Proc. Natl. Acad. Sci. USAVol. 92, pp. 11456-11460, December 1995Biochemistry

Identification of a hexapeptide inhibitor of the humanimmunodeficiency virus integrase protein by using acombinatorial chemical libraryRAMON A. PURAS LUTZKE*, NOOR A. EPPENS*, PATRICIA A. WEBERt, RICHARD A. HOUGHTENt,AND RONALD H. A. PLASTERK*§*Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; tHoughten Pharmaceuticals, Inc.,San Diego, CA 92121; and tTorrey Pines Institute for Molecular Studies, San Diego, CA 92121

Communicated by Martin Gellert, National Institutes of Health, Bethesda, MD, August 4, 1995 (received for review May 25, 1995)

ABSTRACT Integration of human immunodeficiency vi-rus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN proteinis a suitable target for antiviral strategies. To find a potentHIV IN inhibitor, we screened a "synthetic peptide combina-torial library." We identified a hexapeptide with the sequenceHCKFWW that inhibits IN-mediated 3'-processing and inte-gration with an IC50 of 2 ,uM. The peptide is active on INproteins from other retroviruses such as HIV-2, feline immu-nodeficiency virus, and Moloney murine leukemia virus,supporting the notion that a conserved region of IN is targeted.The hexapeptide was also tested in the disintegration reaction.This phosphoryl-transfer reaction can be carried out by thecatalytic core of IN alone, and the peptide HCKFWW was foundto inhibit this reaction, suggesting that the hexapeptide acts at ornear the catalytic site of IN. Identification of an IN hexapeptideinhibitor provides proof of concept for the approach, and,moreover, this peptide may be useful for structure-functionanalysis of IN.

Most pharmaceutical drugs used in current medical practiceare derived from compounds found in nature. Previously, theywere often recognized as drugs (or drug leads) by trial anderror; more recently, they have been identified by large-scalesystematic screens of compound libraries. There is, however,no a priori reason to limit the set of compounds that arescreened to those found in nature, derived by evolution forreasons other than their application as drugs. Recent devel-opments in organic chemistry and molecular biology havemade it feasible to generate a much wider range of complexcompounds, independent of any functional bias, and screenthose for potential drug lead compounds.To identify low molecular weight peptides for drug discovery

or functional studies, several types of peptide libraries havebeen developed. Examples are "phage display libraries" (1),peptides on resin beads (2), and chemically synthesized peptidecombinatorial libraries (3); each methodology has its owncharacteristic pitfalls and applications (4).

In this study we have used the "synthetic peptide combina-torial library" approach (SPCL; ref. 3). Hexapeptides consist-ing of natural L-amino acids are chemically synthesized asmixtures in which each individual hexapeptide is present atapproximately equimolar concentration. Since these peptidesare in solution, they can be assayed for any effect on anyreaction occurring in solution.The selection of an active (not necessarily the most active)

peptide is achieved by an iterative selection process, until aunique hexapeptide is identified (ref. 3 and see legend to Fig.1). We have applied this strategy to find an inhibitor of the

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human immunodeficiency virus (HIV) integrase (IN) protein.This protein is required for integration of the viral DNA intothe host chromosome and is essential for viral replication (5,6). Two other HIV proteins involved in viral replication,reverse transcriptase and protease, have previously been usedas targets to develop antiviral drugs. Highly active inhibitorsagainst reverse transcriptase (e.g., 3'-azido-3'-deoxythymi-dine) and protease eventually lose their value during chemo-therapy, since resistant viral variants are selected (7). It wouldtherefore be useful to find inhibitors of additional HIVenzymes, to be applied in multidrug therapy, and IN would bea good target, since it has no obvious cellular counterpart.IN performs two reactions in the viral replication cycle: it

