REVIEWS

INTEGRASE INHIBITORS TO TREAT HIV/AIDS

Yves Pommier*, Allison A. Johnson and Christophe Marchand Abstract | HIV is a rational target for treating HIV infection and preventing AIDS. It took approximately 12 years to develop clinically usable inhibitors of integrase, and Phase I clinical trials of integrase inhibitors have just begun. This review focuses on the molecular basis and rationale for developing integrase inhibitors. The main classes of lead compounds are also described, as well as the concept of interfacial inhibitors of –nucleic-acid interactions that might apply to the clinically used strand-transfer inhibitors.

Acquired immunodeficiency syndrome (AIDS) is one sustained effort to analyse and understand viral-resistance HIGHLY ACTIVE ANTIRETROVIRAL THERAPY of the greatest challenges to humankind. AIDS and HIV mechanisms, which can be overcome by rational drug (HAART). A therapeutic infection represent global health hazards, complex development and combination therapy. The story of the regime that consists of a scientific puzzles, obvious targets for drug discovery and development of anti-HIV therapies is a good example of protease inhibitor (PI) or a vaccination, and both have enormous social, economical rapid drug development and for turning a dreadful non-nucleoside inhibitor and ethical ramifications. First reported in 1981 in a disease into a manageable chronic infection. There have 1–3 (NNRTI) in combination small number of patients , AIDS has now become a been six major factors that have led to effective HIV with two nucleoside reverse major epidemic with more than 38 million people therapies: first, early recognition of the severe popula- transcriptase inhibitors. infected worldwide, including approximately 1 million tion health problem posed by AIDS; second, adequate in the United States (for details see REF. 4), 580,000 in government prioritization and funding for basic Western Europe and more than 25 million in Sub- research; third, elucidation of the genetics and life cycle Saharan Africa (www.unaids.org). Since AIDS was first of HIV; fourth, identification of viral-specific drug clinically identified, scientific and therapeutic progress targets and the development of screening assays for has been extraordinary. It took less than 6 years to iden- drug discovery; fifth, the development of clinical tests tify the pathogenic , HIV,that caused AIDS, develop for measuring and therefore evaluating thera- sensitive tests to detect infected people during the latency peutic efficacy; and sixth, drug combinations to over- period and to introduce the first rationally designed come innate and acquired drug resistance resulting effective therapy, AZT.However, AIDS remains out of from the high-mutator phenotype of . control4,especially in developing countries where societal FDA-approved therapies target three steps of the factors are a major hurdle to combating the epidemic. HIV life cycle: reverse , proteolytic mat- Laboratory of Molecular The prognosis of AIDS patients who have full access uration and fusion (FIG. 1; TABLE 1).Triple therapy, com- , to current therapies has completely changed since the monly referred to as HIGHLY ACTIVE ANTIRETROVIRAL THERAPY Center for Cancer Research, first cases of AIDS were reported. Today, the median (HAART), is now the standard for treatment. It consists National Cancer Institute, survival for HIV-positive patients receiving treatment of a protease inhibitor (PI) or a non-nucleoside reverse National Institutes of Health, Bethesda, Maryland. exceeds 8 years. The median survival for current combi- transcriptase inhibitor (NNRTI) in combination with To whom correspondence nation therapies has not been determined, because two nucleoside reverse transcriptase inhibitors (NRTI; should be addressed at these combinations were only introduced 8 years ago. TABLE 1) (for details see REF.5). HAART, however, is often Bldg 37, Room 5068, NIH, The life expectancy for AIDS patients was less than 1 not well-tolerated by the patients. It requires discipline, Bethesda, MD 20892-4255. year before AZT was introduced in 1987. This dramatic is expensive and leads to multidrug resistance6.There- e-mail: [email protected] doi:10.1038/nrd1660 change is due to the development of effective therapies, fore, additional therapeutic approaches are warranted. Published online 24 February 2005 to early detection of HIV-positive individuals, and to a One such approach is to target the third viral ,

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integrase, and recent studies from the Merck group have reverse transcriptase inhibitors (Zerit; Bristol- demonstrated the feasibility and efficacy of integrase Myers Squibb), (Epivir; GlaxoSmithKline), inhibitors in animal models7. tenofovir (Viread; Gilead) and (Sustiva; Harnessing the anti-HIV drug market with new Bristol-Myers Squibb) was between US$400 and $600 therapeutic approaches could also be lucrative. In 2003, million. Sales for Trizivir and Combivir, which consist the estimated worldwide sales for each of the single of three and two NRTIs, respectively, combined in the same formulation, were between US$680 and $880 million (TABLE 1) (for details see REF. 8). The first integrase inhibitors were reported approx- Envelope imately 10 years ago9–11.Integrase inhibitors are now core reaching early clinical development. The most advanced Integrase Reverse compounds from Merck and GSK–Shionogi belong to transcriptase a Fusion a class of compounds known as strand-transfer inhibitors Virus adsorption inhibitors (diketo aryl (DKA) and DKA-like inhibitors). These compounds share mechanistic and structural features RNA that will be reviewed. Virus– fusion Requirement of integrase for HIV replication Approximately 40–100 integrase molecules are pack- aged within each HIV particle. The primary role of inte- Receptor and co-receptor grase is to catalyse the insertion of the viral cDNA into the of infected cells, although integrase can also Uncoating act as a cofactor for reverse transcription12,13.Integration is required for , because transcription of Reverse the viral genome and the production of viral proteins transcription DNA requires that the viral cDNA is fully integrated into a chromosome14.Following reverse transcription, the d 3′ Processing viral cDNA is primed for integration in the b Reverse- ′ transcriptase by integrase-mediated trimming of the 3 -ends of the PICs inhibitors viral DNA (FIG. 1d).This step is referred to as 3′-PROCESSING. It requires both fully functional integrase and the Integrase integrity of the last 10–20 base pairs at both ends of the inhibitors e Integration (strand transfer) viral cDNA (the att sites; see FIG. 2a and BOX 1, figure part a). 3′-processing consists of the endonucleolytic cleavage of the 3′-ends of the viral DNA (green arrows in FIG. 2a and BOX 1, figure part a). This cleavage occurs 3′-PROCESSING Transcription ′ Integration requires two immediately 3 to a conserved CA dinucleotide motif consecutive steps that are (underlined in BOX 1, figure part a). Alterations of this catalysed by integrase: sequence prevent integrase from catalysing 3′-processing. 3′-processing and strand This reaction generates CA-3′-hydroxyl DNA ends, transfer. 3′-processing which are the reactive intermediates required for STRAND corresponds to an endonucleolytic cleavage of TRANSFER (see below; FIG. 2c and BOX 1, figure part a). the 3′-ends of the viral cDNA. Following 3′-processing, integrase remains bound to This cleavage is sequence- Translation the viral cDNA as a multimeric complex that bridges specific and occurs immediately both ends of the viral DNA within intracellular particles 3’ to a conserved CA dinucleotide motif. Polypeptide called PRE-INTEGRATION COMPLEXES (PICs). Isolated PICs c contain both viral and cellular proteins in addition to STRAND TRANSFER Protease Proteolytic processing The second step of the inhibitors by viral protease integration reaction, which Figure 1 | The HIV replication cycle and drug targets. corresponds to the ligation of Viral proteins and Current therapies target attachment/fusion of HIV to the host ′ the viral 3 -OH cDNA ends RNA assemble at cell outer membrane (a) and the viral reverse ′ the cell membrane (generated by 3 -processing) to transcriptase (b) and protease (c). Integrase, the third viral ′ the 5 -DNA phosphate of an enzyme, catalyses two steps in the viral replication cycle. First, acceptor DNA (physiologically Budding integrase catalyses the processing of the 3′-ends of the viral a host ). cDNA (3′-processing step) (d); integrase then remains bound in a complex with the viral cDNA ends in the pre-integration PRE-INTEGRATION COMPLEX (PIC). A macromolecular complexes (PICs). Following nuclear translocation of the PICs, complex formed during and integrase catalyses the insertion (strand-transfer step) of the after 3′-processing and carrying viral cDNA ends into host (e) (FIG. 2). The diketo the 3′-processed viral cDNA ends aryl (DKA) integrase inhibitors preferentially block the strand- with viral and cellular proteins to transfer step, whereas other inhibitors (FIG. 4) block both the the nucleus, prior to integration. 3′-processing and strand-transfer steps.

