Discovery and development of non-nucleoside reverse transcriptase inhibitors
Encyclopedia
Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) are antiretroviral drugs used in the treatment of human immunodeficiency virus (HIV
). NNRTIs inhibit reverse transcriptase
(RT), an enzyme
that controls the replication
of the genetic material of HIV. RT is one of the most popular targets in the field of antiretroviral drug development.
Discovery and development of NNRTIs began in the late 1980s and in the end of 2009 four NNRTI had been approved by regulatory authorities and several others were undergoing clinical development. Drug resistance
develops quickly if NNRTIs are administered as monotherapy and therefore NNRTIs are always given as part of combination therapy
, the highly active antiretroviral therapy (HAART).
) is one of the leading causes of death in the world. It was identified as a disease in 1981. Two years later the etiology
agent for AIDS, the HIV
was described. HIV is a retrovirus and has two major serotypes, HIV-1 and HIV-2. The pandemic
mostly involves HIV-1 while HIV-2 has lower morbidity rate and is mainly restricted to western Africa.
In the year 2009 over 40 million people were infected worldwide with HIV and the number keeps on growing. The vast majority of infected individuals live in the developing countries.
HIV drugs do not cure HIV infection but the treatment aim at improving the quality of patients´ lives and decrease mortality
. 25 antiretroviral drugs were available in 2009 for treatment of HIV infection. The drugs belong to six different classes that act at different targets. The most popular target in the field of antiretroviral drug development is the HIV-1 reverse transcriptase (RT) enzyme. There are two classes of drugs that target the HIV-1 RT enzyme, nucleoside
/nucleotide
reverse-transcriptase inhibitors (NRTIs/NtRTIs) and non-nucleoside reverse-transcriptase inhibitors (NNRTIs). Drugs in these classes are important components of the HIV combination therapy called highly active antiretroviral therapy, better known as HAART.
In 1987 the first drug for treatment of HIV infection was approved by the U.S. Food and Drug Administration (FDA). This was the NRTI zidovudine
. In the late 1980s, during the development of NRTIs, the field of NNRTIs discovery began. The development of NNRTIs improved quickly into the 1990s and they soon became the third class of antiretroviral drugs, following the protease inhibitors. The NNRTIs are HIV-1 specific and have no activity against HIV-2 and other retroviruses. The first NNRTI, nevirapine was discovered by researchers at Boehringer Ingelheim and approved by the FDA in 1996. In the next two years two other NNRTIs were approved by the FDA, delavirdine
in 1997 and efavirenz
in 1998. These three drugs are so called first generation NNRTIs. The need for NNRTIs with better resistance profile led to the development of the next generation of NNRTIs. Researchers at Janssens Foundation and Tibotec
discovered the first drug in this class, etravirine
, which was approved by the FDA in 2008. In addition to these four NNRTIs several other are in clinical development.
of HIV and other retroviruses. The enzyme has two enzymatic functions. Firstly it acts as a polymerase
where it transcribes
the single-stranded RNA
genome
into single-stranded DNA
and subsequently builds a complementary strand of DNA. This provides a DNA double helix which can be integrated in the host cell's chromosome
. Secondly it has ribonuclease H
(Rnase H) activity as it degrades the RNA strand of RNA-DNA intermediate that forms during viral DNA synthesis.
1000-amino acid
heterodimer composed of p66 (560 amino acids) and p51 subunits (440 amino acids). The p66 subunit has two domains, a polymerase and ribonuclease H. The polymerase domain contains four subdomains, which have been termed “fingers”, “palm”, “thumb” and “connection” and it is often compared to a right hand (figure 1). The role of the p66 subunit is to carry out the activity of RT whereas it contains the active site
s of the enzyme. The p51 is believed to play mainly a structural role.
from the catalytic site in the palm domain of the p66 subunit site of the enzyme. The NNRTI binding pocket (NNIBP) contains five aromatic (Tyr-181, Tyr-188, Phe-227 and Trp-229), six hydrophobic (Pro-59, Leu-100, Val-106, Val-179, Leu-234 and Pro-236) and five hydrophilic (Lys-101, Lys-103, Ser-105, Asp-132 and Glu-224) amino acids that belong to the p66 subunit and additional two amino acids (Ile-135 and Glu-138) belonging to the p51 subunit. Each NNRTI interacts with different amino acid residues in the NNIBP.
An important factor in the binding of the first generation NNRTIs, such as nevirapine, is the butterfly-like shape. Despite their chemical diversity they assume very similar butterfly-like shape. Two aromatic rings of NNRTIs conform within the enzyme to resemble the wings of a butterfly (figure 2). The butterfly structure has a hydrophilic centre as a ‘body’ and two hydrophobic moieties representing the wings. Wing I is usually a heteroaromatic ring and wing II is a phenyl or allyl substituent. Wing I has a functional group
at one side of the ring which is capable of accepting and/or donating hydrogen bonds with the main chain of the amino acids Lys-101 and Lys-103. Wing II interacts through π-π interactions with a hydrophobic pocket, formed in most part by the side chains of aromatic amino acids. On the butterfly body a hydrophobic part fills a small pocket which is mainly formed by the side chains of Lys-103, Val-106 and Val-179. However many other NNRTIs have been found to bind to RT in different modes. Second generation NNRTIs such as diarylpyrimidins (DAPYs), have a horseshoe-like shape with two lateral hydrophobic wings and a pyrimidine
ring which is the central polar part.
