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When HIV-1 was characterized and identified as the causative agent of AIDS in 1983,70,71 scientists from all over the world joined in the search for a prevention or cure for the disease. Mapping the HIV-1 genome and elucidating the replication cycle of the virus have supplied key information.72 Biochemical targets, many of which are proteins involved in the replication cycle of the virus, have been cloned and se-quenced. These have been used to develop rapid, mechanism-based assays for the virus to complement tissue culture screens for whole virus. Several of the biochemical steps that have been characterized have served as targets for clinical candidates as well as for successfully licensed drugs.73,74

Despite the many advances in the understanding of the HIV virus and its treatment, there is not yet a cure for the infection. Emergent resistance75 to clinically proven drugs such as the RT inhibitors and the PIs has complicated the picture of good therapeutic targets. The idea of using a vaccine as a therapeutic tool has been complicated by the fact that the vaccine apparently can modulate its antigenic structures in its chronic infectious state.76


The chronology of vaccine development and use in the 20th century is nothing short of a medical miracle. Diseases such as smallpox and polio, which once ravaged large populations, have become distant memories. The technique of sensitizing a human immune system by exposure to an antigen so that an anamnestic response is generated on subsequent exposure seems quite simple on the surface. Hence, it is natural that a vaccine approach to preventing AIDS be tried. The successes achieved so far have involved live/attenuated or killed whole-cell vaccines and, in more recent times, recombinant coat proteins.

Successes with vaccines of the live/attenuated (low-virulence), killed whole virus or the recombinant coat protein types have primarily involved acute viral diseases in which a natural infection and recovery lead to long-term immunity.

This type of immunity is of the humoral or antibody-mediated type, and it is the basis for successes in immunizing the human population. Causative organisms of chronic infections do not respond to vaccines. The AIDS virus causes a chronic disease in which infection persists despite a strong antibody response to the virus (at least initially, HIV can circumvent the humoral response to infection by attacking and killing CD4+ T cells). These T cells, also known as T-helper cells, upregulate the immune response. By eradicating the CD4+ cells, the HIV virus effectively destroys the immune system. Cell-mediated immune responses are critical to the prevention and treatment of HIV infection. To be effective, a vaccine against HIV must elicit an appropriate cellular immune response in addition to a humoral response. In other words, the vaccine must have the potential to act on both branches of the immune system.

The initial work on vaccine development focused on iso-typic variants of the HIV envelope glycoprotein gp120 obtained by recombinant DNA techniques. This target was chosen because of concerns about the safety of live/attenuated vaccines. The gp120 glycoprotein is a coat protein, and if great care is taken, a virus-free vaccine is obtainable. Moreover, glycoprotein gp120 is the primary target for neutralizing antibodies associated with the first (attachment) step in HIV infection.77 Early vaccines were so ineffective that the National Institutes of Health suspended plans for massive clinical trials in high-risk individuals.78 There are several reasons why the vaccine failed.79 There are multiple subtypes of the virus throughout the world; the virus can infect by means of both cell-free and cell-associated forms; the virus has demonstrated its own immunosuppressive, im-munopathological, and infection-enhancing properties of parts of the envelope glycoprotein; and vaccines have not been able to stimulate and maintain high enough levels of immunity to be effective.

The failure of the first generation of AIDS vaccines led to a reexamination of the whole AIDS vaccine effort.79 As a guide for research efforts, several criteria for an "ideal" AIDS vaccine have been developed. The ideal AIDS vaccine should (a) be safe, (b) elicit a protective immune response in a high proportion of vaccinated individuals, (c) stimulate both cellular and humoral branches of the immune system, (d) protect components against all major HIV subtypes, (e) induce long-lasting protection, (f) induce local immunity in both genital and rectal mucosa, and (g) be practical for worldwide delivery and administration. It is not yet known how well the second-generation AIDS vaccines will satisfy the previously described criteria or when one might receive approval for widespread use in humans.

A new era in the treatment of AIDS and ARC was ushered in with the advent of some clinically useful, potent inhibitors of HIV. For the first time in the history of AIDS, the death rate reversed itself. There are several different classes of drugs that can be used to treat HIV infection. These are the NRTIs, the NNRTIs, the HIV PIs, the HIV entry inhibitors, and the HIV integrase inhibitors (IN). At present, at least 14 antiretroviral agents belonging to three distinct classes (NRTIs, NNRTIs, PIs) have been licensed for use in patients in the United States. All of these agents are limited by rapid development of resistance and cross-resistance; so commonly, three drugs are used at the same time, each acting at a different point in HIV replication. These drugs can effect dramatic reductions in viral load, but eventually, as resistance develops, the virus reasserts itself.

