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From: Scientific American www.sciam.com\2001\1101issue\1101haseltine.html Beyond Chicken Soup The antiviral era is upon us, with an array of virus-fighting drugs on the market and in development. Research into viral genomes is fueling much of this progress By William A. Haseltine Back in the mid-1980s, when scientists first learned that a virus caused a relentless new disease named AIDS, pharmacy shelves were loaded with drugs able to treat bacterial infections. For viral diseases, though, medicine had little to offer beyond chicken soup and a cluster of vaccines. The story is dramatically different today. Dozens of antiviral therapies, including several new vaccines, are available, and hundreds more are in development. If the 1950s were the golden age of antibiotics, we are now in the early years of the golden age of antivirals. This richness springs from various sources. Pharmaceutical companies would certainly point to the advent in the past 15 years of sophisticated techniques for discovering all manner of drugs. At the same time, frantic efforts to find lifesaving therapies for HIV, the cause of AIDS, have suggested creative ways to fight not only HIV but other viruses, too. A little-recognized but more important force has also been at work: viral genomics, which deciphers the sequence of "letters," or nucleic acids, in a virus's genetic "text." This sequence includes the letters in all the virus's genes, which form the blueprints for viral proteins; these proteins, in turn, serve as the structural elements and the working parts of the virus and thus control its behavior. With a full or even a partial genome sequence in hand, scientists can quickly learn many details of how a virus causes disease--and which stages of the process might be particularly vulnerable to attack. In 2001 the full genome of any virus can be sequenced within days, making it possible to spot that virus's weaknesses with unprecedented speed. The majority of antivirals on sale these days take aim at HIV, herpesviruses (responsible for a range of ills, from cold sores to encephalitis), and hepatitis B and C viruses (both of which can cause liver cancer). HIV and these forms of hepatitis will surely remain a main focus of investigation for some time; together they cause more than 250,000 cases of disease in the U.S. every year and millions in other countries. Biologists, however, are working aggressively to combat other viral illnesses as well. I cannot begin to describe all the classes of antivirals on the market and under study, but I do hope this article will offer a sense of the extraordinary advances that genomics and other sophisticated technologies have made possible in recent years. Drug-Search Strategies The earliest antivirals (mainly against herpes) were introduced in the 1960s and emerged from traditional drug-discovery methods. Viruses are structurally simple, essentially consisting of genes and perhaps some enzymes (biological catalysts) encased in a protein capsule and sometimes also in a lipid envelope. Because this design requires viruses to replicate inside cells, investigators infected cells, grew them in culture and exposed the cultures to chemicals that might plausibly inhibit viral activities known at the time. Chemicals that reduced the amount of virus in the culture were considered for in-depth investigation. Beyond being a rather hit-or-miss process, such screening left scientists with few clues to other viral activities worth attacking. This handicap hampered efforts to develop drugs that were more effective or had fewer side effects. Genomics has been a springboard for discovering fresh targets for attack and has thus opened the way to development of whole new classes of antiviral drugs. Most viral targets selected since the 1980s have been identified with the help of genomics, even though the term itself was only coined in the late 1980s, well after some of the currently available antiviral drugs were developed. After investigators decipher the sequence of code letters in a given virus, they can enlist computers to compare that sequence with those already identified in other organisms, including other viruses, and thereby learn how the sequence is segmented into genes. Strings of code letters that closely resemble known genes in other organisms are likely to constitute genes in the virus as well and to give rise to proteins that have similar structures. Having located a virus's genes, scientists can study the functions of the corresponding proteins and thus build a comprehensive picture of the molecular steps by which the virus of interest gains a foothold and thrives in the body. That picture, in turn, can highlight the proteins--and the domains within those proteins--that would be good to disable. In general, investigators favor targets whose disruption would impair viral activity most. They also like to focus on protein domains that bear little resemblance to those in humans, to avoid harming healthy cells and causing intolerable side effects. They take aim, too, at protein domains that are basically identical in all major strains of the virus, so that the drug will be useful against the broadest possible range of viral variants. After researchers identify a viral target, they can enlist various techniques to find drugs that are able to perturb it. Drug sleuths can, for example, take advantage of standard genetic engineering (introduced in the 1970s) to produce pure copies of a selected protein for use in drug development. They insert the corresponding gene into bacteria or other types of cells, which synthesize endless copies of