DOI: 10.1002/cmdc.201300204

Identification of Hck Inhibitors As Hits for theDevelopment of Antileukemia and Anti-HIV AgentsCristina Tintori,[a] Ilaria Laurenzana,[b] Francesco La Rocca,[b] Federico Falchi,[a, c]

Fabio Carraro,[d] Alba Ruiz,[e] Jos� A. Est�,[e] Miroslava Kissova,[f] Emmanuele Crespan,[f]

Giovanni Maga,[f] Mariangela Biava,[g] Chiara Brullo,[h] Silvia Schenone,[h] andMaurizio Botta*[a, i]

Introduction

Hematopoietic cell kinase (Hck) is a member of the Src familyof non-receptor protein tyrosine kinases (SFKs), the function ofwhich comprises various signaling pathways involved in the

regulation of several processes. The Src family comprises ninemembers (Src, Lck, Hck, Fyn, Fgr, Yes, Blk, Lyn, and Yrk) whichshare a common regulatory mechanism, but differ in cellularexpression and localization.[1, 2] Src, Fyn, and Yes are ubiquitous-ly expressed, whereas the others have tissue-specific expres-sion. Hck is expressed in cells of hematopoietic origin—specifi-cally myelomonocytic cells and B lymphocytes—and partici-pates in phagocytosis, adhesion, migration, and regulation ofprotrusion formation on the cell membrane, lysosome exocyto-sis, podosome formation, and actin polymerization. SFKs arecharacterized by a common domain structure (Figure 1) thatincludes five distinct regions: a unique N-terminal region withsequences for lipid attachment, the regulatory SH3 and SH2domains and a kinase domain, followed by a negative regula-tory C-terminal tail.[3, 4] In detail, from the N terminus, each pro-tein consists of a small region known as the SH4 region (9–12residues) which is required for membrane attachment.[5] This isfollowed by a unique region of 40–70 residues, which mayimpart distinct localization properties to individual familymembers. Subsequent to the unique region are the highly con-served Src homology domains SH3 and SH2, which bind specif-ic proline-rich and phosphotyrosine (pTyr) motifs, respectively.The kinase domain (~260 residues) is responsible for catalyticactivity and consists of two lobes that form a cleft representingthe active site of the kinase. The small N-terminal lobe is com-posed of five b sheets and a single a helix and is primarily in-volved in anchoring and orienting the ATP substrate. The largeC-terminal lobe, which is predominantly a helical in nature andcontains the regulatory activation loop (A-loop), is responsiblefor binding the protein substrate. The two lobes move relative

Hematopoietic cell kinase (Hck) is a member of the Src familyof non-receptor protein tyrosine kinases. High levels of Hck areassociated with drug resistance in chronic myeloid leukemia.Furthermore, Hck activity has been connected with HIV-1.Herein, structure-based drug design efforts were aimed atidentifying novel Hck inhibitors. First, an in-house library ofpyrazolo[3,4-d]pyrimidine derivatives, which were previouslyshown to be dual Abl and c-Src inhibitors, was analyzed bydocking studies within the ATP binding site of Hck to selectthe best candidates to be tested in a cell-free assay. Next, the

same computational protocol was applied to screen a databaseof commercially available compounds. As a result, most of theselected compounds were found active against Hck, with Ki

values ranging from 0.14 to 18.4 mm, confirming the suitabilityof the computational approach adopted. Furthermore, selectedcompounds showed an interesting antiproliferative activityprofile against the human leukemia cell line KU-812, and onecompound was found to block HIV-1 replication at sub-toxicconcentrations.

