Instructor: Patrick M. Woster, Ph.D.
Note: These notes are largely excerpts from my chapter in Foye's Principles of Medicinal Chemistry, 6th Edition, Chapter 43.
The Virus as a Drug Target
Viruses are the smallest of the human infectious agents, and range in size from about 20 nm to about 300 nm in diameter. They contain one kind of nucleic acid, either RNA or DNA, as their entire genome, which codes for a variety of enzymes and other proteins used in replication and transmission of the organism. Although viruses are simple organisms, they are a significant causative agent for numerous human diseases, and as such represent one of the major challenges in the area of drug discovery. Agents that are used clinically for a variety of viral diseases act by targeting processes that are specific to the virus, such as a unique viral enzyme or a necessary process such as transcription. However, to date, no drug has been discovered that is truly curative for viral infection. In addition, because viruses have the ability to undergo mutations, resistance to existing therapies can develop. The discovery of new antiviral agents is thus an important ongoing effort in medicinal chemistry.
Viral Structure and Taxonomy
Numerous species of virus that infect bacteria, plants and animals have been identified, and they exhibit a remarkable range of diversity. All viruses exist as obligate cellular parasites, and as such they do not need to possess the complex biochemical machinery that is characteristic of higher organisms. However, they do have a defined macromolecular structure that is designed to protect them from the environment and facilitate their entry into cells. The basic subunit of a virus is its genome, which can be made up of either DNA or RNA. The nucleic acid portion of a virus can be single or double stranded, and may be present in linear or circular form. Viral genomic DNA or RNA is often associated with basic nucleoproteins, and may be surrounded by a symmetrical protein known as a capsid. The capsid is made up of repeating structural units known as protomers, which themselves are made up of non-identical protein subunits. The combination of the nucleic acid core and the capsid is termed the nucleocapsid, and in some cases this comprises the entire virus. In other cases, the nucleocapsid structure is surrounded by a lipid-containing membrane that is derived during viral maturation, when the virus undergoes budding through the host cell membrane. The complete virus particle, with or without an envelope, is termed the virion. Viral architecture can be grouped into three types based on the arrangement of morphologic subunits, and each virus exhibits cubic (icosahedral) symmetry, helical symmetry or has a complex structure. Icosahedral virions are symmetrical structures that contain 20 surfaces, each of which is an equilateral triangle. A sufficient number of capsid structural units must be employed in the icosahedron to make a capsid large enough to encapsulate the viral genome. Morphologic units called capsomeres are seen on the surface of icosahedral virus particles. These structures are clusters of polypeptides, but they do not necessarily correspond to the chemically defined structural units. Some viruses arrange their structural subunits into a standard helical formation. In viruses with helical symmetry, protein subunits systematically bind to the viral nucleic acid, ultimately forming a nucleocapsid helix. The filamentous nucleocapsid is then coiled inside a lipid-containing envelope. Unlike icosahedral virions, regular, periodic interaction between capsid protein and viral nucleic acid prevents the formation of "empty" helical particles. Finally, some virus particles, such as the large and complex poxviruses, do not exhibit cubic or helical symmetry, but instead form more complicated structures that can be spherical, brick-shaped or ovoid. A subset of complex viruses are termed pleomorphic, in that they assume multiple complex morphologies.
Viral taxonomy is complex, and viruses are classified according to a number of factors, including morphology, properties of the genome (i.e. DNA vs. RNA, single strand vs. double strand, linear or circular, sense or antisense), physicochemical properties, structure of associated proteins, replication strategy and so on. Viruses are separated into major groups called families, with names that end in the suffix –viridae, and then into genera that end in –virus. Thus pox viruses are in the family Poxviridae, and in the genus poxvirus.
Viral Replication
During viral replication, all of the macromolecules required by the virus are synthesized in a highly organized sequence. The replication cycle (see below) begins when the intact virion binds to a host cell through electrostatic adsorption to a specific “receptor” site. This process is known as the attachment phase. Attachment is most likely a fortuitous event resulting from structural complementarity between the exterior structure of the virion and a normal cell surface structure on the host cell. For example, human immunodeficiency virus binds to the CD4 receptor on cells of the immune system, rhinoviruses bind ICAM-1, and Epstein-Barr virus recognizes the CD21 receptor on ß-cells. When attachment has been achieved, the virion enters the penetration phase, the process by which it gains entry into the host cell. Penetration may occur by receptor-mediated endocytosis, fusion of the viral envelope with the cell membrane, or in some cases by direct penetration of the membrane. Following penetration of the cell, viruses must be uncoated, resulting either in the naked nucleic acid, or in the nucleocapsid form which usually contains polymerase enzymes. After they have been uncoated, viruses are no longer infectious.
Once the virus has penetrated the cell and uncoated, it enters a segment of its life cycle known as the eclipse period, the length of which varies with the type of virus. It is during this time that the virus utilizes host resources to replicate and produce necessary viral proteins. Cells that can support viral reproduction are termed permissive, and as a result the infection is known as productive, since it results in new viral particles. When new infectious viral particles are produced, host cellular metabolism may be completely directed to the production of viral products, resulting in destruction of the cell. In other cases, host cell metabolism is not dramatically altered, and the infected cell can survive. During viral reproduction, up to 100,000 new virions can be produced, and the replication cycle can vary from a few hours to more than 3 days. Some cells types, called non-permissive, are unable to support the reproduction of an infective virion, resulting in an abortive infection. Abortive infections also occur when the virus itself is defective. Either situation can lead to a latent infection, where the viral genome may persist in a surviving host cell. As will be described below, such an infection can lead to the transformation of a cell from normal to malignant.
