine) This drug is also a chain terminator. It is phosphorylated by a cell kinase so it can be used against viruses without their own kinase. Reverse transcriptase (RNA-dependent DNA polymerase) is more sensitive to the drug than human DNA-dependent DNA polymerase accounting for the specificity but there are severe toxicity effects. It is used as an anti-HIV drug. Because of the use of RNA polymerase II in the synthesis of the viral genome and the consequent high rate of mutation of the virus, the selective pressure of the presence of the drug rapidly leads to the emergence of resistant viral mutants. All of these have changes in reverse transcriptase.

Other sugar modifications:

dideoxyinosine (DDI, Didanosine). Licenced for use with HIV in AZT-resistant patients and in combination drug treatments along with AZT.

Dideoxycytosine (DDC). Also licensed for use with AZT in HIV patients. Both of these drugs exhibit the same problems as AZT: pronounced toxicity and the rapid emergence of resistant HIV mutant strains.

Base modifications.

DU - iodo-deoxyuridine, Bromovinyl deoxyuridine (BVDU) These are pyrimidine analogs that are incorporated into DNA. They form unstable base pairs and mutant proteins.

Trifluorothymidine. Similar to BVDU. It also is activated by a cellular enzyme

(3) Non-nucleoside inhibitors of reverse transcriptase.

Because of the problems with AZT and the other nucleoside analogs in the treatment of HIV, interest has grown in another approach to inhibiting the same enzyme, reverse transcriptase. Alternative drugs might be useful in combination therapy since there is a limit to the number of mutations that reverse transcriptase can bear without losing function. Clearly, mutations resistant to a non-nucleoside non-competitive inhibitor of reverse transcriptase would be at a different site in the enzyme from the mutation that makes the enzyme resistant to a competitive nucleoside analog.

They are the most potent and selective reverse transcriptase inhibitors that we have, working at nanomolar concentrations. They have minimal toxicity in tests with cultured cells (anti-viral activity at 10,000 to 100,000-fold lower concentration than cytotoxic concentration) and have been shown to work synergistically with nucleoside analogs such as AZT. Moreover, they work against nucleoside-analog resistant HIV, at least in vitro. Thus, these drugs have high therapeutic index and also show good bioavailablity so that anti-viral concentrations are readily achievable

Non-competitive reverse transcriptase inhibitors

These drugs are non-competitive reverse transcriptase inhibitors.

Not surprisingly, since these drugs target reverse transcriptase, resistant mutants rapidly emerge, even after only a few passages in cultured cells. In clinical trials, resistant mutants also arose rapidly. They are therefore of little use in monotherapy; however, although resistant virus strains are cross resistant to other non-nucleoside reverse transcriptase inhibitors, they are not to nucleoside analog inhibitors. There is also some evidence that the drugs may be able to overcome resistance at the high concentrations that seem to be achievable.

There is now a collection of such agents that are chemically distinct:

Nevirapine (NVP or BIRG-587). In monotherapy, gives initial fall in HIV but resistance sets in and virus titers rise again to a high level. High concentrations that are achieved in the bloodstream may be of some use. This drug has been approved for therapy in AIDS patients.

Pyridinone derivatives (e.g. L-697,661). Trials in combination with AZT show delay in emergence of resistant virus but resistance is NOT prevented. Disappointing results mean that this drug is unlikely to be developed.

Bis (heteroaryl) piperazine compounds (e.g. atervidine and delavirdine (DLV)).

Considerable increases are observed in CD4+ cells in combination therapy (with AZT and 3TC). There have been promising results in patients with very low CD4+ cells that have prior treatment with AZT. In combination with AZT and 3TC, DLV may delay emergence of resistance to AZT. The drug is absorbed rapidly. This drug may be of great use in combination with a nucleoside analog such as AZT and the protease inhibitors discussed below.

Efavirenz (Sustiva)

(brand name Sustiva, formerly known as DMP-266), used in combination with other treatment, can suppress viral load at least as well as the protease inhibitor indinavir in the equivalent combination with nucleoside reverse transcriptase inhibitors. In a comparison of viral load reduction with efavirenz plus AZT plus 3TC, vs. a standard-of-care control group treated with indinavir plus AZT plus 3TC, the efavirenz combination suppressed viral load to below 400 copies in a significantly higher proportion of the volunteers than the control arm, at all time points between week 2 and week 24.

