Bioorganic & Medicinal Chemistry Letters 22 (2012) 3109–3114

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Discovery of small molecule HIV-1 integrase dimerization inhibitors

Cristina Tintori a,�, Jonas Demeulemeester b,�, Luigi Franchi a, Silvio Massa a, Zeger Debyser b,Frauke Christ b, Maurizio Botta a,c,⇑a Dipartimento Farmaco Chimico Tecnologico, University of Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italyb Molecular Medicine, Katholieke Universiteit Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgiumc Biotechnology, College of Science and Technology, Temple University, Biolife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, PA 19122, USA

a r t i c l e i n f o

Article history:Received 21 January 2012Revised 15 March 2012Accepted 15 March 2012Available online 21 March 2012

Keywords:HIV-1 integraseDimerizationAlphaScreenProtein–protein interactions

0960-894X/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.bmcl.2012.03.064

⇑ Corresponding author. Tel.: +39 0577 234306; faxE-mail address: botta.maurizio@gmail.com (M. Bo

� These authors contributed equally to this work. CTscreening and selection of the compounds. JD cloneexpression, purified the proteins and developed the Al

a b s t r a c t

Human immunodeficiency virus-1 integrase (HIV-1 IN) inserts the viral DNA into host cell chromatin in amultistep process. This enzyme exists in equilibrium between monomeric, dimeric, tetrameric and highorder oligomeric states. However, monomers of IN are not capable of supporting its catalytic functionsand the active form has been shown to be at least a dimer. As a consequence, the development of inhib-itors targeting IN dimerization constitutes a promising novel antiviral strategy. In this work, we success-fully combined different computational techniques in order to identify small molecule inhibitors of INdimerization. Additionally, a novel AlphaScreen�-based IN dimerization assay was used to evaluate theinhibitory activities of the selected compounds. To the best of our knowledge, this study representsthe first successful virtual screening and evaluation of small molecule HIV-1 IN dimerization inhibitors,which may serve as attractive hit compounds for the development of novel anti-HIV

� 2012 Elsevier Ltd. All rights reserved.

Replication of HIV-1, the causative agent of acquired immuno-deficiency syndrome (AIDS), depends on the insertion of a dsDNAcopy of the viral genome into the host genome. This step is cata-lyzed by the viral integrase (IN), making it an appealing targetfor antiretroviral therapy. The integration process, as catalyzedby IN, occurs in two spatially and temporally separated reactionsknown as 30-processing and strand transfer. First IN removes a30-terminal GT dinucleotide from the long terminal repeats (LTRs)of the viral dsDNA. This step is usually completed in the cytoplasm,shortly after reverse transcription. The formed pre-integrationcomplex is then transported into the nucleus,1 where the strandtransfer reaction takes place, covalently linking the 30ends of theviral DNA to the 50 ends of the host cell DNA.2 Catalytically activeHIV-1 IN exists in a dynamic equilibrium between monomeric,dimeric, tetrameric and higher order oligomeric states. Severalstudies have established that a dimer is sufficient for 30-processingwhereas a tetramer is required for strand transfer catalysis.3

Raltegravir was the first IN inhibitor to be approved by the FDA(US Food and Drug Administration) for use in treatment-experienced patients in 2007.4 In 2009, approval was also grantedfor its use in treatment-naïve patients.5 As an IN strand transferinhibitor (INSTI), raltegravir exerts its effect by binding to theactive site of the 30-processed DNA–protein complex, where it

ll rights reserved.

: +39 0577234333.tta).

did the modeling, the virtuald the constructs for proteinphaScreen� assays.

displaces the viral DNA 30adenosine.6 The drug displays an excel-lent safety profile and efficacy. However, raltegravir-resistantstrains have already emerged in the clinic, underscoring theimportance of developing next-generation IN inhibitors.7 Conse-quently, recent efforts in IN drug discovery are looking beyondthe active site, towards allosteric inhibitors.8

Figure 1. Binding site at the monomer–monomer interface. White spheres repre-sent the grid points identified by PocketPicker which are located between a1 anda5 helixes (dark blue).

Figure 2. IN dimerization AlphaScreen� assay dose–response curves for the INH5peptide and compounds 3, 4. Photon counts were normalized using dimethylsulfoxide and blank controls and plotted as normalized signal (%).

