hiv integrase inhibitors as therapeutic agents in aids

Rev. Med. Virol. 2007; 17: 277–295.Published online 15 May 2007 in Wiley InterScience

(www.interscience.wiley.com)Reviews in Medical Virology DOI: 10.1002/rmv.539

HIV integrase inhibitors as therapeuticagents in AIDSVasu Nair* and Guochen ChiDepartment of Pharmaceutical and Biomedical Sciences, The Center for Drug Discovery, Universityof Georgia, Athens, GA, USA

SUMMARY

HIV-1 integrase is a protein of Mr 32 000 encoded at the 30-end of the pol gene. Integration of HIV DNA into the hostcell chromosomal DNA apparently occurs by a carefully defined sequence of DNA tailoring (30-processing (30P)) andcoupling (integration) reactions. Integration of HIV DNA into human DNA represents the biochemical completion ofthe invasion of the human cell (e.g., T-cell) by HIV. Unlike major successes seen in the development of clinicallyapproved anti-HIV agents against HIV reverse transcriptase and HIV protease, there are no FDA-approved anti-HIVdrugs in clinical use where the mechanism of action is inhibition of HIV integrase. This review summarises some keyadvances in the area of integrase inhibitors with the major focus being on new generation inhibitors. Special emphasisis placed on diketo acids with aromatic and heteroaromatic moieties, diketo acids with nucleobase scaffolds, bis-diketo acids, functionalised naphthyridines and other isosteres of diketo acids. Data pertaining to integrase inhibitionand in vitro anti-HIV activity are discussed. Mention is made of drugs in clinical trials, both past (S-1360, L-870,810and L-870,812 and present (GS-9137 and MK-0518). Other promising drugs, including those from the authors’laboratory, are referred. Resistant mutants arising from key integrase inhibitors and cross-resistance are indicated.Copyright # 2007 John Wiley & Sons, Ltd.

Received: 2 February 2007; Revised: 14 March 2007; Accepted: 19 March 2007

INTRODUCTIONThe pol gene of HIV encodes three key viralenzymes for the replication of this virus that canbe exploited for the development of chemothera-peutic agents [1–6]. Two of these enzymes, HIVreverse transcriptase and HIV protease, havereceived much attention in terms of the develop-

ment of clinically useful inhibitors [4–10]. How-ever, the third enzyme of the pol gene, HIVintegrase [2,3,11–13], has received relatively lessattention in terms of inhibitors, in large partbecause of the difficulty associated with the dis-covery of therapeutically significant inhibitors[14–16]. There are no FDA-approved anti-HIVdrugs in clinical use where the mechanism ofaction is inhibition of HIV integrase. This reviewwill focus on new generation HIV integrase inhibi-tors that includes diketo acids, their isosteres andrelated compounds.

HIV INTEGRASE

The viral enzyme and its catalysisof the integration processHIV-1 integrase is a protein of Mr 32 000 encodedat the 30-end of the pol gene (Figure 1) [2,3,11,12].Integration of HIV DNA into the host cell genomeapparently occurs by a carefully defined sequenceof DNA cleavage (30-processing (30P)) and coupl-ing (joining or integration) reactions [11,12,17–25].

RR EE V II E W

Copyright # 2007 John Wiley & Sons, Ltd.

*Corresponding author: Vasu Nair, Department of Pharmaceuticaland Biomedical Sciences, The Center for Drug Discovery, Universityof Georgia, Athens, GA 30602, USA. E-mail: [email protected]

Abbreviations usedAZT, 30-azido-30-deoxythymidine; CC50, concentration for 50%reduction of cell viability (cytotoxicity); CCD, catalytic core domain;CTD, carboxyl terminal domain; ddCMP, dideoxycytidine monopho-sphate; EC50, concentration for 50% inhibition of virus replication;EC95, concentration for 95% inhibition of virus replication;5FddCMP, 5-fluorodideoxycytidine monophosphate; 5FddCTP, 5-fluorodideoxycytidine triphosphate; IC50, concentration for 50% inhi-bition of the catalytic activity of the viral enzyme; LTR, long terminalrepeat; Mr, relative molecular mass; Nef, negative factor; NTD, N-terminal domain; p, phosphate monoester or diester; pIsodApdC, dinu-cleotide of IsodA and dC containing a 50-phosphate; 30P, 30-processing;RRE, rev response element; SAR, structure-activity relationship; ST,strand transfer; Tat, transacting transcriptional activator; TI, thera-peutic index (CC50/EC50); Vif, viral infectivity factor; Vpu, viral pro-tein U; Vpr, viral protein R.

