hiv protease
Before we discuss HIV protease inhibitors, let’s take a look at the life cycle of HIV to help us understand the role of HIV protease in virus replication.
HIV protease is a virus specific enzyme involved in the entire life cycle of HIV. And protease inhibitors work by blocking virus maturation.
AIDS virus itself is a virus particle, which enters the blood through sexual intercourse, blood transfusion or vertical channels (from mother to baby). In order to allow virus particles to penetrate host cells, the envelope spike protein gp120 comes into contact with CD4+on the T cell membrane. Here gp120 represents a glycoprotein with a molecular weight of 120K Dalton on the HIV envelope protein.
This process of contact is called tropism. Once gp120 and CD4 come into contact, the viral particles, with the help of another envelope glycoprotein gp41, inject their RNA genetic information into the host cell. But RNA is not active alone. Its companions include three enzymes: reverse transcriptase, protease, and integrase. Inhibition of reverse transcriptase and protease has been proved to be the most successful and effective treatment, and most approved AIDS drugs target these two enzymes.
Once the HIV virus enters a cell, it removes its protein shell and releases the genetic information of the RNA chain along with reverse transcriptase into the cytoplasm. Within the cytoplasm, viruses can utilize the advantages of substrates to reverse transcribe RNA genomes into double stranded DNA copies through reverse transcriptase.
HIV protease is an enzyme necessary for the production of new viruses, responsible for processing multi protein gene products into mature proteins. Essentially, some large viral proteins must be broken down by proteases into small proteins with regulatory functions. It plays a crucial role in virus replication and is the optimal feature of viral proteins in both function and structure. It is an aspartic protease with a catalytic mechanism similar to human renin.
Other human aspartate proteases include pepsin, gastrin, and tissue protease. HIV protease is formed by symmetric dimerization of monomer subunits, each subunit having a catalytic aspartic acid residue. Therefore, it is not surprising that many pharmaceutical companies initially screened their renin inhibitors (used to regulate blood pressure) in search of HIV protease inhibitors. The design of protease inhibitors significantly promotes the usability of X-ray crystal structures, allowing for direct observation of inhibitors bound to enzymes.
Proteases act on the last step of the virus’s lifecycle. Protease inhibitors were discovered shortly after the discovery of nucleoside reverse transcriptase inhibitors. In 1996, with the emergence of the first generation HIV protease inhibitors saquinavir, ritonavir, and indinavir, the era of HIV protease inhibitors began.
First generation HIV protease inhibitors
1 Shaquinavir
Roche’s saquinavir (Inviase) is the first HIV protease inhibitor on the US market. As early as 1986, Roche launched an ambitious international cooperation to address the issue of HIV protease.
A chemistry team led by Ian Duncan and Sally Redshaw in Welwyn, UK, used a “transition state simulation” strategy to design inhibitors after selecting colorimetric analysis as an in vitro trial. This strategy was previously very successful in producing potent renin inhibitors. Due to the fact that the transition state of the reaction has the highest thermodynamic energy, transition state mimetics can inhibit enzyme function.
Shaquinavir relies on the hydroxyl group as an isomorphic tetrahedral transition state, a design element derived from extensive exploration of renin inhibitors, another mammalian aspartic protease. Chemists quickly achieved an important milestone result: by defining the smallest simulated peptide, an acceptable level of inhibition was achieved. They found that tripeptides are ideal in terms of efficacy and bioavailability. Therefore, tripeptides have become a common theme followed by many protease inhibitors.
The Roche team has made tremendous efforts in fine-tuning tripeptides, systematically exploring their lead compounds by modifying each amino acid residue one by one. Their hard work paid off in 1991, and saquinavir was approved by FDA in December 1995, becoming the first HIV protease inhibitor used to treat AIDS on the market.
