BiochemJ 2010 527 Broad HIV Inhibitors

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Biochem. J. (2010) 429, 527532 (Printed in Great Britain) doi:10.1042/BJ20091645 527

Identification of broad-based HIV-1 protease inhibitors from combinatoriallibraries

Max W. CHANG*1, Michael J. GIFFIN*1, Rolf MULLER, Jeremiah SAVAGE*, Ying C. LIN, Sukwon HONG, Wei JIN,Landon R. WHITBY, John H. ELDER, Dale L. BOGER and Bruce E. TORBETT*2

*Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines, La Jolla, CA 92037, U.S.A., Department of Immunology and Microbial

Science, The Scripps Research Institute, 10550 N. Torrey Pines, La Jolla, CA 92037, U.S.A., and Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines,

La Jolla, CA 92037, U.S.A.

Clinically approved inhibitors of the HIV-1 protease functionvia a competitive mechanism. A particular vulnerability ofcompetitive inhibitors is their sensitivity to increases in substrateconcentration, as may occur during virion assembly, budding andprocessing into a mature infectious viral particle. Advances inchemical synthesis have led to the development of new high-diversity chemical libraries using rapid in-solution syntheses.These libraries have been shown previously to be effective at

disrupting proteinprotein and proteinnucleic acid interfaces.We have screened 44000 compounds from such a library to

identify inhibitors of the HIV-1 protease. One compound wasidentified that inhibits wild-type protease, as well as a drug-resistant protease with six mutations. Moreover, analysis of thiscompound suggests an allosteric non-competitive mechanism ofinhibition and may represent a starting point for an additionalstrategy for anti-retroviral therapy.

Key words: anti-retroviral therapy, high-diversity chemical lib-

rary, high-throughput screening,HIV-1, kinetics, non-competitiveinhibitor.

INTRODUCTION

HIV infection continues to be a worldwide health crisis, with over33 million infected people worldwide [1]. Despite improvementsin anti-retroviral therapeutic development, drug resistanceremains a major obstacle to the effective control of infectionin HIV-infected patients. Numerous advances and improvementshave been made in drugs targeting the viral protease, which isrequired for maturation of virions into infectious particles [2].

However, common mutations associated with proteaseinhibitordrug resistance appear in drug-experienced patients and, withcertain subtypes of the virus, in drug-nave patients as well[3], often leading to virologic failure and onset of diseaseprogression. Drug resistance complicates the use of therapeuticsin the treatment of HIV infection, necessitating an ongoing searchfor novel therapeutics targeting the viral protease.

The HIV-1 protease is a 22-kDa homodimeric aspartic proteaseconsistingoftwo99-residuepolypeptidechainsthatself-assembleto form the enzymatically active dimer. Currently, all U.S.A.FDA (Food and Drug Administration)-approved PIs (proteaseinhibitors) are in the same mechanistic class, i.e. they arecompetitive inhibitors that bind to the active site of the protease,preventing the association of the protease with substrates and

resulting in disruption of virion maturation [2]. One drawback ofcompetitive inhibitors is that similar active-site mutations candeleteriously affect small molecule binding in the active site,leading to an increased risk of cross-resistance to other com-petitive inhibitors. An additional potential pitfall is thatcompetitive inhibitorsare sensitive to substrate concentrations[4].An alternative to competitive inhibitors has been the identificationof inhibitors that target non-active-site regions of the protease,such as the dimer interface [5,6], flaps [7,8] or other non-substrate active-site regions [9]. Moreover, inhibitors that utilize

non-competitive,uncompetitiveormixed-modemechanismshavealso been identified [58,10,11]. A potential advantage of anon-competitive mechanism will be the insensitivity to substrateconcentrations, whichmay bettermaintain a therapeutic thresholdin the substrate-rich virion. The improved therapeutic thresholdand potential insensitivity to current resistance mutations maymean such inhibitors are more efficacious at inhibiting viralreplication and the emergence of drug resistance.

