Viral Entry Denied
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《新英格兰医药杂志》
Until recently, antiviral drugs were both uncommon and not terribly potent. This has changed: during the past decade, more than 30 antiviral drugs have been licensed, and many of them are very effective. Most of the drugs inhibit the activity of viral enzymes, but a new class of agents that block entry of the virus into the cell is being developed. The development of entry inhibitors is driven by the identification of the cell-surface receptors to which viruses bind and by new findings about viral protein structures that bind receptors and mediate viral entry. These advances offer exciting opportunities for the development of agents that block viral transmission and treat viral infections, as well as for vaccine development.
Most entry inhibitors under clinical development are directed against viruses that are surrounded by a lipid membrane — the so-called enveloped viruses. Regardless of the type of enveloped virus, the fundamental steps of entry are the same. First, the virus attaches to the cell surface, often engaging a specific viral receptor. Viral receptors play a critical role in mediating the entry of the virus into the cell, and the distribution of receptors across specific cell types helps to determine viral tropism. Thus, most strains of the human immunodeficiency virus (HIV) need to engage CD4 and the chemokine receptor CCR5 sequentially to enter the cell, which largely restricts viral infection to certain T cells and macrophages.
Second, binding to the receptor induces the viral-envelope protein to undergo conformational changes that mediate fusion between the viral and cellular membranes (see Figure) in one of two ways. For some viruses, receptor binding leads to endocytosis of the viral particle and delivery to an acidic compartment. There, the low-pH environment triggers conformational changes that lead to membrane fusion. Influenzavirus, West Nile virus, and rabies virus are examples of viruses that use this pathway. For other viruses, the mere process of binding to one or more receptors leads to the needed conformational changes. These pH-independent viruses can fuse at the cell surface; HIV is the best-characterized example.
Figure. The HIV-Entry Process as a Model for Class I Fusion Proteins.
The HIV envelope protein is a trimer, with each monomer containing glycoprotein 120 and glycoprotein 41 subunits (Panel A). Thus, there are three glycoprotein 120 and three glycoprotein 41 subunits in the envelope protein trimer. The glycoprotein 41 subunit has an N-terminal hydrophobic domain termed the fusion peptide, followed by two helical regions, helical region 1 (green cylinder) and helical region 2 (blue spiral). The glycoprotein 120 subunit binds to cell-surface CD4 (Panel B), which induces a conformational change in glycoprotein 120 that exposes a domain important for binding to the coreceptor (purple structure) (Panel C). Coreceptor binding is the final trigger for fusion-inducing conformational changes and is thought to release the fusion peptide of glycoprotein 41 (black spiral), which is inserted into the membrane of the cell. Helical regions 1 and 2 of the glycoprotein 41 protein then fold back and bind to each other, bringing the membrane of the cell and the membrane of the virus into very close proximity (Panel D), resulting in membrane fusion and viral entry. (In Panel D, two trimers are shown.) Enfuvirtide, a peptide whose sequence is based on a portion of helical region 2, binds to helical region 1 after it is made accessible by CD4 binding and prevents the formation of the helical bundle that leads to membrane fusion. Small-molecule inhibitors that are being tested in clinical trials can prevent other steps of the viral-entry pathway, including CD4 and coreceptor binding.
Since each step of the viral-entry pathway is a potential target for antiviral agents, entry inhibitors fall into several categories, depending on which step they target (see Figure). The first category includes compounds that bind to viral receptors. Small-molecule inhibitors that target the HIV receptor CCR5, which are under clinical development, have been shown to reduce dramatically the levels of circulating virus in HIV-infected patients. Because they target an invariant cellular protein, use of these compounds obviates the difficulty of attempting to target a virus that has considerable genetic variability. The success of these compounds in early clinical trials, coupled with resistance to HIV infection in people who lack CCR5, fueled efforts to identify receptors that are engaged by other viruses. The recent identification of angiotensin-converting enzyme 2 as the receptor for the coronavirus that causes the severe acute respiratory syndrome1 has already led to the identification of antibodies that prevent receptor binding and may lead to the discovery of small-molecule inhibitors as well.