Table 1Structure and activity of compounds selected by the application of the first VS protocol

ID Structure Inhibition at 100 lM Quenching at 100 lM

1S

N

NH

O

O

No effect n.d.

2 O

O

NH

O NH

O

14% ± 2 n.d.

3

OH

NH

ON

HN

O

O

50% ± 0 None

4

OH

NH

ON

HN

O

O

O83% ± 2 None

5 O

NNN

NO

HH

HNo effect n.d.

6HN

OO N

O

NH

ONo effect n.d.

7 ON

NS

NN

O

H

H13% ± 1 n.d.

8

N

OO

H

H N

No effect n.d.

n.d., not determined.

3110 C. Tintori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3109–3114

Different strategies have been proposed.9 As HIV has a minimalgenome and therefore depends on cellular co-factors for replica-tion,10 one possibility is to disrupt the protein–protein interactionsbetween IN and these co-factors.11 This is exemplified by recentreports on the rational design of small molecule inhibitors of theinteraction between lens epithelium-derived growth factor(LEDGF/p75) and IN.12–14 Another approach is to disrupt the INoligomerization process. In this regard, a molecule of sucrose, orig-inating from the cryoprotectant, has been observed occupying anallosteric binding site at the IN catalytic core dimer interface.15

This symmetrical binding site is lined by residues Glu96, Lys103,Cys130, Trp131, Trp132, Gln168, Lys173, Thr174 and Met178 ofboth IN monomers. Interestingly, the same pocket was previouslydescribed by various research groups as the target for several smallmolecules.16–18 Remarkably, a second binding site at the dimer

C. Tintori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3109–3114 3111

interface was recently identified by fragment-based screening andcocrystallization.19 This novel fragment binding pocket is formedby residues Ala105, Gly106, Arg107, Trp108, Pro109, Val110,Lys111, Ala133, Gly134, Ile204, Thr206 and Ile208 from one mono-mer and Tyr83, Trp108, Asn184, Phe185, Lys186, Arg187, Ser195,Gly197, Glu198, Ile200 and Val201 from the other monomer. Asopposed to targeting pockets formed by the dimer interface, it isalso possible to disrupt the monomer–monomer interaction itselfto block IN oligomerization. This was proven to be a bona fidestrategy, as peptides derived from the dimer interface (calledINH1 and INH5) have been shown to inhibit through this mecha-nism of action.20,21 Recently, the reducing agent dithiothreitolwas also reported as a weak inhibitor of the interaction betweenIN monomers.22

In this work, pursuing our research in the field of HIV-1 IN,23 abinding pocket at the dimerization interface of the IN monomerwas pinpointed by combining molecular dynamics (MD) with aPocketPicker analysis. Next, we applied a structure-based virtualscreening (VS) approach to identify small molecules from theAsinex databases which potentially interfere with the IN subunitinteraction. As a result, eight molecules were initially selectedand tested in a novel in vitro IN dimerization assay. One of thesemolecules (4) inhibited the association of the two IN monomerswith an IC50 of 38.4 lM. Overall IN activity assays were performedand the mechanism of action was further elucidated. A secondstructure-based VS was then applied, taking into account thefavorable interactions established by compound 4 within the bind-ing site. Other hit molecules were identified from several commer-cial databases.

All catalytic activities of HIV-1 IN are strictly correlated withmultimeric forms (at least a dimer) of this retroviral enzyme.2

Accordingly, in this work we show that inhibition of IN catalyticactivity through disruption of the dimer interface represents anattractive strategy for the development of novel small moleculeinhibitors. Our goal was reached through the following steps: (i)identification of a hypothetical binding pocket at the monomer–monomer interface; (ii) application of a structure-based VS proto-col to the binding site previously identified, with the selection of afirst group of potential inhibitors; (iii) development and applica-tion of an AlphaScreen�-based IN dimerization assay and an over-all IN activity assay for biological evaluation of the compounds; (iv)application of a second VS, taking into account the results of thefirst one; (v) evaluation of the second series of compounds in thedimerization assay (for an overview, see Fig. 1S).

