clejathe

cDepartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143, United Statesd Small Molecule Discovery Center, University of CaliforneDepartment of Pharmaceutical Chemistry, University o

a r t i c l e i n f o

mutations in theor expansion of

r to lowerif mutati

riers live long enough, they will all die of the diseaAlternatively, prions can be transmitted from one personother, as in the case of kuru in New Guinea natives or iatrCJD due cadaveric human growth hormone.12,13 Transmission ofBSE, CWD, and scrapie prions among animals and BSE to humansis well described.1419

Prion diseases are rapidly progressive and invariably fatal in hu-mans and animals, and no effective treatments are available. Dis-covery of new small molecule-based therapies has relied ontesting in dividing cultured cell lines that propagate prions as wellas in animal models infected with prions. Because the brain is

Corresponding author. Address: Institute for Neurodegenerative Diseases,University of California, 675 Nelson Rising Ln, Room 318, San Francisco, CA94143, United States. Tel.: +1 415 476 4482; fax: +1 415 476 8386.

E-mail address: stanley@ind.ucsf.edu (S.B. Prusiner). Present address: ELMEDTECH, LLC, San Francisco, CA 94123, United States. Present address: Global Blood Therapeutics, Inc., South San Francisco, CA 94080,

United States. Present address: Endocyte, Inc., West Lafayette, IN 47906, United States. Present address: Department of Biology, University of Rochester, Rochester, NY

Bioorganic & Medicinal Chemistry 21 (2013) 79998012

Contents lists availab

Bioorganic & Med

journal homepage: www.e14627, United States.ders. In the uniformly fatal diseases caused by the prion protein(PrP), prions are formed when the cellular prion protein (PrPC) isrefolded into an alternative isoform designated PrPSc.4,5 The accu-mulation of PrPSc, which is b-sheet-rich, causes CNS dysfunction.

dominantly inherited PrP diseases are caused byPrP gene, resulting in amino acid substitutionsthe octarepeat region.79 Mutations in PrP appeaergy barrier for spontaneous PrPSc formation, and0968-0896/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmc.2013.09.022the en-on car-se.10,11

to an-ogenicindole in stationary-phase and dividing ScN2a-cl3 cells were 7.5 and 1.6 lM, respectively. Unexpectedly,the number of hits in stationary-phase cells was 10% of that in dividing cells. The explanation for thisdifference remains to be determined.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

A growing body of evidence argues that different proteins canassemble into prions and cause distinct neurodegenerative disor-

13

The PrP prion diseases can present as sporadic, inherited, andinfectious disorders. Epidemiological studies argue that the major-ity of CreutzfeldtJakob disease (CJD) cases are sporadic, whichseem to occur spontaneously through a stochastic process.6 TheArticle history:Received 17 April 2013Revised 30 August 2013Accepted 7 September 2013Available online 18 September 2013

Keywords:Antiprion compoundsPrPSc

Dividing and stationary-phase brain cellsia, San Francisco, CA 94143, United Statesf California, San Francisco, CA 94143, United States

a b s t r a c t

During prion diseases, a normally benign, host protein, denoted PrPC, undergoes alternative folding intothe aberrant isoform, PrPSc. We used ELISA to identify and conrm hits in order to develop leads thatreduce PrPSc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells.We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. Thisled to 3100 HTS and 970 single point conrmed (SPC) hits in dividing cells, 331 HTS and 55 conrmedSPC hits in stationary-phase cells as well as 36 conrmed SPC hits active in both. Fourteen chemical leadswere identied from conrmed SPC hits in dividing cells and three in stationary-phase cells. From morethan 682 compounds tested in concentrationeffect relationships in dividing cells to determine potency(EC50), 102 had EC50 values between 1 and 10 lM and 50 had EC50 values of

medicals (Solon, OH); and guanidine isothiocyanate from RPI (Mt.

the corresponding vendor. For SAR expansion, analogs of vali-

d. Ccomposed of post-mitotic cells, we decided to undertake high-throughput screening (HTS) using stationary-phase and dividingcells. Only a small number of cell lines supports prion replication,the most well-studied of which are from mice; these include theN2a, GT1, PK1, CAD5, and LD9 mouse cell lines.2022 PK1 cells area subclone of N2a cells; the LD9 line is a subclone of L929 cells.The most permissive mouse-passaged prion strain seems to beRML that was derived from sheep prions.23

One therapeutic target for prion disease is to lower levels ofPrPSc, which can be accomplished by decreasing its formation,increasing its clearance, or some combination of both. Manyantiprion compounds have been discovered using low-throughputscreening of small sets of known drugs approved for other indica-tions, or small chemical sets, most frequently using RML- or ME7-infected N2a or GT1 cells that are dividing.2427 The drugs andcompounds discovered to date include many chemical leads,including acridines (e.g., quinacrine and structurally related tricy-clic antidepressants);2830 analogues of quinacrine;31,32 statins;33

pyrazolones;34 indole-3-glyoxylamides35,36 and pyridyl hydra-zones,37,38 including Compound B. In addition, larger polyanionicor polycationic molecules (e.g., dendritic polyamines of PAMAM)have been reported to exhibit antiprion activity in cells.39,40 It isunlikely that such species could be used therapeutically becausethey are likely to suffer from poor oral absorption, low bloodbrain-barrier (BBB) penetration, and high toxicity.