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Table 1 | Anti-AIDS therapies approved by the FDA FDA approval Brand name Generic name Manufacturer Fusion inhibitors 2003 Fuzeon (T-20) Roche Pharmaceuticals & Trimeris Nucleoside reverse transcriptase inhibitors (NRTIs) 1987 Retrovir (AZT) GlaxoSmithKline 1991 Videx (ddI) Bristol-Myers Squibb 1992 Hivid (ddC) Roche Pharmaceuticals 1994 Zerit Stavudine (d4T) Bristol-Myers Squibb 1995 Epivir Lamivudine (3TC) GlaxoSmithKline 1997 Combivir Lamivudine+ Zidovudine GlaxoSmithKline 1998 Ziagen GlaxoSmithKline 2000 Trizivir Abacavir + lamivudine + zidovudine GlaxoSmithKline 2000 Videx EC Didanosine (ddI) Bristol-Myers Squibb 2001 Viread 2003 Emtriva (FTC) Gilead Sciences 2004 Epzicom Abacavir+ Lamivudine GlaxoSmithKline 2004 Truvada Emtricitabine+ Tenofovir Gilead Sciences Non-nucleoside reverse transcriptase inhibitors (NNRTIs) 1996 Viramune Boehringer Ingelheim 1997 Rescriptor (DLV) Pfizer 1998 Sustiva Efavirenz Bristol-Myers Squibb Protease inhibitors (PIs) 1995 Invirase Roche Pharmaceuticals 1996 Norvir Abbott Laboratories 1996 Crixivan (IDV) Merck 1997 Viracept Pfizer 1997 Fortovase Saquinavir Mesylate Roche Pharmaceuticals 1999 Agenerase GlaxoSmithKline 2000 Kaletra + Ritonavir Abbott Laboratories 2003 Reyataz Bristol-Myers Squibb 2003 Lexiva GlaxoSmithKline

the integrase–DNA complexes. The viral proteins reverse access the chromosomes of infected cells, HIV-1 PICs transcriptase (RT), matrix (Ma), nucleocapsid (Nc) and are able to cross the nuclear envelope. The karyophilic can contribute to the transport of PICs through the property of the PICs enables HIV to replicate in non- nuclear envelope. Some cellular proteins packaged proliferative cells, such as macrophages23. within PICs can bind to integrase (for review see REF.15) Once in the nucleus, integrase catalyses the insertion and stimulate the enzymatic activities of integrase. of the viral cDNA ends into host chromosomes (FIG. 1). These proteins include interactor 1 (INI1)16 (the first This ‘strand transfer’ reaction (FIG. 2c,d) consists of the integrase-binding protein discovered), lens epithelium- ligation of the viral 3′-OH DNA ends (generated by derived growth factor (LEDGF, also known as p75)17, 3′-processing) to the 5′-DNA phosphate of a host embryonic ectoderm-development protein18 and heat- chromosome. Integrase can also catalyse the reverse shock protein 60 (HSP60)19.Promyelocytic leukaemia reaction, referred to as DISINTEGRATION24.Physiological (PML) protein also colocalizes and co-migrates with integration requires the concerted joining of both ends DISINTEGRATION PICs20.Two cellular proteins, high-mobility group pro- of the viral cDNA on opposite DNA strands of the target The reverse of the strand tein A1 (HMGA1, also known as HMG1(Y)) and barrier (ACCEPTOR DNA) host chromosome with a canonical five- transfer reaction catalysed by the integrase catalytic core. to auto-integration factor (BAF), regulate integration by base-pair stagger (FIG. 2d).The five-base stagger indicates binding to DNA directly. HMGA1 stimulates integrase that each viral cDNA end attacks the chromosomal ACCEPTOR DNA activity21,22 (for a review see REF.15); BAF stimulates inter- DNA across its major groove (FIG. 2d).Completion of The DNA into which the molecular integration and suppresses auto-integration. integration requires ligation of the 5′-end of the viral donor DNA is integrated, By contrast to other , such as the oncoretro- DNA. This last step of integration can only take place which physiologically is host chromosomal DNA. Also murine Moloney virus and , after trimming of the last two nucleotides at the proviral termed ‘target DNA’. which require mitotic nuclear-envelope breakdown to DNA 5′-ends and extension (gap filling) from the 3′-OH

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abremodelling, and transcription complexes, such as those bound to integrase in the PICs (and described above), are implicated in the selection of the HIV integration sites within transcribing genes. The fully integrated viral att 3′-processing genome is also referred to as the .

