Discovery and development of integrase inhibitors
Encyclopedia
The first human immunodeficiency virus (HIV
) case was reported in the 1980s. Many drugs have been discovered to treat the disease but mutation
s in the virus and resistance
to the drugs make development difficult. Integrase
is a viral enzyme
that integrates retroviral DNA into the host cell genome
. Integrase inhibitors are a new class of drugs used in the treatment of HIV. The first integrase inhibitor, raltegravir, was approved in 2007 and other drugs were in clinical trials in 2011.
. The body uses its immune system
to protect itself from bacteria, viruses and other disease-causing beings, and when it fails to do so immunodeficiency diseases occur. One such disease is acquired immunodeficiency syndrome (AIDS
) which is most commonly a result of an infection by the human immunodeficiency virus (HIV).
Two closely related types of HIV have been identified, HIV-1 and HIV-2. While HIV-2 is spreading in India and West Africa, HIV-1 is more virulent
and the number one cause of AIDS worldwide. Though some of the patients have different results in most cases people infected with HIV go on to develop AIDS and ultimately die of opportunistic infection
s or cancer
.
Integration to the retroviral genome is critical for gene expression and viral replication
. The viral genome is reversely transcribed into the DNA of the infected cell by viral reverse transcriptase
, the DNA is then integrated into the host-cell chromosome
s with the aid of the viral integrase. RNA transcripts are produced from integrated viral DNA and serve both as mRNAs to direct the synthesis
of viral proteins and later as RNA genomes of the new viral particles.Viral particles escape from the cell by budding from the plasma membrane, each enclosed in a membrane envelope
.
In this process HIV-1 integrase is essential and therefore a very promising target for anti-AIDS drug design. Selective drug design is a possibility as HIV-1 integrase has no known cellular equivalent. Many integrase inhibitors have been discovered and designed but only a few of the molecules were developed further and got as far as phase II or phase III of clinical trial
s. Raltegravir (brand name Isentress®) was granted accelerated approval
from FDA in October 2007 and from EMEA (now EMA
) in December 2007. It was marketed as an antiretroviral drug
(ARV) for HIV-1 infected adults who had already been exposed to a minimum of three ARV classes and showed multi-drug resistance. In general there are two main groups of integrase inhibitors; Integrase Strand Transfer inhibitors (INSTI) and Integrase Binding Inhibitors (INBI). INSTIs restrain the binding of pre-integration complex (PIC
) and host DNA and INBIs restrain integrase and viral DNA binding. Raltegravir is an INSTI integrase inhibitor which inhibits both HIV-1 and HIV-2 replication. It is more potent than other previously known integrase inhibitors as well as causing less side effects. Raltegravir is currently the only HIV-1 integrase inhibitor being used to treat HIV infections but other drugs are in clinical trials, e.g. elvitegravir
and S/GSK1349572
.
as they do not interfere with normal cellular processes.
IN is composed of 3 structurally independent, functional domains (see figure 1).:
1. The N-terminal domain (NTD) encompasses amino acid
s 1-50 and contains two histidine residues (His12 and His16) and two cysteine residues (Cys40 and Cys43), all of which are absolutely conserved and form a HHCC zinc-finger motif. Single mutations of any of these four residues reduce IN enzymatic activity. The HHCC zinc-finger motif chelates one zinc atom per IN monomer
. The NTD is required for higher order multimer formation which appears to be its primary role. The multimerization requires zinc atom that stabilizes the fold.
2. The catalytic core domain (CCD), which encompasses amino acids 51- 212, contains the active site of IN but it can´t catalyze
integration in the absence of NTD and CTD (the C-terminal domain). CCD contains three absolutely conserved negatively charged amino acids; D64, D116 and E152. These amino acids form the DDE motif that coordinate divalent metal ions (Mg2+ or Mn2+). These metal ions are essential for the catalysis of integration. CCD has a mixed β and α structure with five β-sheets
and six α helices
that are linked by flexible loops. The flexible loops allow conformational changes that are required for 3´processing of the viral DNA and strand transfer (STF) reactions which are two key steps of the integration reaction. CCD is essential for these steps and substitution of any of the residues in the DDE motif dramatically inhibits the activity of IN.
3. The C-terminal domain (CTD), which encompasses amino acids 213-288, binds DNA nonspecifically and its interaction with NTD and CCD is required for IN 3´-processing and strand-transfer activities. CTD is the least conserved of the three domains.
IN acts as a multimer and dimerization is required for the 3´-processing step, with tetramer
ic IN catalyzing the strand-transfer reaction.