Except for PAMAM, none of the approved drugs or experimentalcompounds has been reported to lower levels of PrPSc in station-ary-phase cells. PAMAM is active in both dividing and stationary-phase cells.41 Whereas Compound B is active in dividing cells, itseffects in stationary-phase cells have not been reported, yet it sig-nicantly extended survival in RML prion-infected mice, in astrain-dependent manner.37 The hypothesis positing that an effec-tive drug needs to be active in stationary-phase cells for it to ex-tend survival in vivo has not been tested. It has, however, beenproposed that quinacrine failed in vivo, not only because of its lackof activity in stationary-phase cells, but also because of the rapiddevelopment or selection of drug-resistant prion strains uponchronic dosing.41

To identify new hits and conrm chemical leads, we developedELISA for both in dividing and stationary-phase cells, and screened53,000 compounds by HTS in each. We identied HTS hits and re-tested them in single point conrmation (SPC), which includes anassessment of cell viability. In dividing cells, conrmed SPC hitsrepresented 14 chemical leads. Concentrationeffect relationships(EC50) performed on these conrmed SPC hits and related analogsin dividing cells yielded 50 with EC50 values

ed. C2.3. Compound selection

To decide which compounds available at the SMDC would bemost useful to screen in the PrPSc assays, we sought to develop ametric that prioritized compounds that were more likely to hitand not narrowly focused in a congeneric series. We assessed thecompounds using two metrics: predicted bioactivity and mutualdissimilarity. One point was assigned for each compound thathad at least one prediction of bioactivity using SEA based on ChEM-BL release 01 and using an E-value cutoff of 1010 (http://www.ncbi.nlm.nih.gov/pubmed/21948594). To calculate dissimi-larity, we used a modied version of the method of Voigt et al.43

(http://cactus.nci.nih.gov/subset/). For each plate, we sorted themolecules by ascending molecular weight and scored one pointfor any molecule that differed from all previously accepted com-pounds by a Tanimoto coefcient (Tc) of P0.7, based on defaultDaylight ngerprints. The combined score (bioactivity + dissimilar-ity) was used to compute a combined gure of merit for each plate,which was used to rank them, and from which we selected53,000 compounds for HTS analysis.

2.4. Antiprion activity and cellular toxicity assays

Mouse N2a neuroblastoma cells (ATCC) were transfected withfull-length mouse PrP and infected with the Rocky Mountain Labo-ratory (RML) strain of mouse-adapted scrapie prions, yieldingScN2a-cl3 cells.44 ScN2a-cl3 cells were maintained in tissue cultureasks (175 cm2) containing 32 mL of lter-sterilized (0.2 lm)MEMwith Earles salts and L-glutamine, supplemented with 10% FBS,250 lg/mL Geneticin, 50 I.U./mL penicillin, and 50 lg/mL strepto-mycin (supplemented MEM) in a humidied and CO2-enriched(5%) environment at 37 C. On day 1, the growth medium (supple-mented MEM) was aspirated from the asks, the cells were washedtwice with 10 mL of calcium- and magnesium-free Dulbeccos PBS,and then detached by addition of 3 mL of cell dissociation buffer(Millipore #S-014-B) after incubation at room temperature (rt) for5 min. The dissociation bufferwas aspirated and the cells suspendedin10 mLof growthmediumbefore countingusingaCellometerAutoT4 (Nexcelom Biosciences; Lawrence, MA). ScN2a-cl3 cells wereseeded either into new, 175-cm2 tissue culture asks for continuedcell culture (9 106 cells into 32 mL growth medium) or onto 96-well, tissue culture-treated, white polystyrene plates (Greiner Bio-One; Monroe, NC) for treatment with test compounds (40,000cells/well in 100 lL of growth medium for dividing ScN2a cells;150,000 cells/well in 80 lL of growth medium for stationary-phaseScN2a cells). Stationary-phase ScN2a-cl3 cells were allowed to ad-here for 1 h at 37 C before cell division arrest was induced by addi-tion of 20 lL of 35 mM sodium butyrate in growth medium (7 mMnal concentration), and the plates incubated for 24 h prior to com-pound addition. Dividing ScN2a-cl3 cellswere allowed to adhere for4 h at 37 C before compound addition. Test compounds (100 lL)were added to each well to attain a nal concentration of 10 lM.Three positive controls were used: simvastatin, quinacrine, and PA-MAM-G4. Simvastatin and quinacrine (10 mM in 100% DMSO) wereadded, thendiluted to anal concentrationof 10 lMingrowthmed-ium (0.1%DMSO, nal concentration). PAMAM-G4was diluted froma 1% stock solution (in MeOH) to achieve a nal concentration of10 lg/mL. As a negative control, 0.1% DMSO in growth mediumwas used.Mediawas aspirated on day 5, and cellswerewashedwithPBS (250lL/well) and aspirateddry. The cellswere lysed byadditionof 20 lL of lysis buffer (10 mM Tris HCl, 150 mMNaCl, 0.5% sodiumdeoxycholate, 0.5% NP-40) containing 7.5 U/mL benzonase; plateswere placed on a shaker at 37 C for 1 h. Proteinase K [PK; 5 lL of