HIV integrase structure and functional domains Retroviruses encode their three enzymes (protease, reverse transcriptase and integrase) within the POL gene, which is translated as the Pol polyprotein. Protease is at the 5′-end and integrase at the 3′-end of c d the POL gene (BOX 1).Integrase is generated during virus maturation (FIG. 1) by cleavage of the Pol poly- protein by HIV protease. HIV integrase is a 32-kDa Strand transfer protein comprising three structural domains: the amino-terminal domain (NTD), the catalytic core domain (CCD) and the carboxy-terminal domain (CTD) (BOX 1, figure part b). The atomic structure of each of these domains has been determined by X-ray diffraction or solution nuclear magnetic resonance (NMR) (for review see REF. 32). FIGURE 3 shows the structures of the CCD in association with the CTD33, 5′ 5′ 5′ 5′ and of the NTD with the CCD34. e CCD, which encompasses residues 50–212, forms a 5′ dimer in all the structures examined. It is, structurally, remarkably similar to other retroviral (MLV 5′ and avian sarcoma virus (ASV)), to the Tn5 and mu , to RNase H, to the Holiday junction Figure 2 | The two integrase catalytic reactions (3′-processing and strand transfer). RuvC35 and to the PIWI domain of The figure shows the viral DNA recombination (att) sites. 3′-processing takes place in the Argonaute36, the RNase associated with Dicer in RNA cytoplasm following reverse transcription (FIG. 1). It is a water-mediated endonucleolytic interference (for review on structural similarities see cleavage (green arrow in a and BOX 1, figure part a) of the viral DNA immediately 3′ from the REF. 37). This family of DNA-processing enzymes (poly- conserved CA dinucleotide (BOX 1, figure part a). 3′-processing generates reactive 3′-hydroxyls at both ends of the viral DNA (red circles (b); other 3′-hydroxyl ends and 5′-phosphate ends are nucleotide ) contains a canonical three- shown as red and green dots, respectively). Integrase multimers (not shown) remain bound to the amino-acid DDE MOTIF formed in HIV-1 integrase by the ends of the viral DNA as the pre-integration complexes (PICs) translocate to the nucleus. The catalytic triad D64, D116 and E152 (BOX 1; FIG. 3).These second reaction (c to d) catalysed by integrase is strand transfer (3′-end joining), which inserts residues are highly conserved in all integrases and both viral DNA ends into a host-cell chromosome (acceptor DNA in blue). Strand transfer is retrotransposases (DD[35]E motif). Mutation of any of ′ coordinated in such a way that each of the two 3 -hydroxyl viral DNA ends (red circles) attacks a these three acidic residues abolishes integrase’s enzy- DNA phosphodiester bond on each strand of the host DNA acceptor with a five-base-pair stagger across the DNA major groove (d). Strand transfer leaves a five-base, single-stranded gap matic activities and viral replication. The two D64 and at each junction between the integrated viral DNA and the host acceptor DNA, and a two-base D116 residues form a coordination complex (chemical flap at the 5′-ends of the viral DNA (d and e). Gap filling and release of the unpaired 5′-ends of the bonds) with a divalent metal (Mg2+ or Mn2+). Because a viral DNA (arrows in e) are carried out in coordination with cellular repair enzymes. second metal has been observed in an ASV integrase crystal structure38, and because of the two-metal structure for polynucleotide transferases35,39, it has been proposed that a second metal (Mg2+ or Mn2+) can end of the genomic DNA (FIG. 2e).It is likely that cellular be coordinated between D116 and E152 once HIV-1 enzymes/ pathways are involved in this 5′-processing, integrase binds its DNA substrate(s)40,41.It is therefore although their identity remains uncertain25,26. likely that the metal(s) coordinate(s) integrase and the By contrast to the strict DNA-sequence requirement phosphodiester backbone of the DNA substrate(s) for the viral DNA ends (see the att sites in BOX 1, figure during the 3′-processing and strand-transfer steps. In part a), HIV integration-site selection shows minimal most structures, the CCD contains a short disordered sequence selectivity with regard to the chromosomal loop (encompassing residues 141–150), the structure of sequence (‘target’ or ‘acceptor’) in which integration which can be stabilized by DNA. Although the CCD DDE MOTIF takes place27,28.This is also the case with recombinant contains the enzyme catalytic site(s), in the absence of Catalytic triad consisting of two 29 aspartate (DD) amino-acid integrase or PICs in the presence of pure acceptor DNA the NTD and CTD it can only catalyse the disintegration 30 residues and one glutamate (E). or nucleosomes .Nevertheless, HIV integrates preferen- reaction — the reverse of the strand-transfer reaction DDE motifs are conserved tially inside transcribed genes28,whereas murine — in vitro24,42.Although the physiological significance among integrase, leukaemia virus (MLV) integrates preferentially in trans- of this reaction is not known. Disintegration is the and phosphoryltransferase cription start regions31.This difference might contribute only reaction catalysed by the isolated CCD24.To cata- enzymes. The HIV-1 integrase ′ DDE motif consists of residue to the greater oncogenicity of MLV compared with HIV. lyse 3 -processing and strand transfer, the CCD needs D64, D116 and E152. It is plausible that cellular factors, such as chromatin both the NTD and CTD in a dimeric complex43.

