Today, small molecules are commonly used as antiviral drugs, generally leading to the inhibition of some viral protein or enzyme. When we say ‘small molecule’ we are referring to molecules of a low molecular weight. Small molecules are not the only option in drug therapy. There are lots of high molecular weight molecules used, like biologics. Biologics are derivatives of natural products used to treat diseases. They include proteins, enzymes, vaccines, blood products, antibodies, etc. So, why use small molecules for antiviral drug therapy and not large molecules?
Small molecules offer many advantages over large molecules. Small molecules are easily manufactured and have a low production cost. Large molecules, on the other hand, can have very difficult, time-consuming, expensive methods of production/collection. Small molecules have good oral bioavailability. Proteins (large molecules) are subject to proteolytic degradation, and have low oral bioavailability. Biologics often must be injected directly. Small molecules diffuse easily, giving them access to intracellular targets. Large molecules do not diffuse easily. Another advantage that small molecules have over large ones is that they do not elicit an immune response. The half-life of many large molecules is drastically reduced by anti-drug antibodies. Also, small molecules are species independent and can be tested on rodents. Because biologics are species-specific, toxicity studies relevant to humans should be performed on other primates. These advantages are summarized in the table that follows.
Advantages of Small Molecules Over Large Molecules:
|Small Molecules|| Large Molecules/ Biologics
|Low cost and easy to produce||Difficult and costly to manufacture|
|Oral bioavailability||Subject to proteolytic degradation, so they may need to be injected directly into the blood stream|
|Diffuse easily (access to intracellular targets)||Low rate of diffusion|
|No immune response||Immunogenicity (Anti-drug antibodies can reduce the half-life)|
|Species independent (facilitates testing on rodents)||Species-specific (testing relevant to humans requires primates)|
Small molecules aren’t perfect though. One of the main disadvantages of small molecules is that they are readily metabolized, and their metabolites can often be active and/or toxic. Because small molecules are processed by the biliary and renal systems, they can often be very hard on the liver and kidneys.
The most common antivirals available today are against human immunodeficiency virus (HIV), herpesvirus, hepatitis B and C viruses, and influenza A and B. While many other disease-causing viruses are completely without antivirals, even these highly studied ones have no cure. Effective treatments for these and many other viruses have yet to be discovered. This is not as easy as it sounds. Besides overcoming the difficulties of finding inhibitors that bind the active sites of proteins, maximizing bioavailability, and avoiding toxicity to the host, which are problems that occur in all drug design, antivirals face their own unique problems. Viruses have a very short replication cycle. In only hours (or even minutes), one virus can produce hundreds of infectious copies of itself. This extremely fast replication allows viruses to mutate constantly and for new strains to become prevalent very quickly. Mutated viruses, if still functional, have the advantage of avoiding the host immune system (if antibodies no longer recognize a viral antigen, for example) or drugs that were once effective inhibitors. This leads to the major problem with antiviral drug therapy: drug-resistance. When a virus has mutated to the point that a once effective drug no longer works, that virus strain is said to be drug-resistant. A single virus strain can become resistant to several different drugs. This is termed multidrug-resistance. Another major problem facing antiviral drugs is cross-resistance. When a virus becomes resistant to a drug after contact with a similar drug, it is displaying cross-resistance to a series of drugs.
Techniques used in design of anti-viral therapies
There are many techniques used during the process of finding and evaluating new compounds for use as small molecule treatments for viral disease. Through automation, many techniques that were once obsolete are now the preferred and standard procedures used in labs around the world. To begin, high-throughput screening (HTS) is a very powerful tool for medicinal chemists to find new lead compounds for potential drug development. Basically, HTS works by allowing a large number of compounds to react with a specific entity for a relatively short period of time, and then the reactivity of the compounds with the entity is measured. The reactivity of the compound with the entity is referred to as bioactivity, and bioactivity can be measured numerous ways depending on the entity that is being tested on.
Enzyme/substrate interactions: If the entity that is being tested is an enzyme and you are testing compounds for inhibitory activity against this enzyme, one would begin the HTS by placing standardized quantities of the enzyme into multi-welled plates. Then one would add the potentially inhibitory compound as well as modified enzyme substrate. The substrate is generally modified in such a way that after interaction with the enzyme, it either fluoresces, radiates, or gives off some kind of measureable reading. Thus, by using a spectrophotometer, or other third party device that can be incorporated directly into the HTS apparatus, one can obtain a measurement of how well a given compound can inhibit the enzyme. In other words, the fluorescence emitted from a given well is inversely proportional to the tested compounds ability to inhibit the entity. So, the more fluorescence the worse the inhibitory activity of the compound.