3´processing IN binds to a short sequence located at either end of the long terminal repeat
(LTR) of the viral DNA and catalyzes endonucleotide cleavage. This results in elimination of a dinucleotide from each of the 3´ends of the LTR. Cleaved DNA is then used as a substrate for integration or strand transfer. Strand transfer is a trans-esterification reaction involving a direct nuleophilic
attack of the 3´hydroxy group of the two newly processed viral 3´-DNA ends on the phosphodiester backbone of the host target DNA. This leads to covalent insertion of viral DNA into the genome of the infected cell. Strand transfer occurs simultaneously at both ends of the viral DNA molecule, with an offset of precisely five base pairs between the two opposite points of insertion. The integration reaction is completed by removal of unpaired dinucleotides from the 5'- ends of the viral DNA, repair of the single-stranded gaps created between the viral and target DNA molecules and ligation of 3'-ends to 5'-ends of the host DNA. Divalent metals, Mg2+ or Mn2+, are required for 3'-processing and strand transfer steps as well as for assembly of IN onto specific viral donor DNA to form a complex that is competent to carry out either function. Because Mg2+ abundance over Mn 2+ is 1,000,000-fold in cells it's a more reasonable cofactor for integration.
Raltegravir and elvitegravir share the same mechanism of action against integrase which is to bind to the active site of Mg2+ ions. Inhibitors compete directly with viral DNA for binding to integrase in order to inhibit 3‘-end processing. In doing this the inhibitors completely block the active site from binding to target DNA. This way of inhibitions is called strand transfer inhibition.
) is a host protein that binds to integrase and is crucial for viral replication. The mechanism of action is not precisely known but evidence suggest that LEDGF/p75 guides integrase to insert viral DNA into transcriptionally active sites of the host genome. Inhibitors of this protein are already being developed and patented. They are likely to be highly target specific and less prone to the development of resistance.
as it implies blockage of a specific integrase-viral DNA complex. This results in selective inhibition of the strand-transfer reaction, with no significant effect on the 3´-processing reaction. INSTIs may therefore be more specific and bind selectively to the target DNA binding site and hence be less toxic than bifunctional inhibitors that are able to bind to both the donor and target binding sites.
INBIs also bind to IN but the mechanism of action is unknown so the binding can not be detailed.
benzyl
moiety that buries into a highly hydrophobic pocket near the active site; and chelating triad that binds with two Mg2+ ions in a rather hydrophilic
region, anchoring the inhibitor onto the protein surface (see figure 3). In fact, all potent integrase inhibitors possess a substituted benzyl component that is critical for maintaining 3‘end joining potency. Removal of the benzyl group prevents inhibitory function. Lipophylic substituents are therefore beneficial for the strand transfer inhibition, in particular the thiophenyl
, furan
yl and (thiophen-2-yl)phenyl substitutions. Heteroaromatic amine and amide also cause increase in 3‘ processing inhibitory action.
When catechol-based inhibitors of IN were researched it was observed that maintaining a planar relationship with the bis-hydroxylated aryl ring increases potency. The inhibitory activity could be further optimized by including a meta-chloro substituent, enhancing the interaction of the benzyl group with the adjacent hydrophobic pocket (see figure 4: Structures A-G).
A benzyl substituted hydroxyl group (fig. 4 H) improves metal-chelating capability (compared to structure J in fig. 4) while a methoxy group (I) is much less potent due to steric clash by the additional methyl group with the catalytic metals.
When researching diketo
derivates, the central pyrrole ring
of structure K in fig. 4 was replaced by a series of aromatic systems having various substitution patterns. That provided optimum relative orientation of the benzyl and diketoacid (DKA) site chain. Structure L in fig. 4 resulted in 100 fold increase in potency.
Benard et al (2004) synthesized INIs with a quinoline
subunit and an ancillary aromatic ring linked by functionalized spacers such as amide, hydrazide
, urea
and hydroxyprop-1-en-3-one moiety. They found that the amide group containing dervates were the most promising ones. By synthesizing series of styrylquinones researchers found out that a carboxyl group at C-7, a hydroxylgroup at C-8 in the quinoline subunit and an ancillary phenyl ring (Figure 4: Structure M) are required for inhibition, although alterations of the ring are tolerated. Two hydroxyl groups on the ancillary phenyl ring are also required for inhibitory potency.
for its inhibition. Wang et al (2010) hoped that by studying the SAR and pharmacophore of a dual inhibitor scaffold, focusing both on integrase and reverse transcriptase (RT) it would be possible to observe anti-integrase activity. By studying the SAR of HIV integrase inhibitors it was possible to find that for optimal integrase inhibition the pharmacophore requires a regiospecific (N-1) DKA of a specific length. A DKA functionality or its heterocyclic
bioisostere
that selectively inhibit strand transfer seem to be present in all major chemotype
s of integrase inhibitors. As detailed in the SAR discussion above the two necessary structural components of INI are a benzyl hydrophobic moiety and a chelating triad to bind the Mg2+ ions. For the triad to bind the Mg2+ ions has to be ionized (see fig. 5) and thus a pharmacophore bioisostere has to be ionized too and the benzyl pharmacophore bioisostere must to be very hydrophobic.