The strategies used by various viruses to replicate vary widely, but all are characterized by the need to transcribe mRNA that is suitable for translation of viral proteins. There are several pathways leading to the required mRNA, after which the host enzymes and raw materials are used to make viral proteins. Early viral proteins used in replication are synthesized immediately after infection, while late proteins used to produce the complete virion structure are synthesized after viral nucleic acid synthesis. Most DNA viruses contain double stranded DNA as their genome, and thus can replicate using host cell machinery to produce mRNA directly. Papillomavirus, adenovirus and herpesvirus are replicated in the host nucleus, and thus use transcriptional enzymes of the host (i.e. DNA-dependent RNA polymerase) to synthesize mRNA. This mRNA is then translated to form proteins needed by the virus, including enzymes (e.g. DNA-dependent DNA polymerase) used to produce progeny DNA copies. These progeny DNA strands are infectious. By contrast, poxviruses replicate in the cytoplasm using a mechanism that is not well understood, wherein the genome is initially transcribed by a viral enzyme in the virion core. Parvoviruses contain a single-stranded DNA genome, and must synthesize double-stranded DNA in the host nucleus prior to synthesis of mRNA and translation of proteins. This process may or may not require a helper virus such as herpes simplex. The hepatitis B viral genome, comprised of double-stranded DNA, contains numerous gaps that must be repaired using a DNA polymerase packaged in the virion before transcription to form mRNA.
Compared to DNA viruses, those viruses with RNA-based genomes have evolved a wide variety of reproductive strategies. The single stranded RNA viruses may be divided into three groups that differ in the method by which the RNA genome is utilized. In all three groups, the RNA genome must serve two functions: to be translated to form protein and to be replicated to form progeny RNA strands. The first group is comprised of viruses such as picornaviruses, flaviviruses and togaviruses that have an RNA genome that can be used directly as mRNA. Viral RNA that can be used as mRNA is by convention termed (+) or sense-strand RNA. In most cases (e.g. picornoviruses) this sense-strand RNA binds to the host ribosome shortly after entering the cell, where it is read and used to produce a single polypeptide called the polyprotein. The polyprotein is then processed by autocatalysis and various proteolytic enzymes to produce the required viral proteins. In some cases (e.g. togaviruses), only a portion of the RNA genome is available to be translated by the host ribosome. Following the initial translation of the sense strand, it serves a second function, namely to serve as a template for the synthesis of a (-) or antisense strand via an RNA-dependent RNA polymerase. This antisense strand then can be used to produce additional sense strand RNAs that are infectious, and can also serve as mRNA. These progeny sense RNA strands are then packaged into an intact virion prior to transmission to another host cell.
The second group of single stranded RNA viruses, including orthomyxoviruses, bunyaviruses, arenaviruses, paramyxoviruses, filoviruses and rhabdoviruses, all contain an antisense RNA genome that can only be used for transcription of new RNA. All antisense RNA viruses contain an RNA transcriptase as part of their virion, because the host cell does not have this type of RNA-dependent RNA polymerase. In the first round of genome expression, a series of short sense strand RNAs are made, and then translated to form the required viral enzymes for replication. Ultimately, these enzymes are used to produce a full-length sense RNA strand, which is then used to make multiple copies of the antisense viral genome. The progeny antisense DNA strands by themselves are not infectious, because they have not yet been packaged with the required RNA transcriptase. When the progeny antisense RNA has been synthesized, it is packaged into an intact virion, in which form it becomes infectious prior to transmission to another cell.
The third group of RNA viruses are the retroviruses, in which single stranded RNA exists as a dimer of a sense- and antisense strand. The genomic RNA strands can be base-paired, although the structure of this complex is not well understood, or the strands can be hydrogen bonded to other macromolecules in the virion. Retroviral genomic RNA serves a single function, namely, to act as a template for the formation of double-stranded viral DNA. Host cells do not contain an enzyme which can form DNA from viral RNA, and thus the virion of a retrovirus must contain a reverse transcriptase enzyme, as well as various host tRNA molecules. Transcription of the genome begins when a complex of reverse transcriptase and tRNA binds to the viral genome. A complimentary DNA strand is then synthesized using one of the host tRNAs as a primer, and the original RNA strand is digested by RNAse H, and viral ribonuclease packaged in the virion. A complimentary DNA strand is then synthesized, and the resulting double-stranded DNA is translocated to the nucleus, where viral enzymes incorporate genome-length viral DNA into the host genome. In some cases, the viral portion of the genome can remain dormant for long periods, or it may be immediately used to make progeny viral RNA, a process that is catalyzed by host RNA polymerase II. Transcription produces both shortened segments that are used to make polyproteins, and full length progeny RNA. The polyproteins are processed to form various viral proteins, while the full length RNA is packaged into an infectious virion.
In addition to replication of the viral genome, a number of other structures associated with the complete virion can also be made. A number of viral proteins may be synthesized that have important functions in the structure, transmission and survival of the virus. These proteins can protect the genetic material in the virus from destruction by nucleases, participate in the attachment process and provide structure and symmetry to the virion. In addition, in certain cases where the virus requires an enzymatic process for which there is no host enzyme, a virion may include enzymes such as RNA polymerases or a reverse transcriptase. Some viruses require a lipid envelope that contains transmembrane proteins specifically coded for by the virus, and which envelopes the genetic material during viral budding. Viral envelopes contain glycoproteins that are involved in cell recognition during attachment to the host cell. These glycoproteins often reflect the composition of glycoproteins in the host cell. They are a determinant of the antigenic nature of viruses, and thus facilitate recognition by the immune system of the host. However, depending on their composition, they can also help the virus elude neutralization by the immune system.