[a] Dr. C. Tintori, Dr. F. Falchi, Prof. M. BottaDipartimento Biotecnologie, Chimica e FarmaciaUniversit� degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena (Italy)E-mail : botta.maurizio@gmail.com

[b] I. Laurenzana, F. La RoccaIRCCS-Centro di Riferimento Oncologico Basilicata (CROB)Laboratory of Preclinical and Translational ResearchVia Padre Pio 1, Rionero in Vulture 85028 Potenza (Italy)

[c] Dr. F. FalchiDepartment of Pharmacy and Biotechnology, Alma Mater StudiorumUniversit� di Bologna, Via Belmeloro 6, 40126 Bologna (Italy)

[d] Prof. F. CarraroDipartimento di Medicina Molecolare e dello SviluppoUniversit� degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena (Italy)

[e] A. Ruiz, Prof. J. A. Est�Irsicaixa, Hospital Germans Trias i PujolUniversitat Aut�noma de Barcelona, 08916 Badalona (Spain)

[f] M. Kissova, Dr. E. Crespan, Prof. G. MagaIstituto di Genetica MolecolareIGM-CNR, Via Abbiategrasso 207, 27100 Pavia (Italy)

[g] Prof. M. BiavaDipartimento di Chimica e Tecnologie del FarmacoUniversit� La Sapienza, Piazzale Aldo Moro 5, 00185 Roma (Italy)

[h] Dr. C. Brullo, Prof. S. SchenoneDipartimento di Scienze FarmaceuticheUniversity of Genoa, Viale Benedetto XV 3, 16132 Genova (Italy)

[i] Prof. M. BottaBiotechnology College of Science and TechnologyTemple University, Biolife Science BuildingSuite 333, 1900 North 12th Street, Philadelphia, PA 19122 (USA)

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to each other and can open or close the cleft.[6, 7] In responseto cellular signals, SFKs undergo large conformational changesto switch between distinct inactive and active states, the struc-tures of which have been characterized by X-ray crystallogra-phy.[3, 8–12] Tyrosine phosphorylation controls the activity of Srckinases in two opposing ways: phosphorylation of Tyr527 inthe C-terminal tail region suppresses tyrosine kinase activity. Incontrast, phosphorylation of Tyr416 within a centrally locatedactivation segment results in the acquisition of catalytic activi-ty. Hck is involved in immune signaling and cell proliferation inhematopoietic cells and is linked to cancer, like other membersof the Src family. High levels of Hck are associated with drugresistance in chronic myeloid leukemia (CML),[13–16] and its con-stitutively active isoforms induce solid tumors in mice.[17] Fur-thermore, Hck activity has been associated with viral infectionsincluding HIV-1.[17] In particular, Hck is activated by the HIV-1 ac-cessory protein negative regulatory factor Nef, a multifunctionalHIV-1 protein that accelerates progression to acquired immunedeficiency syndrome (AIDS) and enhances the infectivity ofprogeny viruses. Nef binding to Hck determines the displace-ment of the SH3 domain from its negative regulatory interac-tion with the catalytic domain, leading to kinase activation,which is important in AIDS pathogenesis.[18–23]

In this context, we employed a structure-based drug designstudy with the aim of identifying novel Hck inhibitors(Scheme 1). First, an in-house library of pyrazolo[3,4-d]pyrimi-dine derivatives, which were previously shown to be dual Abland c-Src inhibitors,[24–27] was analyzed by docking studieswithin the ATP binding site of Hck to select the best candi-dates to be tested in a cell-free assay. Next, the same computa-tional protocol was applied to screen a database of commer-cially available compounds. Results of biological studies

showed that most of the selected compounds are activeagainst Hck, with Ki values ranging from 0.14 to 18.4 mm, con-firming the suitability of the computational approach adopted.The most active inhibitors were then selected for further bio-logical evaluation in the KU-812 leukemia cell line under nor-moxic conditions. An interesting antiproliferative activity pro-file was observed. Finally, promising compounds were testedfor anti-HIV-1 activity. Remarkably, one compound was foundto block HIV-1 replication at sub-toxic concentrations.