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The NTD encompasses residues 1–50 and contains NMR structure (not shown), which is indicative of an HHCC motif (BOX 1, figure part b) that is common multiple arrangements of the integrase multimers (for to all retroviral integrases. Binding of one Zn2+ atom to review see REF. 32). The NTD is the preferential binding the HHCC motif stabilizes the folding of the NTD region for two cellular transcription factors in the PICs, domain and is required for integrase activity. Single INI1 and LEDGF/p7516,17. mutations of any of these four residues reduce integrase The CTD, which encompasses residues 212–288 enzymatic activity44.The NTD dimer interface is differ- (BOX 1, figure part b), has an overall SH3 fold (FIG. 3).It ent in the crystal structures (FIG. 3) and the solution binds DNA nonspecifically and is required for integrase 3′-processing and strand-transfer activities. The CTD binds the cellular embryonic ectoderm development 18 Box 1 | The HIV provirus gene structure and integrase domains protein as well as RT, and this interaction seems to be required for reverse transcription13. FIGURE 3 shows the Part a of the figure shows HIV viral cDNA containing the gene. The long terminal structures of the CCD both with the NTD and the CTD. repeats (LTRs) at both ends of the viral cDNA consist of three consecutive elements, Together these two structures indicate the possibility U3–R–U5, repeated in the same orientation. The sequence of the tips of the U3 (in blue) that the NTD is positioned between the CCD and CTD, and U5 (in red) repeats at the ends of the viral cDNA following reverse transcription. next to the extended α-helix joining the CCD and the ′ 96 The 3 -ends can extend further following reverse transcription (not shown) . CTD. The solution NMR dimer interfaces for the NTD Integration requires correct sequences at both ends of the proviral DNA (att sites). and the CTD, which are different from those observed 3′-processing catalyses the resection (green vertical arrows) of the viral DNA ends in the crystallographic structure (as in FIG. 3) might be immediately 3′ from the conserved CA dinucleotide (underlined), thereby generating two used in higher-order complexes (tetramers and/or dinucleotides (5′-GT) (one from each viral 3′-end) and reactive 3′-hydroxyl DNA ends (FIG. octamers), which have been proposed to correspond to 2).The shaded A/T sequences, which start approximately ten base pairs from the viral DNA the active enzyme. ends, are also conserved across HIV strains77.Integrase (p32IN) is encoded at the 3′-end of the pol gene. Part b shows the product of the pol gene as a long polypeptide precursor consisting of the three viral enzymes (protease (PR), reverse transcriptase (RT) and Integrase inhibitors: 10 years to trials integrase (IN)). Integrase is generated by cleavage of the Pol polyprotein by HIV protease The therapeutic rationale for developing integrase during maturation and is packaged within new viral particles. HIV-1 integrase consists of inhibitors has been clear for many years, even before the three structural and functional domains. The amino-terminal domain (NTD) contains four recent pharmacological validation of integrase7.Indeed, essential and conserved amino-acid residues (two histidines (H12 and H16) and two integrase is essential for retroviral replication, and the cysteines (C40 and C43)) that coordinate one zinc atom. The catalytic core domain (CCD) absence of a host-cell equivalent of integrase means that contains the acidic catalytic triad (DDE motif: D64, D116 and E152) coordinating one or integrase inhibitors do not interfere with normal cellular possibly two divalent metals (Mn2+ or Mg2+) (FIG. 3; BOX 3).The carboxy-terminal domain processes, and therefore have a high therapeutic index. (CTD), like the other two domains, forms homodimers and participates in DNA binding. This claim, however, requires a caveat: the DKAs L-708,906 and 5CITEP as well as blocking integrase also a Pol block — albeit at 10–20-fold higher drug concentrations HIV DNA — the activity of the V(D)J RAG1/2 that 45 1 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 Base pairs generate the normal antibody repertoire .Some DKAs can also inhibit RNase H46.This cross-reactivity probably results from the mechanistic and structural similarities between recombinases, RNases and integrases37,although the structure–activity relationship (SAR) for integrase U3 R U5 LTR and other are clearly distinct46. New drugs and novel targets are also needed, because it is well established that anti-HIV drug combinations are much more effective than monotherapies, which is 5′– AC-TGGAAGGGCTAATTTACTCCC...... CAGTGTGGAAAAATCTCTAGCA^GT TG^ACCTTCCCGATTAAATGAGGG...... GTCACACCTTTTTAGAGATCGT-CA– 5′ why HAART regimens have become the standard of REF. 5 att att care for AIDS patients (for details see ). Com- bination therapy also reduces the emergence of drug- b resistant viruses, as the multiple mutations that are needed to overcome drug resistance decrease viral fitness. RT IN Pol polyprotein PR p51 p15 p32 Hopefully, integrase inhibitors will become a potential additive to HAART or a salvage therapy for patients resistant to currently available anti-HIV drugs. DDE motif The screening and discovery of integrase inhibitors D64 D116 E152 generally relies primarily on simple assays that use 50 212 288 Amino acids recombinant integrase and short oligonucleotide sub- 47–50 NTD CDD CTD Integrase strates that mimick the viral DNA ends (BOX 2). Inhibitors of recombinant integrase can be subdivided H12 C43 according to whether they are antiviral, cytotoxic or H16 C40 inactive in cell culture, and whether they target other • Binds zinc • Binds Mn2+/Mg2+ • Binds DNA nonspecifically • HTH fold • RNase H fold • SH3 fold viral processes besides integration (FIG. 4,5).Extensive • Dimer • Dimer • Dimer lists of inhibitors can be found in several reviews51–55.

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a b Presently, the DKAs (discussed separately in the next section) meet all four of these criteria56,58,60,62.Studies are ongoing to determine whether the antiviral activity of

CTD the phenyldipyrimidines (PDPs), such as V-165, and styrylquinolines (SQLs), such as FZ41, (FIG. 4b) is directly NTD linked to integrase inhibition. Phenyldipyrimidines (for example, V-165) are synthetic antiviral compounds active in the nanomolar range against both viral replica- tion and recombinant integrase57.In contrast to DKAs, V-165 inhibits 3′-processing at least as effectively as it inhibits strand transfer because it blocks the formation CCD of integrase–DNA complexes57.V-165 might therefore need to be present in the infected cells prior to the for- mation of PICs (FIG. 1).Time-of-addition experiments and data demonstrating inhibition of cellular integra- tion and the accumulation of 2-LTR circles (BOX 2) are indicative of integrase inhibition by V-165 in infected c d cells. Integrase mutations leading to resistance to V-165 have also recently been identified (M. Witvrouw, per- sonal communication), which indicates that integrase CTD is targeted by V-165 in infected cells. However, it NTD should be noted that V-165 and other PDPs inhibit RT in the same concentration range as integrase57,indicating that this family of integrase inhibitors might have more than one target. SQLs (FZ41) (FIG. 4b) are synthetic compounds like PDPs and DKAs. SQLs were designed63 to chelate the CCD divalent metal (Mg2+ or Mn2+) in the integrase CCD. In common with PDPs, SQLs compete for the binding of viral DNA to integrase and inhibit 3′-processing as well as strand transfer64.They also block 3′-processing Figure 3 | HIV-1 integrase dimer crystal structure. a and b | Side views showing the catalytic equally well in Mg2+ and Mn2+ 63.A recent report showed acidic triad in red (the canonical DDE motif consisting of residues D64, D116, E152; BOX 1, figure that SQLs prevent the nuclear import of recombinant part a) in the catalytic core domain (CCD) of integrase. The two subunits of the dimer are shown integrase65.However, it is not clear whether this effect in yellow and green. c and d | Front views of the same structures (after 90° anticlockwise rotation is relevant for the anti-integrase activity of SQLs. a b a c of panels and , respectively). and | Structure of the CCD–carboxy-terminal domain Interestingly, the most active antiviral SQLs are cate- (CTD) dimer33; (PDB codes: 1EXQ & 1EX4). b and d | Structure of the amino-terminal domain 66 67,68 (NTD)–CCD34 (PDB code: 1K6Y). Combining the structures (a with b; c with d) indicates the chols matching the pharmacophore obtained from positioning of each NTD into the cavity between the CCD and CTD in the full-size integrase the first integrase catechol inhibitors identified 10 years dimer34. The functional structure of integrase is probably tetrameric, and would therefore involve ago9,69.SQL-resistant viruses contain integrase mutations another dimer interface (unknown, and therefore not represented here). (C280Y,V165I and V249I), indicating that integrase is targeted by SQLs in cells. These mutations also reduce viral replicative fitness59.Although these resistance mutations do not confer cross-resistance to a DKA59,it At least four criteria need to be met to conclude that has not been determined whether recombinant integrase integrase is the cellular target of an antiviral inhibitor containing the mutations is resistant to SQLs. SQLs are found to be active against recombinant integrase. on the list of interesting leads for the development of First, time-of-drug-addition experiments must show therapeutic integrase inhibitors. drug efficacy consistent with the integration phase — Some integrase inhibitors with antiviral activity target that is, following reverse transcription and before other steps in the HIV life cycle, and therefore cannot be maturation (between 4 and 16 hours following infec- considered integrase-specific inhibitors (FIG. 5a).For tion)56–59 (FIG. 1).Second, infected cells treated with the instance, guanosine quartet oligonucleotides (AR177 drug must show an accumulation of 2-long terminal (Zintevir; Aronex)), which are among the most potent repeat (LTR) circles56,58,60 and decreased HIV integra- inhibitors of recombinant integrase70,select for gp120 tion into host chromosomes57–60.The 2-LTR circles rather than integrase mutations in cell culture71.The result from the accumulation of viral cDNA and its same is true for L-chicoric acid (LCA) and its derivatives circularization by cellular enzymes. Third, integrase for which the resistance phenotype can be completely mutations must be found in drug-resistant viruses56,59–62. rescued by recombination in the gp120 wild-type And fourth, the drug should be inactive (or markedly gene72.Coumarin derivatives inhibit HIV-1 protease in less active) in biochemical assays against recombinant the same concentration range as integrase73.These integrases bearing the mutations identified in the examples underline the importance of thorough cross- drug-resistant viruses56,60,62. studies to validate integrase as the target,