Viruses use one of two strategies for exiting infected cells. Non-enveloped viruses (picornoviruses, rheoviruses, etc.) complete their maturation by assembling into their corresponding virion within the cell nucleus or the cytoplasm. For example, picornoviruses assemble by clustering 60 copies of each of three viral proteins, called VP0, VP1 and VP3, into a structure called a procapsid. Viral RNA is then packaged into the procapsid, and proteolytic cleavage of VP0 produces two new viral proteins called VP2 and VP4. The resulting conformational change produces a stable and symmetrical structure that shields the genome from degradation by host nucleases. In most cases, destruction of the host cell is required when the virion exits. Enveloped viruses (all antisense RNA viruses, togaviruses, flaviviruses, coronaviruses, hepadnaviruses, herpesviruses and retroviruses) contain proteins that carry signal sequences and markers that cause them to be inserted into the inner and outer surface of the host cell cytoplasmic membrane. Viral proteins on the outer surface are glycosylated using host enzymes, and then displace host cell surface proteins and collect into patches. Viral nucleocapsids that recognize proteins on the inner surface of the membrane, where they bind and are engulfed by the patch area of the membrane. The completed virion exits the cell by budding and release into the extracellular space. Viral egress can have a variety of effects on the host cell, ranging from destruction of the cell to minimal non-cytolytic effects. Herpesviruses differ from pther enveloped viruses in the manner in which they form their envelope. The nucleocapsid is formed in the nucleus, and final maturation of the virion occurs only on the inner surface of the host cell membrane, forming vesicles that are stored in between the inner and outer aspect of the cell membrane. Egress of the herpesvirus vesicle always occurs through destruction of the host cell.
Tumor Viruses
Viruses are etiologic factors in the development of several types of human tumors, most notably cervical cancer and liver cancer. At least 15% of all human tumors worldwide have a viral cause. Tumor viruses can be found in both the RNA and DNA virus kingdoms. The list of human viruses presently known to be involved in tumor development includes four DNA viruses (Epstein-Barr virus (EBV), certain papilloma viruses, hepatitis B virus (HBV), and the Kaposi sarcoma herpesvirus HHV-8), and two RNA viruses, (adult T-cell leukemia virus (HTLV-1) and the hepatitis virus (HCV). Tumor viruses alter cellular behavior through the use of a small amount of genetic information, using two general strategies. The tumor virus either introduces a new "transforming gene" into the cell (direct-acting), or the virus alters the expression of a preexisting cellular gene or genes (indirect-acting). In both cases, normal regulation of cellular growth processes is lost. Viruses alone cannot act as carcinogens, and other events are necessary to disable regulatory pathways and checkpoints in order to produce transformed, malignant cells. The processes used in the transformation of host cells by human tumor viruses are very diverse.
Cellular transformation may be defined as a stable, heritable change in the growth control of cells that results in tumor formation. Transformation from a normal to a neoplastic cell generally requires the retention of viral genes in the host cell. In the majority of cases, this is accomplished by the integration of certain viral genes into the host cell genome. Retroviruses incorporate their proviral DNA, formed through the action of reverse transcriptase, into host cell DNA. By contrast, DNA tumor viruses integrate a portion of the DNA of the viral genome into the host cell chromosome.
All RNA tumor viruses are members of the retrovirus family, and belong to one of two classes. Class I RNA viruses are direct-transforming, and carry an oncogene obtained through accidental incorporation from the host cell. No class I RNA viruses are known to produce tumors in humans. Class II or chronic RNA tumor viruses are weakly transforming, and do not carry host cell-derived oncogenes. The two known cancer-causing retroviruses in humans act indirectly. They often act by inserting their proviral DNA into the immediate neighborhood of a host cellular oncogene. The human adult T-cell leukemia virus HTLV-1 acts in this manner, thus increasing the number of preneoplastic cells and facilitating secondary cellular changes leading to transformation.
DNA tumor virus strains exist among the papilloma-, polyoma-, adeno-, herpes-, hepadna-, and poxvirus groups. DNA tumor viruses encode viral oncoproteins that are important for viral replication, but also affect cellular growth control pathways. For example, inactivation of the Rb and the p53 pathway by viral transforming proteins is a common strategy used papovaviruses and adenoviruses. As was mentioned above, all DNA tumor viruses kill their host cell when the infectious virion is released to infect other cells. Thus, transformation and tumorigenicity are entirely dependent on a host cell interaction with the virus that do not involve viral spread to other cells, and cells transformed by DNA tumor viruses depend on the continued expression of the virally encoded oncogene.
Recent studies have revealed that the human tumor viruses EBV, HHV-8, human papillomavirus (HPV), HBV, HCV, and HTLV-1 express proteins that are targeted to the mitochondria. Because the mitochondria play a critical role in energy production, cell death, calcium homeostasis, and redox balance, these proteins have profound effects on host cell physiology. Further study of these proteins and their interactions with mitochondria will aid in the understanding of the mechanisms of viral replication and tumorigenesis, and could reveal important new targets for anti-tumor therapy.