Results and Discussion

A docking approach was used in this study to identify novelHck inhibitors that target the ATP binding site. First, our in-house library of pyrazolo[3,4-d]pyrimidines (consisting of ~300structurally characterized compounds with >98 % purity) wasanalyzed to select the most promising binders of Hck for bio-logical investigation. Later, the same computational methodol-ogy was applied to screen a library of commercially availablecompounds. For this, we used the crystal structure of Hckin complex with 4-amino-5-(4-methylphenyl)-7-(tert-butyl)-pyrazolo[3,4-d]pyrimidine (PP1), a Src-family-selective tyrosinekinase inhibitor (2.0 � resolution, PDB ID: 1QCF).[10] Indeed, thestructure of PP1 resembles that of our pyrazolo[3,4-d]pyrimi-dine compounds. Furthermore, this crystal structure representsthe auto-inhibited form of Hck; therefore, targeting it ratherthan the active kinase could provide insight into the design ofSrc-kinase-selective inhibitors.[10] Two software packages wereused for simulations: Glide[28] and Gold.[29] Compounds 1–11were initially selected by taking into account the overall matchbetween the binding modes proposed by the two programs,the number of clusters, and the predicted score values andwere initially tested in a cell-free assay to evaluate their affinitytoward Hck.

Simulations allowed the identification of two different bind-ing modes. Compounds 1–9, with an exocyclic amine at C4,showed an interaction pathway similar to that previously

Figure 1. Domain organization (top) and three-dimensional structure(below) of SFKs, with key residues and domains indicated.

Scheme 1. Workflow of the strategy adopted for the discovery of novel Hckinhibitors.

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found within the ATP binding site of c-Src. As an example, theproposed binding mode of compound 1 is shown in Figure 2 a.The pyrazolo[3,4-d]pyrimidine core occupies the adenineregion, the C4 substituent establishes hydrophobic interactionswith Leu325, Phe405, Ile336, Phe307, Met314, and Leu407, (hy-drophobic region I), while the N1 side chain is involved invan der Waals contacts with Phe340, Met341, and Leu273 (hy-drophobic region II). The C6 substituent is directed toward theexternal region, though still involved in nonpolar contacts withVal281 and Lys295. Two hydrogen bond interactions were alsofound, one involving the C4 amino group and the side chainof Thr338, and the other between N2 of the pyrazolo[3,4-d]pyr-imidine nucleus and the backbone NH group of Met341. Ab-sence of the exocyclic NH at C4 in compounds 10 and 11 is re-sponsible for a reorientation of the heterocyclic nucleus withinthe pocket. Figure 2 b shows the binding mode of compound10 as representative of such an alternative pose. Extensive hy-drophobic contacts were observed between the chlorophenyl-ethyl N1 substituent and hydrophobic region I. The piperidinylmoiety is involved in van der Waals interactions with the sidechains of Met341 and Phe340, while the alkylthio group favora-bly interacts with Val281. Two hydrogen bonding interactionsare also present in the predicted binding pose, the first be-

tween N2 and the OH group of Thr338, and the second be-tween N7 and the terminal side chain of Lys295. The structuresand inhibitory activities of compounds 1–11 toward isolatedHck, Src, and Abl kinases are listed in Table 1. All compoundswere found to be active against Hck in the sub-micromolar tolow-micromolar concentration range. The most active inhibi-tors 1–6 were then chosen for testing against the KU-812 leu-kemia cell line under normoxic conditions. Compounds 2–4and 6 showed antiproliferative activity at micromolar concen-trations. The inactivity of compounds 1 and 5 could be due toan unfavorable pharmacokinetic profile.