cleaves two nucleotides from the 3' ends of viral double-stranded DNA (donor cut) in the cytoplasm of infected cells,and subsequently in the nucleus IN joins the newly generated3'-recessed viral DNA ends to phosphate groups in the cellulartarget DNA (for recent reviews, see refs. 8-10). In its simplestform, the integration reaction can be carried out in vitro withpurified recombinant IN protein, which integrates short(20-30 bp) double-stranded oligonucleotides of the sequenceof viral DNA ends into DNA of any sequence (11-16).Extensive biochemical characterization of IN revealed that theprotein functions as a multimer (17-19) and contains threefunctional domains: the N-terminal region, which contains azinc-finger motif, a nonspecific DNA-binding domain in theC-terminus, and the catalytic core, which harbors the active-site residues D64, D116, and E152 (20, 21). This DD(35)Emotif presumably forms a catalytic triad in the presence ofdivalent cations, and it is conserved among retroviral andretrotransposon IN proteins (22, 23). It has been shown thatthe central catalytic domain of IN can catalyze the disintegra-tion reaction (20, 21, 24), and therefore this phosphoryl-transfer reaction provides a nice tool to study the catalyticactivity of IN.We describe herein the identification and characterization

of a hexapeptide that inhibits HIV IN and INs of relatedviruses, with a reasonably low IC50 of 2 ,iM. To our knowledge,this is the first description of an inhibitor of a viral enzymeisolated from a combinatorial library. Although a peptide ofnatural amino acids might itself not be a good drug, the resultprovides a proof of concept for the approach, which can nowbe followed up using other peptide-chemical libraries. More-over, this peptide may be useful for structure-function analysisof the IN protein.

MATERIALS AND METHODSPeptides. Preparation of the SPCL is described elsewhere

(3). Each peptide or peptide mixture has an N-terminal free

Abbreviations: SPCL, synthetic peptide combinatorial library; HIV,human immunodeficiency virus; FlV, feline immunodeficiency virus; IN,integrase; INC, integration-competent nucleoprotein complex; MoMLV,Moloney murine leukemia virus; MBP, maltose-binding protein.§To whom reprint requests should be addressed.

11456

Proc. Natl. Acad. Sci. USA 92 (1995) 11457

amine group and a C-terminal amide group (e.g., 2HN-HCKFWW-CONH2).IN Assays. Peptide mixtures from the SPCL were assayed as

described (20). Since the specific activity of the peptide goesup with the iteration process, we used lower concentrations ofpeptide mixtures at each step. The following peptide concen-trations were used: OOXXXX, 2.8 mM; HCOXXX, 2.2 mM;HCKOXX, 850 ,uM; HCKFOX, 100 ,uM; HCKFWO, 50 AM(in which 0 represents each of the 20 naturally occurringL-amino acids and X represents an equimolar mixture of allL-amino acids). Reaction products were separated by dena-turing gel electrophoresis and detected by autoradiography.The effect of the hexapeptide HCKFWW on other retroviral

IN proteins such as HIV-2 IN, feline immunodeficiency virus(FIV) IN, and Moloney murine leukemia virus (MoMLV) INwas assayed as described (15, 25, 26). In the site-specificcleavage or integration assay, the protein concentration for theIN proteins was 1 ,tM. As a nonspecific peptide, a hexapeptidemixture from the initial screen was used (ANXXXX).

Disintegration reaction was performed as described (25)using maltose-binding protein (MBP)-IN fusion proteins, ei-ther the HIV-1 IN full-length protein (MBP-INI 288) or thecatalytic domain of HIV-1 IN (MBP-INso5237). The concen-tration of HCKFWW or a nonspecific peptide mixture in thereaction was 100 ,uM.

Alanine Scan. A set of six hexapeptides was synthesized inwhich every amino acid position of the peptide HCKFWW wassubstituted, one by one, by the amino acid alanine. Thesepeptides, varying in concentration from 1 to 400 ,AM, wereassayed for inhibition of site-specific cleavage activity ofHIV-1 IN (see above).

Integration Assay. Integration reactions were performed asdescribed (27). Either hexapeptide HCKFWW or a nonspecificpeptide mixture (2 ,uM, 50 ,uM, or 1.25 mM) was preincubatedwith the integration-competent nucleoprotein complex (INC)for 5 min on ice, and the reaction was started upon addition ofMnC2 and target DNA and subsequently incubated for 15 minat 37°C. Reaction products were detected by Southern blotusing a random-primed 1.2-kb 32P-labeled BamHI/Pst I frag-ment of the MoMLV IN gene (pRP 819) and autoradiography.The MoMLV INC was generously provided by M. S. Lee andR. Craigie (27).