However, despite previous success in clinical development (raltegravir), a detailed binding model is lacking so it has proven difficult to structure base the design of integrase inhibitors. When the pharmacophore of salicylic acid and catechol were merged, new chemical scaffolds were created. The adjacent hydroxyl and carboxylic groups on salicylic acid could bind with the metal ions and serve as their pharmacophore. Polyhydroxylated aromatic inhibitors are mostly active against strand transfer reactions and 3‘-processing which suggests a mechanism that targets both steps. This is a very important part of the compound as it can be used to bind to the divalent metal on the active site of IN and as such be effective against viral strains that are resistant to strand transfer specific inhibitors.
and in vitro
resistance. Due to these mutations and development of resistance the inhibitors are less effective against the virus.
Resistance of INI corresponds to those of other ARV drugs. First IN resistance is caused by primary mutations that decrease INI sensitivity in combination with secondary mutations that further reduce virus sensitivity and/or repair decreased fitness of the virus. Secondly there is a genetic barrier to INI resistance, defined by the number of mutations required for the loss of clinical INI activity. Thirdly there is extensive but incomplete cross-resistance
among the INIs.
A loop containing amino acid residues 140-149 is located in the catalytic-core domain and is important for IN function as mentioned before. This loop is flexible and even though its role is not quite known it is thought to be important and its functions critical for DNA binding. This resistance appears within mutations in this IN-coding region.
The resistance to raltegravir and elvitegravir is primarily due to the same two mutation pathways but other primary mutations are also involved for each of the drugs. Some mutations increase resistance to the drugs to a large extent than others. For example one of the most common mutation pathway increases the resistance to raltegravir up to 100 times more than the second most common one.
Resistance to Integrase Inhibitor S/GSK1349572 is still being developed and the resistance has not been fully characterized. When it was assessed alongside the primary mutations of raltegravir and elvitegravir it did not show cross-resistance which means that it could be useful against drug resistant viruses.
Raltegravir has limited intestinal absorption and thus resistance cannot be overcome by prescribing higher doses. Newer drugs are warranted to overcome this pharmacological disadvantage and gain plasma concentrations high enough to target raltegravir-resistant viruses.
. In 2009 this authorization was converted to a full marketing authorization
and in the same year the FDA changed the approval from accelerated to traditional approval and listed the drug as a first line ARV treatment agent. The second INSTI drug, elvitegravir, was identified by Japan Tobacco and clinical trials began in 2005. In 2011 the drug was still in phase three clinical trials, where it is being compared to raltegravir, in treatment experienced subjects and is also in phase two development in naïve subjects as a part of a multidrug treatment. S/GSK1349572 is a integrase inhibitor discovered by ViiV/Shinongi which was entering phase three in clinical trials in 2011. This new drug is promising and seems to be well tolerated and so far shows better results than both raltegravir and elvitegravir.
Since there have been problems with resistance to raltegravir and elvitegravir, scientists have started to work on new second generation integrase inhibitors, such as MK-2048
which in 2009 was developed by Merck. It's a prototype second generation INSTI that remains potent against viruses containing mutations against raltegravir and elvitegravir. The mechanism of action and SAR of MK-2048 is the same as of the other INSTIs, the structure of MK-2048 shown in figure 6 with essential pharmacophore highlighted.
Even though drugs discussed above are promising the development has a long way to go and many things are still unknown about the efficacy, safety and mechanism of action of these drugs.
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...
) case was reported in the 1980s. Many drugs have been discovered to treat the disease but mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
s in the virus and 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...
to the drugs make development difficult. Integrase
Integrase
Retroviral integrase is an enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell...
is a viral 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 integrates retroviral DNA into the host cell 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....
. Integrase inhibitors are a new class of drugs used in the treatment of HIV. The first integrase inhibitor, raltegravir, was approved in 2007 and other drugs were in clinical trials in 2011.
History
In the 1980s an infectious disease started to plague human civilization. The coexistence of viruses and humans is a fight for survival for both because the invaders can kill the human but in doing so eliminate their own hostHost (biology)
In biology, a host is an organism that harbors a parasite, or a mutual or commensal symbiont, typically providing nourishment and shelter. In botany, a host plant is one that supplies food resources and substrate for certain insects or other fauna...
. The body uses its immune system
Immune system
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own...
to protect itself from bacteria, viruses and other disease-causing beings, and when it fails to do so immunodeficiency diseases occur. One such disease is acquired immunodeficiency syndrome (AIDS
AIDS
Acquired immune deficiency syndrome or acquired immunodeficiency syndrome is a disease of the human immune system caused by the human immunodeficiency virus...
) which is most commonly a result of an infection by the human immunodeficiency virus (HIV).