Prions
Although they are not viruses, the infective proteins known as prions have sufficient similarities to viruses to warrant their discussion in this chapter. Prions are small proteins that have been shown to cause a variety of transmissible spongiform encephalopathies (TSEs), which are rare neurodegenerative disorders typified by symptoms in the CNS such as spongiform changes, neuronal loss, glial activation and the accumulation of amyloid aggregates of an abnormally folded host protein. Human prion diseases include Kuru, Creutzfeldt-Jakob disease (variant, sporadic, familial and iatrogenic), Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. The disease in cattle known as bovine spongiform encephalopathy and the related disease scrapie exhibit similar pathologic features. Following exposure, prions accumulate in lymphoid tissue such as the spleen, lymph nodes, tonsils and in Peyer’s patches (specialized lymphoid follicles located in the submucosa of the small intestine. This accumulation of the infectious agent is necessary for invasion of the CNS. In humans, the incubation period of the disease can vary between 18 months and 40 years. Prions appear to be variant, improperly folded versions of a normal cellular protein called PrPc, a 30-35 kD protein with two sites for N-glycosylation that is anchored in the neuronal cell membrane. PrPc protein is contains three α-helices, a short ß-pleated sheet region and a long, unstructured portion that comprises half of the molecule. The variant, infectious form of the protein is known as PrPsc, and is produced autocatalytically from PrPc. Prion diseases are always fatal, with no known cases of remission or recovery. The host shows no inflammatory response or immune response, no production of interferon and there is no effect on host B cell or T cell function. At the present time, there are no effective agents to treat prion diseases.
Antiviral Drug Therapy
The principles involved in the design of antiviral agents are similar to those used in the design of all chemotherapeutic agents. Drugs in this category are targeted to some process in the virus that is not present in the host cell. The earliest examples of antiviral agents did not achieve this goal, and these drugs were toxic at therapeutic levels, or had a limited spectrum of activity. A variety of factors make the design of effective antiviral agents difficult, including the viral ability to undergo antigenic changes, the latent period during which there are no symptoms, and their reliance on host enzymes and other processes. This problem is compounded by the fact that host immunity is not well understood, and that symptoms of viral infection may not appear until replication is complete and the viral genome has been incorporated into infected cells. None the less, the continuing identification of new targets for antiviral agents provides new avenues for the discovery of small molecule therapies. The following section includes information on currently marketed antiviral compounds that have been designed in eight general areas:
The remainder of this section deals primarily with small molecule antiviral agents that have been approved by the United States Food and Drug Administration (FDA) and are clinically effective in the treatment of viral infection. Some compounds used primarily in the treatment of bacterial infections, such as rifampicin, bleomycin, adriamycin, and actinomycin, also inhibit viral replication. However, these antibiotics do not affect the transcription or translation of viral mRNA, and are only effective in high concentrations. Therefore, such antibiotics are not commonly used for viral infections. There is a continuing need for new antiviral agents, primarily because viral infections are not curable after the virus invades the host cell and begins to replicate. With regard to small molecule antiviral agents, the ideal drug would have broad-spectrum antiviral activity, completely inhibit viral replication, maintain efficacy against mutant viral strains and reach the target organ without interfering with normal cellular processes or the immune system of the host.
Agents that Inhibit Virus Attachment, Penetration or Early Replication.
Amantadine hydrochloride (1-adamantanamine hydrochloride) is a symmetric tricyclic primary amine that inhibits penetration of RNA virus particles into the host cell. It also inhibits the early stages of viral replication by blocking the uncoating of the viral genome and the transferring of nucleic acid into the host cell. Rimantadine hydrochloride (α-methyl-1-adamantanemethylamine hydrochloride), is a synthetic adamatane derivative, which is structurally and pharmacologically related to amantadine. It appears to be more effective than amantadine hydrochloride against influenza A virus with fewer CNS side effects. Rimantadine hydrochloride is thought to interfere with virus uncoating by inhibiting the release of specific proteins. It may act by inhibiting reverse transcriptase (RT) or the synthesis of virus-specific RNA but does not inhibit virus adsorption or penetration. It appears to produce a virustatic effect early in the virus replication, and is used widely in Russia and Europe.
Isaacs and Lindenmann discovered interferon in 1957. When they infected cells with viruses, interference with the cellular effects of viral infection were observed. Interferon was subsequently isolated and found to protect the cells from further infection. When interferon was administered to other cells or animals, it displayed biological properties such as inhibition of viral growth, cell multiplication, and immunomodulatory activities. The results led to the speculation that interferon may be a natural antiviral factor, possibly formed before antibody production, and may be involved in the normal mechanism of resistance displayed against viral infection. Some investigators relate interferon to the polypeptide hormones and suggest that interferon functions in cell-to-cell communication by transmitting specific messages. Recently, antitumor and anticancer properties of interferon have evoked worldwide interest in the possible use of this agent in therapy for viral diseases, cancer, and immunodeficiency disorders. Chemical inducers, such as pyran copolymers, tilorone, diethylaminoethyl dextran, and heparin, have also been used. Tilorone is an effective inducer of interferon in mice but it is relatively ineffective in humans. Initial use of interferon and its inducers instilled intranasally after rhinovirus exposure was successful in the prevention of respiratory diseases. The clinical success of interferon and its inducers has not yet been established, although they may play a significant role in cell-mediated immunity to viral infections and cancer. Disadvantages of interferon use include unacceptable side effects, such as fever, headache, myalgias, leukopenia, nausea, vomiting, diarrhea, hypotension, alopecia, anorexia, and weight loss.Interferon consists of a mixture of small proteins with molecular weights ranging from 20,000–160,000. They are glycoproteins that exhibit species-specific antiviral activity. Human interferons are classified into three types: α, ß, and γ. The α-type is secreted by human leukocytes (white blood cells, non-T-lymphocytes) and the ß-type secreted by human fibroblasts. Lymphoid cells (T lymphocytes) which either have been exposed to a presensitized antigen or have been stimulated to divide by mitogens secrete α-interferon. γ-Interferon is also called “immune’’ interferon. Interferons are active in extremely low concentrations.