Immunoassays are another method of indirectly measuring a drug’s activity. They can be used in cases where a drug is suspected to inhibit production of a specific cellular component or cell type, as long as it can generate an immune response. Then, specific antibodies are developed against the cellular component or cell type that you are trying to inhibit (i.e. rabbit antivirus protein). Subsequently, you develop antibodies against the constant regions of the antibodies you developed against the protein you are trying to inhibit. These are referred to as secondary antibodies and are of the form of pig anti-rabbit. You then label these secondary antibodies with a group that can be registered either by radiation, fluorescence, or something else that is measurable. After you have completed these steps, you would proceed with the HTS by placing both entities and potential drugs into a multi-welled plate. Upon sufficient incubation time, you would incubate the drug-exposed entities with the primary antibody, which should bind to any of the protein that you are trying to inhibit. You would then wash your sample, thereby removing excess rabbit antivirus protein antibody, and add the pig anti-rabbit antibody. You would then wash away the excess enzyme once again and take a reading of the intensity of the fluorescence, radiation, or other measurable parameter. This measurement is once again inversely proportional to the effectiveness of the drug as an inhibitor of protein production or proliferation of the specific type of cell in question.
Examples of high-throughput screening apparatuses.
There are also several other computer based techniques that are widely used in the development of new drugs. Particularly, structure activity relationship (SAR) and quantative structure activity relationship (QSAR) studies can be powerful weapons in the arsenal of the medicinal chemist. SAR is used to map the biological activity of a compound to its structure. Thus, by making small changes to a compound’s structure and measuring the change in biological activity associated with that change, scientists can make inferences about the best types of functional groups to be placed at each position on a drug’s skeleton. At first, substitutions of broad groups would be made. One would switch a hydrophobic group for a hydrophilic group to see the effects on the drug’s activity. This will indicate which is more favorable depending on which type of group had the higher activity, but it will not give insight into the best hydrophobic or hydrophilic group to place at that particular position. Hence, one would carry out these types of general substitutions until the general makeup of the most active compound has been mapped. After the general substitutions are completed one can then create a library of the most potentially active compounds with this particular pharmacophore as a map. Then these compounds can be tested for biological activity and side effects.
QSAR is another technique used in the design of small molecules. This is a technique with similar results as a SAR study, but QSAR generally leads to more specific results in a shorter period of time and can also lend more predictive power than SAR. QSAR is a quantitative technique. This means that it uses calculable numbers to find the best possible design of a potential drug. QSAR assumes that there is a series of calculable descriptors that will best define the activity of a drug in a biological setting. The descriptors are features of the potential drug, such as lipophilicity, number of hydrogen bond donors and accepters, electronic properties, and hydrophilicity. These are used to determine how the drug will act in a biological system. The structure part of “QSAR” comes from the fact that the numbers representing each descriptor depend on the structure of the compound. Thus, a scientist can manipulate a QSAR study to find the best set of possible descriptors to represent the most appropriate features that a drug would need to interact within a given biological system. These descriptors are then used to build an equation, and the equation can be optimized to find the structure of the drug that would be most active in the setting.
Hepatitis C Virus
Hepatitis C virus (HCV) is a member of the Flaviviridae family. It is an enveloped virus that has a plus-sense, single-stranded, RNA molecule as its genetic material. The hepatocytes of the liver are the host cells of HCV, explaining why HCV can cause liver cirrhosis and liver cancer. Furthermore, because hepatocytes are the host cells, the blood of an infected individual still carries a viral load; thus, if one comes into contact with infected blood, there is a high probability of horizontal transmission.
New Treatments for HCV
Ideally, an anti-HCV treatement should target a process only preformed by HCV. This minimizes the chances of the drug having cytotoxic effects on host cells. This being said, there are serveral candidate HCV proteins being studied as potential targets for anti-viral therapy. One of these proteins is non-structural protein 5 B (NS5B), the viral polymerase which uses RNA as a template and makes a complimentary RNA strand. This process is completely foreign to hepatocytes and all other cells in the human body, and as such, it makes a prime candidate for drug research.