Two closely related types of HIV have been identified, HIV-1 and HIV-2. While HIV-2 is spreading in India and West Africa, HIV-1 is more virulent
Virulence
Virulence is by MeSH definition the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism - its ability to cause disease - is determined by its...
and the number one cause of AIDS worldwide. Though some of the patients have different results in most cases people infected with HIV go on to develop AIDS and ultimately die of opportunistic infection
Opportunistic infection
An opportunistic infection is an infection caused by pathogens, particularly opportunistic pathogens—those that take advantage of certain situations—such as bacterial, viral, fungal or protozoan infections that usually do not cause disease in a healthy host, one with a healthy immune system...
s or cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
.
Integration to the retroviral genome is critical for gene expression and viral replication
Viral replication
Viral replication is the term used by virologists to describe the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. From the perspective of the virus, the purpose of viral replication is...
. The viral genome is reversely transcribed into the DNA of the infected cell by viral 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...
, the DNA is then integrated into the host-cell 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...
s with the aid of the viral integrase. RNA transcripts are produced from integrated viral DNA and serve both as mRNAs to direct the synthesis
Biosynthesis
Biosynthesis is an enzyme-catalyzed process in cells of living organisms by which substrates are converted to more complex products. The biosynthesis process often consists of several enzymatic steps in which the product of one step is used as substrate in the following step...
of viral proteins and later as RNA genomes of the new viral particles.Viral particles escape from the cell by budding from the plasma membrane, each enclosed in a membrane envelope
Viral envelope
Many viruses have viral envelopes covering their protein capsids. The envelopes typically are derived from portions of the host cell membranes , but include some viral glycoproteins. Functionally, viral envelopes are used to help viruses enter host cells...
.
In this process HIV-1 integrase is essential and therefore a very promising target for anti-AIDS drug design. Selective drug design is a possibility as HIV-1 integrase has no known cellular equivalent. Many integrase inhibitors have been discovered and designed but only a few of the molecules were developed further and got as far as phase II or phase III of 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. Raltegravir (brand name Isentress®) was granted accelerated approval
FDA Fast Track Development Program
The FDA Fast Track Development Program is a designation of the United States Food and Drug Administration that accelerates the approval of investigational new drugs undergoing clinical trials with the goal review time of 60 days...
from FDA in October 2007 and from EMEA (now EMA
European Medicines Agency
The European Medicines Agency is a European agency for the evaluation of medicinal products. From 1995 to 2004, the European Medicines Agency was known as European Agency for the Evaluation of Medicinal Products.Roughly parallel to the U.S...
) in December 2007. It was marketed as an antiretroviral drug
Antiretroviral drug
Antiretroviral 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...
(ARV) for HIV-1 infected adults who had already been exposed to a minimum of three ARV classes and showed multi-drug resistance. In general there are two main groups of integrase inhibitors; Integrase Strand Transfer inhibitors (INSTI) and Integrase Binding Inhibitors (INBI). INSTIs restrain the binding of pre-integration complex (PIC
Pre-integration complex
The pre-integration complex is a nucleoprotein complex of viral genetic material and associated viral and host proteins. The PIC forms after uncoating of a viral particle after entry into the host cell...
) and host DNA and INBIs restrain integrase and viral DNA binding. Raltegravir is an INSTI integrase inhibitor which inhibits both HIV-1 and HIV-2 replication. It is more potent than other previously known integrase inhibitors as well as causing less side effects. Raltegravir is currently the only HIV-1 integrase inhibitor being used to treat HIV infections but other drugs are in clinical trials, e.g. elvitegravir
Elvitegravir
Elvitegravir is an investigational new drug for the treatment of HIV infection. It acts as an integrase inhibitor. It is undergoing Phase III clinical trial conducted by the pharmaceutical company Gilead Sciences, which licensed EVG from Japan Tobacco in March 2008.According to the results of...
and S/GSK1349572
Dolutegravir
Dolutegravir is an experimental new drug under investigation for the treatment of HIV infection. Dolutegravir is an integrase inhibitor. Also known as S/GSK1349572 or just "572", the drug is under development by GlaxoSmithKline . Studies have shown dolutegravir to be effective in patients with...
.
The HIV-1 integrase enzyme
The HIV-1 integrase (IN) is a key enzyme in the replication mechanism of retroviruses. It is responsible the for transfer of virally encoded DNA into the host chromosome which is a necessary event in retroviral replication. Since IN has no equivalent in the host cell, integrase inhibitors have a high therapeutic indexTherapeutic index
The therapeutic index is a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes death or toxicity ....
as they do not interfere with normal cellular processes.