B. M. Silber et al. / Bioorg. M125 lg/mL ina Tris buffer (10 mM Tris HCl, 20 mM calcium chloride, 50% glyc-erol)] was added and incubated at 37 C for 1 h, with shaking. PKdigestionwas stopped by addition of 5lL of cold (4 C) 20 mMPMSFin ethanol. After 10 min at rt, 10 lL of 5 M guanidine isothiocyanatewas added at 37 C for 1 h (with shaking) to denature the protein.The lysate in each well was diluted with 120 lL of PBS, and 150 lLfrom each well transferred to 96-well polystyrene ELISA plates pre-viously coated with D18 antibody (5 lg/mL/well in 300 lL of acidi-ed PBS overnight at rt in a humidied chamber45), the plates sealedand incubated overnight at 5 C. The next day, the plates werewashed 3 times with TBST buffer (20 mM Tris HCl, 137 mM NaCl,0.05% Tween-20, pH 7.5), the contents of each well aspirated com-pletely. Then 100 lL of a 1:1000 dilution of HRP-conjugated D13antibody was added and incubated at 37 C for 1 h. The plates werewashed4 timeswith TBSTbuffer, the contents aspirated completely,and 100 lL of ABTS peroxidase substrate added to each well. After15 min of development at rt, the enzymatic reaction was stoppedby addition of 100 lL of ABTS stop solution, and the plates immedi-ately loaded onto a SpectraMaxM5plate reader (MolecularDevices;Sunnyvale, CA) for measurement of absorbance at 405 nm.

For cell viability assays, mouse N2a-cl3 cells were seeded into96-well, black polystyrene plates (Greiner) and treated with com-pound as described above for the ELISA plates. After 5 days, thegrowth media was aspirated, the plates washed once with PBS(250 lL/well), and the plates aspirated dry. Calcein-AM (100 lL/well, 5 lg/mL solution in calcium- and magnesium-free PBS) wasadded, and the plates were incubated at 37 C for 45 min. Fluores-cent emission intensity was quantied using a Spectramax M5plate reader, excitation/emission spectra of 485 nm/530 nm.

2.5. Western blot analysis of ScN2a-cl3 cells

ScN2a-cl3 cells, cultured as described above, were seeded onto6-well, tissue culturetreated dishes at a density of 1.54 106cells/well in 6.2 mL of supplemented MEM and allowed to adherefor 4 h at 37 C. Test compounds diluted in supplemented MEM(described above) were added to the plate (0.8 mL/well) to attainnal concentrations ranging from 1 nM to 32 lM. After 5 days,the media was aspirated from each well, and the plates washedone time with PBS (7 mL/well). The cells were lysed by additionof 0.35 mL of lysis buffer (20 mM Tris HCl, pH 8.0; 100 mM NaCl;0.5% NP-40; 0.5% sodium deoxycholate; and 7.5 U/mL benzonase).The total protein in the lysate was measured using a bicinchoninicprotein assay (Pierce). A total of 0.06 mg protein was treated withPK (total protein:enzyme ratio = 50:1) in 0.1 mL PBS, and the sam-ple incubated at 37 C for 1 h. The proteolytic digestion was termi-nated by the addition of PMSF to a nal concentration of 3 mM. Thesamples were centrifuged at 16,000g for 1 h, the supernatant dis-carded, and the pellets resuspended in 15 lL of reducing SDS sam-ple buffer. The PrPSc-containing samples were denatured byheating at 80 C for 5 min and run in a 412% Tris glycine SDSgel (Invitrogen). The gels were transferred to nitrocellulose mem-brane using a Nupage apparatus (Invitrogen), and the membranesblocked with 5% (w/v) nonfat milk/TBST overnight. The mem-branes were immersed for 1 h at rt in a 1:10,000 dilution of D13antibody (1 mg/mL) conjugated to horseradish peroxidase (HRP),washed 3 times with TBST buffer before development (1 min) withEnhanced Chemiluminescent Western Blot reagent (GE Health-care). Imaging and quantication of the blots were done using aGeneGnome (Syngene) equipped with Gene Tools software.

To quantify actin, 200 lg of total protein from lysate was di-luted 1:5 with cold ethanol and incubated overnight at 20 C.The next day, the sample was centrifuged for 60 min at 16,000g,the protein pellet dried and dissolved in Nupage loading buffer.The sample was loaded onto a 412% BisTris gel system (Invitro-

hem. 21 (2013) 79998012 8001gen), transferred to a nitrocellulose membrane, and blocked with5% (w/v) nonfat milk/TBST for 30 min at rt. The membranes wereimmersed in a 1:10,000 dilution of rabbit anti-actin polyclonal

dissolved in a formulation containing 20% propylene glycol, 5% eth-anol, 5% labrosol, and 70% PEG400 and administered by oral gavageto female FVB mice weighing 25 g. At specied time points afterdosing (0.25, 0.5, 1, 2, 4, 6, and 24 h), 2 animals were euthanized byCO2, and 1 mL blood (by cardiac puncture) and brain sampleswere collected. The heparinized blood samples were centrifugedto obtain plasma, which was stored at 80 C until analysis. Brainsamples were weighed, diluted fourfold with water, and thenhomogenized using a Precellys 24 tissue homogenizer. Brainhomogenates (20% wt/vol) were stored at 80 C until analysis.Pharmacokinetic protocols involving animals were all reviewedand approved by the UCSF institutional animal care and use com-mittee (IACUC).