Binding site identification: A binding pocket was pinpointed atthe dimerization interface of the IN monomer by combining the re-sults derived from an MD simulation on the IN dimer and an explo-ration of receptor cavities by PocketPicker.24 After assembly of thecomplete IN dimer structure, an MD simulation was performed to

Figure 3. (a) Binding mode of compound 4. (b) Graphical representation of the pharmacacceptor (green) and two hydrophobic features (cyan). Excluded volumes are represente

optimize the system and to identify residues that have a dominantrole in IN dimer formation, the so-called hot spots (see Fig. 2S). TheAMBER 10 suite25 and the parm94 force field were used for MDsimulation while the MM-GBSA (molecular mechanics-generalizedBorn surface area)26 approach was chosen to discern the hot spots.Most of these hot spots lay close to one of the binding pocketsidentified by PocketPicker. We therefore selected this region as apossible binding site to design IN dimerization inhibitors. The sitewas located on the IN catalytic core domain between the a1 and a5helices (Fig. 1), comprising residues Tyr83, Glu85, Ala86, Tyr99,Phe100, Lys103, Trp108, Gln177 and Asn184. Interestingly, whilethis manuscript was in preparation a paper was published suggest-ing the same binding pocket for the design of ligands targeting thedimerization interface.27

First VS approach: A structure-based VS combining shape analy-sis with docking studies was applied to identify small moleculeswhich might interfere with the IN subunit interaction by bindingto the pocket previously identified. A receptor-based shape wasapplied as the first filter in DiscoveryStudio 2.528 to screen theAsinex Gold and Synergy Databases (> 260,000 commercially avail-able compounds). The number of rotatable bonds was limited to 10or less in a second filter to remove too flexible chemicals. Theresulting selection of 5683 compounds, was then subjected todocking studies, performed with the software GOLD 4.1.29 Dockingcalculations were carried out in two consecutive steps to optimizethe balance between the quality of docking and the time requiredfor calculations. In the first phase, compounds were analyzed usingthe Search Efficiency parameter set to 50% by using both Chem-Score and GoldScore as scoring functions. Compounds that showedboth ChemScore > 30 and GoldScore > 50 (about 300 compounds)were then analyzed in a second docking run, for which the SearchEfficiency parameter was set to 100%. The top scoring hits werevisually inspected and, among them, a small subset of eight puta-tive IN dimerization inhibitors was selected for further analysis.We excluded however molecules for which the binding modes ob-tained with ChemScore and GoldScore were in disagreement andthose for which many clusters of solutions were found.

Biological evaluation of the first series of compounds: The primaryassay used for evaluation of selected compounds was built uponthe well-established AlphaScreen� technology platform. Full de-tails on the development and characterization of this novel INdimerization assay are reported elsewhere.30 Briefly (see SI), gluta-thione S-transferase (GST)-tagged IN is mixed with His6-tagged IN.Incubation allows monomer exchange between any pre-existingdimers and establishment of a steady-state population containinghomo- (GST/GST and His6/His6) as well as heterodimers (GST/His6). When finally Ni2+-chelate acceptor and glutathione donorAlphaScreen� beads are added, the heterodimers will bring bothbeads together. These complexes will generate the AlphaScreen�

ophore hypothesis Hypo1, consisting of two hydrogen bond donors (magenta), oned as gray spheres.

Table 2Structure and activity of compounds selected in the second VS

ID Structure Inhibition at 100 lM Quenching at 100 lM ID Structure Inhibition at 100 lM Quenching at 100 lM

9OH

NH

ON

HN

OO

54% ± 3 None 18NH

N

OHN

ONH

ON No effect n.d.

10O

Cl

NClNH

OHO

49% ± 0 48% 19 NH

N HN

ONH

OOO No effect n.d.

11

NH

O

OHN

O

NH

O

NH

73% ± 0 None 20

OH

NH

O

N

N

No effect n.d.

12

NH

Cl

Cl

O

N NH

O HO

No effect n.d. 21

N+-O O

NH

NH

O HN

OO No effect n.d.

13 ClO N

NH

ONH

49% ± 2 None 22 ONH

O

NH

S

ClOH

23% ± 3 n.d.

14OH

NH

O

N

S

O

32% ± 2 35% 23

OH

NH

O HN

O OH

No effect n.d.