Structure
IN belongs, both mechanistically and structurally, to the superfamily of polynucleotidyl transferases 10 and is composed of 288 amino acids that form the 32 kDa protein. Retroviruses encode their enzymes (protease, reverse transcriptase and integrase) with the POL gene with the 3´end encoding for IN.IN is composed of 3 structurally independent, functional domains (see figure 1).:
1. The N-terminal domain (NTD) encompasses 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...
s 1-50 and contains two histidine residues (His12 and His16) and two cysteine residues (Cys40 and Cys43), all of which are absolutely conserved and form a HHCC zinc-finger motif. Single mutations of any of these four residues reduce IN enzymatic activity. The HHCC zinc-finger motif chelates one zinc atom per IN monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
. The NTD is required for higher order multimer formation which appears to be its primary role. The multimerization requires zinc atom that stabilizes the fold.
2. The catalytic core domain (CCD), which encompasses amino acids 51- 212, contains the active site of IN but it can´t catalyze
Enzyme catalysis
Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes. Catalysis of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions....
integration in the absence of NTD and CTD (the C-terminal domain). CCD contains three absolutely conserved negatively charged amino acids; D64, D116 and E152. These amino acids form the DDE motif that coordinate divalent metal ions (Mg2+ or Mn2+). These metal ions are essential for the catalysis of integration. CCD has a mixed β and α structure with five β-sheets
Beta sheet
The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...
and six α helices
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
that are linked by flexible loops. The flexible loops allow conformational changes that are required for 3´processing of the viral DNA and strand transfer (STF) reactions which are two key steps of the integration reaction. CCD is essential for these steps and substitution of any of the residues in the DDE motif dramatically inhibits the activity of IN.
3. The C-terminal domain (CTD), which encompasses amino acids 213-288, binds DNA nonspecifically and its interaction with NTD and CCD is required for IN 3´-processing and strand-transfer activities. CTD is the least conserved of the three domains.
IN acts as a multimer and dimerization is required for the 3´-processing step, with tetramer
Tetramer
A tetramer is a protein with four subunits . There are homotetramers such as glutathione S-transferase or single-strand binding protein, dimers of hetero-dimers such as hemoglobin , and heterotetramers, where each subunit is different.-Subunit interactions in tetramers:The interactions between...
ic IN catalyzing the strand-transfer reaction.
Function
HIV-1 integration occurs through a multistep process that includes two catalytic reactions: 3´endonucleolytic processing of proviral DNA ends (termed 3´processing) and integration of 3´-processed viral DNA into cellular DNA (referred to as strand transfer). In3´processing IN binds to a short sequence located at either end of the long terminal repeat
Long terminal repeat
Long terminal repeats are sequences of DNA that repeat hundreds or thousands of times. They are found in retroviral DNA and in retrotransposons, flanking functional genes...
(LTR) of the viral DNA and catalyzes endonucleotide cleavage. This results in elimination of a dinucleotide from each of the 3´ends of the LTR. Cleaved DNA is then used as a substrate for integration or strand transfer. Strand transfer is a trans-esterification reaction involving a direct nuleophilic
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
attack of the 3´hydroxy group of the two newly processed viral 3´-DNA ends on the phosphodiester backbone of the host target DNA. This leads to covalent insertion of viral DNA into the genome of the infected cell. Strand transfer occurs simultaneously at both ends of the viral DNA molecule, with an offset of precisely five base pairs between the two opposite points of insertion. The integration reaction is completed by removal of unpaired dinucleotides from the 5'- ends of the viral DNA, repair of the single-stranded gaps created between the viral and target DNA molecules and ligation of 3'-ends to 5'-ends of the host DNA. Divalent metals, Mg2+ or Mn2+, are required for 3'-processing and strand transfer steps as well as for assembly of IN onto specific viral donor DNA to form a complex that is competent to carry out either function. Because Mg2+ abundance over Mn 2+ is 1,000,000-fold in cells it's a more reasonable cofactor for integration.
Mechanism of action
There are several ways to target integrase but strand transfer inhibition is the most common one. Other targets include, for example, the protein domains beyond the active site of IN. The domains interact with viral or host DNA and are important for binding to the enzyme. It is possible to hamper functions of the enzyme by disrupting or removing these bindings. PIC is a multimeric protein structure inside the host cell, composed of both viral and host proteins. Integrase is a part of PIC‘s viral component. PIC‘s viral and host proteins are believed to modulate intrinsic activity of the enzyme, shuttle PIC to the nucleus and direct integration of viral DNA into a transcriptionally active region of the host genome. If it were possible to exclude certain proteins from the PIC it would block the ability of the virus to integrate into the host genome. The process where the retroviral RNA is transcribed to DNA and then integrated into the host cell's genome is shown in figure 2.IN strand transfer inhibitors (INSTIs)
Mg2+ and Mn2+ are critical cofactors in the integrase phase and if they are chelated it can cause functional impairment of IN. This gives the opportunity to design and develop highly efficient IN inhibitors (INIs). In fact, all small molecule HIV-1 INIs that are now being researched contain a structural motif that coordinates the two divalentmagnesium ions in the enzyme’s active site.Raltegravir and elvitegravir share the same mechanism of action against integrase which is to bind to the active site of Mg2+ ions. Inhibitors compete directly with viral DNA for binding to integrase in order to inhibit 3‘-end processing. In doing this the inhibitors completely block the active site from binding to target DNA. This way of inhibitions is called strand transfer inhibition.