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and the requirement that putative anti-integrase Box 2 | Examples of biochemical assays for screening inhibitors meet the four criteria listed above before Parts a and b show the short oligonucleotides derived from the U5 concluding that integrase is indeed their antiviral target. (LTR) DNA ends (BOX 1, figure part a) that are generally used. The choice of divalent For some drugs, the cross-studies, especially the gener- metal — Mg2+ or Mn2+ — is important, because antiviral inhibitors are generally active ation of drug-resistant viruses, have not been reported. 2+ 2+ 2+ either in Mg or Mn ,whereas compounds active only in Mn are generally cytotoxic This is often because the drugs’ therapeutic indexes are 41,97 32 and not antiviral .In part a,the 21-mer oligodeoxynucleotide is radiolabelled with P too low to generate drug-resistant mutants. Examples of ′ (in red) at the 5 -terminus. Recombinant integrase can use the same oligonucleotide antiviral integrase inhibitors with potential specificity species as both donor and acceptor. Release of the GT dinucleotide (BOX 1, figure part a and which might represent leads for further studies are and FIG. 2b) at the 3′-end of the radiolabelled strand generates a 19-mer oligonucleotide presented in FIG. 5b.Many of these inhibitors are poly- that can be readily separated from the 21-mer substrate by electrophoresis. The strand- transfer reaction generates a series of products longer than 21 nucleotides (b+c, lane + hydroxylated derivatives. Thiazolothiazepines have a low IN, STP). In parts b and c differential effects of 3′-processing and strand-transfer cytotoxicity and do not inhibit HIV RT or protease, 74 inhibitors are shown. Note the 3′-processing inhibition in parts b but not in part c. virus attachment or nucleocapsid formation .Caffeic Parts d and e illustrate high-throughput assays, which are generally used for screening acid phenyl ester (CAPE) is a natural product present in integrase inhibitors. The donor DNA (generally derived from the U5 LTR) is immobilized propolis, also called ‘bee glue,’which is the resinous sub- on a micro-titre well plate (d) or bound to a magnetic bead (Dynabead) (e). Integrase 3′- stance bees use to construct and maintain their hives. processing is required to activate the donor DNA, which is then able to react with biotin- CAPE was one the first integrase inhibitors reported and labelled target DNA. Integration can be detected after isolation of the bound donor DNA. exhibits weak antiviral activity9,69. CAPE was also the Inhibition is measured as signal extinction. Pre-integration complex (PIC) assays detect first illustrative example of an inhibitor selective for the the inhibition of integrase within PICs isolated from infected cell extracts. Such assays strand-transfer step, because CAPE inhibits strand 98 are generally cumbersome and use biological materials from HIV-infected cells . transfer at concentrations up to tenfold lower than those 99 Nevertheless, a high-throughput assay has been proposed . required to inhibit 3′-processing9,69.Thalassiolin A, an 21 32p antiviral natural flavone isolated from the Caribbean sea a 5′–32p–GTGTGGAAAATCTCTAGCAGT grass Thalassia testudinum, inhibits both steps of inte- CACACCTTTTAGAGATCGTCA gration in vitro at low micromolar concentration75. CA 32p Long-term passage of cells with thalassiolin A did not 75 GT Strand lead to resistant viruses .Rolitetracycline was identified 3′ processing Or by using a pharmacophore derived from the flavones Transfer 32p CA and CAPE to search the National Cancer Institute’s 19 three-dimensional chemical database67.It has been pro- 5′–32p–GTGTGGAAAATCTCTAGCA–3′OH posed that catechols, tetracyclines and mercaptosalicyl- CACACCTTTTAGAGATCGTCA 32p hydrazides inhibit integrase via metal CHELATION in the bc3′-P inhibitor ST inhibitor enzyme active site9,76; this type of inhibition mechanism

DNA + IN DNA+ IN will be discussed further in the next section on DKAs. Lexitropsins are antiviral synthetic polyamides that inhibit integrase binding to the A/T-rich sequence at the tip of the viral LTR DNA77 (see att sites in BOX 1, figure

STP part a). Natural products with a broad spectrum of activity have also been reported to inhibit HIV-1 inte- grase — examples include granulatine67 and hypericin78, 21 both of which are derived from lichen extracts; cur- cumin79,one of the main components of turmeric; the 19 ground root of Curcuma longa of the ginger family (used for curry powder); and, as recently demonstrated, the antimicrobial cationic peptide indolicidin80.It is d notable that diketo or/and β-hydroxy-keto functions

CAGT CA CA were identified as key functional motifs for many nat- + IN ural derivatives even before the discovery of the DKAs and DKA-like derivatives in which these functionalities play such a crucial role (for example, dihydroxynaphtho- quinones9, curcumin79,granulatine81,hypericin78 and 75 Detection thalassiolin A ). + Target DNA DKA derivatives: synthetic interfacial inhibitors DKAs and their derivatives are all synthetic compounds e discovered both by Shionogi & Co. Ltd82 and the Merck CAGT CA CA Research Laboratories56. Shionogi’s DKA (5CITEP) + IN (FIG. 4a) was a breakthrough because it was co-crystallized with the CCD of integrase in close association with the catalytic DDE triad82.Before the discovery of 5CITEP, Immobilization substrate Magnetic bead Biotin the only other reported co-crystal was a bisulphonate

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a Diketo aryls (Shionogi) Diketo acids Naphthyridine carboxamides (Merck) O OH Cl O OH O N OOH COOH N H N N NH F HN N N O R

5CITEP L-708,906

N O OOH OOH H3C O O N N CH3 N COOH S O N NH 3 R = O N CH3 S-1360 MA-DKA L-870,810 L-870,812

F F OOH N COOH

L-731,988

b NO2

OH OH HO O N CH3 N N OOH OH HSN ON SH OH V-165 (pyranodipyrimidine) FZ41 (styrylquinoline)