The NNIBP is elastic
and the conformation
depends on the size, specific chemical composition and binding mode of the NNRTI. The total structure of RT has segmental flexibility
that depends on the nature of the bound NNRTI. It's important for the inhibitor to have flexibility to be able to bind in the modified pockets of a mutant target. Inhibitor flexibility may not affect the inhibitor-target interactions.
to the RT enzyme (figure 3). The binding causes conformational change in the three-dimensional structure of the enzyme and creates the NNIBP. Binding of NNRTI to HIV-1 RT makes the p66 thumb domain hyper extended because it induces rotamer
conformation changes in amino acid residues Tyr-181 and Tyr-188. This affects the catalytic activity of the enzyme and blocks the HIV-1 replication by inhibiting the polymerase active site of the RT's p66 subunit. The transcription of the viral RNA is inhibited and therefore the replication rate of the virus reduces. Although the exact molecular mechanism is still hypothetical this has been demonstrated by multiple studies to be the primary mechanism of action.
In addition to this proposed primary mechanism of action it has been shown that the NNRTIs have other mechanisms of action and interfere with various steps in the reverse transcriptase reaction. It has been suggested that the inhibition of reverse transcription by the NNRTIs may be due to effects on the RT Rnase H activity and/or template/primer
binding. Some NNRTIs interfere with HIV-1 Gag-Pol
polyprotein processing by inhibiting the late stage of HIV-1 replication.
It is important to gain profound understanding of the various mechanism of action of the NNRTIs in order to develop next-generation NNRTIs and for understanding the mechanism of drug resistance.
DNA synthesis. The specificity of the NNRTIs for HIV-1 is considered the hallmark of the NNRTI drug class.
Three NNRTI compounds of the first generation have been approved by the FDA for treating HIV-1 infection. Nevirapine was approved in 1996, delavirdine in 1997 and efavirenz in 1998 (table 1). Two of these drugs, nevirapine and efavirenz, are cornerstones of first line HAART while delavirdine is hardly used nowadays. The structure of these three drugs show the wide array of rings, substituents, and bonds that allow activity against HIV-1 RT. This diversity demonstrates why so many non-nucleosides have been synthesised but doesn't explain why only three drugs have reached the marked. The main problem has been the potency of these compounds to develop resistance.
Crystal structure
analysis showed that the first generation NNRTIs (for example TIBO, nevirapine and α-APA) bind HIV-1 RT in a “butterfly-like” conformation. These first generation NNRTIs were vulnerable against the common drug-resistance mutations like Tyr-181C and Tyr-188L/H. This triggered the need for finding new and more effective NNRTIs. ITU (imidoylthiourea), a promising series of NNRTIs emerged from α-APA analogs (figure 4). The ITU compounds were obtained by extending the linker that binds the aryl side groups of the α-APA. A potent ITU compound, R100943, was obtained by an arrangement of the chemical composition of the side groups based on structure-activity relationship
s (SAR). A crystal structure of the HIV-1/R100943 complex demonstrated that ITU compounds are more flexible than α-APA compound. The ITU compounds showed distinct mode of binding where they bound with "horseshoe" or "U" mode. The 2,6-dichlorophenyl part of R100943 which corresponds chemically to the wing II 2,6-dibromophenyl part of the α-APA occupied the wing I part in the NNIBP whereas the 4-cyanoanilino part of R100943 occupies the wing II position in the NNIBP.
R100943 inhibited HIV-1 and was considerably effective against a number of key NNRTI-resistant mutants like G190A mutation, which caused high-level resistance to loviride (α-APA) and nevirapine. G190A mutation was thought to cause resistance by occupying a part of the binding pocket that would otherwise be filled by the linker part of the butterfly shaped NNRTIs. R100943, in the horseshoe mode of binding, is located at a distance of approximately 6.0 Å from G190. When compared with nevirapine and loviride which bind in the butterfly shape the ITU derivatives
revealed improved activity against Tyr-181C and Tyr-188L mutants. A structural study suggested that a potent TIBO compound could partly supplement for the effects of the Tyr-181C mutation by moving itself in the non-nucleoside inhibitor binding pocket (NNIBP) of the mutant RT. In this context, R100943 has torsional freedom that enables the conformational alternations of the NNRTI. This torsional freedom could be used by the ITU derivate to bind to a mutated NNIBP and thus compensating for the effects of a resistance mutation. Nevertheless, the potency of R100943 against HIV-1 resistant mutants was not adequate for it to be considered as an effective drug candidate
. Additionally, the chemical stability of the imidoylthiourea part of the ITU derivative was not favorable for an oral drug.