Following reverse transcription, the viral cDNAis first tailored in the cytoplasm prior tointegration in the nucleus. This first step of inte-gration requires fully functional HIV integraseand viral cDNA. Integrase recognises specificsequences in the long terminal repeats (LTRs) ofviral DNA. In the first step of integration, whichis referred to as 30P, there is site specific endonu-clease activity and two nucleotides are removedfrom each 30-end of the double helical viral DNAto produce new 30-hydroxyl ends (CAOH-30) thatare recessed thus by two nucleotides. After 30P,the viral cDNA remains bound to integrase as amultimeric pre-integration complex, which istransported through the nuclear envelope intothe nucleus, where integrase catalyses the inser-tion of the processed viral cDNA ends into hostchromosomal DNA. This insertion is a transesteri-fication reaction, which involves a staggered clea-vage of 4–6 bp in host DNA and the joining ofprocessed CAOH-30 viral DNA ends to the 50-phosphate ends of the host DNA. The joining reac-tion produces a gapped intermediate and repairmay be accomplished by host cell enzymes,although a role here for the integrase is also possi-ble [11,12]. In the initial 30P reaction, integrase acti-vates the phosphodiester bond towards cleavage,and in the DNA strand transfer (ST) reaction, inte-grase plays the same role as well as positioning the

30-OH end of the viral DNA for nucleophilic attackon the phosphodiester bond in the host DNA. Boththe 30P and ST steps require divalent metal ioncofactors.

Structural domains of HIV-1 integraseThe crystal structure of the catalytic core domain(CCD) of HIV-1 integrase (residues 50–212) at aresolution of 2.5 A was reported over a decadeago [13]. Other crystal structures have also beenreported [26–28]. The structure of the core domain(Figure 2) has a five-strand �-sheet (at the center)and six helices. Crystal structure data suggest thatthe core domain (amino acids 50–212) of the inte-grase is related to the family of polynucleotidyltransferases that includes RNase H. HIV integrase,like other DNA processing enzymes, possesses aDDE motif which is a catalytic triad of D64, D116and E152 (Figure 2). The active site region is iden-tified by the position of two of the conserved car-boxylate residues (Asp64 and Asp116) which areessential for catalysis. A third conserved aminoacid residue in the central core domain of HIVintegrase is Glu152. Mutation of any one of thesethree residues results in loss of catalytic activity[21,25]. HIV integrase is composed of two otherfunctional domains; the N-terminal region whichis characterised by a HHCC ‘zinc finger’-likesequence and the C-terminal domain which

tat

vif

LTR 3'LTR5'

gag pol tat nefvpu

gp 120 gp 41

env RRE

kbp

rev

vpr

0 1 2 3 4 5 6 7 8 9

prot.

Mr 32KMr 117KMr 10K

rev. transcrip. integ.

Figure 1. HIV-1 genome showing the viral-encoded enzymes of the pol gene

NTD CCD CTD Integrase

Amino acids50D116D64 E152 212 288

H12H16C40

C43

Binds zinc Binds Mn2+/Mg2+ Binds DNA nonspecificallyHTH fold RNase H fold SH3 foldDimer Dimer Dimer

Structural Domains

Figure 2. Structural domains of HIV-1 integrase

278278 V. Nair and G. ChiV. Nair and G. Chi

Copyright # 2007 John Wiley & Sons, Ltd. Rev. Med. Virol. 2007; 17: 277–295.DOI: 10.1002/rmv

appears to be important for binding to the HIVLTR DNA region [29,30]. Amino acid residuesD64 and D116 form a coordination complex withdivalent metal ions (Mg2þ or Mn2þ). Suggestionhas been made that a second divalent metal ion(Mg2þ or Mn2þ) may be coordinated throughE152, after integrase binds to its DNA substrate[11,12,28]. Thus, it appears that divalent metalion involvement with integrase and the phospho-diester backbone of the DNA substrate may beimportant for both 30P and ST. Also, in order forthe CCD to provide catalysis for the 30P and STsteps, it requires the involvement of both the N-terminal domain (NTD, residues 1–50 containingthe HHCC motif) and the carboxyl terminaldomain (CTD, residues 212–228). In the absenceof the NTD and CTD, CCD can only catalyse thedisintegration reaction (reverse of ST).

EARLIER STUDIES OF INHIBITORSOF HIV INTEGRASEMany of the studies on inhibitors of HIV integrasehave been done using radiolabelled assays witholigonucleotide substrates designed to mimicboth the 30P and ST processes [14–16,31,32]. Themost active mononucleotides for inhibition ofboth the 30P and DNA ST steps of HIV integrase(IC50 40–70 mM) are L-ddCMP, L-5FddCMP and L-

5FddCTP [33]. Interestingly, pyridoxal phosphate,which has been used as a nucleotide binding probefor some DNA polymerases, inhibits both the 30Pand ST steps of integrase with good activity [33].A number of oligonucleotides and oligonucleo-tide-intercalator conjugates are known to inhibitHIV-1 integrase [34–38]. The observation thatseveral DNA binding agents [e.g., doxorubicin 1(Figure 3), ellipticine and ethidium bromide] inhi-bit both the cleavage and integration steps of theintegrase has resulted in some interest in smallmolecule non-nucleotide inhibitors of this enzyme[39]. However, the activity of these compoundsmay be due to non-specific interaction with theDNA binding domain of HIV-1 integrase. Thedata also suggest that integrase inhibition is notsimply dependent on DNA binding as some com-pounds with little ability to bind DNA such assome heteroaromatic systems containing mediumchain amines, appear to be weak to moderate inhi-bitors of HIV integrase [39]. Other compoundswith medium chain amines but without heteroaro-matic systems, such as minor groove binders, sper-mine and spermidine, are not inhibitors. Bis- orpolyhydroxylated aromatic compounds (e.g.,dihydroxynapthaquinone), molecules containingcatechol components, such as �-conidendrol 2and quercetin 3 (Figure 3), have been suggested