Inhibition of the LEDGF/p75- integrase interaction
Lens epithelial derived growth factor (LEDGF/p75PSIP1
PC4 and SFRS1 interacting protein 1, also known as lens epithelium-derived growth factor , dense fine speckles 70kD protein or transcriptional coactivator p75/p52, is a protein which in humans is encoded by the PSIP1 gene.- Function :...
) is a host protein that binds to integrase and is crucial for viral replication. The mechanism of action is not precisely known but evidence suggest that LEDGF/p75 guides integrase to insert viral DNA into transcriptionally active sites of the host genome. Inhibitors of this protein are already being developed and patented. They are likely to be highly target specific and less prone to the development of resistance.
IN binding inhibitors
Another class of INIs could be IN binding inhibitors (INBIs) such as V-165. V-165 is a compound shown to inhibit integration but without obvious effect on viral DNA synthesis. When the mechanism of action was studied it showed that V-165 interferes with viral DNA-IN complex formation. Due to its interfering action it is classified as an IN binding inhibitor. Other compounds, such as styrylquinolines share similar mechanism by competing with the LTR substrate for IN binding.Binding
INSTIs bind tightly and specifically to the IN that is associated with the ends of the DNA by chelating the divalent metal ions (Mg2+) which is coordinated by the catalytic triad i.e. the DDE motif. The DDE motif is located in the CCD of IN and is the active site of the enzyme and hence INSTIs are so called active site inhibitors. INSTIs bind to a specific site close to the DDE motif of IN, a site that is present only in the conformation that occurs after processing of the 3´ viral DNA ends. Viral DNA may well form a part of the inhibitor binding site. The binding is a form of allosteric inhibitionAllosteric regulation
In biochemistry, allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site . Effectors that enhance the protein's activity are referred to as allosteric activators, whereas those that decrease the protein's activity are...
as it implies blockage of a specific integrase-viral DNA complex. This results in selective inhibition of the strand-transfer reaction, with no significant effect on the 3´-processing reaction. INSTIs may therefore be more specific and bind selectively to the target DNA binding site and hence be less toxic than bifunctional inhibitors that are able to bind to both the donor and target binding sites.
INBIs also bind to IN but the mechanism of action is unknown so the binding can not be detailed.
Structure activity relationship (SAR)
Two structural components are necessary for integrase binding: a hydrophobicHydrophobe
In chemistry, hydrophobicity is the physical property of a molecule that is repelled from a mass of water....
benzyl
Benzyl
In organic chemistry, benzyl is the term used to describe the substituent or molecular fragment possessing the structure C6H5CH2-. Benzyl features a benzene ring attached to a CH2 group.-Nomenclature:...
moiety that buries into a highly hydrophobic pocket near the active site; and chelating triad that binds with two Mg2+ ions in a rather hydrophilic
Hydrophile
A hydrophile, from the Greek "water" and φιλια "love," is a molecule or other molecular entity that is attracted to, and tends to be dissolved by water. A hydrophilic molecule or portion of a molecule is one that has a tendency to interact with or be dissolved by, water and other polar substances...
region, anchoring the inhibitor onto the protein surface (see figure 3). In fact, all potent integrase inhibitors possess a substituted benzyl component that is critical for maintaining 3‘end joining potency. Removal of the benzyl group prevents inhibitory function. Lipophylic substituents are therefore beneficial for the strand transfer inhibition, in particular the thiophenyl
Thiophene
Thiophene is a heterocyclic compound with the formula C4H4S. Consisting of a flat five-membered ring, it is aromatic as indicated by its extensive substitution reactions. Related to thiophene are benzothiophene and dibenzothiophene, containing the thiophene ring fused with one and two benzene...
, furan
Furan
Furan is a heterocyclic organic compound, consisting of a five-membered aromatic ring with four carbon atoms and one oxygen. The class of compounds containing such rings are also referred to as furans....
yl and (thiophen-2-yl)phenyl substitutions. Heteroaromatic amine and amide also cause increase in 3‘ processing inhibitory action.
When catechol-based inhibitors of IN were researched it was observed that maintaining a planar relationship with the bis-hydroxylated aryl ring increases potency. The inhibitory activity could be further optimized by including a meta-chloro substituent, enhancing the interaction of the benzyl group with the adjacent hydrophobic pocket (see figure 4: Structures A-G).
A benzyl substituted hydroxyl group (fig. 4 H) improves metal-chelating capability (compared to structure J in fig. 4) while a methoxy group (I) is much less potent due to steric clash by the additional methyl group with the catalytic metals.