Quantitations. For the peptide screen, activities were quan-tified by densitometry using an Ultrascan XL enhanced laserdensitometer (LKB). Activities of IN reactions containingpeptides from the alanine scan and the MoMLV integrationassay were quantified using a phosphoimager (Fuji Bas).

RESULTSScreening an SPCL. To find an IN inhibitor we screened an

SPCL (3). The inhibitory effects of hexapeptide mixtures froman SPCL were tested in functional IN assays (20).A schematic representation of the iterative steps to select the

most effective peptide is shown in Fig. 1. In the first selectionstep, the two N-terminal positions of a hexapeptide weredefined. Therefore, 400 peptide mixtures were tested for theirability to inhibit IN activity. Peptide mixture HCXXXX causedthe strongest inhibitory effect among the 400 peptide mixtures.Two other inhibiting peptide mixtures were 2 and 5 times lessactive than HCXXXX (data not shown). Fig. 2 shows thesecond, third, fourth, and fifth steps of the iterative selectionprocess. In the second step, 20 new peptide mixtures weresynthesized in which the third position of the hexapeptide wasdefined (HCOXXX). Lysine had the strongest inhibitory effectat this position, followed by tryptophan, phenylalanine, andthreonine, whereas methionine, aspartic acid, and glutamicacid were less active in reducing IN activity (Fig. 2;HCOXXX). In the third selection step, phenylalanine was thefavored amino acid defining the fourth position (HCKOXX).

IN assay1) ooxxOO ==

400 160,000

2) HCOXXX Cz>20 8,000

3)

4)

HCKOXX ,z>20 400

HCFKOX >z>20 20

HCXXXX

HCKXXX

HCKFXX

HCKFWX

5) HCKFWO => HCKFWW20

FIG. 1. Concept of SPCL (3) and iterative detection of a hexapep-tide that inhibits HIV-1 IN. In step 1, 400 peptide mixtures were testedthat differ in the amino acid combination in the two N-terminalpositions of a hexapeptide [00; (20)2 possible combinations of allnaturally occurring L-amino acids]. The remaining positions (X) aremixtures of all other possible combinations of L-amino acids [(20)4;16,000] in an equimolar representation. Once an active peptidemixture was identified (HCXXXX), we tested, in the second step, 20peptide mixtures to define the third position of the hexapeptide. Thecomplexity of the remaining amino acid combinations decreases to8000 possible combinations. This iterative process was continued untilall positions were defined and the sequence of the most inhibitinghexapeptide was determined.

The inhibitory effects of most of the amino acids in thisposition do not differ significantly, except for isoleucine,glutamic acid, and tryptophan. The best amino acid candidatefor position five (HCKFOX) is tryptophan, followed by argi-nine and cysteine. In the last selection step, tryptophan,isoleucine, and phenylalanine at the C-terminal end of thehexapeptide were found to show the strongest inhibitory effect,with approximately similar efficiency. To define which of thelatter three amino acids is the most inhibitory, we determinedthe ICs0 values of the corresponding hexapeptides. HCKFWWwas the most effective peptide with an IC50 of 2 .tM followedby HCKFWI and HCKFWF (4 and 7 ,tM, respectively). Wealso tested the effect of the peptide mixtures on IN-mediatedintegration reaction. Peptides were found to have similareffects on site-specific cleavage as on integration (data notshown).The IC50 values of the best peptide mixtures in each selection

step are shown in Fig. 3A, compared to a nonspecific peptidemixture. One expects that the IC50 value drops during theiterative process, because of ongoing reduction of the peptidemixtures' complexity, and this is clearly seen in Fig. 3A. Themost inhibitory hexapeptide detected in this procedure wasHCKFWW with an IC50 of 2 ,uM.The IN protein does not seem to contain such an amino acid

sequence, suggesting that HCKFWW is not a mimetic peptidefragment of an IN region.