H3CO

O

O

OH

OH

OH

O

CH2OH

O

O

H3C

H2NOH

O

O

HO

HOOH

HOOHO

OHHO

H

H

O

OH

OH

NHO2COH

OH

OH

OH

CHO

H2C

O

CH3CO2H

O(CH2)3CH3

O

CH3CH3

PO

OO

O

P OO

O

O

O

OH

O

N

NN

NNH2

N

N

NH2

O

PO

OO

O

P OO

O

O

O

OH

O

NH

N

O

O

N

N

NH2

O

PO

OO

O

P OO

O

O

O

OH

O

N

NN

NNH2

N

N

NH2

O

1 2 3 4

5 6 7 8

Figure 3. Representative examples of earlier HIV integrase inhibitors discussed in the text

HIV integrase as therapeutic agents in AIDSHIV integrase as therapeutic agents in AIDS 279279


as interacting with the catalytic domain of the inte-grase by interfering with the coordination of metalions that are required for the phosphoryl transferreaction [40–42]. However, these catechol deriva-tives do not exhibit significant antiviral activityand/or specificity in cell culture. Styrylquinolinesand integric acids (e.g., 4 and 5) show both ST andanti-HIV activity. Screening of combinatoriallibraries of synthetic peptides has revealed a hex-apeptide as an inhibitor of integrase activity[15,16].In some other work, Nair, Pommier, their cow-

orkers and collaborators have discovered that cer-tain deoxydinucleotide 50-monophosphates showinhibitory activity against wild type HIV integrase[15,16,43–54]. The active compounds in the serieswere pdApdC 6, pdApdT and pdCpdT of whichthe most active was pdApdC. In the integraseassay, this compound showed IC50 values of 6and 3 mM for the 30P and ST steps, respectively.The charged phosphodiester linkage is essentialfor activity. However, in addition to the cellularpermeability problems associated with chargedmolecules, the internucleotide bond of the naturaldinucleotides is cleaved rapidly by 50-exonu-cleases, which renders these dinucleotides of lim-ited therapeutic or probative interest. Utilisinginformation on the mechanism of action of HIVintegrase and molecular modelling, my laboratorydesigned and synthesised new nuclease-stable,non-natural dinucleotides as probes for the inhibi-tion of this enzyme [44–54]. Our inhibitors arerecognised by HIV-1 integrase and block both the30P and ST steps of integrase activity.One of these inhibitors is a conceptually novel

dinucleotide 50-phosphate [54], synthesisedthrough the coupling of an L-related, non-naturalisodeoxynucleoside and a natural D-deoxynucleo-side (pIsodApdC 7, Figure 3). This optically puredinucleotide exhibits good inhibitory activityagainst wild type HIV-1 integrase in reproducibleassays (IC50 19 mM for 30P and 25 mM for ST) [54].The activity is much greater than for dideoxynu-cleoside monophosphates but is a little lowerthan the activity of the corresponding ‘naturaldinucleotide’ pdApdC 6 [43]. The anti-integraseactivity of the unusual dinucleotide, pIsodApdCand its natural analogue suggests base sequenceselectivity. This sequence selectivity is consistentwith the catalytic mechanism of 30P in which endo-nuclease activity produces a truncated viral DNA

with a terminal CA dinucleotide. Molecular recog-nition by the integrase of the ultimate and penulti-mate bases at the 50-end of the minus strand ofnon-cleaved viral DNA may result in stable com-plex formation prior to the ST step. In addition,other data [43,54] also suggest that two neighbour-ing bases may fulfil a substantial part of the essen-tial interaction requirements when integraserecognises its viral DNA substrate. The terminal50-phosphate appears to be essential for activityas the precursor of pIsodApdC, that is the dinu-cleotide that is devoid of the 50-phosphate group,is not active. Another interesting aspect of thistype of isodinucleotide structure is that the 30,50-internucleotide phosphate bond is resistant tocleavage by 30- and 50-exonucleases [49]. Amongother examples of nuclease-stable dinucleotidesfrom our laboratory with anti-HIV-1 integraseactivity was pdCpIsodU 8 [51], which showedeven stronger inhibitory activity against wildtype HIV-1 integrase in reproducible assays [IC50

7.5 mM (30P); IC50 5.8 mM (ST)]. While the anti-HIV integrase activity of our dinucleotide phos-phates provided new probative information onHIV-1 integrase, discovery of the nuclease resis-tance of the unusual internucleotide phosphatediester bonds in these compounds was also verysignificant [16,49,54].

NEWER GENERATION HIVINTEGRASE INHIBITORS

Beta-diketo acidsWhile many structurally diverse compounds havebeen reported to be inhibitors of HIV integrase,only a few compounds of one group, the �-diketoacids, and their related compounds, represent themost convincing, biologically validated inhibitorsof this viral enzyme. Some representative exam-ples of these compounds are identified in thetables that follow. This section will focus on themost active compounds in this group in terms ofintegrase data, inhibition of HIV replication incell culture and pre-clinical and clinical trial infor-mation. HIV-1 integrase data vary somewhatdepending on whether the assay was MnCl2 orMgCl2 based, but the differences are not very sig-nificant.One of the earliest examples of a diketo acid

with HIV-1 integrase inhibitory activity is the aro-matic diketo acid, 9 (L-708,906, Table 1) [55,56].