Plasma and brain homogenate samples were extracted using aprotein-precipitation method and analyzed by specic LC/MS/MSmethods developed for each compound dosed in vivo. The analyt-ical method accuracy and precision were monitored by analyzingquality control (QC) samples that were prepared and treated usingthe same methods as calibration standards for the plasma or brainhomogenate samples.

The data were used to calculate the area under the concentra-tiontime curve (AUClast) by noncompartmental analysis withsparse sampling performed using Phoenix WinNonlin 6.1 software(Pharsight, Mountain View, CA).

3. Results

3.1. Chemical libraries screened

d. Chem. 21 (2013) 79998012antibody (Sigma) in 5% (w/v) nonfat milk/TBST for 1 h, and thenwashed three times with TBST buffer. The membranes were thenincubated with a 1:10,000 dilution of goat anti-rabbit HRP-conju-gated secondary antibody (Biorad) in 5% (w/v) nonfat milk/TBSTfor 1 h, washed three times with TBST buffer, and developed withEnhanced Chemiluminescent Western Blot reagent. Imaging andquantication were performed as described above.

2.6. Data analysis

Absorbance (PrPSc ELISA) and uorescence (calcein cell viability)data were exported from the plate readers as text les and normal-ized either to positive controls (PAMAM-G4 for PrPSc, quinacrine forcalcein). Data were processed and stored in a Collaborative DrugDiscovery (CDD) web-based database (www.collaborativedrug.com). Inhibition curves were generated using nonlinear regressionemploying the Levenberg Marquardt algorithm programmed intothe CDD database.46 Search results were exported to Excel asstructure data les (SDF) or comma-separated values (CSV) lesfor further data manipulation and SAR analysis.

2.7. Analyzing assay performance

Z0 (dividing cell assay) and Z score (stationary-phase cell assay)to assess the precision, accuracy, and robustness were calculatedusing the following equation47:

Z0 1 3s:d:pos:control s:d:neg:control=jmeanpos:control meanneg:controlj

Z 1 3s:d:background s:d:neg:control=jmeanbackground meanneg:controlj;where the positive control is 10 lg/mL PAMAM-G4 and the nega-tive control is 0.1% DMSO in growth medium. In the case of the sta-tionary-phase ScN2a-cl3 ELISA, since no puried protein wasavailable, the background level was set to the same level as the po-sitive control in the dividing ScN2a-cl3 ELISA.

2.8. Structureactivity relationships

SAR analyses were performed using SARvision (Altoris Inc.),permitting the visualization, mining, and organization of chemicaldata. Chemical structure and biological assay data were combinedin SDF les. SARvision was used to generate a list of lead(s) andorganize them into hierarchical tree structures using the IDENTIFYLEADS feature. Leads were also drawn manually by selectingDRAW LEAD under the TREE dropdown menu. Additions or dele-tions by column/rowwere accomplished by selecting the appropri-ate function item under the TABLE dropdown menu, specically tolter data by lead type or any associated data, such as HTS results,SPC data, EC50 values, and physicochemical information. SARvisionwas also used to generate R-group tables to better visualize SAR foreach chemical lead. The sorted data and table were then exportedto MS Word, MS Excel, plain text, HTML, and SDF formats.

2.9. Physicochemical parameters

Qikprop (Schrdinger, New York, NY)48,49 was used to estimatea variety of pharmaceutically relevant physiochemical properties,including calculated log of octanol:water partition coefcient(c logP), polar surface area (PSA), log bloodbrain-barrier (logBBB)permeability, Caco-2 and MDCK cell permeability, and the numberof hydrogen bond acceptors (HBA) and donors (HBD).

2.10. In vivo pharmacokinetic studies

8002 B. M. Silber et al. / Bioorg. MeSixteen of 28 compounds were available to purchase at the 20-mg level. For in vivo pharmacokinetic studies, compounds wereThe source and number of diverse chemical compoundsscreened in each assay are summarized (Table 1). In dividingScN2a-cl3 cells, 54,250 compounds were tested (22,700 fromChemBridge, 30,100 from SPECS, and 1420 FDA-approved drugs).Using stationary-phase ScN2a-cl3 cells, 50,850 total compounds

Table 1Source and number of compounds tested in each HTS assay

Assay Chembridgea SPECSb FDA-approved

Total

Dividing ScN2a-cl3 cells 22,703 30,127 1420 54,250Stationary ScN2a-cl3

cells19,327 30,103 1420 50,850

a Chembridge library = 23,861 compounds.b SPECS library = 30,256 compounds.Figure 1. Z0 (dividing cells, open circles) and or Z (stationary-phase, lled circles)scores for 200 assay runs in ScN2a-cl3 cells.

ed. CB. M. Silber et al. / Bioorg. Mwere screened: 19,300 from ChemBridge, 30,100 from SPECS,and 1420 FDA drugs.

3.2. PrPSc HTS

When we ranked the 180,000 compounds available for screen-ing by their combined gure of merit, including predicted bioactiv-ity and mutual dissimilarity, we found the highest scoringcompounds derived primarily from the ChemBridge and SPECSchemical libraries; from these two libraries, we selected andscreened 53,000 compounds in the PrPSc assays.