For virtual screening, the Asinex database was analyzed bydocking studies to select potential Hck inhibitors with a com-pletely new scaffold. The computational protocol can be divid-ed into two sequential steps: first, all compounds were dockedwithin the ATP binding site of the auto-inhibited form of Hckby using Glide software, which allowed very rapid analysis ofthe entire compound library. The first 2000 compounds interms of Glide SP score were then selected for a second run ofdocking calculations with the Gold program. Known Hck inhib-itors were added to calculations in this second phase to im-prove the enrichment performance of molecular docking.[30–32]

A consensus docking approach was finally applied for com-

Figure 2. Graphical representation of the predicted binding modes of compounds a) 1 (orange), a) 10 (deep teal), c) 29 (violet), and d) 37 (warm pink) in theATP binding site of Hck. For the sake of clarity, only a few key residues are labeled, hydrogen atoms are omitted, and hydrogen bonding interactions are rep-resented by black dashed lines. Compounds and key amino acids are shown in stick representation.

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pound selection by using three scoring functions: Glide SP,GoldScore, and ChemScore (rank-by-rank approach). The struc-tures and inhibitory activities of compounds 12–37 toward iso-lated Hck are listed in Table 2. Remarkably, most of the testedcompounds showed inhibitory activity against Hck, confirmingthe suitability of the computational approach applied. Predict-

ed binding modes for the best compounds 29 and 37 (Ki =

0.26 and 0.22 mm, respectively) are shown in Figure 2 c,d. Thepreferred binding pose of 29 showed that the benzothiazolemoiety perfectly fills hydrophobic region I, being involved inextensive hydrophobic contacts with Leu325, Phe405, Ile336,Phe307, Met314, and Leu407. Furthermore, the central phenol

Table 1. Structure and inhibitory activity of compounds 1–11 toward isolated Hck.

Ki [mm] IC50 [mm]

Compd R R1 R2 Hck[a] c-Src[b] Abl[b] KU-812[c]

1 NHCH2CH2C6H5 S-cyclohexyl CH2CHClC6H5 0.14 1.31 NA[d] >1002 NHCH2C6H4oCl SCH3 CH2CHClC6H4pBr 0.42 ND ND 21.09�5.253 NHCH2C6H4mF S-cyclopentyl CH2CHClC6H5 0.55 0.1 0.1 37.09�2.124 NHC6H4mCl SCH2CH2-4-morpholinyl CH2CHClC6H5 0.65 0.19 0.12 16.54�2.235 NHCH2C6H5 S-cyclopentyl CH2CHClC6H5 0.84 NA 0.06 >1006 NHCH2CH2C6H5 SCH2CH2-4-morpholinyl CH2CHClC6H5 1.27 NA 0.272 33.45�1.727 NHCH2CH2C6H5 S-cyclopentyl CH2CHClC6H5 1.99 4 0.07 ND[e]

8 NHCH2C6H5 SCH2CH2-4-morpholinyl CH2CHClC6H5 2.20 2.9 0.089 ND9 NHCH2C6H4pF SCH2CH2-4-morpholinyl CH2CHClC6H5 3.87 1.21 0.6 ND

10 1-piperidinyl SCH3 CH2CHClC6H5 2.46 2.4 NA ND11 4-morpholinyl SCH3 CH2CHClC6H4pCl 7.50 6.5 0.5 ND

[a] Values are the mean of at least two experiments. [b] Ki values toward isolated c-Src and Abl kinases as published elsewhere.[24–27] [c] IC50 values are themeans �SD of five experiments, each performed in triplicate. [d] NA: not active at 100 mm (the highest concentration tested). [e] ND: not determined.

Table 2. Structure and inhibitory activity of compounds 12–37 toward isolated Hck.