Screening for Important Amino Acid Positions of the In-hibitory Peptide. Knowing the sequence of an inhibitorypeptide, we defined which positions are important and whichpositions are less essential for the inhibition of IN. Therefore,we synthesized a set of six hexapeptides, based on HCKFWW,in which in every individual peptide a single amino acid wassubstituted by alanine (alanine scan; e.g., ACKFWW, HAK-FWW, etc). These peptides were tested in IN assays asdescribed above. Fig. 3B shows the IC50 values of the hexapep-tides from the alanine scan compared to the original peptide.All alanine substitutions show a decrease of inhibition com-pared to the original peptide HCKFWW. The IC50 values ofthe corresponding peptides reflects the relative importance ofevery amino acid position. This is shown by the relatively highIC50 values of HCKFAW and HCKFWA (150 and 210 j,M,respectively), indicating that the last two positions are impor-tant for the overall inhibitory effect. In contrast, amino acidphenylalanine in position 4 is less important as indicated by the

Biochemistry: Puras Lutzke et al.

11458 Biochemistry: Puras Lutzke et al.

1 On_HCOXXX HCKOXX

I,u I c U - -

100 100

80. 80-

60- 60

40. 40

c 20- 200

AC D E FG KLM N PQRSTVWY 0 AC D E FG H I KLMN PQ RSTVWY

Z HCKFOX HCKFWO

120 120-

10 100.80- 0

60- 0

40 40li20 2

ACDEFGH IKLMNPQRSTVWY ACDEFGHIKLMNPQRSTVWYAmino acid

FIG. 2. Screening of the SPCL forIN-inhibiting peptide mixtures. Importantamino acids in the second (HCOXXX),third (HCKOXX), fourth (HCKFOX),and fifth (HCKFWX) iterative selectionsteps are shown. Each bar represents therelative IN inhibition by a peptide mixturedefined in the "O" position by one of the20 L-amino acids. They axis represents thepercentage of IN inhibition (site-specificcleavage). The best amino acid is indicatedby a star. Peptide concentrations used forthis screen are given in Materials andMethods.

effect of peptide HCKAWW (IC50; 8 jiM), suggesting that thisposition is a "wobble" position in the hexapeptide. Obviously,this procedure is biased by the use of alanine as a substituent,but it indicates important positions and provides an additionalcontrol for the specificity of the peptide.

Specificity of the IN Inhibiting Peptide. To determine thespecificity-of the HIV-1 IN-specific hexapeptide HCKFWW,we tested whether this peptide can inhibit the catalytic activityof other IN proteins and endonucleases.

A 10o,ooo

::l

(B

1 000

100

10

1 2 3 4Peptide

5 6

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1

B

200-

2 150

0 100-

50-

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AO Al A2 A3 A4 A5 A6Peptide

FIG. 3. (A) The IC50 values of the most inhibiting peptide mixturesof each selection step are represented. Bars: 1, nonspecific peptidemixture at 8 mM; 2, HCXXXX at 2.2 mM; 3, HCKXXX at 600 ,uM;4, HCKFXX at 450 t,M; 5, HCKFWX at 25 t,M; 6, HCKFWW at 2,uM. (B) Alanine scan of HCKFWW: presentation of the IC50 valuesof each alanine substitution compared to the original hexapeptideHCKFWW. Each bar shows the corresponding IC50 value. Bars: AO,HCKFWW at 2 jiM; Al, ACKFWW at 51 ,uM; A2, HAKFWW at 30,uM; A3, HCAFWW at 49 ,uM; A4, HCKAWW at 8 ,uM; A5,HCKFAW at 150 ,uM; A6, HCKFWA at 210 ,uM.