Table 1. Selected aryl or heteroaryl diketo acid integrase inhibitors

Compound Strand transfer Anti-HIV data Cytotoxicity CC50 References(ST) IC50, mM EC50 or EC95(mM) or CC95 (mM) and

and cell line therapeutic index (TI)

0.10 EC50, 2.0 55,56H-9 cellsEC50, 5.5 CC50, 88.3MT-4 cells TI¼ 16

0.35 EC50, 0.6 — 57Mn2þ 293T cells

< 0.10 EC95, 0.52 — 58MT-4 cells

< 0.10 EC95, 1.11 CC95, > 50 58MT-4 cells (TI> 45)

< 0.10 EC95, 0.10 CC95, > 50 58MT-4 cells (TI> 500)

1.53 EC50, 2.1 CC50, > 50 59Mn2þ 293T cells (TI> 24)

OH

O

O OHBnO

OBn

9

L-708,906

OH

O

O OHBnO

10

OH

O

O OHF

11

OH

O

O OH

12

OH

O

O OH

O

H3C CH3

13

OH

O

O OH

N3

14

(Continues)



This compound is a selective inhibitor of the STstep of HIV-1 integrase with an IC50 of 0.1 mM. Itshows in vitro anti-HIV activity in HIV infectedH9 cells with an EC50 of 2.0 mM. The monobenzylanalogue, 10, also exhibits ST activity and anti-HIVactivity [57]. All of the other aromatic-based com-pounds (Table 1, compounds 11–15) were alsoselective for inhibition of the ST step and exhibitedin vitro anti-HIV activity [58,59]. Compounds in

which the diketo acid moiety is attached to aheterocyclic system (16–19) were also found tohave ST inhibitory activity and a few of thesecompounds were found to be anti-HIV active[57,58,60,61]. Perhaps the best known of this seriesof compounds is S-1360 (compound 19), whichinhibited HIV-1 integrase with an IC50 of 0.02 mM[61]. Anti-HIV assays in PBMC showed that S-1360 was very active (EC50 0.14 mM; CC50 110 mM)

2.4 EC50, 5 CC50, > 50 59Mn2þ 293T cells (TI> 10)

0.17 EC95, 9.6 — 58MT-4 cells

7.0 EC50, 1.5 61 60MT-4/KB cells TI¼ 41

0.65 — — 57Mn2þ

0.02 EC50, 0.14 CC50, 110 61PBMC (TI¼ 786)

OH

O

O OH

N3

N3

15

NO OH

OH

O

F

16

L-731,988

NO O

O

OH

17

HN

O O

NNH

NN

Cl

185ClTEP

OO O

F

N

NHN

19

S-1360

Table 1. (Continued)

Compound Strand transfer Anti-HIV data Cytotoxicity CC50 References(ST) IC50, mM EC50 or EC95(mM) or CC95 (mM) and

and cell line therapeutic index (TI)



with a therapeutic index (TI) of 786. The majormetabolite of S-1360 in in vitro cytosolic studieswas the NADPH-dependent reduction of the eno-lic group, which appears to involve aldo-ketoreductases [62]. This is one possible clearancepathway for the compound. Pharmacokineticdata from phase II clinical trials showed verylow plasma concentrations of S-1360 in a majorityof subjects, which, at least in part, may be due tothe rapid reduction of S-1360 followed by glucur-onidation. The development of S-1360 by Shionogi& Co. and GlaxoSmithKline has been disconti-nued.

Diketo acids with nucleobase scaffoldsIn our laboratory, we discovered conceptually new�-diketo acids with nucleobase scaffolds that arepotent inhibitors of both the 30P and ST steps ofHIV integrase [63–67]. Our data suggest that thenucleobase scaffold, the substituents and the speci-fic spatial relationship of substituents in the scaf-fold, are critical for potent integrase inhibitoryactivity. The discovery makes these compoundsunique among diketo acids, not only in terms ofintegrase activity, but also because of their remark-ably potent anti-HIV activity (discussed below).Our work involved examples of both pyrimidineand purine scaffolds. The discovery is best illu-strated with one notable example, in which thestructural features are a pyrimidine nucleobasescaffold that bears three specific substituents in adefined spatial relationship: hydrophobic benzylgroups at N-1 and N-3 and a diketo acid at C-5(enolic form predominant). This compound, 4-(1,3-dibenzyl-1,2,3,4-tetrahydro-2,4-dioxopyrimi-din-5-yl)-2-hydroxy-4-oxo-but-2-enoic acid (20),was synthesised in our laboratory from readilyavailable ethyl �-ketobutyrate.