Surface glycoprotein hemagglutinin (HA), a protein important for viral binding to host cell receptors via a terminal sialic acid residue, and neuraminidase (NA), an enzyme involved in various aspects of activation of influenza viruses, have emerged as drug targets. Freshly shed virus particles are coated with sialic acid residues. NA is found in both influenza A and B viruses and is thought to be involved in catalytically cleaving glycosidic bonds between terminal sialic acid residues and adjacent sugars on HA. The cleavage of sialic acid bonds facilitates the spread of viruses by enhancing adsorbtion to cell surface receptors, and thus increases the infective level of the virus. In the absence of sialic acid cleavage from HA, viral aggregation or inappropriate binding to hemagglutinin will occur, interfering with the spread of the infection. NA also appears to play a role in preventing viral inactivation by respiratory mucus. Since NA plays such an important role in the activation of newly formed viruses, it is not surprising that the development of NA inhibitors has become an important potential means of inhibiting the spread of viral infections. It is believed that the hydrolysis of sialic acid proceeds through a oxonium cation stabilitized carbonium ion as shown above. Mimicking the transition state with novel carbocyclic derivatives of sialic acid has led to the development of transition-state-based inhibitors. The first of these compounds, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), was found to be an active neuraminidase inhibitor but lacked specificity for viral neuraminidase. Upon determination of the crystal structure of neuraminidase, more sophisticated measurements of the binding site for sialic acid lead to the development of zanamivir and later oseltamivir. Crystallographic studies of DANA bound to NA defined the receptor site to which the sialic acid portion of the virus binds. These studies suggested that substitution of the 4-hydroxy with an amino group or the larger guanidino group should increase binding of the inhibitor to NA. The 4-amino derivative was found to bind to a glutamic acid (Glu 119) in the receptor through a salt bridge while the guanidino was able to form both a salt bridge to Glu 119 and a charge-charge interaction with a glutamic acid at position 227. The result of these substitutions was a dramatic increase in binding capacity of the amino and guanidino derivatives to NA leading to effective competitive inhibition of the enzyme. The result has been the development of zanamivir as an effective agent against influenza A and B virus. Zanamivir is effective when administered via the nasal, intraperitoneal, and intravenous routes. Structure-activity relationship studies showed that maximum binding occured to NA when C-6 was substituted with the 3-pentyloxy side chain such as the one found in oseltamivir. In addition, esterification with ethanol gave rise to a compound that is orally effective. Oseltamivir was approved as the first orally administered NA inhibitor used against influenza A and B. The drug is indicated for the treatment of uncomplicated acute illness due to influenza infection. Recently, the manufacturer has submitted a request for approval of the drug for prevention of influenza A and B for use in adults, adolescents, and children one year of age and older. The drug is effective in treating influenza if administered within 2 days after onset of symptoms.
Entry inhibitors, also known as fusion inhibitors, are a new class of drugs for the treatment of HIV infection, and enfuvirtide is the first compound of this family to be approved for clinical use. Enfuvirtide is an oligo peptide consisting of 36 amino acids (N-acetyl-TYR-THR-SER-LEU-ILE-HIS-SER-LEU-ILE-GLU-GLU-SER-GLN-ASP-GLN-GLN-GLU-LYS-ASP-GLU-GLN-GLU-LEU-LEU-GLU-LEU-ASP-LYS-TRY-ALA-SER-LEU-TRY-ASP-TRY-phenylalaninamide). It is a synthetic peptide that mimics an HR2 fragment of gp41, blocking the formation of a six-helix bundle structure which is critical in the fusion of the HIV-1 virion to a CD4-positive T-lymphocyte. Enfuvirtide is used in combination with other antiretrovirals and works against a variety of HIV-1 variants, but it is not active against HIV-2. Resistance to enfuvirtide can develop when the virus produces changes in a 10-amino acid domain between residues 36 to 45 in the gp41 HIV surface glycoprotein. The HIV chemokine coreceptor CCR5 is of particular interest as a drug target because a natural mutation (CCR5-δ32) leads to reduced or absent expression of CCR5 in heterozygous or homozygous genotypes, respectively, with no apparent deleterious consequences. Individuals homozygous with respect to CCR5-δ32 have a high degree of resistance to HIV infection, whereas heterozygotes have a reduced rate of disease progression. CCR5 antagonists represent a promising new class of entry inhibitors under development for the treatment of HIV-1 infection. The CCR5 antagonist maraviroc has potent anti-HIV activity in vitro and is safe and well-tolerated at multiple doses up to 300 mg twice daily (BID) in healthy volunteers. It was recently approved by the FDA (Selzentry®, Pfizer)
Agents that Interfere with Viral Nucleic Acid Replication.