In analogy, the structure of NS5B, the HCV RNA polymerase, is often compared to a hand. The palm region carries the catalytic polymerase activity of the protein, and the “thumb and fingers” of the protein interact with the template RNA and the newly synthesised RNA. There are two loop regions on the “fingers” that interact with the “thumb” to confer the protein into a barrel-like conformation. NS5B can also adapt to a more open conformation where the “thumb and “finger” regions do not interact, but this inactivates the protein.
One new series of drugs being developed for inhibiton of this protein is anthranilic acid derivatives. They are allosteric inhibitors of the protein, meaning that they do not function by blocking the active site of NS5B like a competitive inhibitor, but rather they work by binding to a different area on the protein and causing a conformational change that disrupts its function. Typically allosteric inhibition of a protein is more effective than competitive inhibition, as the allosteric inhibitors do not have to “fight” with the natural substrate of the enzyme (i.e. there is less chance of the allosteric inhibitor not being able to bind to the enzyme).
In particular, the work being done by Nittoli et al. is very promising, as their last publication yielded two allosteric inhibitors of this class that have in vitro IC50s in the 10-17 nM range. They began with a lead compound refered to as “3a”, which was identified through high-thoughput screening of the Wyeth compound library.
From this lead compound, many analogues were developed. These compounds bind approximately 7.5 Å from the NTP binding site of the NS5B active site; NTPs are nucleotide triphosphates, the natural substrate used by NS5B to make the sister strand to the RNA that it is copying. Through the use of SAR techniques, Nittoli et al. were able to determine the most important, potency enhancing characteristics of the molecules.
Diagram exhibiting the quasi-tricyclic nature of the compounds.
They discovered that much of the potency of this class of inhibitors was due to the drug being in a quasi-tricyclic conformation, held by intramolecular hydrogen bonding between the H of the amino group, the O of the Carboxylic acid, and the linker atom located at the Y position. Thus, removing or changing any of these groups or atoms can have severe consequences for the potency of the inhibitor. This can be seen in the IC50 values of other derivitives where the carboxylic acid group is moved from the 2 position, as in 3b and 3c, and also when the H-bonding ability of the carboxylic acid is compromised, as in 3d. Also, by comparing 3a, 3f, 13, and 14, they were able to evaluate the best linker molecules as being N > O and O > S.
They also studied the effect of substituting the methylene linker and found that S methylation of the linker, as in compound 17a, was very favorable when compared with no methylation, as in 3a, and also that R methylation was unfavorable due to strong steric interactions with the enzyme. This is also the reasoning behind the low activity of 3h.
Through similar reasoning processes, they discovered that the most active inhibitor was one that would be tri-subsituted on the phenoxy ring with 3 electro-negative groups or atoms. They found the most potent inhibiton to be derived from a 2, 4, 5 tri-chloro substitution, as this phenoxy ring sits in a spherical binding pocket and chloro-substituents at these locations provided the stongest binding of the drug into the allosteric site.
From all of these observations, they were able to design two very potent inhibitors of the NS5B polymerase. These two compounds embody all of the favorable SARs that they documented in the course of their experiments and as predicted had the lowest IC50 of all the compounds designed. They have a carboxylic acid located at the 2 position on the “B” ring and have a N for the linker atom. They are also tri-substituted on the anilino-ring; however, they noticed that placing an othro-acetyl group on the anilino ring significantly increased activity of the compound, as did having the anilino ring hetero disubstituted with chloro and fluoro groups or bromo and fluoro groups.
Human immunodeficiency virus type-1 Representation of HIV-1 Structure
Human Immunodeficiency Virus Type-1 (HIV-1) is a retrovirus of the lentivirus family. The name lentivirus comes from the Latin word ‘lentis’, meaning slow, and refers to the slow progression of disease. HIV-1 infects cells of the immune system, including macrophages and helper T cells. As the host immune system becomes progressively weakened, the host develops an increased susceptibility to opportunistic infections and is then said to have acquired immunodeficiency syndrome (AIDS).
The Joint United Nations Programme on HIV/AIDS (UNAIDS) reports that as of 2005, roughly 40 million people were infected and living with HIV. Since the discovery of the virus in 1981, 65 million people have been infected, and 25 million people have died from AIDS-related illnesses. As of 2007, HIV-1 newly infects 14000 people every day.