Although saquinavir is a peptide like drug that can be easily metabolized, it was later found to be more beneficial when used in combination with another protease inhibitor, ritonavir, as a drug enhancer. Due to the inhibition of cytochrome P450 3A4 (CYP3A4) enzymes in the liver by ritonavir, the combination of the two drugs can significantly increase the plasma level of saquinavir. CYP3A4 is the most abundant CYP isoenzyme in the liver. In addition, the emergence of saquinavir marks the beginning of cocktail drug combination therapy that introduces protease inhibitors into clinical practice. [1]
2 Litonavir
Abbott’s ritonavir (Norvir) is the second protease inhibitor on the market. The environment in which Litonavir was discovered is very unique. The Abbott team is led by X-ray crystallographer John Erickson and pharmaceutical chemist Dale Kempf. It is rumored that Merck has 30 chemists involved in their HIV protease inhibitor project, while Kempf only has 3.
Abbott did not screen for their renin inhibitors like most pharmaceutical companies, but instead utilized Erickson’s X-ray crystallography of HIV proteases, which will prove to be helpful for their drug design. By combining Structure Based Drug Design (SBDD) and traditional drug chemistry, they prepared a series of symmetry based inhibitors to match the C2 symmetry properties of HIV proteases.
Because their inhibitors bind to both sides of the enzyme, Kempf refers to them as molecular peanut butter. Through this method, they found tetrapeptide A-77003 containing pyridine. Although A-77003 is effective in binding and cell assays, it has a very high human biliary clearance rate. They achieved an increase in bioavailability by reducing molecular weight and replacing existing amino acids with more soluble ones.
Finally, it was found that the pyridine end was oxidized to N-oxide by liver cytochrome P450. Simply replace pyridine with thiazole, which has stronger metabolism, and make some minor adjustments. The bioavailability of ritonavir is 78%, while the bioavailability of pyridine analogue A-77003 is 26%. In March 1996, Abbott’s ritonavir was approved by the FDA. [2]
Abbott’s discovery of ritonavir is undoubtedly impressive. But the two things that happened after discovering it were equally interesting. One of them involves its crystal morphology, and the other involves cobicistat, a similar compound of ritonavir discovered by Gilead, used as a bioavailability enhancer.
In 1996, Abbott’s ritonavir was approved by the FDA and sold under the trade name Norvir. The active pharmaceutical ingredient (API) of the drug was produced using the original crystal I. Due to the lack of bioavailability of ritonavir in its solid state, it is prepared as an oral solution or semi-solid capsule, both in an ethanol water solution.
In early 1998, two years after the launch of Litovina, problems began to arise in the production of active pharmaceutical ingredients: many batches began to transform into a new, different, and extremely insoluble crystal form II, although crystal form I was initially also difficult to dissolve. The new crystal form II has a broad intermolecular hydrogen bonding network, which is thermodynamically more stable than the original I. Once there is a crystal seed of II, no matter how small and microscopic it is, nature will always convert the API into a thermodynamically more stable crystal form II.
A group of scientists who had been exposed to Crystal II visited Abbott’s manufacturing plant in Italy and investigated the mass production process of Litonavir. Later, in the production process of the factory, a large amount of crystal form II began to appear in the production process of raw materials. This situation has indeed plunged Abbott into a marketing crisis. Abbott’s pharmaceutical chemists put in a lot of effort to come up with a method of producing only the original dosage form I: in order to prevent the formation of dosage form II, semi-solid capsules or oral dosage forms need to be refrigerated before use. [3]
Another story after the success of Litonavir takes place in Gilead, Foster City, California. Interestingly, when many other protease inhibitors are metabolized by CYP3A4, ritonavir effectively inhibits CYP3A4 and P-glycoprotein (Pgp) by delivering the drug out of the cell. Therefore, it can be used in combination with CYP3A4 protease inhibitors or other drugs to increase plasma levels.