Compound HckKi [mm][a]

KU-812IC50 [mm][b]

Compound HckKi [mm][a]

KU-812IC50 [mm][b]

12 4.2 25 3.5

13 NA[c] 26 3

14 NA 27 18.4

15 2.2 28 1.81 >100

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Table 2. (Continued)

Compound HckKi [mm][a]

KU-812IC50 [mm][b]

Compound HckKi [mm][a]

KU-812IC50 [mm][b]

16 NA 29 0.26 >100

17 5.4 30 NA

18 NA 31 NA

19 5 32 6

20 4.8 33 12.4

21 34 13.8

22 0.94 92.9�2.0 35 0.83 92�1.2

23 1.48 >100 36 0.3 64.5�17.5

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ring undergoes hydrogen bond interactions with Glu339 andThr338, whereas the nitronaphthalen-2-ol group occupies hy-drophobic region II, where it establishes van der Waals con-tacts with Phe340, Met341, and Leu273 as well as polar con-tacts with the backbone of Met341 by means of its hydroxygroup. Analysis of the binding mode of 37 showed that the 8-thia-4,6-diazatricyclo scaffold occupies the adenine region andundergoes a hydrogen bond interaction with Thr338 and hy-drophobic contacts with the residues of hydrophobic region II.A second hydrogen bond was found between the ligandamide group and Lys295. Furthermore, the complex is stabi-lized by hydrophobic interactions of the phenylethyl moiety of37 within hydrophobic region I. The morpholine moiety is sol-vent exposed. Biological evaluation of the best Hck inhibitorson the KU-812 leukemia cell line showed only weak activity forcompounds 22, 35, 36, and 37 (Table 2). On the other hand,because Hck was recently shown to be a novel target for HIV,compounds 22, 23, 28, 29, and 35–37 were tested for anti-HIV-1 activity in primary monocyte-derived macrophages(MDM) as described earlier (Figure 3).[33, 34] Among the testedcompounds, 37 blocked viral replication with an EC50 value of12.9 mm without significant cytotoxicity. Furthermore, to assessits specificity against Hck, compound 37 was tested against

a panel of kinases including other Src family members (Hck,Blk, Fgr, Fyn, cSRC, Lck, Lyn, Syk, and Yes), tyrosine kinases (Abland Abl-T315I), as well as some kinases important for HIV-1 (CDK9, FAK, Itk, JAK3, Ron).[35–39] The percentage of enzymaticactivity was measured for each kinase by using 37 at 10 mm

(Millipore) ; results are listed in Table 3. Remarkably, 37 provedto be more efficient against Hck than toward the other investi-gated kinases, confirming such a compound as a useful probeto study Hck function.

Conclusions

In this work, a structure-based computational study led to theidentification of new ATP-competitive Hck inhibitors character-ized by structures distinct from any class of Hck inhibitors re-ported thus far. These compounds show inhibitory activitytoward isolated Hck in the sub-micromolar to low-micromolarconcentration range and are endowed with an interesting anti-proliferative activity profile against the human leukemia cellline KU-812. Furthermore, one compound was identified ashaving significant anti-HIV-1 activity in cells, supporting thefinding that Hck is a potential antiviral target. On the basis ofthese considerable results, further studies are in progress to

develop second-generation compounds with im-proved activity which may serve as potential leads todevelop new drugs against HIV-1 and leukemia.

Experimental Section

Dataset collection : A series of ~300 pyrazolo[3,4-d]pyri-midines was collected from previous work and werebuilt with the Schrçdinger Maestro 9.1 graphical inter-face.[40] Compounds were then processed with the Schrç-dinger LigPrep tool to generate separate files for all pos-sible enantiomers and protonation states at physiologi-cal pH. OPLS_2005 was used as force field. We retrievedthe three-dimensional structures of the commerciallyavailable compounds from the ZINC[41] database by se-lecting Asinex as vendor; 703 200 compounds were ana-lyzed.

Molecular docking calculations : The three-dimensionalcoordinates of Hck were obtained from the Protein DataBank (PDB ID: 1QCF) and were energy-minimized to

Table 2. (Continued)

Compound HckKi [mm][a]

KU-812IC50 [mm][b]

Compound HckKi [mm][a]

KU-812IC50 [mm][b]

24 NA 37 0.22 66.5�11.8

Figure 3. Antiviral activity of Hck-targeting agents. The white bar represents p24 antigenproduction from 100 % of infected control, grey bars represent p24 antigen productionfrom MDMs treated with the control drugs AZT and TAK779 in a dose–response manner,and black bars represent p24 antigen production from assayed compounds as indicated.*Identification of 37 as a compound that significantly inhibits HIV-1 replication.