To identify inhibitors that may target other features ofthe protease structure, to inhibit its function, we screened alibrary of compounds shown previously to include inhibitorsof proteinprotein and proteinnucleic acid interactions [12,13].One compound, compound (1), was found to inhibit wild-type protease from the NL4-3 strain of HIV-1 in the lowmicromolar range.Moreover, compound (1)also inhibiteda MDR(multidrug resistant) protease containing six mutations associatedwith PI resistance [14]. The kinetics of the wild-type proteasedemonstrated a mechanism of inhibition consistent with non-competitive inhibition and cross-competitive inhibition studies,with compound (1) and pepstatin A, a competitive inhibitor,implicated a non-active-site binding effect. Taken together, thesefindings suggest thatcompound (1) functionsas a non-competitiveallosteric PI.

MATERIALS AND METHODS

Enzyme activity assays

HIV-1 protease enzymatic activity was assayed as describedpreviously [15], using the fluorescently labelled anthranilylprotease substrate Abz (aminobenzoyl)-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (H-2992; Bachem) [16]. In brief, bacteriallypurified HIV-1 protease was mixed with inhibitor compounds

Abbreviations used: Boc, t-butoxycarbonyl; ESI, electrospray ionization; MDR, multidrug resistant; PI, protease inhibitor.1 These authors contributed equally to this work.2

To whom correspondence should be addressed (email [email protected]).

c The Authors Journal compilation c 2010 Biochemical Society

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528 M. W. Chang and others

in a reaction buffer containing 25 mM Mes, pH 5.6, 200 mMNaCl, 5% (v/v) DMSO, 5% (v/v) glycerol, 0.0002% TritonX-100 and 1 mM dithiothreitol in a pre-warmed 96-well plate.All clones used for protease bacterial expression were generatedfrom the NL4-3 wild-type or a MDR protease containingsix mutations (L24I/M46I/F53L/L63P/V77I/V82A), termed 6X,associated with resistance to saquinavir, nelfinavir, ritonavir and

TL3 [14]. The enzymesubstrate reaction was started by theaddition of fluorescently labelled substrate and the reactionprogress was measured by fluorescence intensity using an FLx-800 fluorescence plate reader (BioTek). For IC50 determinations,the final reaction concentrations were 25 nM protease, 30 Msubstrate (the approx. Km) and 0.001600 M inhibitor.

Chemical library

Boger et al. [17] have established and reported previouslyon a collection of chemical libraries consisting of approx.66000 compounds, which was prepared by using solution-phasetechnology with liquidliquid acidbase extraction purification,evaluated for composition and purity, and stored for further

assessment [18,19]. Figure 1 shows a representative diagram ofchemical scaffolds and substitutions used to generate the library.From the original library, 44000 compounds were evaluated inthe present study. The lead compounds (1) and (2) (Figure 2)identified from the screening were synthesized, then evaluatedfor composition and purity (below) before use [18,19].

Compound (1): 1H-NMR (400 MHz, DMSO-d6, 25C) d 11.84(s, 1H), 10.74 (s, 1H), 9.86 (s, 1H), 8.39 (d, J= 1.9, 1H), 8.14(dd, J= 8.3, 1.6 Hz, 2H), 8.08 (s, 1H), 7.92 (d, J= 8.8, 1H),7.83 (dd, J = 8.9, 1.9, 1H), 7.39 (s, 1H), 3.84 (s, 3H), 1.49(s, 9H); MS-ESI (electrospray ionization) (m/z) calculated for[C24H22N4O7S2+Cl] 577.1; found: 577.1.

Compound (2): 1H-NMR (400 MHz, DMSO-d6, 25C) d 11.81(s, 1H), 10.85 (s, 1H), 8.41 (d, J= 1.8 Hz, 1H), 8.08 (dd, J=

15.1, 1.4 Hz, 2H), 8.03 (s, 1H), 7.94 (d, J= 8.6 Hz, 1H), 7.84(dd, J= 8.7, 1.6 Hz, 1H), 7.69 (m, 2H), 7.55 (m, 1H); MS-ESI(m/z) calculated for [C18H12N4O5S2+H]+ 429.0; found: 429.0.