Therefore, dual protease inhibitors have been proven to be highly effective treatment options in terms of efficacy and reducing drug resistance. Low dose ritonavir is now mainly used as a pharmacokinetic (PK) enhancer for HIV inhibitors, also known as a drug enhancer. Drug enhancers are enzymes that typically do not have activity towards therapeutic targets, but can inhibit the metabolism of active drugs. Therefore, drug enhancers can improve the PK spectrum of antiviral drugs, achieving sufficient trough concentrations (the lowest concentration reached by the drug before the next administration) at lower doses and fewer times.
Gilead collaborated with Japanese company Tobacco to develop the HIV integrase inhibitor elvitegravir (Vitekta, 2014). Unfortunately, although elvitegravir is an effective integrase chain transfer inhibitor (INSTI), it is widely metabolized by CYP3A4 in the liver and intestines and cannot be treated once a day. Although the use of sub therapeutic doses of ritonavir may lead to resistance to other protease inhibitors, there is a better increase in drug exposure when administered together with ritonavir.
Therefore, Gilead decided to produce a drug that can only improve bioavailability by inhibiting CYP3A4 mediated metabolism, but does not inhibit HIV protease at all; Meanwhile, high water solubility and good physicochemical properties are also beneficial for drug preparation. Although the water solubility of ritonavir is poor, under the leadership of Manoj Desai, the head of pharmaceutical chemistry, a team led by Lianhong Xu has developed an effective and highly bioavailable selective CYP3A4 inhibitor cobicistat, starting from ritonavir.
Scientists knocked out a hydroxyl group on Litonavir because it mimics the transition state of amide hydrolysis: by forming hydrogen bonds with two amino acids in the catalytic domain of the HIV protease active site, molecules without critical hydroxyl groups are almost unable to bind to HIV protease. Cobicistat was approved by the FDA in 2012, and Gilead sold it under the trade name Typost, which has been used in combination with various antiretroviral drugs. [4]
In drug discovery, drug drug interaction (DDI) occurs when two drugs are metabolized by the same liver CYP enzyme. They are often considered a “liability” with sufficient justification. For example, Bayer’s statin drug cerivastatin (Bayer) can cause serious liver damage when taken in combination with cholesterol lowering fiber drugs. Because both drugs are metabolized by CYP3A4, the liver does not have enough CYP3A4 enzymes to metabolize both drugs simultaneously, resulting in toxicity.
But with drug enhancers like ritonavir and cobicistat, we can turn the drug’s “debt” into an “asset.”. When drug enhancers keep the CYP enzyme in the liver busy, antiviral drugs can focus on killing the virus without worrying about themselves. Cobicistat can make daily monotherapy containing protease inhibitors possible.
3 Indinavir
Merck’s indinavir (Crixivan) is the third protease inhibitor on the market. Merck began research on HIV protease inhibitors in 1986, with Irving Sigal, senior director of the molecular biology department, as the project leader. They initially screened for the inhibitory effect of renin inhibitors on HIV protease, and then used the known crystal structure of HIV protease for rational drug design, which was first discovered by Merck and optimized by NIH scientists.
In 1990, chemist Wayne Thompson discovered L-689502, which effectively inhibits HIV protease but lacks renin activity. However, it is only effective by injection and has no bioavailability. Subsequently, inspired by the feasible oral drug saquinavir in the literature, Joseph Vacca successfully implanted a fragment of saquinavir into L-689502.
In 1989, Bruce Dorsey, a new employee of the Vacca team, and his colleague Rhonda Levin successfully synthesized indinavir. Although in monotherapy trials, approximately 40% of patients showed RNA levels below 400 copies per milliliter after 6 months of taking the drug, in some patients, the HIV virus developed resistance to indinavir. Fortunately, they found that indinavir combined with AZT or epivir can effectively inhibit viral levels.
Merck’s research on combination therapy has been the first to demonstrate the efficacy of cocktail therapy and has become an industry standard. After submitting an application to the FDA in January 1996, indinavir was approved during the accelerated review process in March 1996. [5]
Other important early protease inhibitors include Agouron (now Pfizer)’s nifinavir (approved in March 1997) and Vertex’s amprenavir (approved in April 1999). These are collectively referred to as the first generation protease inhibitors.