Acyclovir is a synthetic analogue of deoxyguanosine in which the carbohydrate moiety is acyclic. Because of this difference in structure as compared to other antiviral compounds (idoxuridine, vidarabine, and trifluridine), acyclovir possesses a unique mechanism of antiviral activity. The mode of action of acyclovir consists of three consecutive mechanisms. The first of these mechanisms involves conversion of the drug to active acyclovir monophosphate within cells by viral thymidine kinase. This phosphorylation reaction occurs faster within cells infected by herpesvirus than in normal cells, because acyclovir is a poor substrate for the normal cell thymidine kinase. Acyclovir is further converted to di- and triphosphates by a normal cellular enzyme called guanosine monophosphate kinase. In the second mechanism, viral DNA polymerase is competitively inhibited by acyclovir triphosphate with a lower IC50 concentration than that for cellular DNA polymerase. Acyclovir triphosphate is incorporated into the viral DNA chain during DNA synthesis. Because acyclovir triphosphate lacks the 3’-hydroxyl group of a cyclic sugar, it terminates further elongation of the DNA chain. The third mechanism depends on preferential uptake of acyclovir by herpes-infected cells as compared to uninfected cells, resulting in a higher concentration of acyclovir triphosphate and leading to a high therapeutic index between herpes-infected cells to normal cells. Valacyclovir hydrochloride is an amino acid ester prodrug of acyclovir, which exhibits antiviral activity only after metabolism first in the intestine walls or liver to acyclovir and then conversion to the triphosphate. The related analogue 6-deoxyacyclovir is a pro-drug form of acyclovir which is activated through metabolism by xanthine oxidase. Adefovir dipivoxil is an orally active pro-drug indicated for the treatment of chronic hepatitis B. The drug is hydrolyzed by extracellular esterases to produce adefovir, which in turn is phosphorylated by adenylate kinase to adefovir diphosphate, which inhibits hepatitis B virus (HBV) DNA polymerase. Incorporation of adefovir into viral DNA also leads to DNA chain termination. Cidofovir is a synthetic acyclic purine nucleotide analog of cytosine. It is a phosphorylated nucleotide which is additionally phosphorylated by host cell enzymes to its active intracellular metabolite, cidofovir diphosphate. This reaction occurs without initial virus-dependent phosphorylation by viral nucleoside kinases. It has antiviral effects by interfering with DNA synthesis and inhibiting viral replication.Cytarabine is a pyrimidine nucleoside related to idoxuridine. It is used primarily as an anticancer rather than an antiviral agent. Cytarabine acts by blocking the utilization of deoxycytidine, thereby inhibiting the replication of viral DNA. The drug is first converted to mono-, di- and triphosphates, which interfere with DNA synthesis by inhibiting both DNA polymerase and the reductase that promotes the conversion of cytidine diphosphate into its deoxy derivatives.
Ribavirin, a guanosine analogue, has broad-spectrum antiviral activity against both DNA and RNA viruses. It is phosphorylated by adenosine kinase to the triphosphate resulting in inhibition of viral specific RNA polymerase, disrupting messenger RNA and nucleic acid synthesis.Famciclovir is a synthetic purine nucleoside analogue related to guanine. It is the diacetyl 6-deoxy ester of penciclovir (PCV) which is structurally related to ganciclovir. Its pharmacologic and microbiologic activities are similar to acyclovir. Famciclovir is a pro-drug of penciclovir, which is formed in vivo by hydrolysis of the acetyl groups and oxidation at the 6-position by mixed function oxidases. Penciclovir and its metabolite penciclovir triphosphate possess antiviral activity resulting from inhibition of viral DNA polymerase.Foscarnet sodium is a trisodium phosphoformate hexahydrate that inhibits DNA polymerase of herpes viruses including CMV and retroviral RT. It is not phosphorylated into an active form by viral host cell enzymes. Therefore, it has the advantage of not requiring an activation step before attacking the target viral enzyme.Ganciclovir sodium is an acyclic deoxyguanosine analogue of acyclovir. Ganciclovir inhibits DNA polymerase. Its active form is ganciclovir triphosphate, which is an inhibitor of viral rather than cellular DNA polymerase. The phosphorylation of ganciclovir does not require a virus-specific thymidine kinase for its activity against CMV. The mechanism of action is similar to that of acyclovir; however, ganciclovir is more toxic to human cells than is acyclovir. Ganciclovir has greater activity than acyclovir against CMV and EB virus infection in immunocompromised patients. It is also active against HSV infection and in some mutants resistant to acyclovir. In AIDS patients, ganciclovir stopped progressive hemorragic retinitis and symptomatic pneumonitis related to CMV infection. Idoxuridine is a nucleoside containing a halogenated pyrimidine and is an analogue of thymidine. It acts as an antiviral agent against DNA viruses by interfering with their replication based on their similarity of structure. Idoxuridine is first phosphorylated by the host cell virus-encoded enzyme thymidine kinase to an active triphosphate form. The phosphorylated drug inhibits cellular DNA polymerase to a lesser extent than HSV DNA polymerase, which is necessary for the synthesis of viral DNA. The triphosphate form of the drug is then incorporated during viral nucleic acid synthesis by a false pairing system that replaces thymidine. When transcription occurs, faulty viral proteins are formed, resulting in defective viral particles.Other halogenated uridine derivatives can also be used as antiviral agents, as shown above. Fluorodeoxyuridine has in vitro antiviral activity but is not used in clinical practice. Bromodeoxyuridine is used in subacute sclerosing panencephalitis, a deadly virus-induced CNS disease. This agent appears to interfere with DNA synthesis in the same way as idoxuridine. The 5’-amino analogue of idoxuridine (5-iodo-5’-amino-2,’5’-dideoxy-uridine) is a better antiviral agent than idoxuridine and it is less toxic. It is metabolized in herpesvirus-infected cells only by thymidine kinase to di- and triphosphoramidates. These metabolites inhibit HSV-specific late RNA transcription, causing reduction of less infective abnormal viral proteins.
