Anna Eriksson
email: anna.eriksson@medsci.uu.se

Martin Höglund
email: martin.hoglund@medsci.uu.se

Elin Lindhagen
email: elin.lindhagen@medsci.uu.se

Anna Åleskog
email: anna.aleskog@medsci.uu.se

Sadia Bashir Hassan
email: sadia.hassan@medsci.uu.se

Carina Ekholm
email: carina.ekholm@biovitrum.com

Karin Fhölenhag
email: karin.fholenhag@biovitrum.com

Annika Jenmalm Jensen
email: annika.jenmalm_jensen@orgfarm.uu.se

Agneta Löthgren
email: agneta.lothgren@biovitrum.com

Martin Scobie
email: martinscobie@tele2.se

Rolf Larsson
email: rolf.larsson@medsci.uu.se

Vendela Parrow
email: vendela.parrow@akinion.com

Aminopyrazine Tyrosine Kinase Inhibitor, With Significant Preclinical Activity In Acute Myeloid Leukemia

Keywords: acute myeloid leukemia, new drug development, tyrosine kinase inhibitor, FLT3

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A c c e p t e d M a n u s c r i p t 3

Introduction

Target based drug discovery has been widely used in the development of new therapies. The idea is to find potent and selective compounds, thus avoiding unwanted off-target effects [START_REF] Janne | Factors underlying sensitivity of cancers to smallmolecule kinase inhibitors[END_REF]. The approach has been successful in developing new drugs for cancer treatment, with imatinib mesylate against chronic myeloid leukemia (CML) being one of the most notable examples [START_REF] Druker | Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia[END_REF].

The success of this compound as antileukemic treatment is based on the inhibition of Bcr/Abl, a mutated tyrosine kinase that is expressed in the CML tumor cells and upon which the CML cell proliferation depends [START_REF] Fausel | Targeted chronic myeloid leukemia therapy: seeking a cure[END_REF]. Although targeted cancer therapy is an appealing approach, the clinical progress has been uneven and sometimes limited to a subset of patients. This has for instance been due to differences in the mutational status of oncogenes among patients or the acquisition of drug resistance [START_REF] Knight | Targeting the cancer kinome through polypharmacology[END_REF]. Thus, an optimal kinase inhibitor may be one that combines targeted inhibition with an effect on other kinases involved in proliferation and/or apoptosis [START_REF] Knight | Targeting the cancer kinome through polypharmacology[END_REF][START_REF] Apsel | Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases[END_REF].

Acute myeloid leukemia (AML) is a heterogeneous group of diseases distinguished by uncontrolled proliferation and maturation arrest of early clonal myeloid cells in the bone marrow, leading to decreased production of normal hematopoetic cells. Despite intense chemotherapy, sometimes including allogenic stem cell transplantation, the majority of patients ultimately succumb to their disease [START_REF] Lowenberg | Acute myeloid leukemia[END_REF]. There is an unmet need for new therapies to improve the outcome for AML patients. Thus, several new and more specific targeted antileukemic agents have been developed and are presently undergoing evaluation in clinical trials [START_REF] Soverini | Imatinib mesylate for the treatment of chronic myeloid leukemia[END_REF] [START_REF] Scholl | Deregulation of signaling pathways in acute myeloid leukemia[END_REF].

The FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (RTK) expressed on early hematopoetic progenitor cells, expression normally being lost upon differentiation. FLT3 is also present at high levels on tumor cells in the majority of acute leukemias [START_REF] Ozeki | Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia[END_REF]. Upon interaction with A c c e p t e d M a n u s c r i p t 4 its ligand, the FLT3 tyrosine kinase domain is activated, causing enhancement of cell proliferation and reduction of apoptosis. Activation of FLT3 affects downstream targets in the AKT pathway, STAT and MAPkinases [START_REF] Knapper | FLT3 inhibition in acute myeloid leukaemia[END_REF]. About 20-30 % of all AML patients display activating mutations in FLT3, resulting in constitutive activation of the receptor in absence of the ligand. Some of the FLT3 mutations are associated with increased relapse rate and reduced overall survival [START_REF] Kottaridis | The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials[END_REF][START_REF] Thiede | Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis[END_REF]. Several FLT3 inhibitors have been developed, some are presently being studied in clinical trials in AML [START_REF] Knapper | FLT3 inhibition in acute myeloid leukaemia[END_REF]. However, used as single agents, the clinical response of hitherto tested putative FLT3-inhibitors has in general been limited [START_REF] Small | FLT3 mutations: biology and treatment[END_REF][START_REF] Sanz | FLT3 inhibition as a targeted therapy for acute myeloid leukemia[END_REF], although recently published case reports suggest that occasional patients may be good responders [START_REF] Safaian | Sorafenib (Nexavar) induces molecular remission and regression of extramedullary disease in a patient with FLT3-ITD+ acute myeloid leukemia[END_REF].

In this study, we have used a screening funnel, combining a target based approach with sequential cellular screens, in order to find the optimal compound showing efficacy in both FLT3 wild type and FLT3 mutated AML, without targeting unrelated cells. With this approach, we identified AKN-032: a promising new antileukemic agent meeting these criteria and suitable for further development.

Materials and Methods

Compounds

Three different high throughput kinase inhibition screens of the Biovitrum's Compound library, directed against serine and threonine kinases, resulted in the identification of several chemical scaffolds with FLT3 inhibitory activity as an off-target effect. A subset of 150 molecules from the compound library was then selected as starting points in order to analyze FLT3 inhibitory activity. The compounds were designed to bind to the ATP-binding pocket. In the current study, we present one of these compounds, AKN-032 (for molecular structure, see fig 1a). For A c c e p t e d M a n u s c r i p t 6

Kinase inhibition panel screen and broad panel safety assessment

A 10 mM stock solution of AKN-032 was sent to Upstate Laboratories (NY, USA) for evaluation of kinase inhibitory activity over a panel of 31 kinases at 10 µM using homogeneous time resolved fluorescence assays (HTRF®) [START_REF] Mathis | HTRF(R) Technology[END_REF] according to Upstate standards. AKN-032 was also tested against a MDS Pharma broad selectivity safety assessment panel consisting of 71 different radioligand binding assays for receptors, ion channels and transporters (MDS Pharma Services, Taipei, Taiwan) at 1 and 10 µM according to MDS Pharma standards.

AKT and ERK protein kinase assay

A radiometric protein kinase assay, using FlashPlatesTM from Perkin Elmer (Boston, MA, USA) in a 50 μl reaction volume according the protocol of ProQinase, (33PanQinase® Activity Assay, ProQinase GmbH, Freiburg, Germany) was used for measuring the kinase activity of AKT1,2,3 and ERK1,2. Grade of inhibition of kinase activity (%) was determined after 60 minutes incubation with 10 and 1 µM of AKN-032. Staurosporine (kindly supplied by ProQinase) was used as a reference compound. (harboring a t(8:21) mutation) [START_REF] Asou | Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation[END_REF] and HL-60 (reported to be FLT3 negative and with ability to differentiate spontaneously) [START_REF] Dalton | HL-60 cell line was derived from a patient with FAB-M2 and not FAB-M3[END_REF][START_REF] Collins | Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture[END_REF][START_REF] Shankar | ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia[END_REF] AML cell samples were examined regarding FLT3 mutation status. For FLT3-internal tandem duplications (FLT3-ITD), genomic DNA was analyzed by PCR followed by capillary electrophoresis on an AB3130XL genetic analyzer using GeneMapper TM software (Life Technologies, Foster City, CA, USA) for fragment analysis. For FLT3-tyrosine kinase domain point mutations in codon D835 (FLT3-TKD), genomic DNA was analyzed by PCR as described previously [START_REF] Thiede | Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis[END_REF]. Clinical characteristics, FLT3 mutation status and karyotype of the patients are summarized in Table1.

Western blot

MV4-11 cells were incubated for 15 hours with one of six concentrations of AKN-032. Protein was extracted from cells using RIPA buffer supplemented with phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate and protease inhibitor cocktail (Santa Cruz Biotech, CA, USA).

Protein concentrations were measured by bicinchoninic acid protein assay [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF] and 1000 g protein from each sample was incubated with anti-FLT3-antibody (#sc-20733, Santa Cruz Tyr, #sc7020, Santa Cruz). A secondary antibody (conjugated with horseradish peroxidase (HRP)) was used for chemiluminiscense detection (Amersham ECL Plus detection kit, GE healthcare, Uppsala, Sweden). The membrane was stripped of antibodies and reprobed using a goat antibody against FLT3 (#F0550, Sigma-Aldrich Co) and a HRP-conjugated rabbit anti-goat secondary antibody (#P0449, Dako Sweden AB, Stockholm, Sweden).

The Fluorometric Microculture Cytotoxicity Assay

The fluorometric microculture cytotoxicity assay (FMCA), described in detail previously [START_REF] Lindhagen | The fluorometric microculture cytotoxicity assay[END_REF], is a method based on the measurement of fluorescence generated from hydrolysis of fluorescein diacetate (FDA) to fluorescein by cells with intact plasma membranes. FDA (Sigma-Aldrich Co)

was dissolved in DMSO and kept frozen (-20˚C) as a stock solution (10mg/mL) protected from light.

For screening of cytotoxic effect, dose-response plates containing compounds were prepared as described previously [START_REF] Rickardson | Screening of an annotated compound library for drug activity in a resistant myeloma cell line[END_REF]. In short, the compounds were transferred to 384-well microtitre plates in duplicate wells in ten-or fivefold serial dilution, starting from 100, 25 or 10 µM. A 1% DMSO control served as vehicle control. The plates were stored at -70˚C until further use. Cells were then seeded into the drug-prepared 384-well plates at varying density per well depending on cell type (AML: 25000 cells/well, NCI-H69 and H69AR: 2500 cells/well and all other cell lines: 5000 cells/well). Plates were incubated at 37˚C for 72 h, and cell density measured using the FMCA.