To establish the antiprion activity and the effects of the com-pounds on cell viability, we used doseresponse curves to calculateEC50 in ELISA and LC50 in calcein-AM assays, respectively, in bothdividing and stationary-phase ScN2a-cl3 cells. When cells(35,000/well) were grown in the presence of 7 mM sodium buty-rate (NaB), total uorescence from calcein dye incorporation (ameasure of the total number of viable cells) remained unchangedover the course of 5 days, suggesting that the cells were not divid-ing. In contrast, cell numbers at 5 days were signicantly higher(100,000; p

cells

d. CTable 2Distribution of EC50 values for the 14 chemical leads identied in dividing ScN2a-cl3

Lead Name Murcko Fragment Compounds tested (n)

AminothiazoleS

NNH2

11

ThiazoleN

S16

BenzoxazoleN

O17

Pyrazole HNN

6

ThienopyridineSN

NH2

54

Piperazine N N 11

ImidazopyridineN

N4

ImidazothiazoleN

S N15

8004 B. M. Silber et al. / Bioorg. MeFrom testing the 1420 FDA-approved drugs in dividing cells, weidentied 130 compounds by HTS, 15 of which were conrmed SPChits (Table 4). When the 15 were tested for concentrationeffectrelationships, two (dextran and Congo Red) had good potency(EC50 = 0.4 lM) and no effects (LC50 >10 lM) on cell viability.Unfortunately, neither is suitable as a therapy because they donot traverse the BBB. Four drugs (ethoxazine, dihydroergotamine,acepromazine and amlodipine) had weak antiprion potency(EC50 = 4.88.2 lM) and no untoward effects on cell viability(LC50 >10 lM). Two drugs (carvedilol and tetrandine) showedmoderate antiprion potency in ELISA (EC50 = 2.2 lM) but alsoaffected viability (LC50 = 5.06.1 lM). Three others (fendiline,tamoxifen, and desloratidine) had weak antipotency in ELISA(EC50 = 4.36.7 lM) and a negative effect on cell viability(EC50 = 4.39.6 lM). The remaining four SPC hits tested were com-pletely inactive (EC50 >10 lM). Together, these data suggest thatnone of the FDA-approved drugs identied by our HTS efforts arelikely to be useful as therapies.

From HTS of 49,430 compounds from the ChemBridge andSPECS libraries using stationary-phase cells, we initially identied331 HTS hits (0.65% hit rate) representing 13 chemical leads. Fifty-ve of 321 were conrmed as SPC hits, representing three chemicalleads, including 11 piperazines, six indoles and six ureas, with theremaining 32 being singletons or duplicates (Table 5). Ten of 331

GuanidineHN

NH

NH 15

QuinolineN

24

QuinazolineN

N8

Benzamide NH

O

95

Benzyl ether

O

9

StilbeneAr

7

Totals:in the PrPSc assay

Compounds with EC50 10 lM). From testing the 1420FDA-approved drugs in stationary-phase assays, we identied sixHTS hits, none of which were conrmed by SPC.

3.3. SAR studies for PrPSc

From 14 leads identied in dividing ScN2a-cl3 cells and chosenfor follow up (Tables 2 and 3), an SAR-by-catalog strategy was ini-tiated. The rst set of 467 compounds was obtained, and their EC50values were measured to conrm potency and establish prelimin-ary SAR. We identied two leads (thienopyridine and benzamide)with 6 compounds having EC50 values of

ed. CB. M. Silber et al. / Bioorg. Mantiprion potencies. For these less active leads, it is possible thatthe analogs selected were suboptimal and do not represent thetrue potential of these leads.

Upon closer examination of the hits across different chemicalleads, several trends became evident. A conjugated aromatic orheteroaromatic ring system was prominent in all lead structures.This ring system is comprised of more than two aryl or heteroarylgroups joined in a fused or linear fashion. In the case of linear

Figure 3. Comparison of EC50 values from ELISAs and Western immunoblots individing ScN2a-cl3 cells (n = 3). (A) EC50 data (in lM) from ELISAs (abscissa) andWestern immunoblots (ordinate) show strong correlation for 28 compounds(R2 = 0.75; p

Table 3The specic structures for the 14 leads identied in dividing ScN2a-cl3 cells, with corresponding EC50 results from ELISA and Western immunoblots (nP3 for all compounds)

Lead (Murcko Fragment) Compound Structure EC50 SEM (lM)

ELISA Western

SN

NH2

IND30410S

NHN

Ph

O 0.25 0.07 0.19 0.06

IND30883S

NHN

O 0.99 0.02 0.57 0.18

N

S

IND24350NH

S

N N 0.27 0.04 0.83 0.35

IND24352

NH

S

N N 3.13 0.64 1.70 0.43

N

O

IND114202 N

OOMe

NH

O

BnO

0.07 0.01 2.57 1.51

IND114138 N

OOMe

NH

O

EtO

0.06 0.01 0.13 0.08

HN N

IND25807OMe

HN N

MeO

HO

0.07 0.03 0.10 0.07

IND28801OMe

HN NHO

0.26 0.02 0.12 0.02

SN

NH2

IND52025HN

OO

SN

NH2

MeO

0.08 0.03 0.06 0.01

IND52851 CNSN

NH2

O

O

0.98 0.08 0.52 0.11

N N

IND18629 ON N

N

2.94 0.55 0.89 0.30

IND19136 ON N

0.71 0.11 0.62 0.21

N

NIND9419

N

N0.71 0.22 0.29 0.11

IND9417N

N9.16 0.89 4.72 1.62

N

S NIND9416

N

S N1.78 0.39 0.52 0.16

IND9413 N

NS6.02 1.10 2.49 0.24

8006 B. M. Silber et al. / Bioorg. Med. Chem. 21 (2013) 79998012

ed. CTable 3 (continued)