Vertex started relatively late in the field of protease inhibitors. Using crystallography and computer-aided drug design, Vertex and their Japanese collaborators at Kissei Company discovered amprenavir, a highly effective Pimol HIV protease inhibitor.
In 1993, Vertex collaborated with Burroughs Wellcome to study the PK characteristics of Kissei’s amprenavir. Burroughs Wellcome, who was overwhelmed by AZT’s success, started relatively late in the field of protease inhibitors and needed to rely on Kissei/Vertex drugs as a springboard. Amprenavir is small enough to penetrate the blood-brain barrier and kill the virus inside. Obviously, they are very satisfied with this result, Burroughs Wellcome has closed its HIV protease project and agreed to pay Vertex $200 million in drug clinical trial fees.
Amprenavir was approved by the FDA in 1999, and Vertex and Burroughs Wellcome (renamed Glaxo Wellcome in 1995 and Glaxo SmithKline in 2000) jointly sold the drug under the trade name Agenerase. Although the drug had 100% bioavailability, it was later discontinued due to its poor water solubility (0.04 g/mL).
It requires a high ratio of excipients to drugs to ensure gastrointestinal solubility and final absorption. Vertex continued to prepare its phosphate prodrug fosamprenavir, which increased its solubility in aqueous solution by 8 times (0.31 g/mL). The prodrug Fosanovir was approved in 2005, and Vertex and GSK sold it under the trade name Lexva.
Protease inhibitors may be the most effective among antiretroviral drugs, partly because they typically have a high resistance gene barrier. The first generation protease inhibitors used in the early stages, such as saquinavir, ritonavir, indinavir, and nefenavir, did not utilize their promoting effect on CYP3A4 inhibition.
However, the pharmacokinetic characteristics of these inhibitors are not ideal. Despite having high genetic barriers, these first generation protease inhibitors ultimately developed resistance. Later, the enhanced pharmacokinetics provided by ritonavir resulted in a decrease in the frequency of administration, with many drugs being able to be administered once a day. Additionally, due to the significant increase in daily plasma trough concentrations, the genetic barrier for drug resistance is higher.
Second generation HIV protease inhibitors
Although the initial ritonavir promotion regimen was carried out in conjunction with the first generation protease inhibitors, the development of more effective and safer second-generation inhibitors heralds the arrival of a new era of cocktail regimens. These second-generation inhibitors include amprenavir (a prodrug of amprenavir), lopinavir, atazanavir, tipranavir, and darunavir.
Many first generation protease inhibitors are peptidogenic drugs. They are designed based on an improved mimetic peptide model, where the divisible bonds of the peptide substrate are replaced by indivisible transition state mimicry. We always need a lot of pharmaceutical chemistry research to systematically modify every detail of peptides in order to obtain an effective and biologically effective drug. Due to the fragility of early protease inhibitors to metabolism, almost all of these drugs must be used together with ritonavir as PK enhancers. UpJohn took a different approach.
UpJohn from Kalamazoo, Michigan does not want to use peptide mimicry. On the contrary, they conducted medium flux screening on 5000 different existing historical compounds in the early 1990s. Compared to today’s routine high-throughput screening (HTS) of millions of compounds, 5000 compounds seem absurdly small. Nevertheless, they still identified the blood diluent warfarin as a weak inhibitor of HIV protease.
Coincidentally, their neighbors in Park Davis, Ann Arbor, Michigan, discovered similar substrates from relevant screening work. Although the efficacy of warfarin is far inferior to that of pepsin inhibitors, it is still attractive due to its wide bioavailability. Essentially, UpJohn is more inclined towards pharmacokinetics rather than efficacy.
Subsequently, they focused on screening compounds similar to warfarin and identified the warfarin analogue Phenprocoumon, which is a known oral active anticoagulant. Phenprocoumon is 30 times more potent than warfarin and has begun to demonstrate antiviral activity in cell experiments. The crystal structure of the Phenprocoumon protease complex is of great help for the drug design of future protease inhibitors.