Trifluorothymidine is a fluorinated pyridine nucleoside structurally related to idoxuridine. It has been approved by the FDA and is a potent, specific inhibitor of replication of HSV-1 in vitro. Its mechanism of action is similar to that of idoxuridine. Like other antiherpes drugs, it is first phosphorylated by thymidine kinase to mono-, di- and triphosphate forms, which are then incorporated into viral DNA in place of thymidine to stop the formation of late virus mRNA and subsequent synthesis of the virion proteins.Vidarabine is an adenosine nucleoside obtained from cultures of Streptomyces antibioticus. Cellular enzymes convert vidarabine to mono-, di- and triphosphate derivatives that interfere with viral nucleic acid replication, specifically inhibiting the early steps in DNA synthesis. This agent was used originally as an antineoplastic drug. Its antiviral effect is, in some cases, superior to that of idoxuridine or cytarabine.Methisazone interferes with the translation of mRNA message at the ribosome, preventing the synthesis of proteins. Ultimately, it produces a defect in protein incorporation into the virus. Although viral DNA increases and host cells are damaged, an infectious virus is not produced. Methisazone is active against poxviruses, including variola and vaccinia. Some RNA viruses, such as rhinoviruses, echoviruses, reoviruses, influenza, parainfluenza, and polioviruses are also inhibited.
Antiretroviral Agents: Nucleoside Reverse Transcriptase Inhibitors and Non-Nucleoside Reverse Transcriptase Inhibitors.
While there can be no permanent cure of AIDS without prevention or elimination of HIV infection, AIDS patients can prolong their life if the disease is diagnosed early, and treatment is promptly initiated. Initial HIV treatment requires specific drugs that inhibit RT and HIV protease. In advanced HIV infection, AIDS is complicated by other organisms that proliferate in immunocompromised hosts, known as opportunistic infections. Such patients are treated symptomatically with a variety of drugs depending upon the opportunistic infections [62–64]. Anti-HIV agents have side effects but patients can be managed by a careful monitoring of the drugs. Opportunistic diseases include infections by parasites, bacteria, fungi and viruses. Neoplasms including Kaposi’s sarcoma and Burkitt’s lymphoma also commonly occur. Anti-HIV agents are classified according to the mode of action. The drugs inhibiting RT interfere with replication of HIV and stop synthesis of infective viral particles. They are further classified into nucleoside and nonnucleoside RT inhibitors. The drugs inhibiting HIV protease (PIs) inactivate RT activity and block release of viral particles from the infected cells. The chemistry of RT inhibitors and PIs are discussed below.
Nucleotiside Reverse Transcriptase Inhibitors (NRTIs)
Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
The FDA has recently approved several nonnucleosides that inhibit RT activity. They are used with nucleoside drugs to obtain synergistic activity in decreasing the viral load and increasing CD4? cell count. These drugs are primarily designed and synthesized by protein structure-based drug design methodologies. Their use as monotherapy may be limited because of rapid onset of resistance and hypersensitivity reactions. However, interaction of non-nucleoside drugs with other protease inhibitors, such as saquinavir, indinavir and ritonavir is being investigated. Also, interaction of these drugs with clarithromycin, ketoconazole, rifabutin and rifampin are under study. Non-nucleosides that inhibit RT activity are discussed below.
Nevirapine and its analogues exhibit antiretroviral effect against AZT-resistant HIV strains. Nevirapine in combination with ZDV and ddI produced approximately 18% higher CD4 cell counts and a decrease in viral load compared with patients who took ZDV and ddI. Nevirapine is recommended with nucleosides for HIV-1 infected patients who have experienced clinical or immunologic deterioration. The significant side effects of nevirapine are liver dysfunction and skin rashes. Delavirdine, a bisheteroarylpiperazine derivative, is a potent nonnucleoside RT inhibitor of activity specific for HIV-1. The FDA has approved this drug in combination with other anti-HIV agents. In phase I/II study trials, it demonstrated sustained improvements in CD4 cell counts, p24 antigen levels and RNA viral load. Promising results were obtained when the drug was used in two or three-drug combination with nucleoside drugs. Combination of delavirdine with ddI, ddC or ZDV demonstrated additive or synergistic effect. However, delavirdine with ZDV was more beneficial in early HIV infection. Combinations of nevirapine and delavirdine had an antagonistic effect on HIV-1 RT inhibition. Efavirenz is a new nonnucleoside RT inhibitor that was recently approved by the FDA. It is a potent inhibitor of wild-type as well as resistant mutants HIV-1 that is inhibited up to 95% in efavirenz concentration of 1.5 mM. In combination with indinavir, a mean reduction in HIV-RNA of 1.68 log, and an increase in CD4 cell counts of 96 cells/mm3 were reported. Co-administration of efavirenz with indinavir reduced indinavir concentration (AUC) by approximately 35%. Efavirenz is administered once a day and can be used as a substitute for indinavir in combination therapy with standard drugs, such as ZDV and 3TC. Since, it is given once a day, it cuts down the number of pills that an AIDS patient has to swallow. In the current cocktail therapy of AIDS patients, efavirenz is a good option for reducing the many side effects of cocktail therapy. It is administered to both adults and children and may be less expensive than indinavir.