Lead (Murcko Fragment) Compound Structure

B. M. Silber et al. / Bioorg. Mto dividing cells. Several possible reasons might explain thefewer HTS and conrmed SPC hits in stationary-phase cells.First, drug-resistant PrPSc may have formed due to division ar-rest or an effect of sodium butyrate independent of cell divi-sion. Second, our assay may have selected only for

HN

NH

NH IND26326 NN

NH

O

IND44723 NN

NH

O

N

IND45193

N

N

Cl

NMeN

IND44355

MeO

MeO N

N

NIND34921

Me2N NHN

N

IND35916

HNN

H2N N

NH

O

IND33145

MeO

O

NH

IND31751 NH

OOH

O IND46380

O

Br

O

Cl

IND34452 HOHN

Br

O

Ar IND47979

F

IND47971

HO

OEC50 SEM (lM)

hem. 21 (2013) 79998012 8007compounds that increase the clearance but do not decreasethe formation of PrPSc, which might result in different hits fordividing and stationary-phase cells. Finally, the cell line usedmay present variations that are responsible for thesedifferences.

ELISA Western

Cl

7.32 0.47 4.97 2.43

Br

2.58 0.63 1.45 0.13

H 1.36 0.10 2.07 1.17

OMe

NH2

OMe

2.56 0.25 1.83 0.07

Cl2.65 0.34 2.01 0.50

OMe 4.02 0.35 5.99 1.06

S

N0.40 0.05 0.16 0.04

OEt

0.13 0.04 0.04 0.01

HNOH

4.83 0.57 2.74 0.88

Cl

Cl

4.30 0.81 1.99 0.29

N

1.52 0.27 0.53 0.03

OH

H

3.06 0.19 0.58 0.16

Our previous study on the effect of cell division on prion accu-mulation provides a possible explanation for at least part of whatwe observed.51 In prion-infected cells, three competing processes

Figure 4. Distribution of antiprion potency in dividing ScN2a-cl3 cells by lead, expressed as a percentage of total compounds tested for each chemical lead (Y-axis). For eachlead, potency is grouped by EC50 values 10 (mg/L) >24.9Congo Red 0.45 0.06 >10 >22Carvedilol 2.28 0.27 6.10 1.55 2.67Tetrandine 2.15 0.72 5.01 0.42 2.33Ethoxazine 4.76c >10 >2.1Dihydroergotamine 6.44c >10 >1.55Acepromazine 6.93 1.45 >10 >1.44Amlodipine 8.22 0.75 >10 >1.22Fendiline 4.35 0.66 4.33 0.68 0.99Tamoxifen 4.39 0.33 4.89 0.17 1.11Desloratidine 6.66 0.56 9.56c 1.44Apomorphine >10 >10 ndb

Amiodarone >10 >10 ndHexadimethrine >10 >10 ndEnoxaparin >10 (mg/L) >10 (mg/L) nd

a Units are all lM, except those indicated by mg/L, since drugs are a mixture ofmolecular weights.

b nd, not determined.c Mean of two measurements; third replicate produced values >10 lM.

Table 5Reduction of PrPSc, cell viability, and calculated physicochemical parameters of 23 conrm

Compound Structure MW Reductio

Dividing

PrPSc V

Piperazine

IND7190 379.3 97 8

IND7486 328.8 84 6

IND17848 352.5 95 1

8008 B. M. Silber et al. / Bioorg. Med. Chem. 21 (2013) 79998012inuence prion accumulation: PrPC-to-PrPSc conversion, PrPSc deg-radation, and cell division. We had previously shown that for in-fected N2a cells, the process of cell division plays an importantrole in establishing the steady-state level of intracellular PrPSc. Asinfected cells divide, the accumulated PrPSc is distributed amongthe daughter cells, resulting in reduced PrPSc levels. Thus, cell divi-sion contributes to an elevated apparent rate of prion clearance incontinuously dividing cell cultures. A compound that inhibits prionformation without affecting its clearance will also appear moreefcacious in dividing cell lines, as both nascent PrPSc will be re-duced and existing PrPSc decreased during cell division. Conversely,in stationary-phase cells, PrPSc catabolism is the sole route of prionclearance since PrPSc will not be reduced by cell division underthese conditions.

The different ability of antiprion drugs to clear prions in divid-ing28,31 and stationary-phase cells has been demonstrated for quin-acrine.41 Whereas quinacrine rapidly cleared dividing cells of PrPSc,it was relatively ineffective in stationary-phase prion-infectedcells. Our results suggested that the reduced rate of clearance instationary-phase cells provides an opportunity for the formationof drug-resistant strains upon continuous exposure to quinacrine.

ed SPC hits in stationary-phase cellsna (%) Calculated physicochemical parametersb

Stationary ClogP PSA HDB/HBA QPCaco-2 QPlogBBB

PrPSc V

N N

34 12 3.64 37.1 0/5 768.1 0.542

31 12 3.67 36.8 0/5 785.8 0.435

41 24 3.81 42.3 0/5.8 767.9 0.167

Table 5 (continued)