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remove unfavorable contacts through the all-atom OPLS force fieldand Polak–Ribiere conjugate gradient method. A continuum solva-tion method, with water as the solvent, was also applied. Extendedcutoffs were used, and convergence was set to 0.05 kJ mol�1 �. Aconsensus docking approach that takes advantage of two widelyused docking programs (Glide and Gold) was used in this study, asit has been previously shown that the use of consensus dockingoutperforms single docking with respect to the accuracy of bind-ing pose prediction and scoring. In particular, Glide was first usedon the whole database because of its speed (703 200 compoundswere analyzed in about 20 days on a Quad Core Intel Xeon2.5 GHz processor with Ubuntu 10.04 as the operating system).Compounds were docked and scored using the Glide standard pre-cision (SP) mode. A grid box of default size was centered on theligand of the X-ray crystal structure. No constraints were includedduring grid generation, while rotation of hydroxy groups was al-lowed. Default parameters were used for the docking runs. Thetop pose based on the Glide docking score was saved for eachligand. Only compounds with a docking score lower than thatfound for the X-ray ligand PP1 (�9.4) were chosen for the follow-ing docking step with Gold (~7000 compounds). GoldScore andChemScore were used as fitness functions (see Ref. [29] for details),and compounds were processed in a week. The GA parameter set-tings of Gold were used, with search efficiency set to 200 %. Finally,results differing by <1.5 � in ligand all-atom RMSD were clusteredtogether. For each inhibitor, the first-ranked solution as well as thelowest-energy conformation of the most populated cluster wereanalyzed by comparing them with the best pose previously ob-tained with the Glide software. Compounds were then selected bytaking into account the overall match between the binding modesproposed by the two programs and the predicted score values(GoldScore and ChemScore values higher than 65 and 35, respec-tively, were considered good). The rank-by-rank approach wasused for the final selection.[42, 43]

Compound analysis : The purity of compounds was reported byAsinex to range from 93 to 99 %. To check this degree of purity, weperformed HPLC analysis of compounds after shipping and stor-age. Solutions were analyzed, finding percent purity values similarto those reported by Asinex. 1H NMR spectra were recorded toconfirm that compounds have the reported structure.

Enzymatic assay on isolated Hck : Active, full-length, recombinantHck was purchased from ProQuinase (Germany). Specific peptidesubstrates (Src substrate peptide, cat. # 12-140) were purchasedfrom Merck–Millipore. Kinase assays were performed in the pres-ence of 50 mm ATP and 50 mm peptide substrate. All inhibitionassays were conducted with 0.01 mg active kinase, 0.33 pmol[g32P]ATP, 10 mm magnesium acetate, 8 mm 3-(N-morpholino)pro-panesulfonic acid (MOPS)/NaOH (pH 7), 1 mm ethylenediaminete-traacetic acid (EDTA), and 10 % DMSO in a final volume of 10 mL.After 10 min at 30 8C, the reaction was stopped by adding 5 mL 4 %phosphoric acid. Aliquots (10 mL) were then transferred into a P30Filtermat (PerkinElmer), washed five times with 75 mm phosphoricacid and once with acetone for 5 min. The filter was dried andtransferred to a sealable plastic bag, and scintillation cocktail(4 mL) was added. Spotted reactions were read in a scintillationcounter. ID50 values were obtained according to Equation (1),where v is the measured reaction velocity, V is the apparent maxi-mal velocity in the absence of inhibitor, I is the inhibitor concentra-tion, and ID50 is the 50 % inhibitory dose.

n ¼ V=f1þ ðI=ID50Þg ð1Þ

Ki values (mm) toward recombinant Hck were calculated accordingto Equation (2) for competitive inhibition toward ATP and peptidesubstrates, in which [SATP] and [Spep] are the concentration of com-peting substrate (ATP and peptide, respectively). Curve fitting wasperformed with GraphPad Prism version 5.00.