For accurate IC 50 determination, 3000-5000 MV4-11 cells/well were seeded in 50 µl culture medium supplemented with 10 or 20% heat-inactivated fetal calf serum into a 96-well plate.

A c c e p t e d M a n u s c r i p t 10 Threefold serial dilution from a DMSO stock of AKN-032, sunitinib or AB200434 was made in serum-free culture medium supplemented with penicillin and streptomycin and then added to the cells, 50 µl of the serial dilutions were added to the cell-suspension. The final concentration ranged from 5 µM to 0.8 nM, or from 500 nM to 0.08 nM, all wells containing 0,5% DMSO. A 0,5% DMSO control served as vehicle control. For comparison, the conventional AML drug cytarabine (Apoteket AB, Sweden) was serially diluted as described above, final concentrations ranging from 5 µM to 0.8 nM. The cells were incubated for 72 hours at 37˚C and the total number of viable cells then measured by use of the FMCA.

For the FMCA, cell survival after exposure to cytotoxic drugs is presented as survival index (SI, %) defined as fluorescence in test wells in percent of control cultures (cells in culture medium), blank values subtracted. FMCA was considered successful only when fluorescence signal in control cultures was >5x of mean blank values, a mean coefficient of variation in control cultures was < 30% and tumor cell fraction surpassed 70% after incubation.

Flow cytometry analysis

The effect of compounds on viability and proliferation was also analyzed using flow cytometry.

Apoptosis assay

A high content screening assay for measurement of apoptosis was used to examine death characteristics of cells exposed to AKN-032, the method has been described in detail previously [START_REF] Lovborg | Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828[END_REF][START_REF] Lindhagen | Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia[END_REF]. Briefly, 12 000 cells/well of AML cell lines MV4-11 and HL-60 were plated in flatbottom 96-well plates and exposed for 24 hours to 4 concentrations of AKN-032 in duplicate wells in tenfold serial dilution starting from 50 µM. A known apoptosis-inducing agent, etoposide (Apoteket AB), was used as a positive control at a concentration of 1 or 10 M. The FLICA probe FAM-DEVD-FMK (part of the CaspaTag apoptosis kit, Millipore, Solna, Sweden), which stains cells with activated caspases, was added one hour before the end of drug exposure at a final concentration of 20 M. Plates were washed in PBS, followed by fixation with 3.7% formaldehyde and 10 M Hoechst 33342 (Sigma-Aldrich Co) for 15 minutes and then washed twice again. Caspase activity was analyzed by use of the ArrayScan TM high content screening system (Cellomics Inc., Pittsburgh, PS, USA), described in detail previously [START_REF] Lovborg | Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828[END_REF], at least 800 cells were analyzed per well.

Pharmacokinetic studies

A solution of 3 mg/ml AKN-032, 10% hydroxypropyl-beta-cyclodextrin, 20 mM sodium acetate and 0.9 % sodium chloride was administered subcutaneously at a dose of 15 mg/kg to nine male C57 black mice (Scanbur, Sollentuna Sweden). Blood samples were collected at 0.08, 0.25, 0.5,

Hollow fiber

Cells from two AML patients (see table 1) and from the AML cell line MV4-11 were cultured inside semi-permeable polyvinylidene fluoride fibers [START_REF] Hollingshead | In vivo cultivation of tumor cells in hollow fibers[END_REF]. The fibers were sealed and incubated for two days before implantation subcutaneously into NMRI male mice (Scanbur). The day following implantation, subcutaneous twice daily administration of either 20 mg/kg or 40 mg/kg of AKN-032, or vehicle only (n=8 animals/group) was started. Body weight was determined daily in connection with the first drug administration. Drug administration lasted for six days, whereupon the fibers were retrieved and cell density evaluated using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)-assay [START_REF] Alley | Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay[END_REF]. This method is based on the conversion of MTT to blue formazan crystals by living cells. The formazan was extracted by DMSO as previously described [START_REF] Jonsson | Determination of drug effect on tumour cells, host animal toxicity and drug pharmacokinetics in a hollow-fibre model in rats[END_REF], and optical density (OD) read at 570 nm. Cell density for

Statistical analysis

The IC 50 values for FLT3 enzyme as well as for MV4-11 and 3T3L1 as measured by the flow cytometry analysis were determined using the equation (A+((B-A)/(1+((C/x)^D)))) where A equals min, B equals max, C equals IC 50 , D equals Hill slope and x equals compound concentration [START_REF] Marquardt | An algorithm for least-squares estimation of nonlinear parameters[END_REF]. ICfix50 equals the concentration of compound resulting in 50 % inhibition, in contrast to the IC50-value described by the equation, corresponding to the the inflection point of the dose-response curve.

For the FMCA, the cytotoxic IC 50 was determined from log concentration-effect curves in Graph Pad Prism (GraphPad software Inc., CA, USA) using non-linear regression analysis.

For comparison between groups in the in vivo hollow fiber studies, Student's t-test was used, p<0.05 was considered significant. ), which is in accordance with data reported in the literature [START_REF] Chow | Sunitinib: from rational design to clinical efficacy[END_REF]. In addition, western blot analysis showed that AKN-032 reduces autophosphorylation of the FLT3-receptor in MV4-

cells (fig 2c).

To evaluate the effect of AKN-032 on some of the targets downstream to FLT3; the compound was also evaluated in AKT and ERK protein kinase assay. No direct inhibition of AKT 1,2,3 or ERK 1,2 was seen at the test concentrations 1 and 10 µM (fig 2 d).

AKN-032 is highly cytotoxic to the AML cell line MV4-11 but not to normal fibroblast 3T3-L1 cells in vitro

A c c e p t e d M a n u s c r i p t 15 All compounds with an IC 50 of 300 nM and lower in the FLT3 enzyme inhibition assay were tested in a Guava cell kill assay against the FLT3 mutated AML cell line MV4-11. To find compounds with a preferred cell kill profile, agents displaying activity in the MV4-11 assay were studied in counter-screen using rapidly growing normal fibroblast 3T3-L1 cells. Sunitinib and AB200434, a compound with a IC 50 of 0.5 nM on the FLT3 enzyme [START_REF] Patel | Discovery of potent and specific FLT3 kinase inhibitors[END_REF], were used as reference compounds. Sunitinib showed cytotoxic effect on both MV4-11 and 3T3-L1, whereas AB200434

and in particular AKN-032, displayed a more MV4-11-specific activity (table 2). Compounds highly effective against MV4-11 but lacking in toxicity against the fibroblasts were taken forward in the screening program and tested against a broader panel of tumor cell lines and primary AML samples.

AKN-032 shows selective cytotoxic activity against AML cell lines MV4-11 and

Kasumi

AKN-032 is cytotoxic to primary AML-cells in vitro

Three of the initial 150 compounds, displaying the preferred cell kill profile described above, were tested in the FMCA against primary AML samples. Cells from 11 AML-patients; 6 mutated and 5 wild type with respect to FLT3 (table 1) were used, MV4-11 cells were used as positive control and sunitinib and AB200434 as reference compounds. All compounds tested were active in MV4-11, sunitinib and AB200434 being the most potent ones, but not all agents showed effect in patient cells. Primary AML cells were in general more resistant to all FLT3-inhibitors tested than MV4-11 and the IC 50 in the cell line did not predict efficacy in patient cells. Notably, the discrepancy between the two cell systems varied depending on the compound. The incongruity between cell systems for some of the other compounds, here exemplified by sunitinib, was much more pronounced in the order of 3 log units (fig 4b). In addition, for some compounds, here exemplified by AB200434, efficacy in MV4-11 cells did not translate to efficacy in primary AML cells (fig 4c). This was also the case for the two other FLT3-inhibitors from the Biovitrum compound library, displaying IC 50 :s of 52 nM and 62 nM on the MV4-11 cells but lacking efficacy in the primary AML samples (data not shown).

AKN-032 inhibits growth of MV4-11 cells and primary AML cells in vivo

To study the pharmacokinetic and cytotoxic properties of AKN-032 in vivo, C57 black mice were given 15 mg/kg of AKN-032 subcutaneously. The AUC was 13µM·h, maximum plasma concentration (C max ), was 18 µM (0.25 h after dose) and t ½ was estimated to 0.44 h. In the hollow-fiber study in NMRI mice, 6-8 fibers were available for evaluation of effect in each dosing regimen. The low dose (20 mg/kg) significantly inhibited net growth in MV4-11. At the higher dose (40 mg/kg), not only an inhibition of net growth, but a clear reduction of the leukemic mass was observed for the MV4-11 cell line (fig 5a). In the first AML sample, the total cell growth was very modest and no significant inhibition was observed. In the second AML sample, net growth was significantly inhibited in response to the higher dose of AKN-032 (fig 5a). The mice in the higher dose group of AKN-032 showed decreased food intake and reduced weight gain (p=0.05 vs. vehicle), whereas no difference in weight gain was observed in the low

Discussion

As a RTK only expressed in the hematopoetic system, with mutations and overexpression linked to disease, in particular AML, FLT3 is a valid target for drug discovery [START_REF] Zarrinkar | AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML)[END_REF]. Therefore, several attempts have been made to find FLT3 inhibitors of use in the clinical setting. Focus has been on optimizing potent enzyme inhibitors with good pharmacological properties. However, clinical effects have so far been limited. As for very potent but multitarget kinase inhibitors like sunitinib or lestaurtinib, the dose required to achieve cytotoxic response through inhibition of FLT3 autophosphorylation has in some cases been associated with toxicity, limiting the clinical dose [START_REF] Sanz | FLT3 inhibition as a targeted therapy for acute myeloid leukemia[END_REF]. Very selective compounds, on the other hand, may not be effective in the general AML patient, but might be useful in targeting a subset of AML-cells, where both proliferation and viability is dependant on high FLT3 activity [START_REF] Zarrinkar | AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML)[END_REF].