Changes in the imidoylthiourea complexes led to the synthesis of a new
class of compounds, diaryltriazine (DATA). In these compounds, the
thiourea part of the ITU compounds was replaced by a triazine ring. The
DATA compounds were more potent than the ITU compounds against common
NNRTI resistant mutant strains. R106168, a prototype DATA compound, was
rather easy to synthesize. Multiple substitutions were made at different
positions on all of the three rings and on the linkers connecting the
rings. In the pocket, most of the DATA derivatives conformed a horseshoe
conformation. The two wings in R106168 (2,6-dichlorobenzyl and
4-cyanoanilino) occupied positions in the pocket similar to that of the
two wings of the derivatives of ITU. The central part of the DATA
compounds, in which the triazine ring replaced the thiourea group of ITU
derivatives, is positioned between the side chains of L100 and V179. This
removed a number of torstional degrees of freedom in the central part
while keeping the flexibility between the triazine ring and the wings.
Chemical substitution or modification in the three-aromatic-ring backbone
of the DATA compounds had substantial effect on the activity. R120393, a DATA analog, was designed with a chloroindole part in wing I to expand
interactions with the side chain of conserved W229 of the polymerase
primer grip loop. R120393 had similar effect as R106168 against most of
the NNRTI-resistant mutants. The cloroindole part interacted with the
hydrophobic core of the pocket and influenced the binding mode of the
R120393 so it went deeper into the pocket compared to the wing I position
of other DATA analogs. Crystal structures showed that the DATA compounds
could bind the NNIBP in different conformations. The capability to bind in
multible modes made the NNRTIs stronger against drug-resistance mutations.
Variability between the inhibitors could be seen when the chemical
composition, size of wing I and the two linker groups connecting the rings
were altered. The potency of the NNRTIs changed when the triazine
nitrogen
atoms were substituted with carbons.
(DAPY), were discovered with the replacement of the central triazine
ring from the DATA compounds, with a pyrimidine
. This new class was more effective against drug resistant HIV-1 strains than the corresponding DATA analogs. The replacement enabled substitutions to the CH-group at the 5-position of the central aromatic ring. One of the first DAPY compounds, dapivirine (with R1= 2,4,6-trimethylanilino, R2 = R3 = H and Y = NH) was found to be effective against drug-resistant HIV-1 strains. Systematic chemical substitutions were made at the R1, R2, R3 and Y positions to find new DAPY derivatives. This led to the discovery of etravirine which has a bromine substitution at the 5-position (R3) of the pyrimidine
ring (with R1 = 2,6-dimethyl-4-cyanoanilino, R2 = NH2 and Y = O) (figure 5). Etravirine was discovered by researchers at the Jansen Research Foundation and Tibotec and approved in 2008 by the FDA. It is used in treatment-expirenced adult patients with HIV infection that is multidrug resistant in combination with other antiretroviral drugs.
NNRTI-resistant HIV-strains have the occurring mutations mainly in and around the NNIBP affecting the NNRTI binding directly by altering the size, shape and polarity
on different areas of the pocket or by affecting, indirectly, the access to the pocket. Those mutations are primarily noted in domains which span amino acids 98-108, 178-190 or 225-238 of the p66 subunit. The most frequent mutations observed in viruses isolated from patients who have been on a failing NNRTI containing chemotherapy are Lys-103N and Tyr-181C. NNRTI resistance has been linked to over 40 amino acid substitutions in vitro
and in vivo
.
Antiretroviral drugs are never used in monotherapy due to rapid resistance development. The highly active antiretroviral therapy (HAART) was introduced in 1996. The treatment regimen combines three drugs from at least two different classes of antiretroviral drugs.
The advance of etravirine over other NNRTIs is that multiple mutations are required for the development of drug resistance. The drug has also shown activity against viruses with common NNRTI resistance associated mutations and cross-resistance
mutations.
, efficacy and/or safety factors.
Currently there are four other NNRTIs undergoing clinical development, rilpivirine
, IDX899, RDEA-428 and lersivirine (table 2).
and it is easier to formulate than etravirine. Etravirine has required extensive chemical formulation work due to poor solubility
and bioavailability. Rilpivirine was undergoing phase III clinical trial
s in the end of 2009.
s. It has similar resistance profile against selected NNRTI resistant HIV-1 strains to other next generation NNRTIs. The candidate entered phase IIb clinical trials in the end of 2009.
family and is another next generation NNRTI in clinical trials developed by the pharmaceutical company Pfizer
. The resistance profile is similar to that of other next generation NNRTIs. In the end of 2009 lersivirine was in phase IIb.
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
). NNRTIs inhibit reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
(RT), an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that controls the replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
of the genetic material of HIV. RT is one of the most popular targets in the field of antiretroviral drug development.
Discovery and development of NNRTIs began in the late 1980s and in the end of 2009 four NNRTI had been approved by regulatory authorities and several others were undergoing clinical development. Drug resistance
Drug resistance
Drug resistance is the reduction in effectiveness of a drug such as an antimicrobial or an antineoplastic in curing a disease or condition. When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance. More commonly, the term is used...
develops quickly if NNRTIs are administered as monotherapy and therefore NNRTIs are always given as part of combination therapy
Combination therapy
Combination therapy or polytherapy is the use of more than one medication or other therapy. In contrast, monotherapy is any therapy which is taken by itself....