The second category of entry inhibitors includes compounds that bind to the virus and prevent it from interacting with its receptors. Although some neutralizing antibodies have long been known to operate by this mechanism, small-molecule inhibitors that accomplish the same feat have been developed more recently. Crystallographic studies have helped enormously. For example, an understanding of the structure of picornaviruses (such as human rhinoviruses) has led to the development of a whole series of compounds that fit into a pocket on the viral surface. Some of these compounds block viral attachment, and several have been tested in clinical trials. A small-molecule inhibitor that prevents binding of the HIV envelope protein to the CD4 receptor is also being tested in clinical trials.
The final category of entry inhibitors prevents the conformational changes needed for membrane fusion and includes enfuvirtide, an inhibitor of HIV membrane fusion that has been licensed by the Food and Drug Administration. Enveloped viruses appear to use two classes of membrane-fusion proteins. Class I fusion proteins — such as those found on HIV, influenzavirus, Ebola virus, and respiratory syncytial virus — are trimers of identical subunits that project from the viral surface. On activation by either acid pH or receptor binding, a series of conformational changes occurs. Part of the fusion protein is inserted into the membrane of the cell, linking the viral and cellular membranes. Fusion is then caused by a conformational change in which the two helical regions of the fusion protein fold back on each other, winching the fusion peptide (inserted in the cell membrane) and the membrane-anchoring region of the viral envelope protein (anchored in the viral membrane) toward each other, a process that brings about lipid mixing (see Figure).2 Enfuvirtide is a peptide that, by binding to one of these helical regions, prevents the conformational change needed for fusion. Structural studies have guided the development of fusion-inhibiting peptides as well as of small-molecule inhibitors for other viruses with class I fusion proteins. Viruses such as the dengue, yellow fever, and West Nile viruses have class II fusion proteins that work in a somewhat different manner, and recent structural studies have suggested ways to inhibit fusion of these viruses as well.3
With the licensing of one entry inhibitor and others being tested in clinical trials, these agents have passed the proof-of-principle stage, a benchmark that is testimony to the value of biochemical and structural studies designed to provide the molecular details of how a virus enters a cell.
Source Information
From the Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia.
References
Sui J, Li W, Murakami A, et al. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci U S A 2004;101:2536-2541.
Moore JP, Doms RW. The entry of entry inhibitors: a fusion of science and medicine. Proc Natl Acad Sci U S A 2003;100:10598-10602.
Modis Y, Ogata S, Clements D, Harrison SC. Structure of the dengue virus envelope protein after membrane fusion. Nature 2004;427:313-319.(Robert W. Doms, M.D., Ph.)
Most entry inhibitors under clinical development are directed against viruses that are surrounded by a lipid membrane — the so-called enveloped viruses. Regardless of the type of enveloped virus, the fundamental steps of entry are the same. First, the virus attaches to the cell surface, often engaging a specific viral receptor. Viral receptors play a critical role in mediating the entry of the virus into the cell, and the distribution of receptors across specific cell types helps to determine viral tropism. Thus, most strains of the human immunodeficiency virus (HIV) need to engage CD4 and the chemokine receptor CCR5 sequentially to enter the cell, which largely restricts viral infection to certain T cells and macrophages.
Second, binding to the receptor induces the viral-envelope protein to undergo conformational changes that mediate fusion between the viral and cellular membranes (see Figure) in one of two ways. For some viruses, receptor binding leads to endocytosis of the viral particle and delivery to an acidic compartment. There, the low-pH environment triggers conformational changes that lead to membrane fusion. Influenzavirus, West Nile virus, and rabies virus are examples of viruses that use this pathway. For other viruses, the mere process of binding to one or more receptors leads to the needed conformational changes. These pH-independent viruses can fuse at the cell surface; HIV is the best-characterized example.
Figure. The HIV-Entry Process as a Model for Class I Fusion Proteins.