We assayed the site-specific cleavage activity using recombi-nant, purified HIV-2, FIV, and MoMLV IN proteins and theirspecific viral DNA ends. A nonspecific hexapeptide had noinhibitory effect on HIV-1, HIV-2, FIV, and MoMLV IN activity(Fig. 4A, lanes 3, 7, 10, and 14, respectively). However, peptideHCKFWW, selected for HIV-1 IN, also inhibited HIV-2, FIV,and MoMLV IN (lanes 4, 8, 11, and 15, respectively).To investigate whether the active peptide inhibits endonu-

cleolytic reactions in general, we tested restriction endonucle-ases BamHI and EcoRI and DNase I in functional assays usingpeptide concentrations ranging from 0.01 to 2.7 mM. Note that0.1 mM HCKFWW would totally abolish IN activities, since itis 50 times the IC50. Neither the IN-specific peptide nor thenonspecific peptide mixture could inhibit DNase I and BamHIand EcoRI endonucleases (data not shown). Recently, it hasbeen shown that the catalytic domain of HIV-1 IN has a similarfold as RNase H, MuA transposase, and RuvC (28). We testedthe inhibitory effect of the IN-inhibiting peptide in an RNaseH in vitro assay. High concentrations (> 1 mM) of the hexapep-tide HCKFWW inhibit RNase H activity, whereas high con-centrations of a nonspecific peptide mixture do so to a lesserdegree (data not shown). RNase H, which shares a commonstructural motif of the catalytic domain with HIV-1 IN,appears slightly sensitive to the peptide.The above results suggest that HCKFWW interacts with a

conserved region of retroviral IN proteins, most likely the cata-lytic core of IN. To address this question further, we testedwhether this peptide can inhibit the IN-mediated phosphoryl-transfer reaction, termed disintegration (24). Previously, it hasbeen shown that the central catalytic core of IN on its own is ableto catalyze disintegration (20, 21). We assayed the disintegrationreaction using either full-length IN protein (MBP-IN1 288) or thecatalytic domain (MBP-IN50-237). As shown in Fig. 4B, theIN-specific peptide can inhibit disintegration mediated by eitherIN1_288 or IN50-237 (lanes 4 and 7), whereas a nonspecific peptidemixture (lanes 3 and 6) has no effect on the disintegration activityof either of these proteins. In addition we did DNA-bindingexperiments (UV crosslink assay) in which we varied the order ofaddition of viral DNA, IN, and the peptide. We found that thepeptide HCKFWW does not compete for DNA binding of IN toviral DNA (data not shown).

In summary, the HIV-1 IN-specific peptide HCKFWW actsalso on HIV-2, FIV, and MoMLV IN proteins with approxi-mately similar efficiency, suggesting that it interacts with a

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Proc. Natl. Acad. Sci. USA 92 (1995)

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Proc. Natl. Acad. Sci. USA 92 (1995) 11459

A HIV-1 IN HIV-2 IN FIVIN MoMLVIN

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3 4 5 6 7 8 91011 12131415

IN 1-288 IN50,237CL XL

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FIG. 4. Specificity of HCKFWW. (A) Inhibition of IN proteins ofvarious retroviruses by HCKFWW. HCKFWW (100 ,LM) or a non-

specific peptide mixture (100 ,uM) was tested in a site-specific cleavageassay using different recombinant IN proteins. Lanes 1-4, HIV-1 IN;lanes 5-8, HIV-2 IN; lanes 9-11, FIV IN; lanes 12-15, MoMLV IN.HIV-1 and FIV IN proteins had a C-terminal 6xHis-tag, HIV-2 IN wasuntagged, and MoMLV IN was a fusion protein to MBP (see alsoreferences in Materials and Methods). Reaction products (26-mer)were separated on a denaturing 12% polyacrylamide gel. -IN, DNAsubstrate (28-mer); -pep., reaction without peptide. (B) Effect ofHCKFWW on the catalytic domain of IN. Both the IN full-lengthprotein (MBP-IN1 288) and the catalytic domain (MBP-IN50-237) weretested in a disintegration reaction using a "dumbbell" DNA oligonu-cleotide substrate as described (25). Lane 1, free DNA substrate(-IN); lanes 2 and 5, the phosphoryl-transfer reaction with eitherIN1 288 or IN50-237 in the absence of peptide (-pep.). Addition ofeither a nonspecific peptide mixture (lanes 3 and 6) or peptideHCKFWW (lanes 4 and 7) to the disintegration reaction of eitherINI 288 or IN50-237 is indicated. S, substrate; P, product.