, the highly active antiretroviral therapy (HAART).
History
Acquired immunodeficiency syndrome (AIDSAIDS
Acquired immune deficiency syndrome or acquired immunodeficiency syndrome is a disease of the human immune system caused by the human immunodeficiency virus...
) is one of the leading causes of death in the world. It was identified as a disease in 1981. Two years later the etiology
Etiology
Etiology is the study of causation, or origination. The word is derived from the Greek , aitiologia, "giving a reason for" ....
agent for AIDS, the HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
was described. HIV is a retrovirus and has two major serotypes, HIV-1 and HIV-2. The pandemic
Pandemic
A pandemic is an epidemic of infectious disease that is spreading through human populations across a large region; for instance multiple continents, or even worldwide. A widespread endemic disease that is stable in terms of how many people are getting sick from it is not a pandemic...
mostly involves HIV-1 while HIV-2 has lower morbidity rate and is mainly restricted to western Africa.
In the year 2009 over 40 million people were infected worldwide with HIV and the number keeps on growing. The vast majority of infected individuals live in the developing countries.
HIV drugs do not cure HIV infection but the treatment aim at improving the quality of patients´ lives and decrease mortality
Mortality rate
Mortality rate is a measure of the number of deaths in a population, scaled to the size of that population, per unit time...
. 25 antiretroviral drugs were available in 2009 for treatment of HIV infection. The drugs belong to six different classes that act at different targets. The most popular target in the field of antiretroviral drug development is the HIV-1 reverse transcriptase (RT) enzyme. There are two classes of drugs that target the HIV-1 RT enzyme, nucleoside
Nucleoside
Nucleosides are glycosylamines consisting of a nucleobase bound to a ribose or deoxyribose sugar via a beta-glycosidic linkage...
/nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
reverse-transcriptase inhibitors (NRTIs/NtRTIs) and non-nucleoside reverse-transcriptase inhibitors (NNRTIs). Drugs in these classes are important components of the HIV combination therapy called highly active antiretroviral therapy, better known as HAART.
In 1987 the first drug for treatment of HIV infection was approved by the U.S. Food and Drug Administration (FDA). This was the NRTI zidovudine
Zidovudine
Zidovudine or azidothymidine is a nucleoside analog reverse-transcriptase inhibitor , a type of antiretroviral drug used for the treatment of HIV/AIDS. It is an analog of thymidine....
. In the late 1980s, during the development of NRTIs, the field of NNRTIs discovery began. The development of NNRTIs improved quickly into the 1990s and they soon became the third class of antiretroviral drugs, following the protease inhibitors. The NNRTIs are HIV-1 specific and have no activity against HIV-2 and other retroviruses. The first NNRTI, nevirapine was discovered by researchers at Boehringer Ingelheim and approved by the FDA in 1996. In the next two years two other NNRTIs were approved by the FDA, delavirdine
Delavirdine
Delavirdine is a non-nucleoside reverse transcriptase inhibitor marketed by ViiV Healthcare. It is used as part of highly active antiretroviral therapy for the treatment of human immunodeficiency virus type 1. It is presented as the mesylate...
in 1997 and efavirenz
Efavirenz
Efavirenz is a non-nucleoside reverse transcriptase inhibitor and is used as part of highly active antiretroviral therapy for the treatment of a human immunodeficiency virus type 1....
in 1998. These three drugs are so called first generation NNRTIs. The need for NNRTIs with better resistance profile led to the development of the next generation of NNRTIs. Researchers at Janssens Foundation and Tibotec
Tibotec
Tibotec is a pharmaceutical company with a focus on research and development for the treatment of infectious diseases such as HIV , and Hepatitis C...
discovered the first drug in this class, etravirine
Etravirine
Etravirine is a drug used for the treatment of HIV. Etravirine is a non-nucleoside reverse transcriptase inhibitor . Unlike the currently available agents in the class, resistance to other NNRTIs does not seem to confer resistance to etravirine. Etravirine is marketed by Tibotec, a subsidiary...
, which was approved by the FDA in 2008. In addition to these four NNRTIs several other are in clinical development.
The HIV-1 reverse transcriptase enzyme
Function
Reverse transcriptase (RT) is an enzyme that controls the replication of the genetic materialGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of HIV and other retroviruses. The enzyme has two enzymatic functions. Firstly it acts as a polymerase
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
where it transcribes
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
the single-stranded RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
into single-stranded DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
and subsequently builds a complementary strand of DNA. This provides a DNA double helix which can be integrated in the host cell's chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
. Secondly it has ribonuclease H
RNase H
The enzyme RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from archaea to bacteria and eukaryota....
(Rnase H) activity as it degrades the RNA strand of RNA-DNA intermediate that forms during viral DNA synthesis.
Structure
The HIV-1 RT is an asymmetricAsymmetric
Something which is asymmetric displays asymmetry. Specific uses of the term may include:*Asymmetric relation for information on such relations in mathematics and set theory*Asymmetric warfare for information and theories of modern war...