In this study, we have identified a new compound, AKN-032, by using a screening funnel combining target inhibition with sequential cellular screens and aimed at identifying compounds with a preferred cell-kill profile from a subset of kinase inhibitors. In this process, the cut off level, both on enzyme inhibition and efficacy in the MV4-11 cell assay was set relatively high.

About 150 compounds identified as hits in other kinase screens were selected for testing in a FLT3 enzyme inhibition assay, compounds with an IC 50 below 300 nM in this assay were then selected for further characterization. Inhibition of MV4-11 proliferation was chosen as the first cellular assay. This cell line has been used extensively when characterizing FLT3 enzyme The aim of this project was to find compounds with selective efficacy towards AML cells with and without FLT3-mutation. Consequently, AKN-032 and two other FLT3-inhibitors from the Biovitrum compound library, as well as the two reference kinase inhibitors sunitinib and AB200434, were tested for dose-response on 11 primary patient cells: 6 harboring mutations and 5 wild type with regards to FLT3 by using the FMCA. MV4-11 was again used as a reference cell line.

Sunitinib showed efficacy on all tumor samples tested, the IC 50 being three log units higher on patient cells than on the MV4-11 cell line. Surprisingly, the very potent and selective reference compound AB200434 did not show efficacy in the primary patient cells. This was also the case for the two other FLT3-inhibitors from the Biovitrum compound library with IC 50 :s of 52 nM and 62 nM respectively on the MV4-11 cells, but lacking efficacy in primary patient cells. In a cell line panel representing several different hematological and non-hematological malignancies, the newly synthesized tyrosine kinase inhibitor AKN-032 showed significant cytotoxic activity in the AML cell lines MV4-11 and Kasumi-1 as measured by the FMCA. In the third AML cell line tested HL-60, a cell line reported to be FLT3 negative [START_REF] Shankar | ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia[END_REF], a full cytotoxic response was achieved, but at noticeably higher concentrations.

As expected, IC 50 for AKN-032 in the FMCA cell kill assay was higher than in our FLT3 kinase assay. This may be due to pharmacokinetic factors related to the cellular models used, including uptake and efflux of the compound (data not shown). Still, the dose-response curves from the MV4-11 experiments followed a sigmoid pattern with an IC 50 of 0.4 µM, a value lower than the results observed for the AML standard drug cytarabine in the same experiment. Exploration of death characteristics show that the AKN-032-induced apoptosis in AML cell lines MV4-11 and HL-60 at least in part has its origin in the activation of caspase-3/7.

MV4-11 is an excellent model system for FLT3 mutated AML [START_REF] Quentmeier | FLT3 mutations in acute myeloid leukemia cell lines[END_REF], but as a cell line, it is likely to have acquired additional genetic changes in becoming immortalized and adapted for continual growth [START_REF] Kennedy | Investigating human leukemogenesis: from cell lines to in vivo models of human leukemia[END_REF]. Although being essential for investigation on mechanism of action of cytotoxic drugs and valuable tools in drug discovery, cell lines may have limitations when it comes to predicting clinical outcome [START_REF] Monks | Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines[END_REF][START_REF] Larsson | Prediction of cytotoxic drug resistance in acute leukemia[END_REF]. Primary patient tumor cells, on the other hand, provide a model well suited for preclinical prediction of tumor-specific activity and direction of phase II trials to suitable patients [START_REF] Nygren | Detection of tumor-specific cytotoxic drug activity in vitro using the fluorometric microculture cytotoxicity assay and primary cultures of tumor cells from patients[END_REF]. This prompted us to include efficacy on primary patient cells in In our primary tumor material, we observed no difference in sensitivity to AKN-032 between FLT3 mutated and wild-type AML cases. There are findings suggesting a role of the wild-type FLT3-receptor in AML [START_REF] Ozeki | Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia[END_REF], others proposing that a variance in quantitative expression of wildtype FLT3, regardless of mutation status, might explain different response to AKN-032 as well as to other putative FLT3-inhibitors [START_REF] Knapper | The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of cytotoxicity varies with dependence on FLT3 signaling in both FLT3-mutated and wild-type cases[END_REF]. Results from the AKT and ERK protein kinase assay show no direct inhibition of these kinases. This along with the cytotoxic effect of AKN-032 in the FLT3 negative AML cell line HL-60 might suggest additional mechanisms of cytotoxic activity of the compound. Studies investigating a possible correlation between levels of wild-type FLT3 expression and the response to AKN-032 as well as other mechanisms of action in AML are underway.

The hollow fiber assay allows studies of the effect of different substances in vitro as well as in vivo. Using the hollow fiber method in animal models makes it possible to study pharmacokinetics, tumor effects and hematological toxicity of anticancer drugs in the same animal [START_REF] Jonsson | Determination of drug effect on tumour cells, host animal toxicity and drug pharmacokinetics in a hollow-fibre model in rats[END_REF]. Thus, we were able to demonstrate an antitumoral effect in this relatively resistant no significant toxic effects were observed, however the animals in the higher dose group showed a reduction in weight gain. We speculate that this may be related to nausea with decreased food intake as a result.

As for pharmacokinetics, subcutaneous injections of AKN-032 in C57 black mice resulted in a short time to peak concentration and relatively short t ½ . Assuming linear pharmacokinetics and no strain differences, one can predict that twice daily injections in NMRI mice in the hollow fiber model result in a similar exposure to the compound to that observed in C57 black mice.

In conclusion, AKN-032 is a new tyrosine kinase inhibitor with significant in vitro cytotoxic activity in several different AML cell types, seemingly irrespective of FLT3-mutation status.

Results from the hollow fiber mouse assay demonstrate in vivo activity and suggest a favorable toxicity profile of the drug. Future preclinical studies will focus on pharmacokinetic properties and toxicity, as well as further clarifying the mechanisms of action of AKN-032 in AML. In vivo activity of low and higher doses of AKN-032 against AML cells (cell line MV4-11 and primary AML cells from two individual patients) in the mouse hollow fiber assay (n=6-8) (a).

Results are presented as net growth, defined as the percent change in cell density in the fibers during the 6 days of in vivo experiment (mean + SEM). Body weight development of the animals during the 6 days of the mouse hollow fiber assay (n = 6-8, mean ± SEM) (b).

Hematological profile in the animals after the hollow fiber study (c). Blood samples were analyzed on the day of collection (day 6) for the following parameters: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrite (HCT) and platelet count (PLT). Results presented as mean ± SEM.

  fig 1 b-c). The compounds were stored at -20 or -70˚C dissolved as a 10 mM stock in DMSO and

2. 5 M a n u s c r i p t 7 CCFR-

 57 Cell culture AKN-032 was tested against a panel consisting of 15 different cell lines. The panel included the myeloma cell line RPMI 8226/S and its sublines 8226/Dox40 and 8226/LR5 (kind gifts from WS Dalton, Department of Medicine, Arizona Cancer Center, University of Arizona, Tucson, AZ, USA), the lymphoma cell line U-937 GTB and subline U-937-vcr [19] (kind gifts from professor K. Nilsson, Rudbeck Laboratory, Uppsala, Sweden), the acute lymphocytic leukemia cell line A c c e p t e d CEM and subline CEM/VM-1 (kind gifts from WT Beck, Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, TN, USA), human retinal pigment epithelial cell line hTERT-RPE1 (Clonetech Laboratories, Palo Alto, CA, USA), the small-cell lung carcinoma cell line NCI-H69 and its subline H69AR, the renal adenocarcinoma cell line ACHN, the cervix adenocarcinoma cell line HeLa, and 3 AML cell lines: MV4-11 (expressing a naturally occurring 30 bp FLT3/ITD mutation in homozygous form) [20, 21], Kasumi-1

  Biotech), separated by NuPAGE (Invitrogen Ltd, Paisley, UK #LC5602), transferred to PVDFmembranes (Bio-Rad Laboratories, Sundbyberg, Sweden) and probed with primary antibody (p-A c c e p t e d M a n u s c r i p t 9

MV4- 11 11 (

 1111 or 3T3-L1 cells were seeded, treated with compounds in serial dilution and incubated as described for the accurate IC 50 determination. After 72 hours, 100 l viability reagent (Guava ViaCount, staining dead cells) diluted in PBS according to manufacturer's instruction, was added to each well in the drug-prepared 96-well plates, resulting in a total volume of 200 l. The 3T3-L1 cells were trypsinised and resuspended in 100 l serum free medium prior to addition of viability reagent. Number of viable cells was determined using Guava 96-well ViaCount assay A c c e p t e d M a n u s c r i p t Guava Technologies, CA, USA) according to manufacturer's instructions. Survival (%) was calculated as number of viable cells per ml compared to vehicle treated cells (0,5% DMSO) at the end of experiment.

A c c e p t e d M a n u s c r i p t 12 1

 12 , 2, 4, 8, 16 and 24 hours following injection (n=3 per time point, each mouse was sampled thrice) and plasma was prepared by centrifugation (~4000 rpm, 5 min). Plasma concentrations of AKN-032 were determined by protein precipitation and electrospray LCMS. Lower limit of quantification (LLOQ) was 0.003 µM. Pharmacokinetic parameters were calculated based on median plasma concentration versus time data using WinNonlin® software version 4.1 (Pharsight Corp., USA). The area under the plasma concentration versus time curve (AUC) was calculated by the linear trapezoidal method and the plasma half-life (t ½ ) was estimated from the slope of the curve between 0.25 and 4 hours. Animals were given food and water ad libitum throughout the experiment. The animals were euthanized by terminal bleeding from vena cava, under sedation with isoflurane. The study was approved by the Animal Ethics Committee in Northern Stockholm (no N94/04).

A c c e p t e d M a n u s c r i p t 13 each

 13 fiber on retrieval day was expressed as net growth, defined as (OD retrieval day -OD implantation day ) / OD implantation day, i.e. the percent change in cell density in the fibers during the trial. The animals were observed regarding behavior and weight gain throughout the experiment. After six days of drug administration, 200 µl blood samples were obtained through the orbital plexus after anaesthetization with isofluran just before euthanasion by cervical dislocation. The blood samples were analyzed for hematological parameters using Micros 60 (ABX Diagnostics, Montpellier, France). Animals were caged four in each cage and fed a commercial diet (Lactamin AB, Sweden), water was given ad libitum. The study was approved by the Animal Ethics Committee in Uppsala (no C243/6).