Integrase inhibition studies were conductedwith recombinant wild type HIV-1 integrase anda 21-mer oligonucleotide substrate, following apreviously described procedure [32,54]. Com-pound 20 and its analogue 21 showed strong inhi-bition of both 30P and ST steps of HIV-1 integrase(Table 2). The observed integrase inhibition is unu-sual, as other reported �-diketo compounds arecommonly inhibitors of only the ST step. Althoughthe same active site residues appear to be involvedin 30P and ST, it is not clear whether the mechan-ism of inhibition of HIV integrase by 20 is the same

for 30P in the cytoplasm and ST in the nucleus, thatis interaction with the DDE motif (Asp64, Asp116and Glu152) and other proximal amino acid resi-dues and possible sequestration of critical metalcofactors in the catalytic site. Participation of theuracil amide carbonyl (4-position) in the bindingof this inhibitor to the active site is suggested byour docking experiments [68]. Some support forthe contribution of the uracil ring in the inhibitionof both steps of HIV integrase comes from the inhi-bition data for L-708,906 that lacks the nucleobasescaffold [IC50 for L-708,906: > 1000 mM (30P) and0.48 mM (ST), TI¼ 16, MT-4 cells] [55,56]. It hasbeen proposed that an additional Mg2þ (orMn2þ) ion can be coordinated between Asp64and Glu152 once HIV-1 integrase binds its DNAsubstrates [69,70]. Thus, it appears that one Mg2þ

ion may be involved at the 30P stage in the cyto-plasm and two Mg2þ ions may be involved atthe ST stage in the nucleus. Figure 4 illustratesour docking results of inhibitor 20 with HIV inte-grase-viral DNA complex. It is likely that inhibitor20 functions by competing with the LTR of viralDNA for the active site of integrase.A suggested mechanism for the inhibition of

HIV integrase is shown in Figure 5. Prior to thefirst step of integration, which occurs in the cyto-plasm, there is recognition and binding of HIVintegrase and viral DNA. Integrase recognises spe-cific sequences in the LTRs of viral DNA. After thefirst 30P step, the viral DNA remains bound to inte-grase as a multimeric pre-integration complex,which is transported through the nuclear envelopeinto the nucleus, where integrase catalyses theinsertion of the processed viral DNA ends intohost chromosomal DNA. As shown in Figure 5,inhibition can occur in the cytoplasm and/or inthe nucleus.Compound 20 and the positive control com-

pound, AZT, were tested in a PBMC cell-based,microtiter anti-HIV assay against the clinical iso-late, HIV-1TEKI (non-syncytium inducing pheno-type) and HIV-1NL4-3 (syncytium inducingphenotype), and in a MAGI-X4 assay againstHIV-1NL4-3 performed with HeLa-CD4-LTR-�-galcells. All antiviral determinations were performedin triplicate with serial log10 dilution of the testmaterials (six to nine concentrations total). Theoverall performance of both assays was validatedby the MOI-sensitive positive control compound,AZT, exhibiting the expected level of antiviral



Table 2. Some representative examples of inhibitors of wild type HIV-1 integrase with anti-HIV activity discovered in our Nair laboratory [63–67]

Compound Integrase Cell lines & EC50 CC50 TIinhibition HIV isolatesdata IC50 (mM)

PBMC3.7 (30P) HIV-1TEKI 50 nM > 200 mM > 40000.2 (ST)Mn2þ assay HIV-1NL4-3 < 20 nM > 200 mM > 10 000

GHOSTX4/R5

4.1 (30P) HIV-1TEKI 0.85 mM > 200 mM > 235< 0.6 (ST)Mn2þ assay HIV-1NL4-3 0.24 mM > 200 mM > 833

PBMC10 (30P) HIV-1TEKI 2.63 mM > 200 mM > 760.5 (ST)Mn2þ assay HIV-1NL4-3 12.2 mM > 200 mM > 16.4

PBMC17 (30P) HIV-1TEKI 2.50 mM > 79.4 mM 31.83 (ST)Mn2þ assay HIV-1NL4-3 2.20 mM > 79.4 mM 36.1

PBMC62 (30P) HIV-1TEKI 151 mM > 200 mM > 1.351 (ST)Mn2þ assay HIV-1NL4-3 24.8 mM > 200 mM > 8.1

N

N

O

O

OOH

OH

O

20

N

N

O

O

OOH

OH

O

F

F

21

N

N

O

O

OOH

OH

O

H

22

N

N

O

O

O OH

NNH

N

23

N

N

O

OOHO

OH

O

24

(Continues)



activity. As summarised in Table 3 [63], in vitroanti-HIV studies against HIV-1 isolates in PBMCshowed that compound 20 (highest test concentra-tion¼ 200 mM) was extremely active with EC50

values in the nM range and with antiviral efficacydata (TI¼CC50/EC50) are: > 4000 (HIV-1TEKI) and> 10 000 (HIV-1NL4-3). The EC90 data for 20, whichwere in the low mM range, were also compelling.Cell viability data showed only mild cellular cyto-

toxicity at higher test concentrations; however, aCC50 (> 200 mM) was not reached. The controlcompound, AZT (highest test concentra-tion¼ 1 mM), gave TI of > 7143 (HIV-1TEKI) and> 5556 (HIV-1NL4-3). Also, in comparison, the anti-viral efficacy data of 20 in PBMC (HIV-1NL4-3)were well over an order of magnitude greaterthan those for the well-known �-diketo integraseinhibitor, S-1360 in PBMC (see Table 1). A number

GHOSTX4/R5

100 (30P)10 (ST) HIV-1TEKI 5.26 mM 97.7 mM 18.6Mn2þ assay

HIV-1NL4-3 7.64 mM 97.7 mM 12.8

PBMC> 333 (30P) HIV-1TEKI 4.0 mM > 200 mM > 5020.2 (ST)Mg2þ assay HIV-1NL4-3 3.24 mM > 200 mM > 61.7