1000-amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
heterodimer composed of p66 (560 amino acids) and p51 subunits (440 amino acids). The p66 subunit has two domains, a polymerase and ribonuclease H. The polymerase domain contains four subdomains, which have been termed “fingers”, “palm”, “thumb” and “connection” and it is often compared to a right hand (figure 1). The role of the p66 subunit is to carry out the activity of RT whereas it contains the active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
s of the enzyme. The p51 is believed to play mainly a structural role.
Binding and pharmacophore
Despite the chemical diversity of NNRTIs they all bind at the same site in the RT. The binding occurs allosterically in a hydrophobic pocket located approximately 10 ÅÅngström
The angstrom or ångström, is a unit of length equal to 1/10,000,000,000 of a meter . Its symbol is the Swedish letter Å....
from the catalytic site in the palm domain of the p66 subunit site of the enzyme. The NNRTI binding pocket (NNIBP) contains five aromatic (Tyr-181, Tyr-188, Phe-227 and Trp-229), six hydrophobic (Pro-59, Leu-100, Val-106, Val-179, Leu-234 and Pro-236) and five hydrophilic (Lys-101, Lys-103, Ser-105, Asp-132 and Glu-224) amino acids that belong to the p66 subunit and additional two amino acids (Ile-135 and Glu-138) belonging to the p51 subunit. Each NNRTI interacts with different amino acid residues in the NNIBP.
An important factor in the binding of the first generation NNRTIs, such as nevirapine, is the butterfly-like shape. Despite their chemical diversity they assume very similar butterfly-like shape. Two aromatic rings of NNRTIs conform within the enzyme to resemble the wings of a butterfly (figure 2). The butterfly structure has a hydrophilic centre as a ‘body’ and two hydrophobic moieties representing the wings. Wing I is usually a heteroaromatic ring and wing II is a phenyl or allyl substituent. Wing I has a functional group
Functional group
In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction regardless of the size of the molecule it is a part of...
at one side of the ring which is capable of accepting and/or donating hydrogen bonds with the main chain of the amino acids Lys-101 and Lys-103. Wing II interacts through π-π interactions with a hydrophobic pocket, formed in most part by the side chains of aromatic amino acids. On the butterfly body a hydrophobic part fills a small pocket which is mainly formed by the side chains of Lys-103, Val-106 and Val-179. However many other NNRTIs have been found to bind to RT in different modes. Second generation NNRTIs such as diarylpyrimidins (DAPYs), have a horseshoe-like shape with two lateral hydrophobic wings and a pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
ring which is the central polar part.
The NNIBP is elastic
Elasticity (physics)
In physics, elasticity is the physical property of a material that returns to its original shape after the stress that made it deform or distort is removed. The relative amount of deformation is called the strain....
and the conformation
Chemical structure
A chemical structure includes molecular geometry, electronic structure and crystal structure of molecules. Molecular geometry refers to the spatial arrangement of atoms in a molecule and the chemical bonds that hold the atoms together. Molecular geometry can range from the very simple, such as...
depends on the size, specific chemical composition and binding mode of the NNRTI. The total structure of RT has segmental flexibility
Flexibility
Flexibility may refer to:* Flexibility , the distance of motion of a joint, which may be increased by stretching* Flexibility , in the field of engineering systems design, designs that can adapt when external changes occur...
that depends on the nature of the bound NNRTI. It's important for the inhibitor to have flexibility to be able to bind in the modified pockets of a mutant target. Inhibitor flexibility may not affect the inhibitor-target interactions.
Mechanism of action
The NNRTIs act by binding non-competitivelyNon-competitive inhibition
Non-competitive inhibition is a type of enzyme inhibition where the inhibitor reduces the activity of the enzyme, by binding not to the active site on the enzyme, but to a different site...
to the RT enzyme (figure 3). The binding causes conformational change in the three-dimensional structure of the enzyme and creates the NNIBP. Binding of NNRTI to HIV-1 RT makes the p66 thumb domain hyper extended because it induces rotamer
Conformational isomerism
In chemistry, conformational isomerism is a form of stereoisomerism in which the isomers can be interconverted exclusively by rotations about formally single bonds...
conformation changes in amino acid residues Tyr-181 and Tyr-188. This affects the catalytic activity of the enzyme and blocks the HIV-1 replication by inhibiting the polymerase active site of the RT's p66 subunit. The transcription of the viral RNA is inhibited and therefore the replication rate of the virus reduces. Although the exact molecular mechanism is still hypothetical this has been demonstrated by multiple studies to be the primary mechanism of action.
In addition to this proposed primary mechanism of action it has been shown that the NNRTIs have other mechanisms of action and interfere with various steps in the reverse transcriptase reaction. It has been suggested that the inhibition of reverse transcription by the NNRTIs may be due to effects on the RT Rnase H activity and/or template/primer
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
binding. Some NNRTIs interfere with HIV-1 Gag-Pol
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
polyprotein processing by inhibiting the late stage of HIV-1 replication.
It is important to gain profound understanding of the various mechanism of action of the NNRTIs in order to develop next-generation NNRTIs and for understanding the mechanism of drug resistance.