Figure 4 | Chemical structures of antiviral integrase inhibitors. a | Diketo aryls (DKAs) and DKA-like strand-transfer- selective inhibitors. 5CITEP is included here for discussion of the phylogeny of DKA, but is not antiviral. b | Potential integrase- specific inhibitors. These compounds are not selective for strand transfer. They induce drug-resistant integrase mutations in HIV infected cells, but can also inhibit other viral targets. The mutations leading to drug resistance have not been confirmed in recombinant integrase.

dimer bound to an ASV integrase dimer interface83.The virus.A closely related derivative, L-870,810 (FIG. 4a), has first report of DKA derivatives from Merck56,84 appeared entered clinical trials, as has the Shionogi derivative at approximately the same time as the crystal structure of S-1360 (FIG. 4a)86. 5CITEP was published82, and it was immediately clear Selective inhibition of the integrase strand-transfer that both the Merck and Shionogi compounds could be step at nanomolar concentrations is a trademark of functionally classified together as DKAs. The Merck DKAs56,84,87; although CAPE was the first strand-transfer DKAs were identified via random screening of a 250,000- inhibitor to be reported9,69.However, the benefits of compound library56.As with 5CITEP,they also repre- such selective inhibition in treating HIV-1 infection sented a breakthrough because of their potent antiviral only emerged with the characterization of the more activity (in the nanomolar range) and well-characterized potent DKAs. Therefore, the DKAs were the first inte- selective targeting of integrase in HIV-infected cells56. grase inhibitors to provide proof of the concept that Further synthetic and optimization efforts by the selective inhibition of strand transfer can effectively Merck group led to the recently disclosed 8-hydroxy- decrease HIV-1 infection7,56. (1,6)-naphthyridine-7-carboxamide derivatives (FIG. 4a), A divalent metal cofactor is required for the binding which can still be considered DKA-like derivatives of DKA to integrase40, and integrase inhibition can be β 40,41 because they contain the -hydroxy-ketone structural divalent-cation dependent .For instance, the IC50 of 2+ CHELATION motif (in red in FIG. 4) and because of their common L-708,906 for 3′-processing is 2.5 µM in Mg and 22 µM Coordination of a metal 2+ mechanism of inhibiting integrase (that is, selective inhi- in Mn ,whereas its IC50 for strand transfer is comparable cofactor. In the case of integrase, bition of strand transfer)7,60,85.L-870,812 is a promising with either metal (~0.06 µM)41.The selectivity for strand strand-transfer inhibitors have been proposed to chelate at least lead for therapeutic development because of its selective transfer can also be divalent metal-dependent. Both one Mg2+ or Mn2+ atom (and anti-integrase and antiviral potency, and because of its L-708,906 and 5CITEP have roughly tenfold higher inhi- probably two) in the DDE motif. favourable pharmacokinetic profile (oral bioavailability bition in the presence of Mn2+ compared with Mg2+ 41. The metal serves normally as a >60% and half-life ~5 hours in rhesus macaques). The Furthermore, 5CITEP is a much better inhibitor in Mn2+ ‘coordination bridge’ between antiviral activity of L-870,812 was recently demonstrated than in Mg2+ (one to two orders of magnitude more the integrase DDE motif, the 40,41 viral donor cDNA and the to suppress viraemia as well as chronic infections in rhe- potent) .In spite of their strand-transfer selectivity, chromosomal acceptor DNA. sus macaques7 infected with simian immunodeficiency DKAs can also inhibit 3′-processing, albeit at 30–70-fold

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higher concentrations41,56.Inhibition of 3′-processing markedly increases the inhibitory activity of the hybrid by DKAs has not been observed in treated cells. compound in Mg2+ 40,41.The carboxylate, therefore, SAR studies revealed the contributions made by the might be important for metal chelation40.The carboxy- acidic and aromatic portions of DKA to the inhibition of late portion is, however, not required for binding to the integrase. The relatively poor activity of 5CITEP in Mg2+ integrase complex40. SAR studies reveal that the aromatic is due to the tetrazole function; this is demonstrated by portion is crucial for potency88 and for strand-transfer the fact that replacement of the tetrazole by a carboxylate selectivity41,87.The aromatic moiety can accommodate a

a O O O O

5′ 3′ HO CCOH G 2 2 T G OO OH OH T T G G OO G G G G OH OH G G G T HO OH

T G OH OH

O O O O

AR177 (Guanosine quartets, Zintevir) L-Chicoric acid (LCA) Tetrameric coumarin

b OH H3C CH3 OH N HO H3COH O OH HO O O HO HO H O – N OSO3 N HO OH O OH O O OH O Caffeic acid phenyl ester Thalassiolin A Rolitetracycline

OO H O O R = N R CCR N N H O H H N S S S N H N S SS CH3 O N CH3 N H H H N N N CH3 O N O N CH3 O O O CH3 Thiazolothiazepine MSH (Mercaptosalicylhydrazides) Lexitropsins

OH O OH CH3 O

O CH3 HO OH

HO CH3 HO O OH H3CO COH3 HO CH3 OO O H H3C O O CH 3 OH O OH Granulatine (depsides) Hypericin Cucurmin

H2N NH2 NH2 HN HN HN O O O H O O O H H N H H N N N N N N O N N N H N N NH2 H2N H O O H H O O O O O HN HN HN HN

H2N NH2 Indolicidin (cationic peptide)

Figure 5 | Chemical structures of antiviral integrase inhibitors. a | Compounds inhibiting other identified viral targets. These compounds are antiviral and inhibit integrase in vitro, yet inhibit other viral targets in HIV infected cells. b | Compounds with no other identified targets. These compounds are antiviral and inhibit integrase in vitro, but antiviral targets have not been confirmed in HIV infected cells. The compounds in a and b do not meet all the criteria to be true integrase inhibitors.