N

N N

NO OH

CO2H

25

N

N

O

O

O

P

OH

O

OHOH

26


Compound Integrase Cell lines & EC50 CC50 TIinhibition HIV isolatesdata IC50 (mM)

Figure 4. Docking of HIV integrase inhibitor, (20), with HIV integrase-viral DNA complex. The De Luca3 model of the integrase-LTR

complex was used for docking. The DDE catalytic triad (Asp64, Asp116 and Glu152) and two Mg2þ ions (spheres) are shown. The com-

pound binds to integrase by coordinating the metal ions present in the catalytic core. The docked position is also stabilised by hydrogen

bonding between Asp64 and the enol hydroxyl group [68]



of analogues of compound 20, which are veryactive but which are not shown in Table 2, havealso been discovered.In order to obtain further supporting informa-

tion for the aforementioned PBMC data and theanti-HIV-1 integrase data, evaluations were per-formed for compound 20 against HIV-1NL4-3 in aMAGI-X4 assay. This assay is designed to detectcompounds that block HIV-1 replication via tar-gets in the viral life cycle up to and includingtransacting transcriptional activator (Tat) transac-tivation (e.g., virus attachment/fusion/entry,uncoating, reverse transcription, nuclear import,integration and LTR transactivation). Inhibitors ofviral targets after LTR transactivation (e.g., Rev,virus egress/packaging/release and protease) donot score well in this assay. Integrase inhibitor,20, and the known HIV replication inhibitors,AZT (reverse transcriptase inhibitor), T-20 (fusioninhibitor) and dextran sulfate (entry inhibitor) all

exhibited anti-HIV activity in the MAGI-X4 assayagainst HIV-1NL4-3 in HeLa-CD4-LTR-�-gal cells.The protease inhibitor, saquinavir, was inactive.In addition, compound 20 did not display any cel-lular cytotoxicity up to the highest concentration(200 mM) tested in this assay. This compound iscurrently undergoing pre-clinical studies.Figure 6 shows the structures of HIV integrase

inhibitors that carry bis-diketo acids that are joinedthrough a variety of linkers. The intent of thiswork was to investigate the ability of these bifunc-tional compounds to bind to the two divalentmetal ions critical for one or both steps of themechanism of action of HIV integrase. Implicit inthis design was the expectation that these com-pounds would have greater anti-HIV activitythan the monofunctional compounds. Their inhibi-tory data and anti-HIV activity results are sum-marised in Table 4 [71,72]. While some of thecompounds listed in Table 4 showed inhibitory

3'-Processing

Donor site Acceptor site

Viral DNA

Host DNA(Acceptor)

Strand Transfer

3' Integrase

Viral DNA(Donor)

Inhibitor

Cytoplasm

Nucleus

Cytoplasm

Multimeric Pre-integrationComplex Transported to Nucleus

Inhibitor

Disintegration Reaction

Figure 5. Mechanistic interpretation of the inhibition of HIV integrase by compound 20 [15,16]

Table 3. Antiviral efficacy of compound 20 and the positive control compound, AZT, in theinhibition of HIV-1TEKI and HIV-1NL4-3 replication in PBMC [63]

Compound High Conc. (mM) HIV-1 isolate EC50 (nM) CC50 (mM) TI

20 200 TEKI 50 > 200 > 400020 200 NL4-3 < 20 > 200 > 10 000AZT 1 TEKI 0.14 > 1 > 7143AZT 1 NL4-3 0.18 > 1 > 5556



activity towards both steps of HIV integrase and invitro anti-HIV activity, the therapeutic efficacies ofthe active compounds were not compelling.

Functionalised naphthyridinesand related compoundsWhile the 1,3-diketo moiety appears to be a criticalfunctional group for the inhibition of HIV inte-grase, efforts have also been directed towards the

discovery of alternative pharmacophores thatwould elicit similar inhibitory activity against inte-grase. structure-activity relationship (SAR) datasuggest that the Z-enolic tautomer (predominantform) of the 1,3-diketo acid moiety is coplanarwith the aromatic or heterocyclic moiety. Thus, itwas suggested [73] that a related arrangementcould be achieved with a carbonyl group conju-gated with a phenolic or related hydroxyl group.Table 5 summarises the integrase inhibition data

N

O O OH

O

OHOOH

O

HO

F

O

O OHF

O

OOH FLinker

HOO

O

HO

OHO

O

OH

Linker

N N

NH

HN

HN

HN

HN

HN

HN

NH

OO

O

O

OO

F

27

28

29

30

31

32

33

34

35

36

Linker

Linker

Figure 6. Structures of representative bis-diketo acids

Table 4. Inhibitors of wild type HIV-1 integrase that are bis-diketo acids [71,72]

Compound 30-P (IC50, mM) ST (IC50, mM) Anti-HIV-1 data EC50 CC50 (mM)Mn2þ assay Mn2þ assay (mM), CEM-SS cells (TI¼CC50/EC50)

(except 27, H-9 cells)