the encoded protein. The resulting protein molecules can then form the basis of rapid screening tests: only substances that bind to them are pursued further. Alternatively, investigators might analyze the three- dimensional structure of a protein domain and then design drugs that bind tightly to that region. For instance, they might construct a compound that inhibits the active site of an enzyme crucial to viral reproduction. Drugmakers can also combine old-fashioned screening methods with the newer methods based on structures. Advanced approaches to drug discovery have generated ideas for thwarting viruses at all stages of their life cycles. Viral species vary in the fine details of their reproductive strategies. In general, though, the stages of viral replication include attachment to the cells of a host, release of viral genes into the cells' interiors, replication of all viral genes and proteins (with help from the cells' own protein-making machinery), joining of the components into hordes of viral particles, and escape of those particles to begin the cycle again in other cells. The ideal time to ambush a virus is in the earliest stage of an infection, before it has had time to spread throughout the body and cause symptoms. Vaccines prove their worth at that point, because they prime a person's immune system to specifically destroy a chosen disease-causing agent, or pathogen, almost as soon as it enters the body. Historically vaccines have achieved this priming by exposing a person to a killed or weakened version of the infectious agent that cannot make enough copies of itself to cause disease. So-called subunit vaccines are the most common alternative to these. They contain mere fragments of a pathogen; fragments alone have no way to produce an infection but, if selected carefully, can evoke a protective immune response. An early subunit vaccine, for hepatitis B, was made by isolating the virus from the plasma (the fluid component of blood) of people who were infected and then purifying the desired proteins. Today a subunit hepatitis B vaccine is made by genetic engineering. Scientists use the gene for a specific hepatitis B protein to manufacture pure copies of the protein. Additional vaccines developed with the help of genomics are in development for other important viral diseases, among them dengue fever, genital herpes and the often fatal hemorrhagic fever caused by the Ebola virus. Several vaccines are being investigated for preventing or treating HIV. But HIV's genes mutate rapidly, giving rise to many viral strains; hence, a vaccine that induces a reaction against certain strains might have no effect against others. By comparing the genomes of the various HIV strains, researchers can find sequences that are present in most of them and then use those sequences to produce purified viral protein fragments. These can be tested for their ability to induce immune protection against strains found worldwide. Or vaccines might be tailored to the HIV variants prominent in particular regions. Bar Entry Treatments become important when a vaccine is not available or not effective. Antiviral treatments effect cures for some patients, but so far most of them tend to reduce the severity or duration of a viral infection. One group of therapies limits viral activity by interfering with entry into a favored cell type. The term "entry" actually covers a few steps, beginning with the binding of the virus to some docking site, or receptor, on a host cell and ending with "uncoating" inside the cell; during uncoating, the protein capsule (capsid) breaks up, releasing the virus's genes. Entry for enveloped viruses requires an extra step. Before uncoating can occur, these microorganisms must fuse their envelope with the cell membrane or with the membrane of a vesicle that draws the virus into the cell's interior. Several entry-inhibiting drugs in development attempt to block HIV from penetrating cells. Close examination of the way HIV interacts with its favorite hosts (white blood cells called helper T cells) has indicated that it docks with molecules on those cells called CD4 and CCR5. Although blocking CD4 has failed to prevent HIV from entering cells, blocking CCR5 may yet do so. Amantidine and rimantidine, the first two (of four) influenza drugs to be introduced, interrupt other parts of the entry process. Drugmakers found the compounds by screening likely chemicals for their overall ability to interfere with viral replication, but they have since learned more specifically that the compounds probably act by inhibiting fusion and uncoating. Fusion inhibitors discovered with the aid of genomic information are also being pursued against respiratory syncytial virus (a cause of lung disease in infants born prematurely), hepatitis B and C, and HIV. Many colds could soon be controlled by another entry blocker, pleconaril, which is reportedly close to receiving federal approval. Genomic and structural comparisons have shown that a pocket on the surface of rhinoviruses (responsible for most colds) is similar in most variants. Pleconaril binds to this pocket in a way that inhibits the uncoating of the virus. The drug also appears to be active against enteroviruses, which can cause diarrhea, meningitis, conjunctivitis and encephalitis. Jam the Copier A number of antivirals on sale and under study operate after uncoating, when the viral genome, which can take the form of DNA or RNA, is freed for copying and directing the production of viral proteins. Several of the agents that inhibit genome replication are nucleoside or nucleotide analogues, which resemble the building blocks of genes. The enzymes that copy