Nonnucleoside Reverse Transcriptase Inhibitors

Cloned HIV-1 RT facilitates the study of the effects of a novel compound on the kinetics of the enzyme. Random screening of chemical inventories by the pharmaceutical industry has led to the discovery of several NNRTIs of the enzyme. These inhibitors represent several structurally distinct classes. The NNRTIs share several common biochemical and pharmacological properties.74,80,81 Unlike the nucleoside antimetabolites, the NNRTIs do not require bioactiva-tion by kinases to yield phosphate esters. They are not incorporated into the growing DNA chain. Instead, they bind to an allosteric site that is distinct from the substrate (nucleoside triphosphate)-binding site of RT. The inhibitor can combine with either free or substrate-bound enzyme, interfering with the action of both. Such binding distorts the enzyme, so that it cannot form the enzyme-substrate complex at its normal rate, and once formed, the complex does not decompose at the normal rate to yield products. Increasing the substrate concentration does not reverse these effects. Hence, NNRTIs exhibit a classical noncompetitive inhibition pattern with the enzyme.

The NNRTIs are extremely potent in in vitro cell culture assays and inhibit HIV-1 at nanomolar concentrations. NNRTIs inhibit RT selectively; they do not inhibit the RTs of other retroviruses, including HIV-2 and simian immunodeficiency virus (SIV). The NNRTIs have high therapeutic indices (in contrast to the nucleosides) and do not inhibit mammalian DNA polymerases. The NRTIs and NNRTIs are expected to exhibit a synergistic effect on HIV, because they interact with different mechanisms on the enzyme. The chief problem with the NNRTIs is the rapid emergence of resistance among HIV isolates.75 Resistance is a result of point mutations in the gene coding for the enzyme. Cross-resistance between structurally different NNRTIs is more common than between NNRTIs and NRTIs. In the future, clinical use of the NNRTIs is expected to use combinations with the nucleosides to reduce toxicity to the latter, to take advantage of additive or synergistic effects, and to reduce the emergence of viral resistance.75,80 The tricyclic compound nevirapine (Viramune),82 the bis(heteroacyl)piper-azine (BHAP) derivative delavirdine (Rescriptor),83 and efavirenz84 have been approved for use in combination with NRTIs such as AZT for the treatment of HIV infection. Numerous others, including the quinoxaline derivative GW-420867X,84 the tetrahydroimidazobenzodiazpinone (TIBO) analog R-82913,85 and calanolide-A84 are in clinical trials.


Nevirapine (Viramune)82 is more than 90% absorbed by the oral route and is widely distributed throughout the body. It distributes well into breast milk and crosses the placenta. Transplacental concentrations are about 50% those of serum. The drug is extensively transformed by cytochrome P450 (CYP) to inactive hydroxylated metabolites; it may undergo enterohepatic recycling.

The half-life decreases from 45 to 23 hours over a 2- to 4-week period because of autoinduction. Elimination occurs through the kidney, with less than 3% of the parent compound excreted in the urine.82 Dosage forms are supplied as a 50 mg/5 mL oral suspension and a 200-mg tablet.


Delavirdine (Rescriptor)83 must be used with at least two additional antiretroviral agents to treat HIV-1 infections. The oral absorption of delavirdine is rapid, and peak plasma concentrations develop in 1 hour. Extensive metabolism occurs in the liver by CYP isozyme 3A (CYP3A) or possibly CYP2D6. Bioavailability is 85%. Unlike nevirapine, which is 48% protein bound, delavirdine is more than 98% protein bound. The half-life is 2 to 11 hours, and elimination is 44% in feces, 51% in urine, and less than 5% unchanged in urine. Delavirdine induces its own metabolism.83 Oral dosage forms are supplied as a 200-mg capsule and a 100-mg tablet.