15 NH

O

O

O

NH NH

OO

O

58% ± 2 20% 24

HO

HN

O

N

OS

N30% ± 1 13%

3112C.Tintori

etal./Bioorg.M

ed.Chem.Lett.22

(2012)3109–

3114

16NH

OO

NHN H

OOO

50%

±2

6%25

OH

H N

ON H

OO

No

effe

ctn

.d.

17NH

O

H N

O

H N

OS

NN

oef

fect

n.d

.

C. Tintori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3109–3114 3113

signal upon irradiation (measured in photon counts per second).Any compound interfering with IN dimerization will change theextent of bead cross-linking and concomitantly the output signal.The degree of signal reduction (expressed as % inhibition) directlycorrelates with the inhibitory potency of the small molecule andallows assessing of the tested compounds. Results are reported inTable 1 (compounds 1–8).

Two candidates (compounds 3 and 4 in Table 1) showed signif-icant activity with percentages of inhibition at 100 lM of 50% and83%, respectively. In order to exclude false positive molecules caus-ing a decrease of signal purely due to interference with the Alpa-Screen� technology itself (e.g., absorption of excitation/emissionlight, singlet oxygen quenching, Ni2+ chelation), we also designeda GST-His6 counterscreen where both affinity tags were covalentlylinked to each other. Both molecules, 3 and 4, failed to decrease theAlphaScreen� signal in this counterscreen (Table 1), demonstratingtheir specific inhibition of IN dimerization.

Dose–response curves were determined in the dimerizationAlphaScreen�, resulting in IC50 values of 128 and 38.4 lM for com-pounds 3 and 4 ,respectively, (95% confidence intervals from 83.7to 197 lM and from 30.0 to 49.3 lM, see Fig. 2). In addition, as aproof of concept, we evaluated the effect of the literature-reportedINH5 peptide in the assay.20 This peptide, derived from the IN di-mer interface, was previously found to inhibit IN dimerizationand also behaves as such in our AlphaScreen�-based assay; dis-playing an IC50 of 4.5 lM (95% confidence interval from 3.4 to6.2 lM, Fig. 2).

The most potent hit in this first selection, compound 4, was fur-ther investigated in an overall IN catalytic activity ELISA (combined30processing and strand transfer activity) that was slightly modi-fied from the standard protocol in literature, namely in the orderof addition. The inhibitor was incubated together with IN beforeaddition of the oligodeoxynucleotide substrate (see SI). In thismodified ELISA assay compound 4 inhibited the overall catalyticreaction of IN with an IC50 of 79 lM.

Second VS approach and evaluation: Analysis of the complex be-tween the active inhibitor 4 and IN was performed in order to gaininsight into the specific chemical features that determine its activ-ity. The predicted binding mode of compound 4 is shown in Figure3a. In detail, compound 4 engages in significantly stabilizinghydrogen bond contacts between its hydroxyl group and the sidechain of Gln177 as well as with the backbone NH group of Ala86.Further hydrogen bonding is observed between the NH of thehydrazone moiety and Glu85 and between the carbonyl oxygenof the amide group and Lys103. Additionally, hydrophobic interac-tions between the naphthyl portion of the ligand and the sidechains of Phe185, Tyr83 and Val180 were found as well as positiveVan der Waals contacts involving Tyr99, Ile89 and the 3,4-dime-thoxyphenyl-ethyl group.

Starting from this protein–ligand complex, the software Ligand-Scout31 was used to generate a pharmacophore model, referred toas Hypo1 (Fig. 3b). This model consists of two hydrogen bond do-nors, one hydrogen bond acceptor, two hydrophobic features andeight excluded volumes. Hypo1 was imported into DiscoveryStudio2.5 and used to screen several commercial databases (Asinex Plat-inum & Emerald, Chembridge, InterBioScreen and Specs, totaling±2million commercially available compounds). Molecules satisfy-ing all features of Hypo1 with a FastFit value >1 (±1500 com-pounds) were submitted to docking experiments using aprocedure identical to the one described above, leading to theselection and analysis of a second set of 17 compounds (Table 2).