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wide range of substituents, including azido groups89 metal-chelating functions, and metal-dependent inhibi- (MA-DKA, FIG. 4a) or biphenyl ketone photo-activable tion by DKAs and DKA-like compounds has been inter- groups, which were designed for drug-crosslinking preted as indicating a direct interaction of these drugs studies90.The azido portion of the azido-containing with the divalent metal in the enzyme catalytic site40,41. DKAs contributes to reduced cytotoxicity58 and could Metal coordination could also be important for shaping have a direct role in metal chelation41.The functional the catalytic pocket of integrase and therefore the diketo or β-hydroxy-keto groups are known to have DKA-binding site40,41,84,87. The molecular binding of DKA to integrase com- plexes has been a focus of research because of the importance of DKAs and DKA-like derivatives as Box 3 | Interfacial inhibition: proposed mechanism of action of DKAs antiviral lead compounds and their unique mecha- One of nature’s strategies for interfering with molecular interactions is to trap nism of action. The high selectivity of DKAs for the undergoing transition states with their partners in dead-end complexes strand-transfer step led to a model in which the two that are unable to complete their biological function. This type of inhibition, which we catalytic sites are organized around the three catalytic refer to as ‘interfacial inhibition’92, has been recently illustrated for two natural inhibitors, DDE residues and the divalent metal(s) within the brefeldin A and camptothecin, whose modes of action have been fully elucidated by integrase–DNA complexes (BOX 3)87.In this model, the structural studies. Interfacial inhibition occurs at the protein–protein interface in the DONOR DNA site binds the donor (viral) DNA end and case of brefeldin A and at the protein–DNA interface in the case of camptothecin. In both catalyses 3′-processing. Consequently, integrase systems, the drug takes advantage of transient structural and energetic conditions undergoes a structural change that allows the binding created by the macromolecular complex, which gives rise to ‘hot spots’ for drug binding. of the acceptor (chromosomal) DNA in the acceptor In addition to these examples, various natural compounds, such as forskolin, tubulin site for strand transfer84,87.DKAs,it is suggested, inhibitors and translation inhibitors, have been shown to target protein interfaces (for would bind selectively to a unique conformation of more details see REF.92). In the model below, diketo aryls (DKAs) inhibit integrase at its the acceptor site following binding of the viral DNA interface with viral DNA and divalent metal. and 3′-processing, which would then produce the The figure shows how DKAs block strand transfer selectively by binding at the interface of the integrase–DNA complexes. Part a shows that integrase has two proposed required change in conformation in the acceptor site binding sites: the donor site for viral DNA (blue circle) and the acceptor site for host for accommodating the DKA ligands. According to DNA (red circle). Part b shows the events following 3′-processing: the integrase–DNA this scheme, divalent metal coordination would be complex undergoes a structural change that renders the acceptor-site competent (red crucial for DKA binding to the acceptor site (BOX 3, rectangle) for binding host (chromosomal) DNA. In part c, and under normal figure part e). This model is supported by scintillation conditions, binding of the host (acceptor) DNA to the acceptor site leads to strand proximity assays with radiolabelled compounds40,84 transfer. In part d, the DKA inhibitor (grey rectangle) can only bind to the acceptor site that demonstrated the binding of DKA to an interme- after 3′-processing. Part e shows details of the hypothetical binding of DKAs (here diate of the integrase PIC in the presence of Mg2+ or MA–DKA; see FIG. 4a) at the interface of the integrase–DNA–divalent metal complex. Mn2+.DKA binding was shown to require functional The processed viral 3′-DNA end (in blue) is bound to integrase (the three acidic catalytic integrase, as mutant and catalytically inactive residues (DDE) are shown in red), ready to attack a host DNA phosphodiester bond enzymes failed to support DKA binding40.Drug bind- (see integrase reactions in FIG. 2). It has been proposed that DKAs chelate the metal in ing also requires viral DNA ends, as nonspecific DNA the integrase catalytic site and stabilize the macromolecular integrase–DNA complex at fails to support binding40,84.Finally, DKAs fail to bind the 3′-processing step of the reaction40,41.DKAs could therefore belong to the emerging in the absence of Mg2+ or Mn2+ 40 and compete with group of interfacial inhibitors, a class of drugs with a unique new mode of action. the strand-transfer target DNA84. The structural features of the DKA-binding site a Donor site Acceptor site bc Host DNA can also be inferred from mapping the amino-acid (Acceptor) residues associated with DKA-resistance in the crystal 3′-processing Strand transfer structure of integrase (FIG. 6).Integrase mutants selected by DKA bear mutations in the CCD56,60,62,86 3′ (and one in the CTD62). The amino-acid residues within the CCD that confer resistance are clustered Viral DNA around the catalytic site defined by the DDE residues (Donor) e Viral DNA (Donor) (FIG. 6a,c).This cluster overlaps with the cluster of DKA Adenine residues involved in DNA binding (FIG. 6b,d). One O residue (Q148) is common to both clusters and seems 5′ O to be involved in the correct positioning of the viral d 91 OH DNA before strand transfer onto the acceptor DNA . O– O O The clustering of the integrase residues involved both – DKA in drug resistance (and therefore probably in DKA O 2+ Me O binding) and in DNA contacts around the catalytic O O triad that coordinate the divalent metal(s) highlights – Me2+ the likelihood that DKAs bind at the interface of the O N3 viral (donor) DNA–integrase–metal ternary complex. O DKA binding would take place specifically at the step of Integrase acidic – residues O the catalytic cycle of integrase action that immediately (D64, D116, E152) precedes the complex switching from 3′-processing to

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a b Conclusions and perspectives Remarkable progress has been made since integrase K156 was recognized as a rational therapeutic target for the Q148 Y143 treatment of HIV infection. Recombinant integrase M154 K136 can be readily produced and used for high-through- S153 Q148 put and molecular pharmacology assays. Several I151 E152 E152 atomic structures of the integrase domains are avail- N155 D64 D64 able for docking studies. Two co-crystal structures of D116 F121 D116 different inhibitors with integrase have been pub- L74 K159 lished (1A5V83 and 1QS482), and two compounds have T125 T72 recently been introduced in clinical trials (S-1360 and T66 L-870,810). The mechanism of inhibition by the specific strand-transfer inhibitors (DKAs and naphthyridine carboxamide derivatives) fits the model of interfacial c d inhibitors of protein–nucleic acid interactions, as F121 I151 these drugs block a transition state of the integrase– M154 DNA complex. Counterscreens with related enzymes, S153 K136 such as HIV RNase H, might be useful to assess the Y143 selectivity of integrase inhibitors, and could lead to Q148 Q148 the discovery of RNase H inhibitors that are thera- E152 E152 N155 K156 peutically active against HIV and AIDS. A different D64 D64 therapeutic application of retroviral integrases is gene D116 D116 T66 K159 therapy. Fusing retroviral integrase to a sequence-spe- L74 cific DNA-binding protein is an attractive approach for T72 T125 delivering exogenous genes into specific chromosomal sites93,94. Figure 6 | HIV-1 integrase mutations leading to resistance to DKA and naphthyridine On the basis of sequence conservation of the viral carboxamide (DKA-like) inhibitors cluster at the interface between the integrase and cDNA att sites, it might be possible to design DNA- DNA. A monomer of the HIV-1 integrase catalytic core domain (CCD)82 (PDB code: 1QS4) is sequence-specific binding molecules that interfere shown both from the side (a and b) and from the front (c and d), with the catalytic acidic selectively with integrase binding and activity at the att residues coloured in red (D64, D116 & E152). The orientations are similar to those in FIG. 3. The metal Mg2+ is shown as a magenta sphere. The amino-acid residues associated with drug sites. Sequence-specific polyamines might represent 95 resistance are coloured in blue in a and c. They cluster around the three catalytic residues (in one such approach .In addition, it might be possible red) and overlap with the amino-acid residues implicated in DNA binding (in green in panels b to develop inhibitors of integrase multimerization or and d). DKA, diketo aryl. inhibitors that affect the interactions between integrase and other proteins present in the PICs or that are required for integration. However, these approaches will require the development of protein-interaction 92 DONOR DNA strand transfer (that is, when the active PIC is assembled) assays that integrate the interfacial inhibition model . The viral cDNA containing (BOX 3,step b leading to steps d and e).Such binding The discovery of novel inhibitors and the optimiza- 3′-hydroxyl ends that act as would stabilize (trap) the 3′-processing intermediate tion of lead compounds remains hampered by the lack nucleophilic donors during the strand-transfer reaction. and prevent strand transfer. We recently proposed that of atomic structures that reveal the atomic interactions this mode of inhibition is a common mechanism for between integrase and its DNA substrates (viral and INTERFACIAL INHIBITOR natural compounds that interfere with macromolec- target DNA) or the atomic structure of the complete Interfacial inhibitors bind at ular protein–DNA complexes (such as camptothecin– enzyme with its three domains. Interfacial inhibitors the interface of two or more topoisomerase I cleavage complex, dexrazoxane and stabilizing 3′-processing might be useful for determining macromolecules (protein– protein or protein– nucleic anthracylines for the topoisomerase II–DNA com- the atomic structure of an intermediate of integrase acid). The drug takes plexes, or antibiotic–ribosome complexes) or pro- bound to its DNA substrate(s), and lead to the devel- advantage of transient structural tein–protein complexes (such as brefeldine A and the opment of more potent and more selective inhibitors. and energetic conditions created Arf–GEP complex or the colchicine–α/β-tubulin com- Finally, the results of clinical trials with the strand- by conformational changes in 92 (BOX 3) the macromolecular complex plex) .We therefore propose that DKAs should transfer inhibitors are eagerly awaited, and will affect that give rise to ‘hot spots’ for be classified as INTERFACIAL INHIBITORS of macromolecular the future development of integrase inhibitors for the drug binding. complexes. prophylaxis and treatment of AIDS.