Efavirenz (Sustiva)84 is also mandated for use with at least two other antiretroviral agents. The compound is more than 99% protein bound, and CSF concentrations exceed the free fraction in the serum. Metabolism occurs in the liver. The half-life of a single dose of efavirenz is 52 to 76 hours, and 40 to 55 after multiple doses (the drug induces its own metabolism). Peak concentration is achieved in 3 to 8 hours. Elimination is 14% to 34% in urine (as metabolites) and 16% to 41% in feces (primarily as efavirenz).84 The oral dosage form is supplied as a capsule.

HIV Protease Inhibitors

A unique biochemical target in the HIV-1 replication cycle was revealed when HIV protease was cloned and ex-pressed86,87 in Escherichia coli. HIV protease is an enzyme that cleaves gag-pro propeptides to yield active enzymes that function in the maturation and propagation of new virus. The catalytically active protease is a symmetric dimer of two identical 99 amino acid subunits, each contributing the triad Asp-Thr-Gly to the active site.86,87 The homodimer is unlike monomeric aspartyl proteases (renin, pepsin, cathepsin D), which also have different substrate specificities. The designs of some inhibitors86,87 for HIV-1 protease exploit the C2 symmetry of the enzyme. HIV-1 protease has active site specificity for the triad Tyr-Phe-Pro in the unit Ser-(Thr)-Xaa-Xaa-Tyr-Phe-Pro, where Xaa is an arbitrary amino acid.

HIV PIs are designed to mimic the transition state of hydrolysis at the active site; these compounds are called analog inhibitors. Hydrolysis of a peptide bond proceeds through a transition state that is sp3 hybridized and, hence, tetrahedral. The analog inhibitors possess a preexisting sp3 hybridized center that will be drawn into the active site (one hopes with high affinity) but will not be cleavable by the enzyme. This principle has been used to prepare hundreds of potentially useful transition state inhibitors.86,87 Unfortunately, very few of these are likely to be clinically successful candidates for the treatment of HIV infection. Because HIV PIs are aimed at arresting replication of the virus at the maturation step to prevent the spread of cellular infection, they should possess good oral bioavailability and a relatively long duration of action. A long half-life is also desirable because of the known development of resistance by HIV under selective antiviral pressure.74,75 Resistance develops by point mutations.

Most of the early PIs are high-molecular-weight, dipep-tide- or tripeptide-like structures, generally with low water solubility. The bioavailability of these compounds is low, and the half-life of elimination is very short because of hydrolysis or hepatic metabolism.88 Strategies aimed at in creasing water solubility and metabolic stability have led to the development of several highly promising clinical candidates. Saquinavir (Invirase),81 indinavir (Crixivan),89 ritonavir (Norvir),90 nelfinavir (Viracept),91 and ampre-navir (Agenerase)92 have been approved for the treatment of HIV-infected patients. Several others are in clinical trials.

There is an important caution for the use of PIs. As a class, they cause dyslipidemia, which includes elevated cholesterol and triglycerides and a redistribution of body fat centrally to cause the "protease paunch" buffalo hump, facial atrophy, and breast enlargement. These agents also cause hyperglycemia.


Saquinavir (Invirase)89 is well tolerated following oral administration. Absorption of saquinavir is poor but is increased with a fatty meal. The drug does not distribute into the CSF, and it is approximately 98% bound to plasma proteins. Saquinavir is extensively metabolized by the firstpass effect. Bioavailability is 4% from a hard capsule and 12% to 15% from a soft capsule. Saquinavir lowers p24 antigen levels in HIV-infected patients, elevates CD4+ counts, and exerts a synergistic antiviral effect when combined with RT inhibitors such as AZT and ddC.93-95 Although HIV-1 resistance to saquinavir and other HIV PIs occurs in vivo, it is believed to be less stringent and less frequent than resistance to the RT inhibitors.96 Nevertheless, cross-resistance between different HIV PIs appears to be common and additive,97 suggesting that using combinations of inhibitors from this class would not constitute rational prescribing. The drug should be used in combination with at least two other antiretroviral drugs to minimize resistance. Dosage forms are Invirase (hard capsule) and Fortovase (soft capsule).

Ritonavir, Amprenavir, and Nelfinavir

Ritonavir (Norvir),98 amprenavir (Agenerase),99 and nelfinavir (Viracept)100 have similar properties and cautionary statements. All cause dyslipidemia, and they have a host of drug interactions, mainly because they inhibit CYP3A4. These agents must always be used with at least two other antiretroviral agents. Used properly, the PIs are an important part of HIV therapy.