  -032 is inhibitory to the FLT3 enzyme AKN-032 was originally identified as a hit in one of three different high throughput kinase inhibition screens of approximately 100 000 compounds from the Biovitrum compound library, performed at Biovitrum AB. The three screens identified chemical scaffolds with FLT3inhibitiory potential and approximately 150 compounds were selected for further analysis and verification of the FLT3 inhibitory activity. A subset of these hits, including AKN-032, was further characterized in a panel screen of 31 kinases at 10 µM. Inhibitory activity for AKN-032 was most pronounced against FLT3 kinase (data not shown). Screening data from other kinases tested (ex. PDGF-R, CK1delta, TRK A/B) indicated IC 50 values low µM range (data not shown). The 150 compounds identified as potentially inhibitory to FLT3 were tested in a dose-response enzyme inhibition assay. The IC 50 value of AKN-032 on the FLT3 enzyme was determined to be 70 nM (fig 2a). Sunitinib was used as a positive control, typically showing an IC 50 value of 0.5 nM (fig 2b

- 1

 1 Of the 15 cell lines tested in the FMCA, MV4-11 was by far the most sensitive to AKN-032 with an IC 50 value of 0.4 µM (fig3a), followed by the AML cell line Kasumi-1 (IC 50 = 2.3 µM). The corresponding IC 50 value for the conventional AML drug cytarabine was 1.7 µM in MV4-11 (fig3a). In HL-60, full cytotoxic response with no surviving cells was achieved, but at considerably higher concentrations than for the other two AML cell lines. In the other cell lines tested, including parental cell lines and drug resistant sublines, AKN-032 displayed cytotoxic activity only at high concentrations (fig3b). No difference between the parental cell lines and their respective drug-resistant sublines was observed.

A c c e p t e d M a n u s c r i p t 16 Potential

 16 off-target effects were studied in a broad selectivity safety assessment panel consisting of 71 different assays for receptors, ion channels and transporters, in which AKN-032 revealed no activity (data not shown).3.4 AKN-032 induces apoptosis in MV4-11 and HL-60 cellsThe flow cytometry analysis showed the presence of dead cells even after exposure to low concentrations of AKN-032 in MV4-11 cells (fig 3c). To further explore the mechanism of cell death induced by AKN-032, cells from the AML cell lines MV4-11 and HL-60 were incubated for 24 hours with different concentrations of the compound and then analyzed in a high content screening assay for measurement of apoptosis. Incubation with AKN-032 caused activation of caspase-3/7 accompanied by fragmentation of the nuclei (fig 3d), indicating that the caspase 3pathway is active in the AKN-032-induced apoptosis in AML cell lines.

A c c e p t e d M a n u s c r i p t 17 Out

 17 of the compounds tested, AKN-032 was selected for further evaluation due to high cytotoxic activity in the primary tumor cells. No difference between FLT3 wild type and mutated cases (meanIC 50 values of 8.5 M and 7.2 M respectively) was observed. The dose-response curve was right-shifted by 1 log unit for the primary tumor samples as compared to MV4-11 (fig4a).

A c c e p t e d M a n u s c r i p t 18 dose

 18 group (fig 5b). As shown by blood counts analyzed on the day of fiber retrieval, no major hematological toxicity was observed in the animals (fig 5c). No organ effects were seen upon macroscopical inspection post mortem.

A c c e p t e d M a n u s c r i p t 19 inhibitors

 19 both in cell-kill assays and in xenografts[START_REF] Shankar | ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia[END_REF][START_REF] Auclair | Antitumor activity of sorafenib in FLT3-driven leukemic cells[END_REF][START_REF] O'farrell | SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo[END_REF]. Compounds with an IC 50 below 400 nM in the MV4-11 cell line were then tested in another cellular assay, using the rapidly proliferating 3T3-L1 fibroblast cell line as a counter-screen. Only compounds showing better efficacy on MV4-11 than on 3T3-L1 cells were brought to the next level of screening. The aim of this counter-screening step was to exclude compounds with a multitarget kinase inhibitory profile. Several very potent compounds, here exemplified by the reference compound AB200434, but also less potent compounds like AKN-032 displayed the preferred cell kill profile. These compounds were then taken to the final level of cellular screening.

  Unexpectedly, we found AKN-032, a FLT3-inhibitor with an IC 50 of 70 nM in the FLT3 enzyme inhibition assay, inhibiting autophosphorylation of FLT3 in MV4-11 cells and with a relatively high IC 50 in the MV4-11 flow cytometry cell proliferation assay, to be more potent on the A c c e p t e d M a n u s c r i p t 20 primary patient cells than for example AB200434. Thus AKN-032 fulfilled the criteria set out in our screening funnel, despite not being the most potent compound.

A c c e p t e d M a n u s c r i p t 21 our

 21 screening funnel, including both FLT3 mutated and wild type AML samples. Our results confirmed that AKN-032 has significant in vitro activity in AML. The more prominent effect (i.e. lower IC 50 ) in the cell line assay reflects a high proliferation rate making the cells more sensitive to cytotoxic drugs in general. The effect of AKN-032 in rapidly growing cell lines like MV4-11 is caused by a combination of absent proliferation and cell death, as shown by the Guava ViaCount assay as well as the caspase activation assay. In contrast to cell lines, most primary AML tumor cells are low proliferating in vitro and the efficacy of AKN-032 in these cells may to a greater extent be caused by cell death.

A c c e p t e d M a n u s c r i p t 22 model

 22 , suggesting a good distribution of AKN-032 to subcutaneous tissue in mice. Importantly,

A c c e p t e d M a n u s c r i p t 23 The

 23 following authors were employees of Biovitrum AB when these experiments were performed or when this manuscript was written and revised: C. Ekholm, K. Fhölenhag, A. Jenmalm Jensen, A. Löthgren, M. Scobie, V. Parrow. V. Parrow is now primary affiliated to and part owner of Akinion Pharmaceuticals AB. M. Scobie is primary affiliated to Orexo AB and A. Åleskog works as medical advisor for Merck Sharp and Dome AB. C. Ekholm is a minor stockholder of Biovitrum AB.

  Fig 1 a-c

Fig

  Fig 5 a-c

Fig

  Fig 3a-d

Figure

  Figure

Fig

  Fig 4a-c

Fig

  Fig 5a-c

Table 2 Inhibitory activity of AKN-032, sunitinib and AB200434 in MV4-11 and 3T3-L1 normal fibroblast cells evaluated by flow cytometry, indicated by IC 50 values (± (SD), n = number of experiment repeats)

 2

	Compound	Cell line IC50 (nM) SD (nM)	N
	AKN-032	MV4-11	307	103	38
		3T3-L1	>5000	NA	11
	Sunitinib	MV4-11	1,5	1,2	13
		3T3-L1	2,2	0,7	6
	AB240034	MV4-11	1	1,4	14
		3T3-L1	44	23,6	7

Table A c c e p t e d M a n u s c r i p

Acknowledgement

We thank Mrs. Lena Lenhammar, Ms Christina Leek, Dr. Monica Hermansson, Dr Christina Kaiser and MSc Kerstin Larsson for skillful technical assistance, Prof. Richard Rosenquist for valuable comments on the manuscript and Prof. Anders Wahlin and assoc. Prof Mats Brune for providing patient samples. This study was supported by Biovitrum AB and Erik, Karin and Gösta Selanders Research Foundation.

Conflict of interest

A c c e p t e d M a n u s c r i p t Graphical Abstract


Deuan C Jones

Irene Hallyburton

Laste Stojanovski

Kevin D Read

Julie A Frearson

Alan H Fairlamb
email: a.h.fairlamb@dundee.ac.uk

Identification of a kappa-opioid agonist as a potent and selective lead for drug development against human African trypanosomiasis

Keywords: Phenotypic screening, African trypanosomiasis, Target identification, Target validation, U50,488

A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 "library of pharmacologically active compounds" against bloodstream forms of T. brucei in vitro identifying 33 compounds with EC 50 values < 1 M. Counter-screening versus normal diploid human fibroblasts (MRC5 cells) was used to rank these hits for selectivity, with the most potent (< 70 nM) and selective (> 700-fold) compounds being suramin and pentamidine. These are well known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)-trans-(1R,2R)-U50,488 having an EC 50 value of 60 nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (-)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were > 70-fold less active or inactive, as were several and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.

gambiense is responsible for the chronic form of the disease found in western and central Africa, accounting for over 90 % of reported cases of the disease, whereas T. b. rhodesiense is responsible for the more acute form of the disease located in eastern Africa [START_REF] Garcia | Human African trypanosomiasis: connecting parasite and host genetics[END_REF]. Only 10-15% of the 60 million people at risk of the disease are under surveillance [START_REF] Garcia | Human African trypanosomiasis: connecting parasite and host genetics[END_REF] and the death rate is currently estimated at 30,000 per annum [START_REF] Stuart | Kinetoplastids: related protozoan pathogens, different diseases[END_REF]. T. b. brucei and other Trypanosoma spp are responsible for related veterinary diseases of economic importance, such as nagana in cattle.