The HIV envelope protein is a trimer, with each monomer containing glycoprotein 120 and glycoprotein 41 subunits (Panel A). Thus, there are three glycoprotein 120 and three glycoprotein 41 subunits in the envelope protein trimer. The glycoprotein 41 subunit has an N-terminal hydrophobic domain termed the fusion peptide, followed by two helical regions, helical region 1 (green cylinder) and helical region 2 (blue spiral). The glycoprotein 120 subunit binds to cell-surface CD4 (Panel B), which induces a conformational change in glycoprotein 120 that exposes a domain important for binding to the coreceptor (purple structure) (Panel C). Coreceptor binding is the final trigger for fusion-inducing conformational changes and is thought to release the fusion peptide of glycoprotein 41 (black spiral), which is inserted into the membrane of the cell. Helical regions 1 and 2 of the glycoprotein 41 protein then fold back and bind to each other, bringing the membrane of the cell and the membrane of the virus into very close proximity (Panel D), resulting in membrane fusion and viral entry. (In Panel D, two trimers are shown.) Enfuvirtide, a peptide whose sequence is based on a portion of helical region 2, binds to helical region 1 after it is made accessible by CD4 binding and prevents the formation of the helical bundle that leads to membrane fusion. Small-molecule inhibitors that are being tested in clinical trials can prevent other steps of the viral-entry pathway, including CD4 and coreceptor binding.
Since each step of the viral-entry pathway is a potential target for antiviral agents, entry inhibitors fall into several categories, depending on which step they target (see Figure). The first category includes compounds that bind to viral receptors. Small-molecule inhibitors that target the HIV receptor CCR5, which are under clinical development, have been shown to reduce dramatically the levels of circulating virus in HIV-infected patients. Because they target an invariant cellular protein, use of these compounds obviates the difficulty of attempting to target a virus that has considerable genetic variability. The success of these compounds in early clinical trials, coupled with resistance to HIV infection in people who lack CCR5, fueled efforts to identify receptors that are engaged by other viruses. The recent identification of angiotensin-converting enzyme 2 as the receptor for the coronavirus that causes the severe acute respiratory syndrome1 has already led to the identification of antibodies that prevent receptor binding and may lead to the discovery of small-molecule inhibitors as well.
The second category of entry inhibitors includes compounds that bind to the virus and prevent it from interacting with its receptors. Although some neutralizing antibodies have long been known to operate by this mechanism, small-molecule inhibitors that accomplish the same feat have been developed more recently. Crystallographic studies have helped enormously. For example, an understanding of the structure of picornaviruses (such as human rhinoviruses) has led to the development of a whole series of compounds that fit into a pocket on the viral surface. Some of these compounds block viral attachment, and several have been tested in clinical trials. A small-molecule inhibitor that prevents binding of the HIV envelope protein to the CD4 receptor is also being tested in clinical trials.
The final category of entry inhibitors prevents the conformational changes needed for membrane fusion and includes enfuvirtide, an inhibitor of HIV membrane fusion that has been licensed by the Food and Drug Administration. Enveloped viruses appear to use two classes of membrane-fusion proteins. Class I fusion proteins — such as those found on HIV, influenzavirus, Ebola virus, and respiratory syncytial virus — are trimers of identical subunits that project from the viral surface. On activation by either acid pH or receptor binding, a series of conformational changes occurs. Part of the fusion protein is inserted into the membrane of the cell, linking the viral and cellular membranes. Fusion is then caused by a conformational change in which the two helical regions of the fusion protein fold back on each other, winching the fusion peptide (inserted in the cell membrane) and the membrane-anchoring region of the viral envelope protein (anchored in the viral membrane) toward each other, a process that brings about lipid mixing (see Figure).2 Enfuvirtide is a peptide that, by binding to one of these helical regions, prevents the conformational change needed for fusion. Structural studies have guided the development of fusion-inhibiting peptides as well as of small-molecule inhibitors for other viruses with class I fusion proteins. Viruses such as the dengue, yellow fever, and West Nile viruses have class II fusion proteins that work in a somewhat different manner, and recent structural studies have suggested ways to inhibit fusion of these viruses as well.3
With the licensing of one entry inhibitor and others being tested in clinical trials, these agents have passed the proof-of-principle stage, a benchmark that is testimony to the value of biochemical and structural studies designed to provide the molecular details of how a virus enters a cell.
Source Information
From the Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia.
References
Sui J, Li W, Murakami A, et al. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci U S A 2004;101:2536-2541.
Moore JP, Doms RW. The entry of entry inhibitors: a fusion of science and medicine. Proc Natl Acad Sci U S A 2003;100:10598-10602.
Modis Y, Ogata S, Clements D, Harrison SC. Structure of the dengue virus envelope protein after membrane fusion. Nature 2004;427:313-319.(Robert W. Doms, M.D., Ph.)