Lopinavir is a protease inhibitor that has been approved for use in combination with ritonavir for patients with HIV who have not responded to other treatment modalities. Lopinavir is used in excess over ritonavir. Ritonavir at amounts given has no antiretroviral activity, Ritonavir inhibits lopinavir's metabolism by CYP3A4, causing a higher level of lopinavir in the system. The combination is the first protease inhibitor approved for patients as young as 6 months of age.


When administered with a high-fat diet, indinavir (Crixivan)90 achieves a maximum serum concentration of 77% of the administered dose. The drug is 60% bound in the plasma. It is extensively metabolized by CYP3A4, and seven metabolites have been identified. Oral bioavailability is good, with a tmax of 0.8 ± 0.3 hour. The half-life of elimination is 1.8 hour, and the elimination products are detectable in feces and urine. Indinavir also causes dyslipidemia. The available dosage forms are capsules of 200 mg, 333 mg, and 400 mg.


Atazanavir is an antiretroviral agent that has been approved by the FDA for use in combination with other anti-RT agents for the treatment of HIV infections. The drug is always used in combination with RT inhibitors.


Fosamprenavir is used in combination with other HIV drugs in adult patients. Like the other PIs, this compound is a prodrug that produces the active drug upon hydrolysis. In this case, the active drug is amprenavir, a peptidomimetic transition state inhibitor. Fosamprenavir is typically administered in combination with RT inhibitors.


Tipranavir is unique among the PIs because it is not a pep-tidomimetic compound. It does appear to bind to the active site of HIV-1 protease the same as the peptidomimetics do. The benefit of this agent is that, because it is a different chemical structure, cross-resistance does not develop to the same extent as seen with the peptidomimetics. The drug is administered with a booster dose of ritonavir. This protocol inhibits CYP3A4, causing the levels of tipranavir to increase.

Several other nonpeptide inhibitors of HIV protease have been developed as a result of two very different approaches. For example, the C2 symmetry of the active site of the enzyme was exploited in the structure-based design of the symmetric cyclic urea derivative DMP-323.101 This inhibitor exhibited potent activity against the protease in vitro, excellent anti-HIV activity in cell culture, and promising bioavailability in experimental animals. In phase I clinical trials, however, the bioavailability of DMP-323 was poor and highly variable, possibly because of its low water solubility and a high fraction of hepatic metabolism. Subsequent synthesis of nonsymmetric derivatives DMP-850101 (page 351) and DMP-851101 yielded in vitro antiviral potency comparable with that of the already-approved PIs. These were selected as clinical candidates on the basis of their favorable pharmacokinetics in dogs. In a second approach, random screening of chemical inventories yielded the 5,6-dihydropyran -2-one-basedinhibitor102 PD-178390 (page 351). This compound, in addition to having good potency against HIV protease and good anti-HIV activity in cell culture, exhibits high bioavailability in experimental animals. PD-178390 appears not to share the resistance profile of the other PIs, and no virus resistant to the compound emerged, even during the prolonged in vitro selection.

Dipeptide PIs containing 2-hydroxy-3-amino-4-arylbu-tanoic acid in their scaffold showed promising preclinical results. JE-2147103, containing the allophenylnorstatin moiety, exhibited potent in vitro anti-HIV activity. JE-2147 appears to fully retain its susceptibility against various HIV strains resistant to multiple approved PIs and exhibits good oral bioavailability and a good pharmacokinetic profile in two animal species. Also, emergence of resistance was considerably delayed with JE-2147.

host cell membrane. In addition to gp120-chemokine receptor interaction, the fusion activity of gp41 is currently being explored as a novel target for antiretroviral therapy. At least one agent from each class is in clinical testing.

Chemokine Receptor Binders

Most HIV-1 isolates rely on the CCR5 coreceptor for entry (R5 strains). In later stages of the disease, however, more pathogenic selection variants of the virus emerge in about 40% of individuals, which use the CXCR4 coreceptor in addition to CCR5 (R5X4 strains) or the CXCR4 receptor only (X4 strains). Bicyclam compound AMD-3100104 was the first compound identified as a CXCR4-specific inhibitor that interferes with the replication of X4 but not R5 viruses. The compound is currently in phase II clinical evaluations. It is used as an injectable agent because of its limited bioavailability.