Of the four drugs that are currently registered for use against HAT, pentamidine and suramin are used against the early stage of the disease; and melarsoprol and eflornithine (difluoromethylornithine, DFMO) are used against the late stage of the disease, when the infection has spread to the central nervous system (CNS). These treatments are beset with problems such as difficulties in administration (none are given orally), cost, duration of treatment, toxicity and resistance [START_REF] Renslo | Drug discovery and development for neglected parasitic diseases[END_REF]. Melarsoprol treatment is highly toxic and responsible for iatrogenic deaths in 5% of patients. Eflornithine therapy is only effective against T.b.gambiense infections and presents severe economic and logistical problems in resource poor settings due to the need for 4 daily intravenous infusions over 14 days [START_REF] Pépin | The treatment of human African trypanosomiasis[END_REF]. A recent clinical trial suggests the duration and frequency of treatment can be reduced by combination of eflornithine with nifurtimox [START_REF] Priotto | Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial[END_REF], which may act as an interim solution until better and safer drugs are developed. The recent failure of the first orally active first stage drug, pafuramidine (DB289) [START_REF] Thuita | Efficacy of the diamidine DB75 and its prodrug DB289, against murine models of human African trypanosomiasis[END_REF], and rumours of increasing failures with eflornithine therapy underlines the urgent need for novel therapeutics.

Whilst many dominant paradigms of drug discovery focus on screening against molecular targets [START_REF] Frearson | Target assessment for antiparasitic drug discovery[END_REF], there has been a resurgence of interest in phenotypic screening against A c c e p t e d M a n u s c r i p t 4 whole parasites [START_REF] Mackey | Discovery of trypanocidal compounds by whole cell HTS of Trypanosoma brucei[END_REF]. Phenotypic screening, particularly when in combination with a mammalian counter-screen, has the distinct advantage of addressing key druggability and toxicity issues early in drug discovery, thereby reducing attrition at later stages in development. In some cases phenotypic screening may identify novel molecular targets thereby accelerating drug development. However, understanding the mode of action of phenotypic screening hits can prove challenging since many drugs act by modulation of multiple intracellular targets ("network pharmacology") [START_REF] Hopkins | Network pharmacology: the next paradigm in drug discovery[END_REF]. Hit identification for novel targets is best approached with a screening library of diverse chemical space, but whole cell assays typically have a much lower throughput than molecular-target-based screens. Hence, we chose to screen a library of known pharmacologically-active compounds against T.brucei cultured in vitro and to counterscreen actives against a human fibroblast cell line (MRC5 cells) to eliminate non-selective inhibitors. Potent and selective hits from such an approach can present exploitable shortcuts, particularly if they have already been used in humans with known dosing and toxicity information. Developing these hits could represent a low-risk, lowcost strategy for tackling orphan diseases of the poor [START_REF] Frearson | Target assessment for antiparasitic drug discovery[END_REF].

The Library of Pharmacologically Active Compounds from Sigma-Aldrich (LOPAC 1280; international version) contains 1268 compounds that are ligands for many enzymes, receptors and ion channels in other organisms. Many are drug-like molecules and some are CNS activean important consideration when seeking a replacement therapy for late-stage trypanosomiasis. Here we report over 30 compounds with EC 50 values against T.brucei less than 1 M, a concentration that should be readily achieved in plasma. Of these, one third have >20-fold selectivity with the -opioid receptor agonist U50,488 showing the greatest potency and selectivity. Some preliminary structure-activity relationships of CNS-active and opioid receptor agonists and antagonists are reported.

Chemicals and materials

The LOPAC 1280 library (international version) was purchased from Sigma-Aldrich (Gillingham, UK). Pentamidine isethionate was obtained from Research Biochemicals International, eflornithine was a gift from Merrell Dow Research Institute (Ohio, USA) and melarsoprol a gift from Rhone-Poulenc (France). U69593, U5449A and naloxone were obtained from Alexxis Biochemicals (Nottingham, UK). Norbinaltorphimine, (-)-U50,488,

(+)-U50,488, naltrexone, DIPPA (2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothio- cyanatophenyl)-2-(1-pyrrolidinyl)ethyl]
acetamide hydrochloride) and 4-P-PDOT (cis-4phenyl-2-propionamidotetralin) were obtained from Tocris Bioscience (Avonmouth, UK). DMSO was purchased from VWR international and HPLC-grade methanol and acetonitrile from Fluka. Resazurin, thioglycerol and PEG400 were obtained from Sigma-Aldrich (Gillingham, UK). Sterile 96-well plates were obtained from Greiner Bio-one (Stonehouse, UK).

Trypanosome culture

Bloodstream form T. brucei brucei cells (strain 427, "single marker") were grown at 37 o C and 5% CO 2 in a modified HMI9 medium [START_REF] Hirumi | Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers[END_REF] (HMI9-T where 0.2 mM 2-mercaptoethanol was replaced with 0.056 mM thioglycerol). Stock cultures were maintained in T75 vented cap culture flasks (Greiner, Stonehouse, UK) and sub-cultured every 2 to 3 days by 500-fold dilution into fresh medium. For microtitre plate assays, cells were counted using a Casy cell counter TT (Sharfe systems) and diluted appropriately.

Mammalian cell culture

Linearity of assay

T. brucei bloodstream-form cells and human fibroblasts were pipetted into 96-well plates to give a range of densities in a total volume of 200 µl culture medium. Resazurin (20 µl of a 500 µM stock; 45.5 M final concentration) was added immediately to T. brucei cells or the next day for human fibroblasts, following incubation overnight to allow attachment to the plastic plates. Fluorescence was then measured at intervals up to 5 h (excitation of 528 nm and emission of 590 nm) on a Biotec Instruments FLX 800 fluorescent plate reader.

DMSO-tolerance of the assay

DMSO was serially diluted across a 96-well plate in 200 µl of the appropriate media. For T. brucei, 100 µl aliquots were then transferred to a plate containing 100 µl cells at 2 x 10 3 ml -1 .

A c c e p t e d M a n u s c r i p t 7 Human fibroblasts were plated at 2 x 10 4 ml -1 in 100 l of medium and incubated overnight to allow cells to adhere before the addition of medium containing DMSO (100 µl) over a range of concentrations. Parasites and mammalian cells were then incubated for 3 days, after which 20 µl 500-μM resazurin was added to each well. Plates were incubated for a further 4 h before measuring fluorescence as above.

EC 50 determinations against T. brucei

Test compounds were dissolved in DMSO at 20 or 10 mM except for eflornithine which was dissolved in water and sterilised by filtration. The control drug (pentamidine) was dissolved in DMSO at 100 µM. HMI9-T media (148.5 µl) was added to column 2 (B2-G2) of a sterile 96well culture plate. HMI9-T +1 % DMSO (100 µl) was added to all remaining wells. Test compound solutions (1.5 µl) were added to column 2 (B2-G2). Pentamidine was placed on row G of all plates as a control. Three-fold serial dilutions were carried out by transferring 50 µl from column 2 to the adjacent column (100 µl). The process was repeated up to column 10. HMI9-T containing 2 x 10 3 trypanosomes ml -1 (100 µl) was added to all wells except column 1. HMI9-T (100 µl) was added to column 1. Column 1 and column 11 served as controls without cells and without test-compound respectively. Cells were incubated for 3 days, after which 20 µl 0.5 mM resazurin was added to each well, before measuring fluorescence after 4 h incubation. Data were processed using GRAFIT (version 5.0.4; Erithacus software) and fitted to a 3-parameter equation, where the data are corrected for background fluorescence, to obtain the effective concentration inhibiting growth by 50% (EC 50 ):

s i y y           50 m ax EC 1 (1)
A c c e p t e d M a n u s c r i p t 8 where y max is the uninhibited fluorescence value, i the inhibitor concentration and s is the Hill slope of the curve.

Measurements for each compound were carried out on 3 separate occasions and the mean weighted to the standard error calculated using the following formulas, where "a" is the standard error of EC 50 determination "A" etc.

) / 1 ( ) / 1 ( ) / 1 ( ) / ( ) / ( ) / ( 2 2 2 2 2 2 c b a c C b B a A mean weighted      ) / 1 ( ) / 1 ( ) / 1 ( ) / 1 ( ) / 1 ( ) / 1 ( 2 2 2 c b a c b a error     
(2)

Production of LOPAC daughter plates.

Latch-racks containing the LOPAC library at 10 mM dissolved in DMSO were stored frozen at -20 o C. Racks were thawed at room temperature for 3 h and centrifuged in a Beckman CS-15R centrifuge at 1,100g for 2 min to return any condensation on the lids to the solution.

Racks were then mixed vigorously for 5 min on a platform shaker. Aliquots (50 µl) of each rack were transferred to v-bottomed polypropylene 96-well plates (Matrix) using a Janus liquid handler (Perkin-Elmer) to create a primary daughter-set. DMSO (98 µl) was plated out to columns 2-11 of fresh v-bottom polypropylene 96-well plates using a Precision 200 liquid handler (Biotek). An aliquot (2 µl) of the primary daughter set was transferred to the DMSOcontaining plates using a Platemate 2X2 liquid handler (Matrix) to create secondary daughter sets at 200 µM. At this stage, 50 l of 200 µM pentamidine was added to wells E1-H1, 50 µl DMSO was added to the remaining wells of column 1 and 12 for use as control wells.

Duplicate daughter sets (set A and set B) created in this way were used for two independent screening runs to control for errors in liquid handling. Daughter sets were sealed using a nitrogen-flushing heat sealer (K Biosystems) and stored at -20 o C.

A c c e p t e d

M a n u s c r i p t 9

Screening of the LOPAC library against T. brucei

Daughter sets were thawed and mixed thoroughly, before 1 µl was transferred to an empty 96well tissue-culture plate (Greiner) using a Platemate 2X2 liquid handler (Matrix). Plates were transferred to a tissue culture hood and 180 µl media added to each well using a WellMate dispenser (Matrix). HMI9-T (19 µl) was added to wells A1 -D1 and 19 µl media containing T. brucei (1.05 x 10 4 ml -1 ) was added to all other wells using a Imact2 pipettor (Matrix).

Wells A1-D1 served as background (no cell) controls; wells E1-H1 served as pentamidine controls (1 M); and column 12 served as a full signal control (cells, no compound). Plates were incubated for 3 days after which 20 µl 0.5 mM resazurin was added to each well. Plates were incubated for a further 4 h before measuring fluorescence as above. Fluorescence signal for each well was background subtracted and expressed as percentage of the full signal controls. Z-prime (Z′) values were calculated for all plates using the following equation [START_REF] Zhang | A simple statistical parameter for use in evaluation and validation of high throughput screening assays[END_REF]:

Z′ = ) ( ) ( ) ( 3 ) ( 3 1 signal background signal full signal background signal full        (3)
where  and  represent the means and standard deviations, respectively 2.9 EC 50 determinations against mammalian cells.