HIV Protease Inhibitors (PIs)
J. Chem. Soc. Perkin Trans. I 2001, 1421-1430 and Organic and Biomolecular Chemistry 2003, 1, 5-14
The HIV genome contains various regions designated as genes, such as the gag and gag-pol genes, that are translated as polyproteins and form immature viral particles. As shown in the diagram above, HIV protease is responsible for processing the gag-pol gene product to generate proteins necessary for virus replication and survival. HIV protease is an enzyme that is essential for viral growth, and mediates the post-translational modification of core proteins into structural proteins. The structural proteins p7, p9, p17 and p24, which play important roles in infectivity of HIV, are products of the pol gene. The pol precursor protein is cleaved by a viral pol-encoded aspartic proteinase to form the desired structural proteins of the mature viral particle. HIV protease also activates RT, and plays an important role in the release of infectious viral particles. Thus, an area of considerable interest has been the development of drugs that act as inhibitors of protease and pol gene. Such inhibitors act on HIV protease and prevent post-translational processing and budding of immature viral particles from the infected cells. This group of drugs represents a major breakthrough in treatment of HIV when used in combination with RT inhibitors, and their development is one of the most significant advances in medicinal chemistry. As shown above, one normal cleavage site for HIV protease is between an aspartate and a proline. The amino acid R-groups are designated P1, P2, etc. on the amino terminus, and P1', P2', etc. on the carboxyl terminus of the cleavage site. The corresponding residues on the enzyme that are responsible for binding arer called S1, S2, S1', S2', etc. In the active site, a pair of aspartates (one on each of the 2 subunits of the protein) work together to complete the reaction. One coordinates the water that performs the hydrolysis, while the other coordinates the carboxyl group on the P1' amino acid. Drugs that inhibit HIV protease are designed as transition-state mimics that align at the active site of HIV-1 protease, as defined by three-dimensional crystallographic analysis of the protein structure. A number of oligopeptide-like analogues have been synthesized that differentially inhibit viral and mammalian aspartic proteases, and the most useful of these are selective for the viral enzyme HIV-1 protease. Structurally, these agents are either peptidomimetic and nonpeptide compounds. Their effectiveness is related to their ability to inhibit the gag-pol gene, which processes p24, p55 and p160. Consequently, the infectivity of HIV-1 is diminished.
Saquinavir mesylate was the first protease inhibitor approved by the FDA in December 1995. It is a carboxamide derivative that is specifically designed to inhibit HIV protease, thus preventing post-translational formation of viral proteins. It contains a hydroxyethylamine moiety rather than the Phe-Pro scissle bond present in the normal substrate for HIV protease. Ritonavir is another HIV PI approved by the FDA in March 1996. Ritonavir is a peptidomimetic inhibitor of both the HIV-1 and HIV-2 proteases. A 50% reduction in viral replication was obtained at a 3.8 to 153 nM concentration of ritonavir. Indinavir sulfate, a pentanoic acid amide derivative, was approved by the FDA in March 1996. The 95% inhibitory concentration against laboratory adapted HIV variants, primary clinical isolates and clinical resistant virus to indinavir analogues, is 25–100 nM in drug combination studies with ZDV and ddI. However, HIV has shown ritonavir resistance in some patients. This resistance is due to mutation of the virus that is correlated with the expression of amino acid substitutions in the viral protease. Cross-resistance to indinavir is observed with other PIs but not with the RT inhibitor. For this reason, indinavir is beneficial with ZDV and other nucleoside drugs. Nelfinavir mesylate is a peptidomimetic drug which is effective in HIV-1 and 2 wild type and ZDV-resistant strains, demonstrating ED50 concentrations ranging from 9–60 nM (95% effective dose was 0.04 mg/mL). Amprenavir is the fifth in a series of protease inhibitors to be approved for marketing in the U.S. While structurally unique from the previous agents, its pharmacologic profile does not appear to differ significantly from the previously marketed agents. Early studies suggest that a different resistance profile may exist and that the drug may be effective against some resistant strains of HIV. Side effects appear to be more common than with other PIs and include nausea, vomiting, paresthesia, depression, and rash. Since amprenavir is a sulfonamide there is some concern for cross-sensitivity with antibacterial sulfonamides. Recently, the FDA has approved the release of lopinavir/ritonavir combination in patients who have not responed to other regimens for treatment of HIV. The product is available in a soft gelatin capsule containing 133.3mg of lopinavir and 33.3mg of ritonavir as well as oral solutions containing 80mg of lopinavir and 20mg ritonavir/mL. The small amount of ritonavir is not expected to have antiretroviral activity, but rather the ritonavir is meant to increase the plasma concentrations of lopinavir by inhibiting lopinavir’s metabolism by CYP3A4. These drugs in combination with other antiretroviral agents have been approved for use in adults as well as patients between the ages of 6 months and 12 years. This is the first PI to be indicated for the very young. Atazanavir is an antiretroviral agent approved for use in combination with other antiretroviral agents for the treatment of HIV infections. Atazanavir is a peptidomimetic transition-state inhibitor which targets HIV-1 protease and reduces viral replication and thus virulence of HIV-1. Similar to saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinavir, the drug is used in combination with reverse transcriptase inhibitors to produce excellent efficacy in AIDS patients. Foramprenavir calcium has been approved for the treatment of HIV in adults when used in combination with other anti-HIV drugs. It is a pro-drug that, upon hydrolysis by serum phosphatases, gives rise to amprenavir, which is a peptidomimetric transition-state inhibitor that targets HIV-1 protease and reduces viral replication and thus, infectiousness of HIV-1. It is commonly administered in combination with reverse transcriptase inhibitors to produce excellent efficacy in AIDS patients.A newly approved protease inhibitor, darunavir, is a smaller inhibitor molecule that was specifically designed to bind to multiple residues in the HIV protease active site. Because it is very specific, it is active at a much lower dose. It is claimed that darunavir is active against drug-resistant strains of HIV, and that resistance to the drug will not develop, although this remains to be seen.
The picture below shows lopinavir bound in the active site cavity of HIV protease. Note that the hydroxyl group in the transition state mimicking core group is coordinated to Asp 25 in both protein strands of the enzyme. Lopinavir, like other HIV protease inhibitors, binds tightly in the active site and produces a potent competitive inhibition of th enzyme.
In the close-up view below, you can see that the aspartate residues are 2.92 and 3.04 angstroms away from the hydroxypropylamine core oxygen moiety.
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