Drug discovery and design
The development of effective anti-HIV drugs is difficult due to wide variations in nucleotide and amino acid sequences. The perfect anti-HIV drug chemical should be effective against drug resistance mutation. Understanding the target RT enzyme and its structure, mechanism of drug action and the consequence of drug resistance mutations provide useful information which can be helpful to design more effective NNRTIs. The RT enzyme can undergo change due to mutations that can disturb NNRTI binding.Discovery
The first two classes of compounds that were identified as NNRTIs were the 1-(2-2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and tetrahydroimidazo[4,5,1-jkj][1,4]benzodiazepin-2(1H)-one and -thione (TIBO) compounds. The discovery of the TIBO compounds led to the definition of the NNRTI class in the late 1980s when they were unexpectedly found to inhibit RT. This finding initiated researches on mechanism of action for these compounds. The HEPT compounds were described before the TIBO compounds and were originally believed to be NRTIs. Later it was discovered that they shared common mechanism of action with the TIBO compounds. Both the HEPT and TIBO compounds were first to be identified as highly specific and potent HIV-1 RT inhibitors, not active against other RTs. These compounds do not interrupt the cellular or mitochondrialMitochondrion
In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 1.0 micrometers in diameter...
DNA synthesis. The specificity of the NNRTIs for HIV-1 is considered the hallmark of the NNRTI drug class.
First generation NNRTIs
After the discovery of HEPT and TIBO, compounds screening methods were used to develop BI-RG-587, the first NNRTI commonly known as nevirapine. Like HEPT and TIBO, nevirapine blocked viral RT activity by non-competitive inhibition (with respect to dNTP binding). This reinforced the idea that the new class of anti-HIV inhibitors was inhibiting the activity of RT but not at the active site. Several molecular families of NNRTIs have emerged following screening and evolution of many molecules.Three NNRTI compounds of the first generation have been approved by the FDA for treating HIV-1 infection. Nevirapine was approved in 1996, delavirdine in 1997 and efavirenz in 1998 (table 1). Two of these drugs, nevirapine and efavirenz, are cornerstones of first line HAART while delavirdine is hardly used nowadays. The structure of these three drugs show the wide array of rings, substituents, and bonds that allow activity against HIV-1 RT. This diversity demonstrates why so many non-nucleosides have been synthesised but doesn't explain why only three drugs have reached the marked. The main problem has been the potency of these compounds to develop resistance.
| Nevirapine | Delavirdine | Efavirenz |
Development from α-APA to ITU
Crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
analysis showed that the first generation NNRTIs (for example TIBO, nevirapine and α-APA) bind HIV-1 RT in a “butterfly-like” conformation. These first generation NNRTIs were vulnerable against the common drug-resistance mutations like Tyr-181C and Tyr-188L/H. This triggered the need for finding new and more effective NNRTIs. ITU (imidoylthiourea), a promising series of NNRTIs emerged from α-APA analogs (figure 4). The ITU compounds were obtained by extending the linker that binds the aryl side groups of the α-APA. A potent ITU compound, R100943, was obtained by an arrangement of the chemical composition of the side groups based on structure-activity relationship
Structure-activity relationship
The structure–activity relationship is the relationship between the chemical or 3D structure of a molecule and its biological activity. The analysis of SAR enables the determination of the chemical groups responsible for evoking a target biological effect in the organism...
s (SAR). A crystal structure of the HIV-1/R100943 complex demonstrated that ITU compounds are more flexible than α-APA compound. The ITU compounds showed distinct mode of binding where they bound with "horseshoe" or "U" mode. The 2,6-dichlorophenyl part of R100943 which corresponds chemically to the wing II 2,6-dibromophenyl part of the α-APA occupied the wing I part in the NNIBP whereas the 4-cyanoanilino part of R100943 occupies the wing II position in the NNIBP.
R100943 inhibited HIV-1 and was considerably effective against a number of key NNRTI-resistant mutants like G190A mutation, which caused high-level resistance to loviride (α-APA) and nevirapine. G190A mutation was thought to cause resistance by occupying a part of the binding pocket that would otherwise be filled by the linker part of the butterfly shaped NNRTIs. R100943, in the horseshoe mode of binding, is located at a distance of approximately 6.0 Å from G190. When compared with nevirapine and loviride which bind in the butterfly shape the ITU derivatives
Derivative (chemistry)
In chemistry, a derivative is a compound that is derived from a similar compound by some chemical or physical process. In the past it was also used to mean a compound that can be imagined to arise from another compound, if one atom is replaced with another atom or group of atoms, but modern...
revealed improved activity against Tyr-181C and Tyr-188L mutants. A structural study suggested that a potent TIBO compound could partly supplement for the effects of the Tyr-181C mutation by moving itself in the non-nucleoside inhibitor binding pocket (NNIBP) of the mutant RT. In this context, R100943 has torsional freedom that enables the conformational alternations of the NNRTI. This torsional freedom could be used by the ITU derivate to bind to a mutated NNIBP and thus compensating for the effects of a resistance mutation. Nevertheless, the potency of R100943 against HIV-1 resistant mutants was not adequate for it to be considered as an effective drug candidate
Drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are discovered or designed.In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery...