27 0.2 0.01 4.3 > 200 (TI> 47)28 2.5 0.3 0.8 11 (TI¼ 14)29 5.8 0.2 > 5 5 (TI< 1)30 5.3 0.2 > 9 9 (TI< 1)31 4.8 0.7 > 12 12 (TI< 1)32 32 2.7 16 124 (TI¼ 8)33 1.8 0.3 39 > 200 (TI> 5)34 7.3 0.4 > 200 > 200 (TI¼ 1)35 1.7 1.9 > 200 > 200 (TI¼ 1)36 1.8 0.2 17 81 (TI¼ 5)



Table 5. Integrase inhibition and anti-HIV data for functionalised naphthyridines andrelated compounds

Compound Strand transfer Anti-HIV data, Cytotoxicity Reference(IC50, nM) EC50 or EC95 CC50(mM) and TI

and cell line

40 EC95, 6.2 mM 12.5 73MT-4 cells

370 EC95, 5 mM 1.25 73MT-4 cells

50 EC95, 2.5 mM 2.50 73MT-4 cells

10 EC95, 0.39 mM > 12.50 73MT-4 cells

15 EC95, 100 nM — 74H-9/IIIB in MT-4 cells

22.8 EC50, 3.6 nM 0.7 75MT-4 cells (TI¼ 194)

40 EC95, 250 nM — 76

N

N

OBn

OH

37

NO

BnOH

38

NO

N

BnOH

39

N

N

OBn

OH

NSO

O

40

N

N

OHO

HN

FN

SO

O

41

L-870,810

N

N

HN

O

FN

OH

H3C

N

O

OCH3

CH3

42L-870,812

(Continues)



1600 EC50, > 30 mM > 30 75MT-4 cells (TI¼ 1)

43.5 EC50, 805 nM > 12 75MT-4 cells (TI> 14.9)

24.2 EC50, 76.3 nM > 15 75MT-4 cells (TI> 197)

9.1 EC50, 17.1 nM 5.3 75MT-4 cells (TI¼ 310)

8.2 EC50, 7.5 nM 14.0 75MT-4 cells (TI¼ 1867)

7.2 EC50, 0.9 nM 4.0 75MT-4 cells (TI¼ 4444)


Compound Strand transfer Anti-HIV data, Cytotoxicity Reference(IC50, nM) EC50 or EC95 CC50(mM) and TI

and cell line

HN

O O

OH

43

HN

O O

OH

FCl

44

N

O O

OH

FCl

HO

45

N

O O

OH

FCl

HOO

46

N

O O

OH

FCl

HOH

47

N

O O

OH

FCl

HOO

H

48

GS 9137, JTK-303



and anti-HIV activity of representative functiona-lised naphthyridines and related compounds, 37–48 [73–76]. Perhaps the best known and earliestdiscovery in this series was 41 (Merck, L-870,810),which is a ST inhibitor (IC50 15 nM using 5 nMDNA) of HIV-1 integrase. Compound 41 showsin vitro anti-HIV activity with a 95% inhibitoryconcentration (EC95) of 100 nM, against the labora-tory-adapted HIV-1 isolate H9/IIIB in MT-4 Tlymphoid cells in 50% normal human serum [74].The integrase and antiviral activities reported bySato et al. [75] were 22.8 and 3.6 nM (MT-4 cells),respectively. However, this compound showed aCC50 of 0.7 mM. Investigational studies of L-870,810 were halted due to liver and kidney toxi-city observed in dogs [77]. The next compoundin this development was L-870,812, which wasalso a ST inhibitor with an IC50 of 40 nM andwith in vitro anti-HIV activity showing an EC95

of 250 nM [76]. The outcome of this drug develop-ment is not known.The design of new bioisosters of the diketo acid

motif have three functional components thatmimic the diketo acid: a ketone, an enolizableketone or equivalent and a carboxyl oxygen allwithin a coplanar conformation. One basic struc-ture is compound 43, which is an inhibitor ofHIV integrase (IC50 1.6 mM). However, this com-pound had poor anti-HIV efficacy [75]. Modifica-tions as illustrated in 43–47 increased the TI from1 to 1867. The most active compound in the serieswas 48 (JTK-303, GS-9137). Compound 48 inhib-ited ST with an IC50 value of 7.2 nM, being three-fold more potent than L-870,810. GS-9137demonstrated remarkable antiviral activity (EC50

0.9 nM) in an acute HIV-1 infection assay usingMT-4 human T lymphoid cells. However, the com-pound also showed a CC50 of 4.0 mM, thus produ-cing a TI of more than 4444 [75]. GS-9137 isundergoing phase II clinical trial by GileadSciences [78].

Other bioisosteres of the diketo acid motifWith SAR considerations related to those used forthe design of compounds of Table 5, Summa et al.[79] designed a series of bioisosters (49–55) of thediketo acid motif, of which compound 49 repre-sents the minimal structural requirements for inte-grase inhibition (Figure 7). While they were all STinhibitors of HIV integrase with IC50 values of 0.1to 0.8 mM and they were active against HIV, thesecompounds had some significant disadvantageswith respect to cellular permeability and proteinbinding, which limited their therapeutic useful-ness (Table 6). Other modifications included theracemic compound 56 and the chiral compounds,57 and 58, all of which were active against HIV.However, these compounds also had issues withrespect to protein binding, cellular permeabilityand potency. The most interesting compound ofthis entire series, in the therapeutic sense, wascompound 59 (Merck, MK-0518). MK-0518 is apromising HIV integrase inhibitor, which is inphase II/III clinical trials. The EC95 is 33 nM in50% human serum [80,81].