When researching diketo
Diketone
A diketone is a molecule containing two ketone groups. The simpliest diketone is diacetyl, also known as 2,3-butanedione. Diacetyl, acetylacetone, and hexane-2,5-dione are examples of 1,2-, 1,3-, and 1,4-diketones, respectively...
derivates, the central pyrrole ring
Pyrrole
Pyrrole is a heterocyclic aromatic organic compound, a five-membered ring with the formula C4H4NH. It is a colourless volatile liquid that darkens readily upon exposure to air. Substituted derivatives are also called pyrroles, e.g., N-methylpyrrole, C4H4NCH3...
of structure K in fig. 4 was replaced by a series of aromatic systems having various substitution patterns. That provided optimum relative orientation of the benzyl and diketoacid (DKA) site chain. Structure L in fig. 4 resulted in 100 fold increase in potency.
Benard et al (2004) synthesized INIs with a quinoline
Quinoline
Quinoline is a heterocyclic aromatic organic compound. It has the formula C9H7N and is a colourless hygroscopic liquid with a strong odour. Aged samples, if exposed to light, become yellow and later brown...
subunit and an ancillary aromatic ring linked by functionalized spacers such as amide, hydrazide
Hydrazide
Hydrazides in organic chemistry are a class of organic compounds sharing a common functional group characterized by a nitrogen to nitrogen covalent bond with 4 substituents with at least one of them being an acyl group. The general structure for an hydrazide is R2-N-N-R3R4. A related class of...
, urea
Urea
Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....
and hydroxyprop-1-en-3-one moiety. They found that the amide group containing dervates were the most promising ones. By synthesizing series of styrylquinones researchers found out that a carboxyl group at C-7, a hydroxylgroup at C-8 in the quinoline subunit and an ancillary phenyl ring (Figure 4: Structure M) are required for inhibition, although alterations of the ring are tolerated. Two hydroxyl groups on the ancillary phenyl ring are also required for inhibitory potency.
Pharmacophore
Since critical structure information is scarce on HIV integrase catalysis it is difficult to find the exact pharmacophorePharmacophore
thumb|right|300px|An example of a pharmacophore model.A pharmacophore is an abstract description of molecular features which are necessary for molecular recognition of a ligand by a biological macromolecule....
for its inhibition. Wang et al (2010) hoped that by studying the SAR and pharmacophore of a dual inhibitor scaffold, focusing both on integrase and reverse transcriptase (RT) it would be possible to observe anti-integrase activity. By studying the SAR of HIV integrase inhibitors it was possible to find that for optimal integrase inhibition the pharmacophore requires a regiospecific (N-1) DKA of a specific length. A DKA functionality or its heterocyclic
Heterocyclic compound
A heterocyclic compound is a cyclic compound which has atoms of at least two different elements as members of its ring. The counterparts of heterocyclic compounds are homocyclic compounds, the rings of which are made of a single element....
bioisostere
Bioisostere
In medicinal chemistry, bioisosteres are substituents or groups with similar physical or chemical properties which produce broadly similar biological properties to a chemical compound. In drug design, the purpose of exchanging one bioisostere for another is to enhance the desired biological or...
that selectively inhibit strand transfer seem to be present in all major chemotype
Chemotype
Introduced by Pierre Franchomme in 1975 and formalized in the European Union in 2006 with the adoption of the regulation REACH, a chemotype is a chemically distinct entity in a plant or microorganism, with differences in the composition of the secondary metabolites...
s of integrase inhibitors. As detailed in the SAR discussion above the two necessary structural components of INI are a benzyl hydrophobic moiety and a chelating triad to bind the Mg2+ ions. For the triad to bind the Mg2+ ions has to be ionized (see fig. 5) and thus a pharmacophore bioisostere has to be ionized too and the benzyl pharmacophore bioisostere must to be very hydrophobic.
However, despite previous success in clinical development (raltegravir), a detailed binding model is lacking so it has proven difficult to structure base the design of integrase inhibitors. When the pharmacophore of salicylic acid and catechol were merged, new chemical scaffolds were created. The adjacent hydroxyl and carboxylic groups on salicylic acid could bind with the metal ions and serve as their pharmacophore. Polyhydroxylated aromatic inhibitors are mostly active against strand transfer reactions and 3‘-processing which suggests a mechanism that targets both steps. This is a very important part of the compound as it can be used to bind to the divalent metal on the active site of IN and as such be effective against viral strains that are resistant to strand transfer specific inhibitors.
Resistance
It has been discovered that over 60 variations of INSTI mutations cause in vivoIn 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...
and 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...
resistance. Due to these mutations and development of resistance the inhibitors are less effective against the virus.
Resistance of INI corresponds to those of other ARV drugs. First IN resistance is caused by primary mutations that decrease INI sensitivity in combination with secondary mutations that further reduce virus sensitivity and/or repair decreased fitness of the virus. Secondly there is a genetic barrier to INI resistance, defined by the number of mutations required for the loss of clinical INI activity. Thirdly there is extensive but incomplete 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...
among the INIs.