Compounds with submicromolar EC 50 values against T. brucei were tested against human fibroblasts as follows. Cells were detached, counted as described above and diluted to 2 x 10 4 ml -1 . Cells (100 µl) were added to columns 1 to 11 and 100 µl media was added to column 12 of a 96 well plate. The plate was then incubated for 24 h to allow attachment. Test compounds and Doxorubicin as a control, were serially diluted in DMSO in a 96-well poly-propylene Vbottomed plate using a Janus liquid handler (Perkin-Elmer). Samples from the test compound dilution series and QC plates (2 µl) were transferred to duplicate tissue culture 96-well plates using a PlateMate 2X2 liquid handler (Matrix). These plates were transferred to a tissue

D C 1 A B           x A y ( 4
)
Where A is the minimum y-value; B is the maximum y-value; C is the EC 50 ; and D is the Hill slope. All test compound curves had floating maximum and minimum and pre-fit was used for all 4 parameters.

Homology searching and protein alignments

Sequences for κ-opioid receptor protein were retrieved from the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) for sheep (accession AAY66995), zebrafish (accession NP_878306), rough skinned newt (accession AAU15126), mouse (accession NP_035141 and AAA56759) and guinea pig (accession AAA67171). Global alignments were carried out using ClustalW and T. brucei GeneDB was searched using the TBLASTN algorithm [START_REF] Altschul | Basic Local Alignment Search Tool[END_REF].

Effect of (+)-U50,488 on cell growth.

Flasks of T. brucei were seeded at 1 x 10 4 cells ml -1 and incubated in the presence of the known inhibitors pentamidine and eflornithine, and the LOPAC hit (+)-U50,488 for 3 days. determined at intervals using a haemocytometer and generation times calculated using GraFit (version 5.0.13; Erithacus software) using the following equation:

g t N N / 0 2  (5)
where N 0 and N are the number of cells at time zero and time t, respectively, and g is the time per generation. For low cell densities, samples were concentrated 150-fold by centrifugation and resuspension in an appropriate volume of medium.

DMPK and efficacy studies

All animal experiments were carried out following local ethical review and under UK regulatory licensing in accordance with the European Communities Council Directive (86/609/EEC). NMRI outbred mice were purchased from Harlan laboratories, UK.

In brief, a 96-well equilibrium dialysis apparatus (HT Dialysis LLC, Gales Ferry, CT) was used to determine the free fraction in plasma for (+)-U50,488. Membranes (12-14 kDa cutoff) were conditioned in deionised water for 60 min, followed by conditioning in 80:20 deionised water:ethanol for 20 min, and then rinsed in isotonic buffer before use. Frozen female mouse plasma was thawed, centrifuged (2100g, 10 min), spiked with (+)-U50,488 (10 µg g -1 ), and 150-μl aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate. Dialysis against isotonic buffer (150 l) was carried out for 5 h at 37ºC using an orbital microplate shaker at 125 revolutions min -1 . At the end of the incubation period, aliquots of plasma or buffer were transferred to Micronic tubes (Micronic B.V., the Samples were allowed to mix for 1 min and then centrifuged at 2100g in 96-well blocks for 10 min. All samples were analysed by UPLC/MS/MS on a Quattro Premier XE Mass Spectrometer (Waters Corporation, USA) and the unbound fraction determined as the ratio of the peak area in buffer to that in plasma.

To determine brain penetration, (+)-U50,488 was administered intravenously to female NMRI mice (n=3) at a dose level of 2mg kg -1 (freshly prepared in 5% DMSO v/v in sterile saline). A blood sample (50 µl) was collected by cardiac puncture from each animal into Micronic tubes containing deionised water (100 µl) at 5 min post-dose and the brain of each animal removed into tared Covaris glass tubes. Blood and brains were stored at -80°C until analysis.

To determine exposure, (+)-U50,488 was administered by oral gavage using a 20gauge feeding tube fitted with a 1-ml syringe to female NMRI mice (n=3) at a dose level of 150 mg kg -1 (freshly prepared in DMSO, PEG400, milliQ water (1:8:11 v/v respectively)).

Blood samples (10 µl) were collected from the tail vein of each animal into Micronic tubes containing deionised water (20 µl) at 0.25, 0.5, 1, 2, 4, 6 and 8 h post-dose and stored at -80°C until analysis.

For analysis, brain tissue was homogenised in 50% v/v methanol in deionised water (1:2 w/v) using a Covaris S2 (K Biosciences, Hoddesdon, UK). Extraction of blood and brain samples was then performed by a method based on protein precipitation using acetonitrile containing a structural analogue of (+)-U50,488 as an internal standard. Levels of (+)-U50,488 were determined in these extracted samples by UPLC-MS/MS on a Quattro Premier XE mass spectrometer (Waters corporation, USA). A calibration curve was constructed in For the efficacy study, groups of three female NMRI mice were infected with the bloodstream form of T. brucei brucei (strain 427) by a single intraperitoneal injection of 10 4 parasites in 0.2 ml HM19-T medium. Three days following infection, mice were either dosed by oral gavage with (+)-U50,488 (150 mg kg -1 , freshly prepared in DMSO, PEG400, milliQ water (1:8:11 v/v respectively)) or left untreated. Dosing of (+)-U50,488 treated mice was then repeated in an identical manner every 4 h for a further 32 h. Thereafter, animals were inspected daily for clinical signs of infection and wet smears of tail blood were examined microscopically as appropriate. Parasitaemia was determined using a Neubauer haemocytometer and mice were monitored for 30 days. Mice that exceeded a parasitaemia >10 8 ml -1 were humanely killed since prior experience indicated that animals would succumb to an overwhelming infection by the following day.

Linearity of the resazurin cell viability assays

Alamar Blue has been used previously as an in vitro viability assay for T. brucei [START_REF] Raz | The Alamar Blue® assay to determine drug sensitivity of African trypanosomes (T.b.rhodesiense and T.b.gambiense) in vitro[END_REF]. This proprietary reagent contains resazurin [START_REF] O'brien | Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity[END_REF], a dye which is metabolically reduced in cells to the highly fluorescent product resorufin. Using the less expensive and widely available reagent resazurin, the linearity and dynamic range of the assay was determined for parasite and human fibroblasts. To determine the dynamic range of the assay, bloodstream forms of T.brucei were incubated at 37 C in culture medium at a range of cell densities containing 440 M resazurin and fluorescence measured at intervals. For each time point fluorescence was proportional to cell density with linear correlation coefficients (r 2 ) >0.997 (Fig. 1A).

Likewise, fluorescence increased linearly with time at any cell density (Fig. 1C, r 2 >0.996).

Thus fluorescence is proportional to cell density up to 2×10 5 cells per well within a 2 to 5 h incubation period. Similar results were obtained for human fibroblasts (Fig. 1B and1D) up to 3×10 4 cells per well within a 2 to 6 h incubation. In further assays, final cell densities and resazurin-incubation times did not exceed these ranges.

DMSO-tolerance of the resazurin cell viability assays

Test-compounds were supplied as 10 mM stocks dissolved in DMSO and so it was necessary to determine the tolerance of the assays for this solvent. Both cell lines showed a similar sensitivity to DMSO with IC 50 values of 0.80  0.06 and 1.04  0.08 % (v/v) for T.brucei and human fibroblasts, respectively (data not shown). DMSO (0.5%) was selected as an acceptable compromise allowing compound addition up to 50 M with an acceptable attenuation of fluorescence of 20% for the T brucei assay and 15% for the MRC5 assay. All subsequent assays contained 0.5% DMSO.

A c c e p t e d M a n u s c r i p t 15

Sensitivity to standard drugs

Five drugs currently used in treatment of HAT were tested for growth inhibition (Fig. 2A).

The most potent of these were pentamidine, suramin and melarsoprol, with EC 50 values in the low nanomolar range, similar to those published for T. brucei rhodesiense and T. brucei gambiense by Raz et al [START_REF] Raz | The Alamar Blue® assay to determine drug sensitivity of African trypanosomes (T.b.rhodesiense and T.b.gambiense) in vitro[END_REF] and for various T. brucei gambiense stocks by Likeufack et al. [START_REF] Likeufack | Comparison of the in vitro drug sensitivity of Trypanosoma brucei gambiense strains from West and Central Africa isolated in the periods 1960-1995 and 1999-2004[END_REF]. Eflornithine was the least effective drug (EC 50 22.4  1.1 M). Since eflornithine induces physiological activation of mitochondrial metabolism [START_REF] Giffin | Physiological activation of the mitochondrion and the transformation capacity of DFMO induced intermediate and short stumpy bloodstream form trypanosomes[END_REF], it could potentially yield erroneous results due to increased reduction of resazurin in drug-treated cells. However, this does not appear to be the case as direct counting of cell growth returned a similar EC 50 of 17.1 ± 0.1 µM. Nifurtimox was intermediate in potency (EC 50 1.8 M) in good agreement with previous reports [17;18]. These results provide further validation of resazurin as a suitable substitute for Alamar blue reagent. Pentamidine isethionate was chosen for use as a "perplate" control for all future assays. Similarly, the anticancer compound doxorubicin [START_REF] Arcamone | Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S.Peucetius var caesius[END_REF] was used as a control for MRC5 assays (Fig. 2B).

Screening of the LOPAC collection

Initially, the LOPAC collection of pharmacologically active compounds was screened against T. brucei at a fixed concentration of 1 µM on two independent occasions. This identified 52 compounds producing a mean signal of less than 50% of the control on at least one occasion: an initial hit rate of 4.3%. Hits were analysed by LC-MS confirming a purity of at least 95% for all compounds (data not shown).