viral DNA or RNA incorporate these mimics into the nascent strands. Then the mimics prevent the enzyme from adding any further building blocks, effectively aborting viral replication. Acyclovir, the earliest antiviral proved to be both effective and relatively nontoxic, is a nucleoside analogue that was discovered by screening selected compounds for their ability to interfere with the replication of herpes simplex virus. It is prescribed mainly for genital herpes, but chemical relatives have value against other herpesvirus infections, such as shingles caused by varicella zoster and inflammation of the retina caused by cytomegalovirus. The first drug approved for use against HIV, zidovudine (AZT), is a nucleoside analogue as well. Initially developed as an anticancer drug, it was shown to interfere with the activity of reverse transcriptase, an enzyme that HIV uses to copy its RNA genome into DNA. If this copying step is successful, other HIV enzymes splice the DNA into the chromosomes of an invaded cell, where the integrated DNA directs viral reproduction. AZT can cause severe side effects, such as anemia. But studies of reverse transcriptase, informed by knowledge of the enzyme's gene sequence, have enabled drug developers to introduce less toxic nucleoside analogues. One of these, lamivudine, has also been approved for hepatitis B, which uses reverse transcriptase to convert RNA copies of its DNA genome back into DNA. Intense analyses of HIV reverse transcriptase have led as well to improved versions of a class of reverse transcriptase inhibitors that do not resemble nucleosides. Genomics has uncovered additional targets that could be hit to interrupt replication of the HIV genome. Among these is RNase H, a part of reverse transcriptase that separates freshly minted HIV DNA from RNA. Another is the active site of integrase, an enzyme that splices DNA into the chromosomal DNA of the infected cell. An integrase inhibitor is now being tested in HIV-infected volunteers. Impede Protein Production All viruses must at some point in their life cycle transcribe genes into mobile strands of messenger RNA, which the host cell then "translates," or uses as a guide for making the encoded proteins. Several drugs in development interfere with the transcription stage by preventing proteins known as transcription factors from attaching to viral DNA and switching on the production of messenger RNA. Genomics helped to identify the targets for many of these agents. It also made possible a novel kind of drug: the antisense molecule. If genomic research shows that a particular protein is needed by a virus, workers can halt the protein's production by masking part of the corresponding RNA template with a custom-designed DNA fragment able to bind firmly to the selected RNA sequence. An antisense drug, fomivirsen, is already used to treat eye infections caused by cytomegalovirus in AIDS patients. And antisense agents are in development for other viral diseases; one of them blocks production of the HIV protein Tat, which is needed for the transcription of other HIV genes. Drugmakers have also used their knowledge of viral genomes to identify sites in viral RNA that are susceptible to cutting by ribozymes--enzymatic forms of RNA. A ribozyme is being tested in patients with hepatitis C, and ribozymes for HIV are in earlier stages of development. Some such projects employ gene therapy: specially designed genes are introduced into cells, which then produce the needed ribozymes. Other types of HIV gene therapy under study give rise to specialized antibodies that seek targets inside infected cells or to other proteins that latch onto certain viral gene sequences within those cells. Some viruses produce a protein chain in a cell that must be spliced to yield functional proteins. HIV is among them, and an enzyme known as a protease performs this cutting. When analyses of the HIV genome pinpointed this activity, scientists began to consider the protease a drug target. With enormous help from computer-assisted structure-based research, potent protease inhibitors became available in the 1990s, and more are in development. The inhibitors that are available so far can cause disturbing side effects, such as the accumulation of fat in unusual places, but they nonetheless prolong overall health and life in many people when taken in combination with other HIV antivirals. A new generation of protease inhibitors is in the research pipeline. Stop Traffic Even if viral genomes and proteins are reproduced in a cell, they will be harmless unless they form new viral particles able to escape from the cell and migrate to other cells. The most recent influenza drugs, zanamivir and oseltamivir, act at this stage. A molecule called neuraminidase, which is found on the surface of both major types of influenza (A and B), has long been known to play a role in helping viral particles escape from the cells that produced them. Genomic comparisons revealed that the active site of neuraminidase is similar among various influenza strains, and structural studies enabled researchers to design compounds able to plug that site. The other flu drugs act only against type A. Drugs can prevent the cell-to-cell spread of viruses in a different way--by augmenting a patient's immune responses. Some of these responses are nonspecific: the drugs may restrain the spread through the body of various kinds of invaders rather than homing in on a particular pathogen. Molecules called interferons take part in this type of immunity, inhibiting protein synthesis and other aspects of viral replication in infected cells. For that reason, one form of human interferon, interferon