The current treatment for HIV infection is highly active antiretroviral treatment (HAART). HAART uses a combination of three or more drugs from multiple drug classes to target several different proteins involved in various stages of the HIV replication cycle. There are currently four FDA-approved drug classes used in HAART:
1) nucleoside reverse transcriptase inhibitors (NRTIs) – Reverse transcriptase is a viral enzyme necessary for replication; it converts the single-stranded RNA genome into double-stranded DNA. There are no host enzymes capable of doing this. NRTIs inhibit reverse transcriptase by acting as nucleoside analogues that, once incorporated into the growing DNA strand, will terminate further polymerization. NRTIs can lead to multi-drug resistant strains of HIV and can be toxic to cell mitochondria.
2) non-nucleoside reverse transcriptase inhibitors (NNRTIs) – NNRTIs inhibit reverse transcriptase by binding to an allosteric site near reverse transcriptase’s active site. NNRTIs have led to many drug-resistant HIV strains.
HIV Reverse Transcriptase
HIV-1 life cycle showing reverse transcription
3) protease inhibitors (PIs) – Many of HIV’s genes are translated into polyproteins. HIV’s protease is an aspartic acid protease required to cleave the polyproteins into individual, functional proteins. This step is necessary for virus production. All of the current FDA approved PIs are peptidomimetic nonhydrolyzable analogues. These drugs have many disadvantages: they increase drug-resistance, they have low oral bioavailability, and they are the most toxic of all available anti-HIV drugs.
4) fusion inhibitors – When HIV’s envelope glycoprotein gp120 has bound the host receptor CD4 and a co-receptor (either CCR5 or CXCR4), the viral glycoprotein gp41 is inserted into the cell membrane and fusion occurs. Enfuvirtide (ENF) or fuzeon is currently the only fusion inhibitor that has gained FDA approval. It inhibits viral entry by binding to one region of gp41, preventing the glycoprotein from binding its other regions, ultimately changing its conformation. ENF is only used as a last resort, because it has low bioavailability and a high production cost.
HIV Protease with Glaxo Wellcome inhibitor in active site. Enfuvirtide
New prospects for the treatment of HIV-1
Integrase inhibitors – Integrase is HIV’s third enzyme that is necessary for replication. The integrase enzyme integrates the new ds-DNA virus genome into the host genome, where the host cellular machinery can replicate the virus. By inhibiting integrase, HIV is unable to replicate. Because nothing similar to integrase can be found in mammalian cells, integrase inhibitors should be expected to have low toxicity to humans. In October of this year, Raltegravir (a small molecule) became the first integrase inhibitor to receive FDA approval, and can now be used as a part of HAART therapy. However, it has only been approved for patients whose infection shows resistance to other HAART drugs. There are other integrase inhibitors in various stages of clinical trials and many others being developed.
Entry inhibitors – Entry inhibitors block virus entry into a cell, thereby preventing the spread of infection. Most entry inhibitors being developed will only prevent the spread of infection within an already infected individual and not from person-to-person. Entry inhibitors can target several different proteins, including viral adhesins, like gp120 and gp41, host cell receptors, like CD4, and host co-receptors, like CCR5 and CXCR4 (both are normally chemokine receptors). In August of 2007, Maraviroc gained FDA approval. It is the first drug of its class to do so. Maraviroc (a small molecule) is a CCR5 antagonist. It binds the CCR5 host receptor and interferes with the host-virus interaction, thereby preventing infection. It is also used in HAART. There are many other entry inhibitors with various targets currently being developed.
Topical microbicides – A new area being researched is small molecule inhibitors that can be used in topical microbicides to prevent the spreading of HIV infection from person-to-person. In order to be used in a topical, these entry inhibitors must be able to inhibit virus entry without interacting with the host cell receptors. Therefore, these small molecules must render a virus particle non-infectious after binding only a viral adhesion. Because 80% of HIV infections are transmitted sexually, the development of anti-HIV topical microbicides could drastically reduce the number of people being newly infected.