conserved region of IN. The inhibition of the disintegrationreaction shows that the catalytic core is the target of the peptideinhibitor. Furthermore, HCKFWW may have a slight effect on

proteins structurally related to IN, as, for example, RNase H.Effects of the Peptide on the Integration Activity of the

MoMLV Preintegration Complex. After reverse transcription,the viral DNA is part of a nucleoprotein complex, derived from

HCKFWW non-spec. pep.

0C.

integratedviral DNA - _ & 4 -

viral DNA * * 4*.4*A46 4

1 2 3 4 5 6 7 8

FIG. 5. Inhibition of integration activity of MoMLV INC by HCK-FWW. Reactions and detection of integrated DNA products were asdescribed in Materials and Methods. Lane 1, viral donor DNA (8.8 kb);lane 2, integration of the viral DNA into OX174 RFI targetDNA resultingin a 14-kb product; lanes 3-5, increasing amounts of HCKFWW wereadded to the integration reaction (2 ,tM, 50 ,uM, and 1.25 mM, respec-tively); lanes 6-8, effect of a nonspecific peptide mixture.

the core of the infecting virion. This complex, recently termedINC (27), is able to perform double-ended integration in vitro(29-35).Can the hexapeptide inhibit the function of the native IN

protein within the preintegration complex? As already shownin Fig. 4A, peptide HCKFWW inhibits the activity of recom-binant MoMLV IN protein. We tested the inhibitory peptideHCKFWW and a nonspecific peptide mixture in an integrationassay using partially purified MoMLV INC derived fromMoMLV-infected NIH 3T3 fibroblasts and linearized OX174RFI as target DNA. The viral donor DNA of 8.8 kb (Fig. 5, lane1) can be integrated into exogenous, linear target DNA(+X174 RFI; 5.4 kb) resulting in an integration product of 14kb (lane 2), with an integration efficiency of 42%. Peptideconcentrations of 2 and 50 ,M HCKFWW had hardly anyeffect on the integration reaction (lanes 3 and 4; 38% and 30%integration efficiency, respectively). However, at 1.25 mMHCKFWW, the integration efficiency decreased to 15% (lane5). A nonspecific peptide mixture had no influence on theintegration reaction (lanes 6-8), even under higher concen-trations (5 mM) (data not shown).

These data show that the hexapeptide HCKFWW is able toinhibit the IN protein within the preintegration complex, al-though the concentration ofHCKFWW required for inhibition ishigher than in IN assays using recombinant IN. Possibly, the INprotein within the preintegration complex is less accessible to thiscompound than purified recombinant IN in solution.

DISCUSSIONOne crucial problem of antiviral drug therapy is the highmutation rate of HIV. Consequently the retrovirus escapesdrug treatment as result of selection of mutated variants, andchemotherapy of AIDS eventually fails (for a recent review,see ref. 7). One potential way to overcome this problem is toapply a combined therapy; therefore, it is useful to discoverpotent inhibitors against different HIV proteins, such as theHIV IN protein. In addition to other HIV pol gene productslike reverse transcriptase and protease, the IN protein attractsmore attention now that the mechanism of IN-mediatedretroviral DNA integration has been, in part, clarified. So far,several IN inhibitors such as topoisomerase inhibitors (36),caffeic acid derivatives (37), nucleotide analogs (38), aurintri-carboxylic acids (39), bis-catechols (40), and phenanthroline-cuprous complexes (41) have been described.We here describe the use of an SPCL to find an IN inhibitor.

This particular library has the advantage over other combina-torial approaches in that the peptide mixtures are in solutionand can be directly applied to in vitro IN assays. We can directly

Biochemistry: Puras Lutzke et al.