MichaelisMenten kinetic measurements

For MichaelisMenten kinetic measurements, the proteasesubstratewas titrated from 1 to 200M.Toassayforpromiscuousinhibition, 0.0010.01% Triton X-100 was added to the reaction.To assess inhibitor specificity horseradish peroxidase (SigmaAldrich) activity was assayed in 14 mM potassium phosphate,pH 6.0, and 0.5% (v/v) hydrogen peroxide with an enzymeconcentration of5 nM.The reactionwasinitiated with theadditionof the substrate O-phenylenediamine at concentrations from

20 M to 100 M in 100 mM sodium phosphate and 50 mMsodium citrate, pH 5.0. Kinetic constants were determined bynon-linear regression of initial reaction velocities as a function ofinhibitor concentration using Prism 5.0c (GraphPad Software).

IC50 values were fitted with the following equation:

Y =100

1+ 10[(log IC50+x)HillSlope]

MichaelisMenten kinetic constants were fitted to the followingequation:

Y =Vmax X

Km + X

Ki constantsforcompounds(1)and(2)werefittedtothefollowingequation:

Vmaxi =Vmax

1+I

Ki

Cross-competitive inhibitor measurements

A variation of Yonetani and Theorell [20] analysis was used toevaluate the binding mode of compound (1) [20,21]. The useof the variation of Yonetani and Theorell analysis, as discussedby Martinez-Irujo et al. [21], takes into account the bindinginteractions, on an enzyme, of competitive and non-competitiveinhibitors. The cross-competitive inhibitor assessment wasaccomplished by using various concentrations of Pepstatin A(Roche), a competitive inhibitor, with a fixed concentration ofcompound (1), a non-competitive inhibitor, while keeping thesubstrate and protease concentrations constant. The experimentalconditions for assessing protease function were identical to thoseused to determine IC50. Pepstatin A was used at concentrations

from 0.6 to 3.0 M, whereas compound (1) was held constantat 45, 30, 20 or 0 M. The determination of the interaction term, which defines the degree to which binding of one inhibitorinfluences the binding of the second inhibitor, was determinedutilizing Prism 5.0c using the following equation [21]:

v0

v1,2= 1+

[I1](IC50)1

+[I2]

(IC50)2+

[I1][I2](IC50)1(IC50)2

Docking studies of compound (1)

The docked conformation of compound (1) with the HIVprotease was generated using AutoDock Vina 1.02 [22]. Ahigh-resolution HIV-1 protease structure (PDB code 2HS1) waschosen as the receptor. Two overlapping search spaces were used,each measuring 25 32 40 (1 = 0.1 nm), which togetherspanned chain A of the structure. The darunavir molecule boundin the active site was preserved. In each docking run, nineconformations were reported and only the most favourable isdetailed below. Three-dimensional co-ordinates for the ligandwere determined using Corina [23]. Other docking parameterswere kept to their default values.

RESULTS AND DISCUSSION

We have screened a library of compounds shown previouslyto inhibit proteinprotein and proteinnucleic acid interactions[12,13,17,24]. A chemically diverse library of 44000 compounds

was synthesized using a solution-phase combinatorial synthesisas described previously [13,18,19,25]. To facilitate synthesisand screening, some compounds were synthesized as part of ascreenedmixture,withsomemixturescontaininguptotenrelated,but distinct, compounds. A representative group of compoundsfrom which the lead compounds emerged is shown in Figure 1.

Compounds were screened initially for the ability to inhibitthe wild-type HIV-1 protease, obtained from the NL4-3 virus,in a real-time kinetics assay using a fluorigenic substrate at aconcentration equal to the Km. Compounds that had significantaffects on the baseline fluorescent signal, which was designatedas 10% above baseline independently of the substrate peptide,were excluded from further screening. Assay conditions werechosen to reduce the possibility of false positives resulting

from promiscuous inhibitors, including minimizing compoundc The Authors Journal compilation c 2010 Biochemical Society


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Non-competitive HIV-1 protease inhibitors 529

Figure 1 Representative group of chemical substituents that are used in the reaction in the context of the compound scaffold

Each substituent is found at the site labelled A, in this case generating ten different compounds. For additional information see the Materials and methods section, and [18,19].