HIV Entry Inhibitors

Entry of HIV into a cell is a complex process that involves several specific membrane protein interactions. Initially, viral glycoprotein gp120 mediates the virus attachment via its binding to at least two host membrane receptors, CD4+ and the chemokine coreceptor. This bivalent interaction induces a conformational change in the viral fusion protein gp41. Protein gp41 acts as the anchor for gp120 in the virus. With the conformational change, the viral envelope fuses with the

Several positively charged 9- to14-mer peptides have been described as capable of blocking the CXCR4 corecep-tor. A small molecule, TAK-779,105 exhibits high-affinity binding to the CCR5 coreceptor, specifically blocking R5 isolates.

Inhibitors of GP41 Fusion Activity

The fusion of the HIV-1 viral envelope with host plasma membrane is mediated by gp41, a transmembrane subunit of the HIV-1 glycoprotein subunit complex. Pentafuside106 (T-20) is a 36-mer peptide that is derived from the C-terminal repeat of gp41. Pentafuside appears to inhibit the formation of the fusion-competent conformation of gp41 by interfering with the interaction between its C- and N-terminal repeat. Pentafuside is a potent inhibitor of HIV-1 clinical isolates, and it is currently in clinical trials.

Integrase Inhibitors

Two closely related types of small molecules that block strand transfer catalyzed by recombinant integrase have been identified. Both types show in vitro antiviral activity. The diketo acids107 inhibit strand transfer catalyzed by recombinant integrase with an IC50 less than 0.1 fim. Mutations that conferred resistance to the diketo acids mapped near conserved residues in the IN enzyme. This finding demonstrates that the compounds have a highly specific mechanism of action. X-ray crystallography of the bound tetrazole108 derivative revealed that the inhibitor was centered in the active site of IN near acidic catalytic residues.

Short-Interfering RNA

RNA interference is a phenomenon that has been recently used as a way to silence genes that are part of viral replication cycles. The siRNAs very specifically interrupt gene function and switch off some aspect of a disease. The siRNAs are found in higher organisms (eukaryotes) and are typically double-stranded duplexes of RNA of about 21 base pairs. In nature, these siRNAs are produced by excision from a parent duplex RNA molecule. The siRNAs instruct the cell to split specific mRNAs that have identical sequences as the siRNAs. As antiviral therapy, the idea would be for the siRNAs to stop synthesis of mRNAs of a pathogenic organism. Antiviral siRNA therapies can be tailored for any given pathogen. The use of siRNAs as potential therapeutic modalities is a highly active area of research at the present time.

Combination Antiviral Therapy

Combination antiviral therapy, in which a mixture of drugs possessing different mechanisms of action, has been shown to be advantageous for several reasons. The antiviral effect of the combination is excellent, toxicities are decreased, and resistance to any drug in the combination is slow to develop. Resistance to single drugs such as amantadine, ganciclovir, and acyclovir is problematic. Administered of a given agent in combination with other types of drugs retards the development of such resistance. Combination treatment is especially important in antiretro-viral therapy.

Typical antiretroviral therapy, as exemplified by HIV treatment, includes combinations of NRTI or NNRTI along with PIs. The key that makes the combination work is that the drugs act to inhibit HIV virus replication at different stages of the viral infective cycle. The RT inhibitors (NRTIs or NNRTIs) prevent RNA formation or viral protein synthesis or inactivate the catalytic site of RT (NNRTIs). The PIs act once the provirus integrates into the host's genes. Protease is necessary to split viral precursor polypeptides into new virus. The combination of RTs and PIs is synergistic. There are many two- and three-drug combinations that have been reported to be useful in various viral infections.

• review questions^

1. What is the difference between the lytic cycle and the lysogenic cycle in viruses?

2. What is an RNA virus? What is a DNA virus?

3. What is the mechanism of action of an antimetabolite antiviral drug?

4. What is chemoprophylaxis? How might you exercise chemoprophylaxis against the influenza virus?

5. What is the mechanism of action of amantadine?

6. Describe the mechanism of action of the IFNs as antiviral agents.

7. Describe the class of agents that we call NRRTIs.

8. What kinds of drugs are typically used in combination therapy of HIV?

9. Describe the HIV entry inhibitors.

10. How do the HIV protease inhibitors exert their mechanism of action?

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