Cell splicing equipment inhibitors – When the HIV genome is transcribed, the initial product is a single strand of genome-length pre-mRNA. This pre-mRNA is then spliced by the host splicing equipment, producing 40 different functional mRNAs. If the host’s splicing machinery has been inhibited, HIV cannot successfully replicate. This theory has lead to a new study researching small molecule inhibitors of splicing equipment. Scientists have begun researching the inhibition of host proteins in an attempt to reduce drug-resistance. The idea is that it would be highly unlikely that a virus could mutate to compensate for a host deficiency. Therefore, a virus couldn’t gain resistance to drugs that inhibit host proteins and enzymes, like splicing equipment. These drugs would be extremely useful for patients with multidrug-resistant HIV infections. However, because these drugs are targeting the host, they have the potential to be highly toxic.
Influenza viruses are members of the Orthomyxoviridae virus family. They are enveloped, negative sense RNA viruses that use the cells of the lungs as host cells. There are 3 types of common influenza viruses that infect humans, deemed influenza A, B, and C. Each of these subtypes can be further classified into specific serotypes, which are classed based on the two types of outer membrane proteins found on the virus. The two outer membrane proteins that determine viral serotype are hemagglutinine and neuraminidase. Because there are several types of both, viral hemagglutinine and neuraminidase, there are many different viral serotypes.
Representation of typical Influenza A Virus structure
Hemagglutinine is used by the virus to gain entry into the host cells. It binds to receptors that contain sialic-acid on the host cell surface and causes the virus to become endocytosed. After the endocytosis the virus cell is able to unwrap from its membrane and begin host infection. Neuraminidase is used by the virus when it is time to bud from the host cell membrane. Neuraminidase is an enzyme that will sever the last remaining sialic-acid residue tying the newly formed progeny virus to the host, thus causing the release of the virus and allowing it to infect a new host cell. Because both of these proteins are located on the viral outer membrane, they commonly illicit a host immune response, and this makes them good targets for anti-viral vaccine treatments. Also, since both of these proteins are critical to viral admission and departure from the cell, they each become superior targets for small molecule anti-viral therapies.
The first class of drugs used to treat viruses of the Orthomyxoviridae family was matrix protein (M2) inhibitors. M2 inhibitors, such as amantadine and rimantadine, block a viral ion channel that is necessary for virus proliferation. However, the efficacy of this drug was short lived, as a very small mutation of this protein instilled the virus with complete immunity to the drug. Some work was done on finding M2 inhibitor analogues that would circumvent this mutation, but after several unproductive years the effort was stopped and focus was turned to other viral targets.
There are currently several different small molecule inhibitors of the influenza neuraminidase. Zanamivir was designed in the late 1960’s and was found to be a very potent competitive inhibitor. However, it had horrible bioavailability and had to be administered through inhalation to direct it to the site of viral infection. Peramivir was another potent competitive inhibitor that was developed some time later, but it also had very low bioavailability; work is currently being done to develop an intravenous treatment with this drug. However, there was one drug that did show promise – Tamiflu. Tamiflu is a potent neuraminidase inhibitor with an effective bioavailability, but there are some down sides to Tamiflu that were underestimated until the late 1990’s, when Tamiflu was being regarded as the number one reactionary drug to a possible influenza pandemic.
Zanamivir Peramivir Tamiflu
The first problem with Tamiflu lays not in the drug itself, but rather in its target, neuraminidase. Neuraminidase is a highly mutagenic protein undergoing antigenic shift on an almost yearly basis. This presents a major problem for any drug that acts as a competitor to it. At the time of a flu pandemic, there will be no guarantee that the pandemic strain of flu will not have mutated outside the influence of Tamiflu, rendering the drug useless. This is not a good scenario for the “most promising” drug in our influenza arsenal.
Tamiflu also has a very delicate synthesis process. Currently, the starting reagent, shikimic acid, is only effectively isolated from the ancient Chinese cooking spice Star Anise. Star Anise is only grown in ~ 6 provinces in China, and 90% of the plant is already being utilized by Roche, the pharmaceutical company responsible for Tamiflu, to synthesize Tamiflu. Hence, another strike against Tamiflu: if there was ever a pandemic, it would be nearly impossible to scale up the production of of the drug to match the demand that would be needed to combat such a large scale viral infection.