Figure 2 Compounds used in the present study

(A) Compound (1) and (B) compound (2). Compound (1) was identified through proteasesubstrate screens of the original chemical library (see the text for more details), whereas compound (2) isa derivative of compound (1) with the Boc and methyl groups removed from the ends of the compound.

aggregate formation by the inclusion of detergent and reducingcompound incubation time [26,27]. Compound groups thatshowed greater than 50% inhibition of the wild-type proteaseat a compound concentration of 20 M were then tested againstthe 6X protease, a MDR protease containing six mutations(L24I/M46I/F53L/L63P/V77I/V82A) associated with resistanceto saquinavir, nelfinavir, ritonavir and TL3 [14].

Compound families showing greater than 50% inhibitionagainst both wild-type and 6X proteases were then deconvolutedand synthesized as individual compounds. These compounds

were then tested individually against the wild-type and MDR6X proteases. Individual compounds again showing greater than50% inhibition at a concentration of 20 M were selected formore detailed kinetics analyses. One compound, compound (1)(Figure 2), was found to inhibit the wild-type protease in thelow micromolar range (Figure 3). Furthermore, compound (1)also showed low micromolar inhibition of the 6X protease. Thehalf maximal inhibitory concentrations (IC50 values) were deter-mined against the wild-type and 6X proteases and found to be17 M against the wild-type protease and 11 M against the 6Xprotease (Figure 3). Thus compound (1) is effective in inhibitingboth wild-type and an MDR 6X protease at a similar IC50.

To address whether compound (1) was a general enzymaticinhibitor we evaluated whether the compound altered horseradish

peroxidase function at various concentrations. No measurable

Figure 3 IC50 titration of compound (1) against wild-type and the 6X MDRproteases

Evaluation of compound (1) (log [I]) against wild-type () and 6X MDR () proteases. Inset:for comparison, titration of TL-3 (log[TL-3]), a protease inhibitor which is effective against thewild-type () protease, but not the MDR 6X protease mutant [14] (), is shown. Results fromnon-linear regressionindicate thatthe IC50s arewithina factorof2 ofeachother forthewild-typeand MDR 6X protease mutant. IC50 curve fitting was performed as described in the Materials

and methods section. Results are given as means +S.E.M. for four experiments.



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effect on the MichaelisMenten kinetics of the reactionwas observed at any of the compound concentrations evaluated,suggesting that compound (1) is not a general enzymatic inhibitor(results not shown). Furthermore, the inhibitory activity ofcompound (1) on HIV-1 protease was not abrogated by theaddition of non-ionic detergents, strengthening further the casethat compound (1) is not a promiscuous inhibitor (results not

shown) [26,27].InordertoidentifytheminimalchemicalmoietiesnecessaryforPI activity, we synthesized a derivative library based on compound(1) and screened each fragment against the wild-type proteaseindependently. Whereas the majority of the derivatives showedsignificantly reduced inhibition of protease activity, with IC50values ranging from 20 to greater than 1000 M, one derivative,compound (2) (Figure 2), showed more potent inhibitory activity.Compound (2) is similar to compound (1), but with the Boc(t-butoxycarbonyl) and methyl groups removed. When theinhibitory activityof compound (2)was compared with compound(1), it showed a slight decrease in both the IC 50 and Ki valuesas determined with the fluorigenic protease substrate assay(Figures 3 and 4). Therefore both compounds were active againstthe wild-type protease and compound (1) demonstrated activityagainst the MDR 6X protease mutant.

We next determined the effect of compounds (1) and (2)on the Km and Vmax of the wild-type protease with reactionsperformed within a range of substrate concentrations from 1 to200 M, centred around the Km of 30 M, at several inhibitorconcentrations, from 0 to 30 M. The values for the initialvelocities were then fitted to a MichaelisMenten model usingnon-linear regression to determine the dose-dependent effects ofthe compounds on the Km and Vmax for the HIV protease, asshown in Figures 4(A) and 4(B). When we measured proteaseactivity as a function of both substrate concentration and inhibitorconcentration, and used non-linear regression to fit the resultinginitial velocities to a MichaelisMenten model, we observeda curvi-linear response in Vmax as a function of increasing

concentration of both compounds (Figure 4C). The results areconsistent with a non-competitive mechanism of inhibition.