Compound Structure MW Reductiona (%) Calculated physicochemical parametersb

Dividing Stationary ClogP PSA HDB/HBA QPCaco-2 QPlogBBB

PrPSc V PrPSc V

IND18785 330.4 85 30 30 20 3.56 36.8 0/5 785.8 0.475

IND19130 328.8 86 5 41 2 3.54 37.0 0/5 776.9 0.393

IND18721 328.5 78 6 55 10 4.24 36.2 0/5 765.6 0.033

IND19672 405 64 7 35 0.1 2.91 69.3 0/9 396.7 0.106

IND2118 382.5 76 25 51 24 5.19 29.6 1/4.5 1300.5 0.468

IND15461 405.3 22 4 48 3 4.46 34.1 1/4.5 1214.7 0.579

IND19847 282.8 42 6 51 3 3.64 7.9 0/3.5 2396.0 1.253

IND7046 373.3 67 14 39 2 3.74 36.8 0/5 785.8 0.447

Urea

IND5942 315.6 91 3 51 8 3.90 46.7 2/2 1717.3 0.327

IND8255 333.6 92 2 61 11 4.04 46.8 2/2 1720.5 0.388

IND8314 264.7 87 7 56 17 3.11 46.7 2/2 1717.7 0.094

IND10851 499.6 83 15 61 19 5.19 114.4 1/4 155.5 1.048

IND19237 299.1 25 2 66 26 3.60 46.7 2/2 1717.6 0.256

IND19238 349.1 17 3 63 9 4.35 46.0 2/2 2005.2 0.485

Indole

(continued on next page)

B. M. Silber et al. / Bioorg. Med. Chem. 21 (2013) 79998012 8009

ctio

d. CTable 5 (continued)

Compound Structure MW Redu

Dividing

PrPSc V

IND5381 282.4 27 12

IND15474 273.3 14 11

IND10094 326.4 81 19

IND4176 348.5 98 9

8010 B. M. Silber et al. / Bioorg. MeWhether the formation of drug-resistant strains in stationary-phase cells is a general phenomenon remains to be established.

One interpretation of the failure of quinacrine to extend sur-vival of prion-infected mice is that antiprion compounds need tobe potent in both dividing and stationary-phase prion-infectedcells in order to be effective in vivo in mice and patients with priondisease. Despite its lack of potency in stationary-phase cells (datanot shown), Compound B was potent in dividing cells and signi-cantly extended survival in prion-infected mice if drug treatmentwas started either immediately or soon after infection with RMLprions.37 However, Compound B is unlikely to be acceptable foruse in humans because it contains a phenylhydrazone moiety thatis metabolically unstable and could lead to reactive intermediatescausing potentially serious adverse effects, drugdrug interactions,or both.5254

One conrmed lead (AMT) was previously reported from ascreen of 10,000 compounds55 and this series continues to be pur-sued.56,57 Most of the initial AMT analogs described here (e.g.,IND30410 and IND30883) are commercially available. More ad-vanced leads from this series were synthesized using Hantzsch-type condensation between bromomethyl ketones and thioureasas previously reported.56,57

In the present work, we performed HTS assays of 53,000 di-verse chemical compounds in dividing ScN2a-cl3 cells, and discov-ered 3100 hits and 970 conrmed SPC hits (1.85% conrmed hitrate). The conrmed SPC hits generally derived from 14 distinctchemical leads and produced 50 compounds (either from the origi-

IND23308c 285.4 88c 30c

IND22103 229.2 81 3

The 23 hits represent three leads, shown with their respective Murcko fragments. Resula Percentage reductions of PrPSc and cell viability (V) measured by ELISA from SPC assa

potency; compounds reducing cell viability 630% are considered to have a safe effect.b Physicochemical parameters calculated using Qikprop (Schrdinger, New York, NY).c EC50 values were 1.58 lM and 7.5 lM in dividing and stationary-phase cells, respecna (%) Calculated physicochemical parametersb

Stationary ClogP PSA HDB/HBA QPCaco-2 QPlogBBB

PrPSc V

48 16 4.32 24.5 2/1.5 1527.6 0.630

39 9 2.01 70.6 2/5.4 776.4 0.795

46 21 5.60 16.9 1/1.5 1471.9 0.551

53 2 5.43 23.0 2/1.5 1696.6 0.610

hem. 21 (2013) 79998012nal screen or related analogs) with submicromolar potency (PrPSc

ELISA with EC50 10 lM) (Table 2). In our search for these hits, we used pre-dictions of bioactivity and a metric of dissimilarity58 to prioritizeour libraries by screening the top 53,000 of 180,000 compoundsavailable. This strategy successfully identied and conrmed manyleads in dividing ScN2a-cl3 cells, but fewer conrmed leads in sta-tionary-phase cells. Furthermore, SAR-by-catalog successfullyidentied many analogs that led to the identication of promisingnew leads: two of four compounds have shown to be absorbed or-ally and reach the brain at high concentrations.

In contrast to the 3100 HTS and 970 conrmed SPC hits in divid-ing cells, we discovered only 331 HTS and 55 conrmed SPC hits instationary-phase cells (0.11% SPC hit rate). The 55 conrmed SPChits included 23 analogs from the three chemical leads. Of these,only one indole analog (IND22308) had a large effect in HTS (70%inhibition) and SPC assays (94% inhibition; Table 5), and hadantiprion potency in both stationary-phase and dividing cells(EC50 = 7.5 and 1.6 lM, respectively) with no effect on cell viability.In contrast, when we tested ve of 55 conrmed SPC hits, whichare part of the 32 not representing any conrmed leads inconcentration-effect assays, they were inactive in the PrPSc ELISA(EC50 >10 lM). The reason for the lack of potency is unknown.