. Additionally, the chemical stability of the imidoylthiourea part of the ITU derivative was not favorable for an oral drug.
Development from ITU to DATA
Changes in the imidoylthiourea complexes led to the synthesis of a new
class of compounds, diaryltriazine (DATA). In these compounds, the
thiourea part of the ITU compounds was replaced by a triazine ring. The
DATA compounds were more potent than the ITU compounds against common
NNRTI resistant mutant strains. R106168, a prototype DATA compound, was
rather easy to synthesize. Multiple substitutions were made at different
positions on all of the three rings and on the linkers connecting the
rings. In the pocket, most of the DATA derivatives conformed a horseshoe
conformation. The two wings in R106168 (2,6-dichlorobenzyl and
4-cyanoanilino) occupied positions in the pocket similar to that of the
two wings of the derivatives of ITU. The central part of the DATA
compounds, in which the triazine ring replaced the thiourea group of ITU
derivatives, is positioned between the side chains of L100 and V179. This
removed a number of torstional degrees of freedom in the central part
while keeping the flexibility between the triazine ring and the wings.
Chemical substitution or modification in the three-aromatic-ring backbone
of the DATA compounds had substantial effect on the activity. R120393, a DATA analog, was designed with a chloroindole part in wing I to expand
interactions with the side chain of conserved W229 of the polymerase
primer grip loop. R120393 had similar effect as R106168 against most of
the NNRTI-resistant mutants. The cloroindole part interacted with the
hydrophobic core of the pocket and influenced the binding mode of the
R120393 so it went deeper into the pocket compared to the wing I position
of other DATA analogs. Crystal structures showed that the DATA compounds
could bind the NNIBP in different conformations. The capability to bind in
multible modes made the NNRTIs stronger against drug-resistance mutations.
Variability between the inhibitors could be seen when the chemical
composition, size of wing I and the two linker groups connecting the rings
were altered. The potency of the NNRTIs changed when the triazine
Triazine
A triazine is one of three organic chemicals, isomeric with each other, whose molecular formula is 333 and whose empirical formula is CHN.- Structure :...
nitrogen
atoms were substituted with carbons.
Next generation NNRTIs
Researchers used multi-disciplinary approach to design NNRTIs with better resistance profile and an increased genetic barrier to the development of resistance. A new class of compounds, diarylpyrimideDiarylpyrimidines
Diarylpyrimidines and diaryltriazines are two closely related classes of molecules resembling the pyrimidine nucleotides found in DNA. They show great potency in inhibiting the activity of HIV reverse transcriptase...
(DAPY), were discovered with the replacement of the central triazine
Triazine
A triazine is one of three organic chemicals, isomeric with each other, whose molecular formula is 333 and whose empirical formula is CHN.- Structure :...
ring from the DATA compounds, with a pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
. This new class was more effective against drug resistant HIV-1 strains than the corresponding DATA analogs. The replacement enabled substitutions to the CH-group at the 5-position of the central aromatic ring. One of the first DAPY compounds, dapivirine (with R1= 2,4,6-trimethylanilino, R2 = R3 = H and Y = NH) was found to be effective against drug-resistant HIV-1 strains. Systematic chemical substitutions were made at the R1, R2, R3 and Y positions to find new DAPY derivatives. This led to the discovery of etravirine which has a bromine substitution at the 5-position (R3) of the pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
ring (with R1 = 2,6-dimethyl-4-cyanoanilino, R2 = NH2 and Y = O) (figure 5). Etravirine was discovered by researchers at the Jansen Research Foundation and Tibotec and approved in 2008 by the FDA. It is used in treatment-expirenced adult patients with HIV infection that is multidrug resistant in combination with other antiretroviral drugs.
Resistance
When treating infection, whether bacterial or viral, there is always a risk of the infectious agent to develop drug resistance. The treatment of HIV infection is especially susceptible to drug resistance which is a serious clinical concern in the chemotherapeutic treatment of the infection. Drug resistant HIV-strains emerge if the virus is able to replicate in the presence of the antiretroviral drugs.NNRTI-resistant HIV-strains have the occurring mutations mainly in and around the NNIBP affecting the NNRTI binding directly by altering the size, shape and polarity
Chemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...
on different areas of the pocket or by affecting, indirectly, the access to the pocket. Those mutations are primarily noted in domains which span amino acids 98-108, 178-190 or 225-238 of the p66 subunit. The most frequent mutations observed in viruses isolated from patients who have been on a failing NNRTI containing chemotherapy are Lys-103N and Tyr-181C. NNRTI resistance has been linked to over 40 amino acid substitutions in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
and in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
.
Antiretroviral drugs are never used in monotherapy due to rapid resistance development. The highly active antiretroviral therapy (HAART) was introduced in 1996. The treatment regimen combines three drugs from at least two different classes of antiretroviral drugs.