To glean further insights into the underlying molecular processof protease inhibition by compound (1), we utilized a variationof Yonetani and Theorell analysis [20,21] to evaluate the bindingmode. As the inhibition by compound (1) is consistent with a non-competitive mechanism, which might predict that the substratemay still bind to the protease active site when compound (1) isbound, pepstatin A was used as a cross-competitive inhibitor forthe analysis. This method of inhibitor cross-competitive analysisallows determination of the degree to which the binding ofcompound (1) to the protease influences the binding of thesecond inhibitor to the active site [4,20,28]. The choice ofpepstatin A was based on the ability to inhibit the protease

[29], the well-established biochemical and structural reports ofits binding location in the active site [30], that it has a competitiveinhibition mechanism [31,32] and the reported use of acetyl-pepstatin A for inhibitor cross-competitive studies for non-active-site inhibitors [5]. The graphical findings from a representativecross-competitive study utilizing compound (1) and pepstatin A isshown in Figure 5. In each case, non-parallel lines were obtained,which converged at the x-axis, consistent with the interpretationthat compound (1) and pepstatin A may bind independent sites[4,20,21]. The interaction term , which defines the degree towhich the binding of one inhibitor to the enzyme influencesthe binding of the second inhibitor, can be determined throughinterpolation of the x-intercept or can be calculated [4,20,21]. Asmall value (1) indicates mutual

Figure 4 MichaelisMenten kinetics of compounds (1) and (2) againstwild-type protease

(A) Compound (1) was used at 0 (), 3 (), 10 () and 30 () M and (B) compound(2) was used at 0 (), 10 (), 15 () and 20 () M over a range of M substrateconcentrations ([S]) for determination of the MichaelisMenten kinetics. A representative resultfrom three independent experiments is shown and the Ki values are given as means+ S.E.M.(C) Non-linear regression of Vmax as a function of compound (1) () or (2) () concentration.A representative experiment is shown (the S.E.M. for individual points varied by less than 10%of the mean; curve fitting is described in the Materials and methods section).

antagonism and in the case that =1 the inhibitors bind tothe enzyme in an independent manner. Calculation of yieldedapproximately 1, consistent with compound (1) binding to aprotease site independent of that of pepstatin A, binding in the

active site. These findings are consistent with compound (1)c The Authors Journal compilation c 2010 Biochemical Society


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Non-competitive HIV-1 protease inhibitors 531

Figure 5 Yonetani and Theorell plot of v/vi against concentration ofpepstatin A and compound (1)

Compound (1) was used at 0 (), 20 (), 30 () and 45 () M with various concentrationsof pepstatin A. Results are means+ S.D. from an experiment performed in triplicate. Assayconditions and curve fitting are described in the Materials and methods section.

Figure 6 IC50 titrations of compound (1) against wild-type and tethereddimer proteases

Compound (1) (log[I]) demonstrates similar inhibitory efficacy against wild-type () and thetethered protease dimer (). A representative experiment is shown (the S.E.M. for individual

points varied by less than 10% of the mean; assay conditions are described in the Materialsand methods section).

binding and providing inhibition through a site independent ofthe active site.

Given our findings from the inhibitor cross-competitive study,indicating that compound (1) was not binding in the active site,we investigatedwhether compound (1) functions as a dimerizationinhibitor. A number of compounds have been reported to promoteinhibition through disruption of protease dimerization [5,6]. Toaddress whether compound (1) disrupts dimerization we utilizeda tethered homodimeric protease, formed by a direct repeat ofprotease monomers linked by a five-residue amino acid sequence[33].TheIC50 ofcompound(1) was found tobe similar for boththe

non-covalent wild-type protease dimerand the covalently tethereddimer protease, as shown in Figure 6. As compound (1) was activeagainst the protease-tethered dimer, this implies that dimerizationdisruption is not required for inhibitory activity.