4) Other non-nucleoside polymerase inhibitors

Foscarnet (PFA, phosphono formic acid). This is a competitive inhibitor of DNA polymerase - it binds to pyrophosphate site. Herpes DNA polymerase is inhibited at 10-100x lower concentration than cell DNA polymerases giving some selectivity.

RNA SYNTHESIS INHIBITORS

Ribavirin. This drug is not a pyrimidine or purine. It inhibits influenza RNA polymerase non-competitively in vitro but poorly in vivo. It may act as guanosine analog and inhibit 5' cap formation on mRNA. The cap normally contains methyl guanosine. However, ribavirin is known to inhibit the production of infectious polio virus and this virus does not have a methyl guanosine cap; so there must be alternative mechanisms for ribavirin action. It is likely that this drug introduces multiple mutations into viral RNA rendering it incapable of a new round of cell infection

An aerosol form is used against RSV (respiratory syncytial virus) and the drug is used intra-venously against Lassa fever. N.B. Ribavirin can antagonize the effect of AZT as was found in some initial combination therapy trials against HIV.

Neoplanocin A (dihydropropyl adenine) also inhibits capping of mRNA.

RNA CLEAVAGE ENZYMES

Ribozymes are RNA molecules that have catalytic properties among which are the specific cleavage of nucleic acids. Heptazyme is a ribozyme that cleaves hepatitis C RNA at highly conserved regions (thereby reducing the possibility of the development of resistance). It recognizes and cuts all known types of the hepatitis C virus, thereby stopping viral replication

PROTEIN SYNTHESIS INHIBITORS

No specific inhibitors are available at this time

The process of retrovirus protease activity in which the protease starts as part of the POL polyprotein and then cleaves the polyprotein

PROTEIN PROCESSING INHIBITORS

a) Protease inhibitors

Many viruses must cleave the proteins that they make. In the case of surface glycoproteins, this is usually carried out by a host protease in the secretory pathway (e.g. in Golgi body). In the case of internal proteins, such as the polymerase or the group-specific antigens (GAGs), there is a viral protease that is encoded in the POL gene of retroviruses and by some other viruses.

Active site-directed inhibitors of the HIV aspartyl protease have been developed as this enzyme is not similar to known host proteolytic enzymes. The action of the HIV protease is crucial to viral infectivity. Now we have the promise of a drug regimen that can suppress indefinitely the progress of disease.

Protease inhibitor brand names (go here for further information on these and other drugs used to treat AIDS)

Saquinavir (Invirase, Fortonase)

Ritonavir (Norvir)

Indinavir (Crixivan)

Nelfinavir (Viracept)

These are all substrate analogs. When used individually they can drive down viral load to between one 30th and one 100th of initial value but sub-optimal doses of these inhibitors when used alone can result in loss of suppression after several months and an accumulation of multiple mutations in the protease gene giving a high level of drug resistance. Note: patients with sustained suppression do not develop the resistant mutations. This seems to be because replication must be maintained for the development of such mutations under the selective pressure of the drug.

Saquinavir (SQ) (Hoffman-La Roche). This is a hydroxyethylamine transition-state analog of the cleavage site on a protein recognized by the HIV protease. It is the least bio-available of the present protease inhibitors and is the least effective. Nevertheless, SQ + AZT + ddC reduced viremia with a rise in T4 cells in individuals with a T4 cell count of 50 - 300/mm3. SQ plus ddC versus any drug alone in individuals with prior AZT treatment showed significant benefit.

Ritonavir (Abbot Labs). Reduces AIDS-defining events and death by 58% compared to placebo. Causes nausea in 25% of patients

Indinavir (Merke). Indinavir plus two anti-RT drugs reduces HIV to such an extent that PCR cannot detect the virus in 85% of patients

Amprenavir (Agenerase) (Glaxo) is another protease inhibitor used in combination therapy

Highly active anti-retroviral therapies (HAART)

The combination therapy that has been very effective consists of zidovudine (AZT) , lamivudine (3TC) and protease inhibitor (Indinavir). Viral RNA levels before treatment start as high as 11 million copies per ml. and are reduced to undetectable levels in few weeks (we can measure as low as 20 copies /ml). The evidence suggests that there is NO replicating virus in these patients. This has been sustained for several years. When treatment is stopped, however, the virus comes back becuase of latent virus in memory T cells and possibly other cells.