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Entrez Gene: Biogs http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene Yves Pommier received his M.D. and Ph.D. degrees from the University of Paris, France, and has been at the National Institutes of Argonaute Health (NIH) since 1981. Pommier is a member of the Molecular http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R Target steering committee at the National Cancer Institute (NCI). He etrieve&dopt=Graphics&list_uids=26523 received an NIH Merit Award for his role in elucidating the function of topoisomerase enzymes as targets for anticancer drugs and Federal BAF Technology Transfer Awards for studies on HIV-1 integrase and http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R DNA topoisomerase inhibitors. Pommier is a programme committee etrieve&dopt=Graphics&list_uids=8815 member of the American Association for Cancer Research, Senior Editor of Cancer Research and Associate Editor of Cancer Research, HMGA1 Molecular Pharmacology, Leukemia, The Journal of Experimental http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R Therapeutics and Oncology, The International Journal of Oncology, etrieve&dopt=Graphics&list_uids=3159 Drug Resistance Updates and Current Medicinal Chemistry.Pommier serves as Chair for 2004–2005 Gordon conferences on the Molecular HSP60 Therapeutics of Cancer. Pommier holds several patents on inhibitors http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R of DNA topoisomerases I and II and HIV-1 integrase inhibitors. etrieve&dopt=Graphics&list_uids=3329 Allison Johnson obtained a Masters degree in plant physiology from INI1 Texas A&M University, USA, in 1996. She subsequently researched http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R the effect of heat shock on enolase in Echinochloa phyllopogon,a rice etrieve&dopt=Graphics&list_uids=6598 paddy weed under Proff. Mary Rumpho-Kennedy, and received her Ph.D. in molecular biology in 2000 from the University of Texas LEDGF under Proff. Kenneth Johnson. Johnson cloned the accessory subunit http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R of the mitochondrial DNA polymerase and kinetically characterized etrieve&dopt=Graphics&list_uids=11168 the polymerase holoenzyme and its interaction with nucleoside ana- logues used to treat HIV infection. As a postdoctoral researcher, Matrix Johnson joined the Pommier laboratory at the NCI in 2001 to work http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R on HIV-1 integrase. Johnson’s research in the Pommier laboratory etrieve&dopt=Graphics&list_uids=155030 includes drug discovery and interactions between integrase and its substrates. PML http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R Christophe Marchand obtained his Ph.D. in molecular pharmacol- etrieve&dopt=Graphics&list_uids=5371 ogy in 1997 from the University Pierre and Marie Curie, Paris, France, in the area of via DNA triple-helix formation POL under the leadership of Proff. Claude Hélène. In 1998, he joined as a http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R postdoctoral fellow the Laboratory of Molecular Pharmacology at etrieve&dopt=Graphics&list_uids=155348 the NCI to work on HIV-1 integrase. Since 2003, he has been a research fellow and focuses on the discovery of novel integrase Reverse transcriptase inhibitors and the study of their mechanism of action. Marchand is http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R co-inventor on several patent applications for integrase inhibitors etrieve&dopt=Graphics&list_uids=155030 and is a recipient of several Federal Technology Transfer Awards. Vpr http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=R etrieve&dopt=Graphics&list_uids=155807 ONLINE

Online Summary • HIV-1 integrase is a rational target for anti-HIV therapy, and the feasibility and efficacy of integrase inhibitors in animal models has been recently demonstrated. • Integrase catalyses the insertion of the viral cDNA ends generated by reverse transcription of the viral RNA genome into host chro- mosomes. The integration reaction consists of two consecutive steps: 3′-processing and strand transfer. • Several structures of retroviral integrases have been solved. Integrase is structurally similar to other DNA-processing polynu- cleotide transferases, including the Tn5 and mu transposases, RuvC recombinase, RnaseH and the RNase component Argonaute. All of these contain a conserved DDE motif required for enzymatic activity. Divalent metals (almost certainly at least one, and probably two, Mg2+ or Mn2+ atoms) coordinate the inte- grase DDE motif, the viral cDNA and chromosomal DNA for the integration reactions. • Integrase can be used for high-throughput screening and a variety of inhibitors from diverse chemical classes have been identified. Criteria required to demonstrate targeting of cellular integrase are reviewed. • Diketo acids and diketo-like acids are the most promising integrase inhibitors. They are referred to as strand-transfer inhibitors because they uncouple the two integrase reactions. They can block strand transfer without affecting 3′-processing by chelating diva- lent cofactors in the integrase active site and by interfering with host (acceptor) DNA binding. • Strand-transfer inhibitors probably bind at the interface of the inte- grase–metal cofactor–viral DNA ternary complex by chelating the divalent metal, and thereby interfering with the binding of the chromosomal target DNA. Strand-transfer inhibitors are candi- date interfacial inhibitors, and represent a new mechanism of action in drug discovery.

Competing interests statement Y.P. and C.M. are inventors on a number of pending and granted patents relating to HIV-1 integrase inhibitors.