As compound (1) was shown to have a distinct binding locationcompared with pepstatin A and not to promote dimer interfacedisruption,possiblebindingmodeswereexploredusingmoleculardocking. The search was focussed on the outside surface of theprotein and a low-energy conformationwas discoveredthat placedcompound (1) in a long solvent-exposed cleft, termed the exo site[34], as shown in Figure 7. The exo site is composed of distinctregions that include the elbow, cantilever and fulcrum componentsof the protease. Molecular dynamic simulations of protease flapmovement relative to the exo site has indicated that the exo site

is compressed when the flaps are open and is extended when the

Figure 7 Docked conformation showing compound (1) bound outside of theHIV-1 protease (2HS1) active site

A space-filling rendering of the exo site showing the location of the solvent-exposed cleft andbinding of compound (1). The exo site is a feature of the protease altered by movement of theflaps. Insert: the area of the protease that is magnified in the Figure. The predicted bindingenergy of this conformation was 7.2 kcal/mol, equivalent to a Ki of 5.2 M.

flaps are closed [34]. Moreover, the exo site has been shown, viaa fragment-based screen, to accommodate small molecules [35].The predicted binding energy from the compound (1) dockingsimulation [7.2 kcal/mol (1 kcal 4.184 kJ)] corresponds to aKi of 5.2 M, very close to the experimentally observed Ki of6.1 M. Together with the biochemical findings from the presentstudy,thedockedcompound(1)conformationsupportsaplausibleallosteric binding mechanism, which is consistent with structuraldata [35]. It is tempting to speculate that binding of compound (1)

to the exo site influences flap dynamics, perhaps by locking theflaps closed and rendering the protease unable to bind substrate.A number of recent reports have implicated novel compoundsthat disrupt flap movement, thereby altering enzymatic function[36,37].

Currently, all approved PIs are competitive inhibitors, whichtarget the active site. Given the rise in PI-resistant HIVs, newinhibitors with novel inhibitory mechanisms are needed. Non-active-site allosteric inhibitors may avoid the selective pressureassociated with active-site inhibitors, which results in drugresistance mutations. The identification of compound (1) froma novel library of diverse compounds was found to inhibitboth wild-type and a MDR protease through a non-competitiveallostericmechanism.Thiscompoundprovidesarationalestartingpoint from which to chemically investigate novel inhibitorymechanismsthatmay provide another avenueof viralsuppression.

AUTHOR CONTRIBUTION

Rolf Muller, Jeremiah Savage and Ying Lin screenedthe combinatorial chemical libraryforHIV-1proteaseinhibitoryactivity. YingLin and Jeremiah Savage produced the protease forthebiochemical assays andproteasescreening. Sukwon Hong,Wei Jinand Landon Whitbysynthesized, determined the composition and purity of the library, and deconvoluted thelibrary. Michael Giffin and Max Chang designed and performed all biochemical analyseson the selected PIs. Max Chang performed all the docking studies. John Elder, Dale Bogerand Bruce Torbett were involved in the design and interpretation of the results. MichaelGiffin, Max Chang and Bruce Torbett were primarily involved in writing the manuscript.

Bruce Torbett and Max Chang edited the manuscript.



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FUNDING

ThisworkwassupportedbytheNationalInstitutesofHealth[grantnumbers5T32AI007354(to M.J.G.), 5T32NSO412119 (to M.W.C.), GM083658, GM48870 and AI40882 (to B.E.T.and J.H.E.) CA78045 (to D.L.B.)]; and by the Center for AIDS Research [grant number3 P30 AI036214-13S1]. This is publication MEM 20132 from The Scripps ResearchInstitute.

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Received 21 October 2009/8 May 2010; accepted 27 May 2010Published as BJ Immediate Publication 27 May 2010, doi:10.1042/BJ20091645


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BiochemJ 2010 527 Broad HIV Inhibitors