Using a minimal cut off of 30% inhibition at 100 lM, the dimer-ization assay identified 7 new hits from this second set (com-pounds 9–11, 13–16 in Table 2). After testing in the GST-His6

counterscreen, 5 showed to be clean hits (% inhibition � % quench-ing > 30%). Interestingly, next to several analog of compound 4 (9,

Figure 4. Superposition of compounds 4 (gray) and 11 (blue) within the bindingsite of interest.

3114 C. Tintori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3109–3114

13, 24), a number of new scaffolds were picked up (11, 15, 16, 22).The most promising one, compound 11, showed a percentage ofinhibition of 73% at 100 lM.

In conclusion, we have successfully combined several computa-tional methodologies and screened large chemical databases in sil-ico, selecting non-peptidic compounds with a potential to inhibitIN dimerization. Eight molecules were selected and evaluated ina newly developed IN dimerization assay based on the Alpha-Screen� technology. 30 Two of the compounds showed significantactivity in this in vitro assay and survived a subsequent counter-screen for nonspecific inhibitors. The most potent compound (4)also inhibited IN catalytic activity as measured in an ELISA-basedassay. Figure 4 shows compounds 4 and 11 superposed withinthe IN binding pocket. It is noteworthy that the two hit com-pounds, despite their different chemical structure, share the samepattern of interactions with the protein.

Knowledge obtained from modeling the complex between thiscompound and IN was used to feed a second round of virtualscreening, selection and evaluation. This again led to the discoveryof several novel promising scaffolds. Further studies on these newIN inhibitor scaffolds are currently underway and will be reportedin due course. We believe that the hits disclosed in this work rep-resent the first small molecule IN inhibitors operating through thismechanism of action and may ultimately be optimized as next-generation integrase inhibitors to overcome resistance to tradi-tional INSTIs

Acknowledgments

This work was supported by the European Union collaborativeprojects ‘THINC’ (Grant No. HEALTH-2007-2.3.2-1) and ‘CHAARM’(Grant No. HEALTH-F3-2009-242135) as well as by the Italian Min-istero dell0Istruzione, dell0Università e della Ricerca, Prin 2008 re-search project (Grant No. 2008CE75SA_004). F.C. is a postdoctoralfellow of the Industrial Research fund (IOF) and J.D. is a doctoralfellow for the Flemish Fund for Scientific Research (FWO). Wethank Nam Joo Van der Veken and Martine Michiels for excellenttechnical assistance.

Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.064.

References and notes

1. Christ, F.; Thys, W.; De Rijck, J.; Gijsbers, R.; Albanese, A.; Arosio, D.; Emiliani,S.; Rain, J. C.; Benarous, R.; Cereseto, A.; Debyser, Z. Curr. Biol. 2008, 18, 1192.

2. Delelis, O.; Carayon, K.; Saïb, A.; Deprez, E.; Mouscadet, J. F. Retrovirology 2008,5, 114.

3. Guiot, E.; Carayon, K.; Delelis, O.; Simon, F.; Tauc, P.; Zubin, E.; Gottikh, M.;Mouscadet, J.-F.; Brochon, J.-C.; Deprez, E. J. Biol. Chem. 2006, 281, 22707.

4. Burger, D. M. Expert Opin. Drug Metab. Toxicol. 2010, 9, 1151.5. Boyd, S. D. Am. J. Health Syst. Pharm. 2011, 68, 991.6. Hare, S.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. Nature 2010, 464,

232.7. Mouscadet, J. F.; Delelis, O.; Marcelin, A.-G.; Tchertanov, L. Drug Resist. Updat.

2010, 13, 139.8. Al-Mawsawi, L. Q.; Neamati, N. ChemMedChem 2011, 6, 228.9. Voet, A.; Maeyer, M. D.; Debyser, Z.; Christ, F. Future Med. Chem. 2009, 1, 1259.

10. Van Maele, B.; Busschots, K.; Vandekerckhove, L.; Christ, F.; Debyser, Z. TrendsBiochem. Sci. 2006, 31, 98.

11. Busschots, K.; De Rijck, J.; Christ, F.; Debyser, Z. Mol. Biosyst. 2009, 5, 21.12. Christ, F.; Voet, A.; Marchand, A.; Nicolet, S.; Desimmie, B. A.; Marchand, D.;

Bardiot, D.; Van der Veken, N. J.; Van Remoortel, B.; Strelkov, S. V.; De Maeyer,M.; Chaltin, P.; Debyser, Z. Nat. Chem. Biol. 2010, 6, 442.