Triplicate independent EC 50 determinations were carried out for each of these hits against T. brucei. The resulting EC 50 values for each hit were in good agreement with A c c e p t e d M a n u s c r i p t 16 coefficients of variation typically less than 20%. Means weighted to standard error for each compound were determined and at this point 19 hits with EC 50 values of greater than 1 µM were rejected (Table 1), leaving 33 confirmed sub-micromolar EC 50 hits against T. brucei (Table 2).

Selective activity against the parasite is an essential feature of drug leads [START_REF] Caffrey | Bis-acridines as lead anti-parasitic agents: structure activity analysis of a discrete compound library in vitro[END_REF]. We therefore determined EC 50 values of our hits against MRC5 cells, a human embryonic lung cell line used in similar previous studies [START_REF] Girault | Antimalarial, antitrypanosomal, and antileishmanial activities and cytotoxicity of bis(9-amino-6-chloro-2-methoxyacridines): Influence of the linker[END_REF]. Coefficients of variation between the resulting 4 determinations were typically less than 40%. Means weighted to standard error were determined and used to calculate the fold-specificity for T. brucei over human fibroblasts (Table 2). Two known trypanocidal drugs, pentamidine and suramin, which happen to be present in the LOPAC library returned specificities of 3,500-fold and >700-fold, respectively.

The highest specificity of the remaining hits (270 fold) was demonstrated by (+)-U50,488, with an EC 50 of 59 nM. The mixture of enantiomers, ()-U50,488, showed a similar specificity, but only half the potency of the 1R,2R trans enantiomer, suggesting that the growth inhibition is largely due to (+)-U50,488. Only two other hits returned specificities greater than 100-fold: dequalinium dichloride, an broad-range antimicrobial known to act against plasmodium in mice [START_REF] Rodrigues | Plasmodium berghei: in vitro and in vivo activity of dequalinium[END_REF] and oligomycin A, a macrolide antibiotic that inhibits membrane associated mitochondrial ATPase [START_REF] Lardy | Antibiotics as tools for metabolic studies. I. A survey of toxic antibiotics in respiratory, phosphorylative and glycolytic systems[END_REF]. Only one compound (vincristine sulphate, an inhibitor of microtubule assembly [START_REF] Jordan | Comparison of the effects of vinblastine, vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation in vitro[END_REF]) demonstrated greater specificity for human fibroblasts compared to T. brucei. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism. The average Z′ scores throughout the testing was 0.66 for T. brucei assays and 0.57 for MRC5 assays, values characteristic of high quality cell-based screening assays.

A c c e p t e d

M a n u s c r i p t 17

Confirmation of inhibition by (+)-U50,488 and other opioid receptor ligands

A sample of (+)-U50,488 and eight other compounds known to act on or -opioid receptors were purchased for EC 50 determination. The mean weighted to standard error was 58.9 nM for the authentic (+)-U50,488 sample compared to the 60 nM value found during the library screening. Of the other opioid receptor ligands with structural similarity to (+)-U50,488, three displayed EC 50 values ~5 M (Fig. 3) and one (U69593) was inactive. Of the compounds showing structural similarity to morphine, only buprenorphine showed weak activity. Thus, the original (+)-U50,488 hit had the highest potency of this group of compounds and was 70times more potent than its trans enantiomer, (-)-U50,488.

κ-Opioid receptor homology searching

Global alignments of the amino acid sequences of the κ-opioid receptor from sheep, zebrafish, rough skinned newt, mouse and guinea pig return similarity scores of 60-99%. TBLASTN searching of the T. brucei GeneDB with these query sequences revealed several predicted genes with only short stretches of alignment. Global alignments of these predicted genes with the known receptor sequences returned similarity scores of only 9 to 19%.

Effect of (+)-U50,488 on T. brucei in vivo

Pharmacokinetic studies on (+)-U50,488 in uninfected mice were performed. The compound is not tightly bound to mouse plasma proteins (fraction unbound is 0.69) and is well tolerated when administered at 150 mg kg -1 via the oral route. The brain to blood ratio was 8.2, an excellent property for a stage 2 candidate HAT drug. The peak free blood concentration of (+)-U50,488 was 800 ng ml -1 1 h after dosing with a half-life of 2.6  0.04 h (Fig. 4). Based on these findings, 3 mice were infected with T. brucei and dosed orally at 150 mg kg -1 every 4

A c c e p t e d M a n u s c r i p t 18 h for 32 h to ensure that the free blood level remained above the EC 99 . However, this regimen was not curative, with only one mouse showing a transient reduction in parasitaemia followed by relapse, in sharp contrast to our recent studies on inhibitors of N-myristoyltransferase [START_REF] Frearson | N-myristoyltransferase inhibitors as new leads to treat sleeping sickness[END_REF] and nitroaromatic drugs [START_REF] Sokolova | Cross-resistance to nitro drugs and implications for treatment of human African trypanosomiasis[END_REF].

Effect of (+)-U50,488 and HAT drugs on T. brucei culture growth

To examine the cause of the poor trypanocidal activity in vivo, (+)-U50,488 was compared with the known HAT drugs pentamidine and eflornithine for their ability to kill or slow the growth of T. brucei cells in culture (Fig. 5). Exposure to pentamidine (10 x EC 50 ) caused the cell density to drop below the limit of detection (1 x 10 3 cells ml -1 ) by 40 h with no viable cells visible by microscopy, indicating a cytocidal effect. Eflornithine arrested cell growth after 30 h and cell density did not change thereafter, indicating a cytostatic effect. In contrast, exposure to (+)-U50,488 at 10 x EC 50 only had a minor effect on growth, reducing the doubling time from 6.8 h to 9.7 h. Further inspection of the EC 50 curves suggested that this unexpected result might be attributed to a low Hill slope for this compound. Based on the EC 50 values and Hill slopes for pentamidine and eflornithine (8 nM, s = 3.1 and 22.4 M, s = 4.0 , respectively) EC 99 values of 37 nM and 77 M can be calculated, values which are less than 10-times EC 50 . In contrast, (+)-U50,488 (EC 50 60 nM, s = 1.8) has a predicted EC 99 value of 740 nM, which is greater than 10-times the EC 50 . Thus, the observed partial inhibition of growth is consistent with exposure to this concentration of drug. In a series of additional experiments the effect on growth of 50-, 100-and 500-times the EC 50 for (+)-U50,488 was examined. These concentrations showed a dose dependent reduction in growth rate, but only at the highest drug concentration (30 M, 500-times EC 50 ) was it possible to achieve a cytocidal effect (Fig. 5, closed squares). Our experiments establish that resazurin can be used as a cheaper substitute for the Alamar blue formulation for growth inhibition assays for bloodstream-form T. brucei or human fibroblasts. However, this assay method does not necessarily indicate cell death. Inhibitors could be cytocidal, cytostatic, or interfering with the reduction of resazurin without effect on cell growth. Lead compounds must therefore be assessed for cytotoxicity by alternative means such as direct cell counting of viable parasites (Fig. 5) or a "live-dead assay" [START_REF] Turner | Human infectivity trait in Trypanosoma brucei: stability, heritability and relationship to sra expression[END_REF]. The HMI9-T media used in this assay has been shown to mask inhibition of certain metabolic pathways such as folate metabolism and thymidylate biosynthesis [START_REF] Sienkiewicz | Chemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomes[END_REF] and consequently these inhibitors are unlikely to be identified with these culture conditions. Given this proviso, our results indicate that this semi-automated medium-throughput screening method against T. brucei is reproducible and robust. With further automation and miniaturisation it could be used to screen much larger compound collections and other special focussed sets available in our Drug Discovery Unit [START_REF] Brenk | Lessons learnt from assembling screening libraries for drug discovery for neglected diseases[END_REF].

The drugs currently used to treat HAT show a wide range of potencies in this assay, ranging from 8 nM for pentamidine and melarsoprol to 1.8 M for nifurtimox and 22 M for eflornithine. Despite many current drug-discovery paradigms being driven by potency -at least at the early stages of lead identification -the example of eflornithine demonstrates that lower potency compounds may still emerge as effective treatments. Any lead offering improvement on either the mode of administration or cost of eflornithine regimen would be an advance in the treatment of HAT.

The EC 50 values determined by Likeufack et al against T. brucei gambiense vary by a maximum of 4.5-fold between stocks [START_REF] Likeufack | Comparison of the in vitro drug sensitivity of Trypanosoma brucei gambiense strains from West and Central Africa isolated in the periods 1960-1995 and 1999-2004[END_REF], and the values we report against T. brucei brucei fall within these ranges. The same is true for the values reported by Raz et al [START_REF] Raz | The Alamar Blue® assay to determine drug sensitivity of African trypanosomes (T.b.rhodesiense and T.b.gambiense) in vitro[END_REF] for T.b.rhodesiense. However, although T. brucei brucei, which is non-pathogenic to humans, is The 33 nanomolar inhibitors of T. brucei identified from the LOPAC collection included pentamidine and suramin which acted as internal controls. The minor differences in EC 50 values between the library screening and our testing of standard compounds could be due to higher purity in the original samples purchased and evaporation in the smaller volumes of the library samples. Original hits that were rejected at the 1 µM cut-off include several compounds that have known pharmacological activity in animal models. Testing these against rodent models of HAT at the already-established maximum tolerated doses could identify additional promising leads.

Six out of the 33 hits with EC 50 values less than 1 M showed more than 100-fold selectivity for T. brucei. Pentamidine and suramin were the most selective compounds identified, illustrating the target selectivity (>1000 fold) that should be aimed for at the final stages of lead development. The most selective compounds previously unknown to inhibit T. brucei were from isomers of U50,488, a known agonist of the κ-opioid receptor [START_REF] Vonvoigtlander | U-50,488 -A selective and structurally novel non-mu-(kappa) opioid agonist[END_REF]. This compound shows good brain penetration and therefore potentially able to access a late-stage HAT infection, a key feature specified by the current target product profile from WHO.