Using computer-assisted Structure Based Drug Discovery Platform (SBDD), UpJohn has obtained their first generation clinical candidate drugs. But later it was replaced by more effective analogues. Their second-generation clinical candidate drug has once again been abandoned because, although it has a very high protein binding affinity, its efficacy is still limited compared to the most active digestive analog drugs of the time.
The third generation was successful. They ultimately developed a third-generation clinical candidate drug, Tepronavir, with better safety and efficacy, under the trade name Aptivus, which was approved by the FDA in 2005. Unfortunately, compared to other protease inhibitors, tepinavir has more serious side effects. [6]
The latest HIV protease inhibitor is Janssen’s darunavir (Prezista), which was approved by the FDA in 2006. Arun Ghosh discovered that the experience of Dalunavir was a long and winding journey.
In 1988, Ghosh completed E J. After receiving postdoctoral training, Corey worked at Merck’s West Point facility in Pennsylvania for 6 years and was involved in the discovery of HIV protease inhibitors. But in 1994, he decided to start his academic career at the University of Illinois at Chicago and went to Purdue University in 2005.
In order to better discover HIV protease inhibitors, Ghosh adopted the design concept of “skeleton binding” to maximize interaction with the active sites of HIV protease, especially promoting extensive hydrogen bonding with protein backbone atoms. His team initially installed a tetrahydrofuran (THF) ring on Roche’s saquinavir. Later, by combining the levorotatory properties of mercinavir, Ghosh obtained a very good protease inhibitor containing tetrahydrofuran.
Meanwhile, Vertex/Kissei also added a similar 3- (S) – THF motif as the P2 ligand for its sulfonamide compound. Interestingly, in reality, Searle had priority over protease inhibitors containing sulfonamide, despite ultimately failing due to protein binding and metabolic issues. However, Vertex had to pay Searle $25 million in 1995 to resolve the patent issue.
In the field of drug discovery, people always learn from each other. Although Vertex borrowed Ghosh’s THF fragment, Ghosh also shamelessly borrowed Vertex’s sulfonamide moiety. For Ghosh, in order to consolidate his intellectual property position and enhance efficacy through more hydrogen bonds, he invented a double THF group to replace THF fragments. In the end, Darunavir was obtained, and the universal name “arun” undoubtedly pays tribute to the inventor. The development of Dalunavir was carried out by Tibotec, a subsidiary of Janssen. The drug was approved in 2006 and Janssen sold it under the trade name Prezista. [7]
The PK enhanced second-generation protease inhibitor covers the initial protease resistance, allowing patients who develop resistance to the first generation drug to continue using protease inhibitors. This is especially true for more effective second-generation inhibitors.
Finally, almost all HIV protease inhibitors, including the latest and best darunavir, need to be taken in combination with pharmacokinetic enhancers such as ritonavir or cobicistat to achieve effective plasma levels at the desired dose and frequency. It is gratifying that the use of protease inhibitors has reduced the mortality of AIDS patients by 70%.
4. Summary
To summarize, ten HIV protease inhibitors currently on the market are:
Sakinavir (Inviase, Roche), 1995
Indinavir (Crixivan, Merck), 1996
Ritonavir (Norvir, Abbott), 1996
Nelfinavir (Viracept, Agouron/Eli Lilly), 1997
Amprenavir (Agerase, Vertex/GSK, discontinued), 1999–2008
Lopinavir (Kaletra with ritonavir, Abbott), 2000
Atazanavir (Reyataz, Ciba Geigy), 2003
Fosamprenavir (a prodrug of amprenavir, Lexiva, GSK/Vertex), 2005
Tipranavir (Aptivus, Upjohn/Pfizer/Boehringer Ingelheim), 2005
Darunavir (Prezista, Janssen), 2006
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