A loop containing amino acid residues 140-149 is located in the catalytic-core domain and is important for IN function as mentioned before. This loop is flexible and even though its role is not quite known it is thought to be important and its functions critical for DNA binding. This resistance appears within mutations in this IN-coding region.
The resistance to raltegravir and elvitegravir is primarily due to the same two mutation pathways but other primary mutations are also involved for each of the drugs. Some mutations increase resistance to the drugs to a large extent than others. For example one of the most common mutation pathway increases the resistance to raltegravir up to 100 times more than the second most common one.
Resistance to Integrase Inhibitor S/GSK1349572 is still being developed and the resistance has not been fully characterized. When it was assessed alongside the primary mutations of raltegravir and elvitegravir it did not show cross-resistance which means that it could be useful against drug resistant viruses.
Raltegravir has limited intestinal absorption and thus resistance cannot be overcome by prescribing higher doses. Newer drugs are warranted to overcome this pharmacological disadvantage and gain plasma concentrations high enough to target raltegravir-resistant viruses.
Current status
The search for new ways to improve treatment of patients infected with HIV is constant. Considering the experience that has been gathered since the 1980s of ARV drug development arrival of INSTIs as a new potent class of ARV signals a new era in the treatment of HIV. Development of a successful INSTI treatment was accomplished when raltegravir was discovered by Merck Sharp & Dohme Limited. A conditional marketing authorization was licensed in December 2007 by the European Commission which was valid throughout the European UnionEuropean Union
The European Union is an economic and political union of 27 independent member states which are located primarily in Europe. The EU traces its origins from the European Coal and Steel Community and the European Economic Community , formed by six countries in 1958...
. In 2009 this authorization was converted to a full marketing authorization
Marketing authorization
Process of reviewing and assessing the dossier to support a medicinal product in view of its marketing , obviously finalized by granting of a document also called marketing authorization...
and in the same year the FDA changed the approval from accelerated to traditional approval and listed the drug as a first line ARV treatment agent. The second INSTI drug, elvitegravir, was identified by Japan Tobacco and clinical trials began in 2005. In 2011 the drug was still in phase three clinical trials, where it is being compared to raltegravir, in treatment experienced subjects and is also in phase two development in naïve subjects as a part of a multidrug treatment. S/GSK1349572 is a integrase inhibitor discovered by ViiV/Shinongi which was entering phase three in clinical trials in 2011. This new drug is promising and seems to be well tolerated and so far shows better results than both raltegravir and elvitegravir.
Since there have been problems with resistance to raltegravir and elvitegravir, scientists have started to work on new second generation integrase inhibitors, such as MK-2048
MK-2048
MK-2048 is a second generation integrase inhibitor, intended to be used against HIV infection. It is superior to the first available integrase inhibitor, raltegravir, in that it inhibits the HIV enzyme integrase 4 times longer. It is being investigated for use as part of pre-exposure prophylaxis...
which in 2009 was developed by Merck. It's a prototype second generation INSTI that remains potent against viruses containing mutations against raltegravir and elvitegravir. The mechanism of action and SAR of MK-2048 is the same as of the other INSTIs, the structure of MK-2048 shown in figure 6 with essential pharmacophore highlighted.
Even though drugs discussed above are promising the development has a long way to go and many things are still unknown about the efficacy, safety and mechanism of action of these drugs.
See also
- Raltegravir
- ElvitegravirElvitegravirElvitegravir is an investigational new drug for the treatment of HIV infection. It acts as an integrase inhibitor. It is undergoing Phase III clinical trial conducted by the pharmaceutical company Gilead Sciences, which licensed EVG from Japan Tobacco in March 2008.According to the results of...
- IntegraseIntegraseRetroviral integrase is an enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell...
- Integrase inhibitorIntegrase inhibitorIntegrase inhibitors are a class of antiretroviral drug designed to block the action of integrase, a viral enzyme that inserts the viral genome into the DNA of the host cell. Since integration is a vital step in retroviral replication, blocking it can halt further spread of the virus...
- HIVHIVHuman 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...
- Reverse transcriptaseReverse transcriptaseIn 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...
- MK-2048MK-2048MK-2048 is a second generation integrase inhibitor, intended to be used against HIV infection. It is superior to the first available integrase inhibitor, raltegravir, in that it inhibits the HIV enzyme integrase 4 times longer. It is being investigated for use as part of pre-exposure prophylaxis...
- DolutegravirDolutegravirDolutegravir is an experimental new drug under investigation for the treatment of HIV infection. Dolutegravir is an integrase inhibitor. Also known as S/GSK1349572 or just "572", the drug is under development by GlaxoSmithKline . Studies have shown dolutegravir to be effective in patients with...