From the limited number of commercially available opioid-receptor ligands, (+)-U50,488 was the most potent (Fig. 4). Other compounds showed either weak or no activity.

The complete lack of activity of norbinaltorphimine against T. brucei suggests that possible CNS side effects of a treatment based on (+)-U50,488 could be reduced by co-administration of a κ-opioid antagonist. Whilst the (-)-U50,488 isomer is a stronger agonist of the κ-opioid receptor than the (+)-U50,488 isomer, the latter is almost 70 times more effective against T. brucei. κ-Opioid receptors typically share high similarity at the amino acid level across species as indicated by our homology searching. However, consistent with the poor activity of brucei genome. An opioid receptor agonist (endomorphin-1; YPWF-NH 2 ) has been shown to inhibit a drug-metabolite transporter from the malaria parasite Plasmodium falciparum [START_REF] Martin | Chloroquine transport via the malaria parasite's chloroquine resistance transporter[END_REF].

Mutations in this transporter confer resistance to chloroquine, but unfortunately the physiological substrates are not known [START_REF] Sanchez | Is PfCRT a channel or a carrier? Two competing models explaining chloroquine resistance in Plasmodium falciparum[END_REF]. Moreover, no convincing homologues could be identified in the T. brucei genome database. Thus the target for U50,488 remains to be identified in T. brucei.

The in vivo and in vitro experiments indicate that further optimization of (+)-U50,488 is required to achieve efficacy. Although the compound has good pharmacological properties, including high CNS penetration, it was not curative using a therapeutic schedule which was predicted to maintain free blood exposure above the predicted EC 99 . The reason for this is not entirely clear. The in vitro studies demonstrated that the compound is cytostatic at 100-times the EC 50 , like eflornithine, rather than cytocidal, like pentamidine. Thus, continuous exposure above the minimum inhibitory concentration may be required to allow sufficient time for the immune system to eliminate non-dividing parasites, as is the case with eflornithine. Alternatively, differences in the physiological environment in vivo and in vitro may be responsible for the lack of efficacy. Further studies on the mode of action of (+)-U50,488 would be informative. Although a cytocidal effect was observed at high concentrations (30 M, 500-times EC 50 ), this could be due to additional off-target effects and, in any case, is above the maximum free blood concentration achievable under the dosing schedule used here.

  African trypanosomiasis (HAT) is a disease endemic to the sub-Saharan region of Africa and is caused by two subspecies of the protozoan parasite Trypanosoma brucei. T. b.

A c c e p t e d M a n u s c r i p t 6

 6 Normal human MRC5 cells (diploid foetal lung fibroblasts) were used as a counter-screen for non-selective inhibitors. Stabilates were obtained from the European Collection of Cell Cultures (ECACC) were grown at 37 o C and 5% CO 2 in a humidified incubator in Eagle"s Minimal Essential Medium (Sigma-Aldrich, Gillingham, UK) supplemented with 10% foetal bovine serum (Invitrogen, Paisley, UK). Stock cultures were maintained in T75 vented cap culture flasks with half the medium changed each day. Cells were split once confluent as follows: medium was removed and the monolayer washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4). Cells were detached by incubation for 2 min with 1 ml of trypsin EDTA solution (0.5 mg ml -1 trypsin, 0.2 mg ml -1 EDTA•4Na in Hanks balanced salt solution supplied by Invitrogen, Paisley, UK). Detachment was confirmed by microscopy. Fresh media was added to the cells to subculture into new flasks at twofold to fourfold the original volume as required.

A c c e p t e d M a n u s c r i p t 10 culture

 10 hood and 200 µl media added to all wells. From these plates, 100 µl was transferred to the cell-containing plates set up the previous day. Plates were incubated for 3 days after which 20 µl 0.5 mM resazurin was added to each well. Plates were incubated for a further 4 h before measuring fluorescence as above. Fluorescence signal for each well was background subtracted and expressed as percentage inhibition of the full signal control wells. EC 50 curve fitting employed a 4 Parameter Logistic dose response curve using IDBS XLFit 4.2 Model 205:

A c c e p t e d M a n u s c r i p t 11 Flasks

 11 were set up in triplicate for each inhibitor at multiples of EC 50 along with 3 control flasks with no inhibitor added. Pentamidine and (+)-U50,488 were added in DMSO in a volume of 0.1% of the culture. Eflornithine was dissolved directly in HMI9-T media and sterilised by 0.22 µM filtration before diluting into the cell culture. Cell densities were

  A c c e p t e d M a n u s c r i p t 12 Netherlands) and the composition in each tube balanced with control fluid, such that the volume of buffer to plasma is the same. Sample extraction was performed by the addition of 400 l of acetonitrile containing a structural analogue of (+)-U50,488 as internal standard.

A

  c c e p t e d M a n u s c r i p t 13 blood and brain homogenate to cover at least 3 orders of magnitude for (+)-U50,488 (range 2 -2000ng ml -1 ).

A c c e p t e d M a n u s c r i p t 20 a

 20 suitable model for HAT, it is important to test advanced lead compounds against these clinically relevant sub-species.

A

  c c e p t e d M a n u s c r i p t 21 other opioid-receptor ligands, we found no convincing homologue of the receptor in the T.

Future

  work will involve further medicinal chemistry to explore structure-activityrelationships, improve potency and the Hill slope. It remains to be seen whether the cytostatic effect of U50,488 is a function of the target(s) or the inhibitor itself. Identifying the intracellular target of U50,488 could provide a new avenue for drug discovery efforts against T. brucei.

1 FIG. 1 . 17 FIG. 2 .

 11172 Figure Legends 1

3 FIG. 3 . 8 FIG. 4 . 14 FIG. 5 .

 3384145 FIG. 3. Structure activity relationships of opioid receptor ligands against T. brucei 4

a 5 -( 1 -

 51 Aziridinyl)-2,4-dinitrobenzamide b 1'-Methyl-5-([2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl] carbonyl)-2,3,6,7tetrahydro-spiro(furo[2,3-f]indole-3,4'-piperidine) c Phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromided 9-Chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine e (-)-(1R,3S)-3-Adamantyl-1-(aminomethyl)-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride hydrate

  N 2 -(cis-2-Aminocyclohexyl)-N 6 -(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine HCl b 4-(3,4-dihydroxyphenyl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine HBr

Figure 3

 3 Figure 1 Jones et al

Figure

  Figure

TABLE 1 . Initial submicromolar hits from the LOPAC library returning EC 50 versus T. brucei greater than 1 µM. Compound EC 50 vs. T. brucei

 1

	(µM)

Table A

 A

	M a n u s c r i p t
	c c e p t e d

TABLE 2 Submicromolar hits against T. brucei ranked by selectivity versus MRC5 cells Compound EC 50 vs. T. brucei (nM) EC 50 vs. MRC5 (nM) Selectivity

 2

	Pentamidine isethionate	3.7 ± 0.10	13 000 ± 6 800	3400
	Suramin hexasodium	68 ± 3.5	>50 000	>710
	(+)-trans-(1R,2R)-U-50488	59 ± 2.2	16 000 ± 4 900	270
	hydrochloride (±) trans-U-50488 methanesulfonate	113 ± 7.6	26 000 ± 8 700	230
	Dequalinium dichloride	43 ± 8.8	6 100 ± 1 500	140
	Oligomycin A	150 ± 3.1	18 000 ± 2 600	120
	Mitoxantrone	2.4 ± 0.20	110 ± 37	46
	8-(4-Chlorophenylthio)-cAMP sodium 400 ± 54	18 000 ± 12 000	44
	Chelerythrine chloride	59 ± 5.7	2 500 ± 480	42
	Pimozide	340 ± 19	14 000 ± 3 300	41
	8-Cyclopentyl-1,3-dipropylxanthine	530 ± 53	17 000 ± 7 900	31
	4-Chloromercuribenzoic acid	690 ± 85	19 000 ± 3 000	27
	Dihydroergocristine methanesulfonate 890 ± 57	20 000 ± 770	23
	Dequalinium analog, C-14 linker	70.0 ± 4.6	1 000 ± 290	14
	Calmidazolium chloride	510 ± 66	6 600 ± 750	13
	Purvalanol A	820 ± 55	10 000 ± 3 200	12
	Beta-Lapachone	850 ± 130	8 600 ± 2 900	10
	Vinblastine sulphate salt	94 ± 6.0	780 ± 910	8.3
	CGP-74514A a	210 ± 8.3	1 500 ± 620	7.1
	Sanguinarine chloride	240 ± 17	1 400 ± 350	5.8
	Diphenyleneiodonium chloride	120 ± 7.6	700 ± 870	5.7
	Sphingosine	820 ± 78	4 600 ± 2 500	5.7
	Quinacrine dihydrochloride	380 ± 13	1 900 ± 440	5.0
	SKF 89145 hydrobromide b	610 ± 44	2 900 ± 200	4.7
	Calcimycin	180 ± 13	750 ± 140	4.2
	Idarubicin	20 ± 1.5	78 ± 29	3.9
	Emetine dihydrochloride hydrate	7.9 ± 0.53	30 ± 3.1	3.8
	Ellipticine	460 ± 45	1 700 ± 620	3.7
	Mevastatin	940 ± 50	2 000 ± 360	2.2
	Taxol	3.3 ± 0.071	6.6 ± 9.8	2
	(S)-(+)-Camptothecin	530 ± 13	800 ± 99	1.5
	Alsterpaullone	400 ± 40	510 ± 170	1.3
	Vincristine sulphate	30 ± 1.0	0.60 ± 1.0	0.02
	a

Acknowledgements

This work was supported by grants from the Wellcome Trust (WT079838, WT077705, WT083481) and the Drugs for Neglected Diseases initiative. We thank Bhavya Rao for technical assistance in the testing of compounds against human fibroblasts, Robert Kime and Suzanne Norval for DMPK studies, Frederick Simeons for assistance with animal efficacy testing and Magnus Alphey for critical reading of the manuscript.