The trouble with all of these complicated drug regimens is compliance. Also the combination of drugs must be taken at certain times. For example, failure to take saquinavir within 2 hours of high fat meal leads to no absorption of drug. On the other hand, Indinavir must be ingested with minimal food intake.

Even when their drug use was being recorded, 80% of participants in trials failed to adhere to the regimen if we include those that were off by a few hours but did not miss a dose. In patients that fail to take the three drugs for a week, there is a marked rise in viral load. Non-compliance with protease inhibitor therapy is of serious concern as the new virus that emerges is resistant to the inhibitor being taken and also resistant to other protease inhibitors. This is a major problem since the new resistant mutants may be transmitted to others. Thus if a patient is known to be likely to be non-compliant he/she should probably not be offered the drugs since resistance can emerge so quickly and can be spread to contacts. The HAART is very expensive, for example the combination of zidovudine/lamivudine/protease inhibitor costs $12,000 per year

Can we cure an HIV infection with drug therapy? Some years ago this would have been scoffed at. The drugs available then reduced viral load only to small extent and a double drug combination was thought to be acting well if it led to a rise in CD4 cells of 50/cu mm and the viral load was down 1.5 logs. Now these are considered to be infinitesimally small changes. If, as seems likely, the triple drug therapy when taken correctly stops all HIV replication in the patient, we might be able to eliminate the virus as cells that harbor it in the latent form are turned over. There is some evidence, however, that this may be difficult because latent reservoirs of HIV undoubtedly exist. When a CD4 cell leaves the thymus it is likely to meet an antigen, activate and subsequently die but a small subset of these cells become memory T cells and revert to a resting state. They may stay in the body for many years and if they are HIV-infected they will harbor the provirus. These cells therefore form a reservoir for HIV in the patient. The infection rate of this subset of cells does not appear to be great, less than 1 in 10,000 harbor latent viral DNA. This means that only some 10,000,000 of the 1000 trillion lymphocytes in the body are latently infected. But these may persist of decades and they will be untouched by the triple therapy, protease inhibitor combination. In individuals that have been treated with the combination therapy for more than 3 years, the rate of latently infected cells remains the same (1 in 10,000). Interestingly, the archival virus had the same resistance patterns as those that infected the patient. This means that in more than 3 years there were probably no new rounds of HIV replication. However, the bad news is that this reservoir of cells may last decades.

PROTEIN MODIFICATION INHIBITORS

(i) Glycosylation
2-deoxyglucose and D-glucosamine interfere with glycosylation in vitro but, not surprisingly, have little effect in vivo. Castanospermine (a natural product derived from a species of Australian chestnut) interferes with glycosylation of HIV and other retroviruses. It leads to a dramatic decrease in syncytia. Interest in this drug as an anti-HIV agent has waned.

(ii) Phosphorylation
No good drugs that target HIV by altering the phosphorylation of its proteins have been found

(iii) Myristoylation
An anti-HIV drug of this type is being tested. Fatty acylation is necessary for viable virus.

(iv) Sialidation. Two glycoproteins are found on the surface of influenza viruses; the hemagglutinin and the neuraminidase (sialidase). The latter has several functions. It allows the virus to move through mucous secretions in the respiratory tract so that it may infect new cells. Since sialic acid is the influenza receptor, it is necessary to remove sialic acid from the surface of the infected cell and of the virus so that viral particles may escape. The neuraminidase is therefore very important for the spread of the virus from cell to cell.

Zanamivir (Relenza), a new antiviral agent for influenza announced in the fall of 1997, is a potent inhibitor of the viral neuraminidase of types A and B influenza viruses. This is important as the previously available drugs such as rimantadine are ineffective against influenza type B. The design of Zanamivir is based on the three-dimensional structure of the neuraminidase. Treatment of community-acquired type A and B influenza with Zanamivir shortens the duration of major symptoms by about one day in the study group as a whole and about three days in the sicker patients if the drug is started early. Since no antiviral drug has been approved for the treatment or prevention of influenza B, Zanamivir could fill a niche in the control of influenza, but type B causes only about 35 percent of cases. Moreover, it has the disadvantage of requiring aerosol delivery to the respiratory tract, an approach that could prove difficult for many.

Another neuraminidase inhibitor, Tamiflu (generically called oseltamivir), a carbocyclic sialic acid analogue can be given orally.