11460 Biochemistry: Puras Lutzke et al.

score for inhibition in a functional assay and not only forbinding of certain compounds to IN, like in solid-supportbound combinatorial libraries or phage display libraries (1, 2).Another advantage of the SPCL approach is that the numberof assays that need to be done to screen the entire universe ofpossible natural hexapeptides (64 million) is only a few hun-dred, and therefore we chose to use the more sensitive andinformative gel analysis, rather than any of the high-throughputmicrotiter plate assays (42, 43). In the iterative process of theSPCL, we were able to identify a hexapeptide inhibitor againstHIV-1 IN with an IC50 value of 2 ,uM. This peptide inhibits boththe donor cut of viral DNA ends and the integration of viral DNAinto any target DNA. In addition, we assayed the peptide in theso-called disintegration reaction (24). This sequence-indepen-dent phosphoryl-transfer reaction (26) is a useful tool to measurethe catalytic activity of IN, since it has been shown that it can becarried out by the catalytic domain of IN alone (20, 21). Disin-tegration reaction mediated by both the IN full-length protein(INI 288) and the catalytic domain (IN50-237) were inhibited byHCKFWW, indicating that the peptide interacts with the catalyticdomain of IN. This finding was supported by the observation thatHIV-1 IN-inhibiting peptide also inhibits IN proteins from re-lated retroviruses like HIV-2, FIV, and MoMLV.

In contrast, other functionally related endonucleases werenot or, in the case of RNase H and MuA transposase, onlyslightly inhibited by the peptide, arguing that we discovered anIN-specific peptide. For example, in the phage Mu transposi-tion reaction, the IN-specific peptide inhibits MuA trans-posase, but not MuB, with an IC50 of -0.5 mM, whereas thenonspecific peptide mixture showed almost no effect. It islikely that the hexapeptide mainly inhibits the stable synapticcomplex assembly (M. Mizuuchi and K. Mizuuchi, personalcommunication).The hydrophobic features and also positive charges of

HCKFWW suggest that it may slip into the hydrophobic coreof IN, which contains the acidic catalytic triad D64, D116, andE152.IN is, a priori, a good target for antiviral strategies, because

there is no obvious (host) cellular counterpart. However, oneshould be aware of the toxicity of IN drugs since the host cellcontains presumably several proteins performing similar chemi-cal reactions (e.g., recombination and DNA repair enzymes).Some of these proteins contain similar structural motifs as hasbeen shown by Dyda et al. (28) for the three-dimensional struc-tures ofMuA transposase (46), RNase H (44), and RuvC (45) andthe catalytic domain of HIV-1 IN. Therefore, it will be importantto determine what effect an IN inhibitor in general would have onthese structurally related proteins.We clearly have identified an IN-specific inhibiting peptide,

but would this compound be a good drug? The hexapeptideHCKFWW consisting of L-amino acids would presumably notbe a suitable drug because natural peptides usually do not passmembranes well and they are rapidly degraded by proteases.However, whether this hexapeptide could be further modifiedin order to develop a potential drug lead remains to be seen.Possible amino acid positions for modifications were deter-mined in the alanine scan (Fig. 3B).

In addition, the peptide may be useful as a tool for theunraveling of the mechanism of action of IN. It could, forinstance, help in attempts to crystallize the entire IN protein.Our demonstration that an inhibitor of an HIV replicationprotein can be derived from a "first generation" combinatoriallibrary provides proof of concept for this approach as well asencouragement to extend these screens to other similar com-binatorial libraries to identify suitable drug lead compounds.

We thank Myung Soo Lee and Bob Craigie for the generous gift ofMoMLV preintegration complexes and Michiyo Mizuuchi and KiyochiMizuuchi for testing the peptides in the Mu transposition assays. We alsothank Fusinita van den Ent, Karin van der Linden, and Kees Vink for

providing IN50-237, FIV IN, and MoMLV IN proteins. We would like toacknowledge Fusinita van den Ent, Chris Vos, Karl H'ard, and Piet Borstfor critical comments on the manuscript. This work was supported by theNetherlands Organization for Scientific Research (Grant 900-502-140).

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33.34.35.36.

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Proc. Natl. Acad. Sci. USA 92 (1995)