Resistant mutantsResistance to integrase inhibitors emerges from theselection of mutant viruses with genetic changesthat confer functional and conformational changesin the 30P and ST catalytic sites compared to thewild type HIV-1 integrase [77]. The degree of resis-tance observed to integrase inhibitors is dependenton the number of integrase mutations, whichappears to be additive. Also, some mutationsmay have more significant effects on the viabilityof viral variants. For example, mutant virusesencoding the C130S mutation were particularlyimpaired with respect to their replication in T-cell lines [82]. Although some cross-resistancehas been seen with integrase inhibitors, it doesappear that cross-resistance may be generally lesssignificant with integrase inhibitors of different

N

N

OO

O

HN

FH3CHN

O

NN

OH3C

H3C CH3

K

R

N

N

OHOH

O

HN

F N

N

OOH

O

HN

FH3C

XN

CH3

*

MK-0518

49−55 56−58 59

Figure 7. Structures of bioisosteres of the diketo acid motif



structural classes. The in vitro mutation selectionsfrom three representative integrase inhibitors areshown in Table 7. Two of these compounds, S-1360 and L-870,810, have not been successful inearly clinical trials and have been abandoned.The drug, GS-9137 (JTK-303), shows four muta-tions, which are different from S-1360 and L-870,810 [74,83,84]. The selection of mutant virusesarising from MK-0518 has not been revealed.

CURRENT AND FUTURE DIRECTIONSAt present there are no FDA-approved anti-HIVdrugs in clinical use where the mechanism ofaction is inhibition of HIV integrase. Two poten-tially viable drugs, that are potent HIV integraseinhibitors (GS-9137 and MK-0518), are in variousstages of clinical trials for development as thera-peutic agents against HIV. Other possible inte-grase inhibitors with clinical relevance are alsobeing extensively investigated, including thosehighly potent compounds discovered in our

laboratory (see Table 2). Research efforts mustalso continue in the area of drug discovery fornew classes of compounds that are specific inhibi-tors of this enzyme.Resistant mutant viruses have been observed

with integrase inhibitors and these mutants appearto have functional and conformational changes inthe 30P and ST catalytic sites compared to the wildtype HIV-1 integrase. Interestingly, cross-resis-tance appears to be a less significant problemwith integrase inhibitors of different structuralclasses. Also, some mutations appear to havemore pronounced effects on the viability of viralvariants.It is clear that the discovery and development of

HIV integrase inhibitors with clinically useful ther-apeutic efficacy has been progressing, albeitslowly, for over a decade. The complexity andincomplete details of the mechanism of action ofHIV integrase has been the source of some of thedifficulties pertaining to earlier efforts in this

Table 6. Integrase inhibition and anti-HIV data for diketo acid bioisosteresa

Compound [79] R or X ST (IC50 mM) Anti-HIV data EC95 Anti-HIV(mM, 10% FBS)b (mM, 50% NHS)c data EC95

49 H 0.06 > 10 > 1050 thiophene 0.01 > 10 > 1051 Me 0.06 10 > 1052 CH2Ph 0.05 5.8 > 1053 CH(Me)NMe2 0.08 0.13 0.5054 C(Me)2NMe2 0.05 0.05 0.1155 2-NMe piperidine 0.20 0.15 0.4056 CH2 (� ) 0.21 0.84 1.1057 O (þ ) isomer 0.02 0.04 0.0758 O (� ) isomer 0.03 0.09 0.1959 MK-0518 [80,81] — — — 0.033

aCC50 values were not given.bFBS¼ 10% fetal bovine serum.cNHS¼ 50% normal human serum.

Table 7. In vitro-selected mutations to HIV integrase inhibitors

Integrase inhibitors Mutations

S-1360 [83] T66I L74M A128T E138K Q146K S153A K160D V165I V201IL-870,810 [74] V72I F121Y T125K V151I — — — — —GS-9137 [84] H51Y E92Q S147G E157Q — — — — —



area. In addition, some of the compounds found tobe inhibitors of integrase in isolated enzyme stu-dies, did not produce anti-HIV activity in cell cul-tures. However, a much greater effort is stillneeded in the future for the therapeutic develop-ment of the very few known classes of biologicallyvalidated integrase inhibitors that are of clinicalpotential. Time of addition studies, 2-LTR PCRassays and cellular ST assays have been particu-larly helpful in providing supporting data for vali-dation of mechanism of inhibition.Clinically useful drugs specifically targeted at

integrase would be exceedingly valuable thera-peutic agents in combination therapy with reversetranscriptase, protease and other inhibitors of HIVreplication.

ACKNOWLEDGEMENTSThe research work described in this review frommy laboratory was supported by the NationalInstitutes of Health (NIAID), by the Terry Endow-ment Fund and by the Georgia Research Alliance.It is with pleasure that I acknowledge the contribu-tions of my many coworkers and collaboratorswhose names appear in the publications refer-enced in this review.

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