Star anise fruits (Illicium verum) Shikimic acid
The next problem with Tamiflu is with the current dosage regime. It is thought by many that the current recommended dosage of Tamiflu, which is commonly prescribed, is much too low, and as a result, Tamiflu is not obliterating viral populations. The remaining viruses may be more resistant to Tamiflu’s mode of action. These more resistant viruses will then be the ones that re-establish infection in a Tamiflu treated host. Thus, this makes a second round of Tamiflu treatment useless if the virus has achieved resistance or immunity.
Finally, Tamiflu has been reported to have some severe psychological effects on teenage recipients, such as hallucination and delirium. However, it is not clear whether these effects are due to the drug or if they are side effects of the influenza infection. In 2006, a study done by a professor at a Japanese university reported that Tamiflu had no apparent psychological side effects on the ~3000 children monitored in the study. It was, however, later found that the Roche had made significant donations to the department of the university where the principal investigator worked, and with this information in mind one must ask him/herself exactly how objective the study was.
Tamiflu (Oseltamivir) pills
Aedes aegypti mosquito
A genus of the family Flaviviridae, flaviviruses contain (+)stranded RNA and replicate in the host cytoplasm. The flaviviruses cause a variety of diseases. They are spread by insect bites or contact with contaminated blood. The genus includes (but is not limited to) the following virus:
Dengue fever virus – Dengue fever causes fever, joint pain, and severe flu-like symptoms. It can often progress to dengue hemorrhagic fever, which is characterized by internal bleeding and circulatory failure. Infection with the virus has a 5% mortality rate. This may seem low, but more than 50 million people are infected every year. The disease is caused from infection by one of four different virus serotypes, so many people remain susceptible to infection even after outbreaks of the disease, and new outbreaks occur roughly every five years.
West Nile virus – West Nile virus (WNV) infects birds and mammals. In humans, infection by WNV can have no symptoms, cause fever and flu-like symptoms, or lead to West Nile encephalitis or West Nile meningitis. Though the majority of people infected show either no symptoms or non-severe ones, one in 150 people develop the far more serious encephalitis or meningitis, which can be fatal. There are currently no drugs to treat West Nile encephalitis.
Yellow fever virus – This virus causes severe flu-like symptoms. 15% of infected patients will develop yellow fever, which is named for the jaundice that occurs with the disease. Along with flu-like symptoms and jaundice, the disease causes haemorrhaging and kidney malfunction. Roughly 7% of infected individuals die. Though there is an effective vaccine, yellow fever is prominent in Africa and South America. There is currently no cure for the disease.
Dengue fever virus West Nile virus Yellow fever virus
World Community Grid
The World Community Grid is a non-profit organization that uses grid computing for scientific research projects that can benefit humanity. Grid computing joins individual computers together into a ‘grid’ to increase computational power. Anyone can register their computer with World Community Grid, and computational analyses will be run on these computers when they are idle.
The World Community Grid launched a new project in August called ‘Discovering Dengue Drugs – Together’. The goal of this project is to find new small molecule inhibitors of viruses in the Flaviviridae family, more specifically Dengue fever virus, West Nile virus, Yellow fever virus, and Hepatitis C virus. These viruses contain a common target for small molecule inhibition, the NS3 protease, which is essential for virus replication. The amino acid sequence and atomic structure of the NS3 protease is very similar in all four viruses, and its structure is known. This allows the computational analyses of one protein structure to be meaningful for all of the viruses.
So how is the Discovering Dengue Drugs project finding new drug leads against the NS3 protease? Their method can be divided into two phases. First, they determine the binding orientation of a given small molecule into the active site of the NS3 protease. They do this using mean-field molecular dynamics algorithms and AutoDock, a docking program. Docking is the process of bringing two molecules together. They determine the orientation of the small molecule by maximizing favourable interactions with the protease’s active site and minimizing unfavourable ones. Millions of small molecules are screened through this process. The molecules that appear to have protease inhibitor qualities are advanced to the next phase.
In the second phase, the molecules are analyzed in CHARMM, a molecular dynamics program. The binding free energies of the molecules and the protein are calculated using the binding orientations determined in phase one. The binding free energy is a thermodynamic measure of the energy difference between the bound and unbound state. This phase is much more precise than phase one, but requires significantly more time. This is why molecules are first screened through phase one. Overall, this process drastically reduces the time required to find potential dengue drug leads. After the second phase is completed any molecules that appear to be good drug leads are tested in labs, where actual antiviral activity is determined.
Yellow fever virus NS3 protease
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