discovery and development of integrase inhibitors discovery and development of integrase inhibitors the first human immunodeficiency virus (hiv) case was reported in the united states in the early 1980s. many drugs have been discovered to treat the disease but mutations 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. history 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 infections 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 chromosomes 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 trials. raltegravir (brand name isentress) was granted accelerated approval from the u.s. food and drug administration (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, elvitegravir, dolutegravir, and bictegravir are the only hiv-1 integrase inhibitor being used to treat hiv infections s/gsk1349572. the hiv-1 integrase enzyme the hiv-1 integrase (in) is a key enzyme in the replication mechanism of retroviruses. it is responsible for the 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 index 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. figure 1: structural domains of the hiv-1 integrase in is composed of 3 structurally independent, functional domains (see figure 1).: 1. the n-terminal domain (ntd) encompasses amino acids 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 tetrameric 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). in 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 nucleophilic 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 the abundance of magnesium (mg2+) versus manganese (mn 2+) in human cells is 1,000,000-fold, magnesium seems a more readily available divalent cofactor for integration. mechanism of action figure 2: integration of viral rna into host cell dna there are several ways to target integrase but strand transfer inhibition is the most intuitively obvious and readily pursued to date. 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 integration phase. inactivating these cofactors (e.g. through chelation) causes functional impairment of in. this concept gives researchers 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 divalent magnesium ions in the enzyme's active site. raltegravir and elvitegravir share the same mechanism of action against integrase: to bind to the active site of mg2+ ions. competitive 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 inhibition is called strand transfer inhibition. inhibition of the ledgf/p75- integrase interaction lens epithelial derived growth factor (ledgf/p75) 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. drug design 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 inhibition 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) figure 3: structure activity relationship of elvitegravir and raltegravir. a benzyl group in a hydrophobic pocket and a triad to chelate the two mg2+ ions. two structural components are necessary for integrase binding: a hydrophobic 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, furanyl and (thiophen-2-yl)phenyl substitutions. heteroaromatic amine and amide also cause increase in 3‘ processing inhibitory action. figure 4: examples of integrase inhibitors. 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