alpha, has been a mainstay of therapy for hepatitis B and C. (For hepatitis C, it is used with an older drug, ribavirin.) Other interferons are under study, too. More specific immune responses include the production of standard antibodies, which recognize some fragment of a protein on the surface of a viral invader, bind to that protein and mark the virus for destruction by other parts of the immune system. Once researchers have the gene sequence encoding a viral surface protein, they can generate pure, or "monoclonal," antibodies to selected regions of the protein. One monoclonal is on the market for preventing respiratory syncytial virus in babies at risk for this infection; another is being tested in patients suffering from hepatitis B. Comparisons of viral and human genomes have suggested yet another antiviral strategy. A number of viruses, it turns out, produce proteins that resemble molecules involved in the immune response. Moreover, certain of those viral mimics disrupt the immune onslaught and thus help the virus to evade destruction. Drugs able to intercept such evasion-enabling proteins may preserve full immune responses and speed the organism's recovery from numerous viral diseases. The hunt for such agents is under way. The Resistance Demon The pace of antiviral drug discovery is nothing short of breathtaking, but at the same time, drugmakers have to confront a hard reality: viruses are very likely to develop resistance, or insensitivity, to many drugs. Resistance is especially probable when the compounds are used for long periods, as they are in such chronic diseases as HIV and in quite a few cases of hepatitis B and C. Indeed, for every HIV drug in the present arsenal, some viral strain exists that is resistant to it and, often, to additional drugs. This resistance stems from the tendency of viruses--especially RNA viruses and most especially HIV--to mutate rapidly. When a mutation enables a viral strain to overcome some obstacle to reproduction (such as a drug), that strain will thrive in the face of the obstacle. To keep the resistance demon at bay until effective vaccines are found, pharmaceutical companies will have to develop more drugs. When mutants resistant to a particular drug arise, reading their genetic text can indicate where the mutation lies in the viral genome and suggest how that mutation might alter the interaction between the affected viral protein and the drug. Armed with that information, researchers can begin structure-based or other studies designed to keep the drug working despite the mutation. Pharmaceutical developers are also selecting novel drugs based on their ability to combat viral strains that are resistant to other drugs. Recently, for instance, DuPont Pharmaceuticals chose a new HIV nonnucleoside reverse transcriptase inhibitor, DPC 083, for development precisely because of its ability to overcome viral resistance to such inhibitors. The company's researchers first examined the mutations in the reverse transcriptase gene that conferred resistance. Next they turned to computer modeling to find drug designs likely to inhibit the reverse transcriptase enzyme in spite of those mutations. Then, using genetic engineering, they created viruses that produced the mutant enzymes and selected the compound best able to limit reproduction by those viruses. The drug is now being evaluated in HIV-infected patients. It may be some time before virtually all serious viral infections are either preventable by vaccines or treatable by some effective drug therapy. But now that the sequence of the human genome is available in draft form, drug designers will identify a number of previously undiscovered proteins that stimulate the production of antiviral antibodies or that energize other parts of the immune system against viruses. I fully expect these discoveries to translate into yet more antivirals. The insights gleaned from the human genome, viral genomes and other advanced drug-discovery methods are sure to provide a flood of needed antivirals within the next 10 to 20 years. -------------------------------------------------------------------------------- Further Information: Viral Strategies of Immune Evasion. Hidde L. Ploegh in Science, Vol. 280, No. 5361, pages 248-253; April 10, 1998. Strategies for Antiviral Drug Discovery. Philip S. Jones in Antiviral Chemistry and Chemotherapy, Vol. 9, No. 4, pages 283-302; July 1998. New Technologies for Making Vaccines. Ronald W. Ellis in Vaccine, Vol. 17, No. 13-14, pages 1596-1604; March 26, 1999. Protein Design of an HIV-1 Entry Inhibitor. Michael J. Root, Michael S. Kay and Peter S. Kim in Science, Vol. 291, No. 5505, pages 884-888; February 2, 2001. Antiviral Chemotherapy: General Overview. Jack M. Bernstein, Wright State University School of Medicine, Division of Infectious Diseases, 2000. Available at www.med.wright.edu/im/AntiviralChemotherapy.html -------------------------------------------------------------------------------- The Author WILLIAM A. HASELTINE, who has a doctorate in biophysics from Harvard University, is the chairman of the board of directors and chief executive officer of Human Genome Sciences; he is also editor in chief of a new publication, the Journal of Regenerative Medicine, and serves on the editorial boards of several other scientific journals. He was a professor at the Dana-Farber Cancer Institute, an affiliate of Harvard Medical School, and at the Harvard School of Public Health from 1988 to 1995. His laboratory was the first to assemble the sequence of the AIDS virus genome. Since 1981 he has helped found more than 20 biotechnology companies. [ 此消息由 特深沉 在 2002-05-10.23:11:24 编辑过 ]