The advance of etravirine over other NNRTIs is that multiple mutations are required for the development of drug resistance. The drug has also shown activity against viruses with common NNRTI resistance associated mutations and cross-resistance
Cross-resistance
Cross-resistance is the tolerance to a usually toxic substance as a result of exposure to a similarly acting substance. It is a phenomenon affecting e.g. pesticides and antibiotics. As an example rifabutin and rifampin cross react in the treatment of tuberculosis. This sort of resistance is also...
mutations.
Current status
Four drugs in the class of NNRTIs have been approved by regulatory authorities. These are the first generation NNRTIs nevirapine, delavirdine and efavirenz and the next generation NNRTI etravirine. Several other NNRTIs underwent clinical development but were discontinued due to unfavourable pharmacokineticPharmacokinetics
Pharmacokinetics, sometimes abbreviated as PK, is a branch of pharmacology dedicated to the determination of the fate of substances administered externally to a living organism...
, efficacy and/or safety factors.
Currently there are four other NNRTIs undergoing clinical development, rilpivirine
Rilpivirine
Rilpivirine is a pharmaceutical drug, developed by Tibotec, for the treatment of HIV infection. It is a second-generation non-nucleoside reverse transcriptase inhibitor with higher potency, longer half-life and reduced side-effect profile compared with older NNRTIs, such as efavirenz.Rilpivirine...
, IDX899, RDEA-428 and lersivirine (table 2).
Rilpivirine
Rilpivirine is a DAPY compound like etravirine and was discovered when further optimization within this family of NNRTIs was conducted. The resistance profile and the genetic barrier to the development of resistance is comparable to etravirine in vitro. The advantage of rilpivirine over etravirine is a better bioavailabilityBioavailability
In pharmacology, bioavailability is a subcategory of absorption and is used to describe the fraction of an administered dose of unchanged drug that reaches the systemic circulation, one of the principal pharmacokinetic properties of drugs. By definition, when a medication is administered...
and it is easier to formulate than etravirine. Etravirine has required extensive chemical formulation work due to poor solubility
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
and bioavailability. Rilpivirine was undergoing phase III clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
s in the end of 2009.
RDEA806
In 2007 a new family of triazole NNRTIs was presented by researchers from the pharmaceutical company Ardea Biosciences. The selected candidate from the screening executed was RDEA806 belonging to the family of triazoleTriazole
Triazole refers to either one of a pair of isomeric chemical compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms.The two isomers are:*1,2,3-Triazole 100px*1,2,4-Triazole 100px...
s. It has similar resistance profile against selected NNRTI resistant HIV-1 strains to other next generation NNRTIs. The candidate entered phase IIb clinical trials in the end of 2009.
IDX899
IDX899 is another next generation NNRTI developed by Idenix Pharmaceuticals. It belongs to the family of 3-phosphoindoles. In vitro studies have shown comparable resistance profile to that of the other next generation NNRTIs. On November 3, 2009 the candidate entered phase II clinical trials.Lersivirine (UK-453061)
Lersivirine belongs to the pyrazolePyrazole
Pyrazole refers both to the class of simple aromatic ring organic compounds of the heterocyclic diazole series characterized by a 5-membered ring structure composed of three carbon atoms and two nitrogen atoms in adjacent positions, and to the unsubstituted parent compound...
family and is another next generation NNRTI in clinical trials developed by the pharmaceutical company Pfizer
Pfizer
Pfizer, Inc. is an American multinational pharmaceutical corporation. The company is based in New York City, New York with its research headquarters in Groton, Connecticut, United States...
. The resistance profile is similar to that of other next generation NNRTIs. In the end of 2009 lersivirine was in phase IIb.
| Drug candidate | Rilpivirine (TMC278) | RDEA806 | IDX899 | Lersivirine (UK-453061) |
|---|---|---|---|---|
| Chemical structure | ||||
| Phase of development | III | IIb | II | IIb |
See also
- Antiretroviral drugAntiretroviral drugAntiretroviral drugs are medications for the treatment of infection by retroviruses, primarily HIV. When several such drugs, typically three or four, are taken in combination, the approach is known as Highly Active Antiretroviral Therapy, or HAART...
- Reverse-transcriptase inhibitor
- Protease inhibitorProtease inhibitor (pharmacology)Protease inhibitors are a class of drugs used to treat or prevent infection by viruses, including HIV and Hepatitis C. PIs prevent viral replication by inhibiting the activity of proteases, e.g.HIV-1 protease, enzymes used by the viruses to cleave nascent proteins for final assembly of new...
- Entry inhibitor
- Discovery and development of HIV-protease inhibitors
- Discovery and development of CCR5-receptor antagonists
- Discovery and development of nucleoside and nucleotide reverse-transcriptase inhibitorsDiscovery and development of nucleoside and nucleotide reverse-transcriptase inhibitorsDiscovery and development of nucleoside and nucleotide reverse-transcriptase inhibitors began in the 1980s when the AIDS epidemic hit Western societies. NRTIs inhibit the reverse transcriptase , an enzyme that controls the replication of the genetic material of the human immunodeficiency virus ....
- Discovery and development of Bcr-Abl tyrosine-kinase inhibitors