13. De Luca, L.; Ferro, S.; Morreale, F.; Chimirri, A. Mini-Rev. Med. Chem. 2011, 11,714.

14. Fenwick, C.; Bethell, R.; Cordingley, M. 51st Interscience Conference onAntimicrobial Agents and Chemotherapy (ICAAC 2011): Chicago 2011.

15. Wielens, J.; Headey, S. J.; Jeevarajah, D.; Rhodes, D. I.; Deadman, J.; Chalmers, D.K.; Scanlon, M. J.; Parker, M. W. FEBS Lett. 2010, 584, 1455.

16. Du, L.; Zhao, Y.; Yang, L.; Zheng, Y.; Tang, Y.; Shen, X.; Jiang, H. Acta Pharmacol.Sin. 2008, 29, 1261.

17. Shkriabai, N.; Patil, S. S.; Hess, S.; Buduhas, S.; Craigie, R.; Burke, T. R., Jr.; LeGrice, S. F. J.; Kvaratskhelia, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6894.

18. Kessl, J. J.; Eidahl, J. O.; Shkriabai, N.; Zhao, Z.; McKee, C. J.; Hess, S.; Burke, T. R.;Kvaratskhelia, M. Mol. Pharmacol. 2009, 76, 824.

19. Wielens, J.; Headey, S. J.; Deadman, J. J.; Rhodes, D. I.; Parker, M. W.; Chalmers,D. K.; Scanlon, M. J. ChemMedChem 2011, 6, 258.

20. Maroun, R. G.; Gayet, S.; Benleulmi, M. S.; Porumb, H.; Zargarian, L.; Merad, H.;Leh, H.; Mouscadet, J.-F.; Troalen, F.; Fermandjian, S. Biochemistry 2001, 40,13840.

21. Zhao, L.; O’ Reilly, M. K.; Shultz, M. D.; Chmielewski, J. Bioorg. Med. Chem. Lett.2003, 13, 1175.

22. Tsiang, M.; Jones, G. S.; Hung, M.; Samuel, D.; Novikov, N.; Mukund, S.;Brendza, K. M.; Niedziela-Majka, A.; Jin, D.; Liu, X.; Mitchell, M.; Sakowicz, R.;Geleziunas, R. Biochemistry 2011, 50, 1567.

23. (a) Rinaldi, M.; Tintori, C.; Franchi, L.; Vignaroli, G.; Innitzer, A.; Massa, S.; Esté,J. A.; Gonzalo, E.; Christ, F.; Debyser, Z.; Botta, M. ChemMedChem 2011, 2, 343;(b) Rajamaki, S.; Innitzer, A.; Falciani, C.; Tintori, C.; Christ, F.; Witvrouw, M.;Debyser, Z.; Massa, S.; Botta, M. Bioorg. Med. Chem. Lett. 2009, 19, 3615.

24. Weisel, M.; Proschak, E.; Schneider, G. Chem. Cent. J. 2007, 1, 7.25. Case, D. A.; Cheatham, T.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev,

A.; Simmerling, C.; Wang, B.; Woods, R. J. Comput. Chem. 2005, 26, 1668.26. Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.; Chong, L.; Lee, M.; Lee,

T.; Duan, Y.; Wang, W.; Donini, O.; Cieplak, P.; Srinivasan, J.; Case, D. A.;Cheatham, T. Acc. Chem. Res. 2000, 33, 889.

27. Sippel, M.; Sotriffer, C. A. J. Chem. Inf. Model. 2010, 50, 604.28. Discovery Studio, version 2.5; Accelrys Inc.: San Diego, CA.29. (a) Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43; (b) Jones, G.;

Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267, 727; (c)Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins2003, 52, 609.

30. Demeulemeester, J.; Tintori, C.; Botta, M.; Debyser, Z.; Christ, F. J. Biomol. Screen.2012. http://dx.doi.org/10.1177/1087057111436343.

31. Wolber, G.; Langer, T. J. Chem. Inf. Model. 2005, 45, 160.