K i ¼ fID50=ð1þ K M ATP=½SATP�Þg=ð1þ K M pep=½Spep�Þ ð2Þ

Cell proliferation assay : Human CML KU-812 cells were obtainedfrom the American Type Culture Collection (ATCC) and were grownin RPMI 1640 medium (Euroclone, Devon, UK), containing 10 %fetal bovine serum (FBS) and antibiotics (100 U mL�1 penicillin and100 mg mL�1 streptomycin). The cultures were free of mycoplasma.Experiments were conducted under normoxic conditions. The com-pounds were dissolved in DMSO and used at various concentra-tions. At the end of the experiments, cells were promptly analyzed.To quantify cell proliferation, starved cells were plated into 96-wellplates in complete medium at the concentration of 1 � 104 cells perwell and then incubated for 72 h, with or without various concen-trations (0.5–150 mm) of the studied compounds, under normoxicconditions. At the end, each culture microplate was centrifuged, in-verted to remove growth medium, and kept at �80 8C. The micro-plates were then thawed at room temperature, and 200 mL of theCyQUANT GR dye/cell-lysis buffer (Molecular Probes, Eugene, OR,USA) was added to each sample well. The concentrated cell-lysisbuffer was diluted 1:20 into ultrapure water, and CyQUANT GR dyewas then diluted 1:400 into lysis buffer. The samples were incubat-ed for 2–5 min at room temperature, protected from light, prior tomeasuring relative fluorescence (485 nm excitation, 520 nm emis-sion) using a fluorescence microplate reader FLUOstar OPTIMA(BMG Labtech, Offenburg, Germany). Data analysis for IC50 calcula-tions was performed with the L5W Data Analysis Package plug-infor Excel (Microsoft). Results are reported as the mean �SEM.

Antiviral activity and cytotoxicity : Anti-HIV-1 activity and cytotox-icity in MDM cells was determined as described before.[33, 34] Briefly,MDM previously stimulated for three days with macrophage-colony stimulating factor (M-CSF) were infected with HIV-1 BaL inthe presence of the corresponding compound. At seven days post-infection HIV p24 antigen production was quantified by ELISA. AZTand TAK779 were used as reference compounds.

Table 3. Kinase profiling of compound 37 (Millipore).

Kinase Residual Activity [%][a]

Hck(h) 38Abl(h) 103Abl(T315I)(h) 83Blk(h) 100CDK9/cyclin T1(h) 104cSRC(h) 67FAK(h) 101Fgr(h) 105Fyn(h) 93Itk(h) 102JAK3(h) 92Lck(h) 120Lyn(h) 91Ron(h) 109Syk(h) 77Yes(h) 83

[a] Residual kinase activity after treatment with 37 at 10 mm, expressed aspercent of basal kinase activity.

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Acknowledgements

This project was supported by the Spanish Ministerio de Econo-m�a y Competitividad (MEC, projects BFU2009-06958, BFU2012-31569 and SAF2010-63622-C02), FIPSE 360783-09 and Galacontra la SIDA, European Union collaborative projects “THINC”(grant no. HEALTH-2007-2.3.2-1) and “CHAARM” (grant no.HEALTH-F3-2009-242135), as well as by the Italian Ministero del-l’Istruzione, dell’Universit� e della Ricerca, PRIN 2010 researchproject (grant no. 2010W2KM5L). A.R. is a research fellow fromMEC.

Keywords: docking · Hck · HIV-1 · kinases · leukemia

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Received: May 9, 2013

Published online on June 28, 2013

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