Whether the identication of lead compounds that inhibit PrPSc

formation or enhance its clearance in stationary-phase cells willmore predictive of drugs that are efcacious in mice and humansremains to be established. To probe why quinacrine was effective

94c 12c 3.45 31.7 2/3.5 289.4 0.684

37 21 2.53 53.4 1/2.8 2945.9 0.017

ts in dividing cells also shown.ys. Compounds reducing PrPSc levels byP30% are considered to have good antiprion

48,49

tively, and LC50 was >10 lM in both.

opy

ed. CTable 6Antiprion activity by ELISA (EC50) and cell viability by calcein (LC50) for selected thien

B. M. Silber et al. / Bioorg. Min ScN2a cells in suppressing PrPSc levels but ineffective in mice,we examined stationary-phase cultures. In those non-dividingcells, quinacrine was found to be ineffective in lowering PrPSc lev-els. In contrast, compound B was ineffective in stationary-phasecultured cells but doubled the incubation time in RML-infectedmice, arguing that stationary-phase cell cultures may not havesuperior predictive value over that of dividing cells. Clearly morestudies are needed to determine the utility of stationary-phase cul-tured cell lines in identifying hits and leads in the quest to developtherapeutics for CJD and other human prion diseases.

Compound R2 EC50 SEM (lM) LC50 (lM) C

IND35860 0.14 0.04 >10

IND37769 0.29 0.13 >10 I

IND35833 >10 >10 I

IND40541 >10 >10 I

IND37771 0.42 0.02 >10 I

Compound R1 EC50 SEM (lM) LC50 (lM) Compoun

IND35860 0.14 0.04 >10 IND24575

IND44746 0.24 0.02 >10 IND52025

IND42063 0.75 0.07 >10 IND24609

Table 7Maximum brain and plasma concentrations (Cmax) and area under the curve [area under thIND52851 after a single dose (10 mg/kg) administered orally to two FVB mice

Compound Matrix Cmax SD (lM)

IND45193 Brain 1.61 0.23Plasma 0.13 0.01

IND52851 Brain 1.02 0.71Plasma 0.41 0.25

Chemical structures shown in Table 3.ridine analogs in dividing ScN2a-cl3 cells (n = 3 for all compounds)

hem. 21 (2013) 79998012 8011Acknowledgments

This work was supported by grants from the National Institutesof Health (Nos. AG002132, AG010770, AG031220, and AG021601)as well as by gifts from the Fight for Mike Homer Program,Sherman Fairchild, Lincy, and Rainwater Charitable foundations.The authors thank Ms. Alejandra Acevedo, Ms. Josephine Lau, andMs. Darlene Groth for their contributions to the HTS assays;Mr. Manuel Elepano and Mr. Phillip Benner for dose preparation,animal dosing, and sample collection in pharmacokinetic studies;

ompound R2 EC50 SEM (lM) LC50 (lM)

ND24576 0.10 0.02 >10

ND24573 0.42 0.06 >10

ND24581 1.83 0.58 >10

ND42103 >10 >10

d R1 EC50 SEM (lM) LC50 (lM)

2.22 0.72 >10

0.06 0.01 >10

>10 >10

e curve values from rst to last measureable time points (AUClast)] for IND45193 and

AUClast SD (lM h) Brain/plasma AUClast ratio

7.36 0.78 4.721.56 0.312.12 0.93 2.590.82 0.31

8012 B. M. Silber et al. / Bioorg. Med. CMs. Ana Serban, Ms. Julia Becker, and Mr. Frederic Letessier for D13and D18 antibodies; Dr. Kurt Giles, Dr. Pierre Lessard, and the staffof the Hunters Point animal facility for expert animal studies; Mr.

Figure 5. Brain (squares) and plasma (circles) concentrations in mice after oraladministration of 10 mg/kg of IND52851 (a) and IND45193 (b). Chemical structuresfor these compounds are shown in Table 3. Data points and bars signify themean SD representing two mice at each time point. Each drug was given in aseparate experiment.Joseph Mulvaney of the SMDC for early discussions involving eval-uation and selection of compounds for HTS; Dr. Christian Laggnerfor helpful suggestions regarding purchased analogs for SAR; Dr.Elena Dolghih for calculation of physicochemical properties usingQikprop (Tables 3 and 5); and Ms. Hang Nguyen for editorial assis-tance. We are grateful to Daylight for the use of their ngerprinttoolkit (www.daylight.com) and to Molinspiration for a license touse their mib software (www.molinspiration.com). M.P.J. is a con-sultant to Schrdinger LLC.

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Antiprion compounds that reduce PrPSc levels in dividing and stationary-phase cells1 Introduction2 Materials and methods2.1 Materials2.2 Chemical library2.3 Compound selection2.4 Antiprion activity and cellular toxicity assays2.5 Western blot analysis of ScN2a-cl3 cells2.6 Data analysis2.7 Analyzing assay performance2.8 Structureactivity relationships2.9 Physicochemical parameters2.10 In vivo pharmacokinetic studies

3 Results3.1 Chemical libraries screened3.2 PrPSc HTS3.3 SAR studies for PrPSc3.4 Pharmacokinetics and brain distribution

4 DiscussionAcknowledgmentsReferences and notes