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News Article | February 28, 2017
Site: www.businesswire.com

BEIJING--(BUSINESS WIRE)--Pharmaron, a fully integrated contract research organization offering R&D services to the life sciences industry, today announced that it has signed a definitive agreement under which Pharmaron will acquire a majority stake in Shin Nippon Biomedical Laboratories Clinical Pharmacology Center, Inc. (“SNBL CPC”) in Baltimore, Maryland, USA. This clinical center is a leading provider of moderate and highly complex Phase I/II clinical development services for the life sciences sector. Current shareholder Shin Nippon Biomedical Laboratories, Ltd. (SNBL) (TSE:2395) will retain a minority stake in the business following the transaction. SNBL CPC is a full-service clinical CRO located on the campus of the University of Maryland BioPark. Since its inception in 2005, over 200 studies have been completed in the purpose-built, clinical pharmacology unit. Many of these completed clinical studies have been submitted in support of global regulatory filings for drug approval for marketing of both small and large molecules. This strategic acquisition allows Pharmaron to be one-step closer to offering a full spectrum of R&D services. Addition of this capability to the Pharmaron Group naturally complements and expands Pharmaron’s existing drug R&D services, further consolidating the clinical development capabilities, through synergistic integration with recently acquired radiolabelled science capabilities, including Quotient Bioresearch’s clinical metabolism in the UK and Xceleron’s AMS-based 14C-microtracer technology in the USA. Mr. Larry Lou, President and COO of Pharmaron commented: “We are delighted to have SNBL CPC join the Pharmaron Group. This is an important milestone for Pharmaron. Once integrated and further developed, the new clinical platform will fuel the corporate engine for business growth in a sustainable manner. This is another testimony of our determination to fully realize our mission to support our clients’ success in discovery, development and commercialization of important medicines and fulfill our vision to be a global leading organization in the life sciences service industry.” Dr. Ryoichi Nagata, Chairman and CEO of SNBL commented: “The mission of SNBL CPC has been to offer complex and innovative clinical pharmacology services in close proximity to leading university medical centers. Through this transaction, we look forward to seeing future growth of CPC as part of Pharmaron Group.” The financial terms of the transaction are not being disclosed. Teneo Capital served as financial advisor to Pharmaron; O’Melveny & Myers LLP served as Pharmaron’s legal advisor. SC&H Capital served as financial advisor to SNBL; Miles & Stockbridge P.C. served as legal advisor to SNBL. Pharmaron is a private, premier R&D service provider for the life sciences industry. Founded in 2003, Pharmaron has invested in its people and facilities, and established a broad spectrum of R&D service capabilities ranging from synthetic and medicinal chemistry, biology, DMPK, pharmacology, safety assessment, radiochemistry and radiolabelled metabolism, clinical analytical sciences to chemical & pharmaceutical development. With over 4,000 employees, and operations in China, the United States, and the United Kingdom, Pharmaron has an excellent track record in the delivery of R&D solutions to its partners in North America, Europe, Japan and China. www.pharmaron.com SNBL CPC is a clinical pharmacology facility located in the University of Maryland BioPark in Baltimore, Maryland, USA. The state of the art facility and equipment is bolstered by a vibrant research community. SNBL CPC specializes in complex Phase I-II trials, including TQT/Phase 1 QT de-risking, first-In-human (FIH), Japanese Bridging and Phase II Proof of Concept (POC) studies in therapeutics areas including immunology/infectious disease, neurology, respiratory, dermatology and more. SNBL CPC conducts clinical trials from multiple sectors, including biopharmaceutical and biotech industry, academia, and the government. SNBL CPC offers full service support of clinical trials through its in-house resources, expert partners from surrounding universities and practices. Proximity and the agreements that SNBL CPC has developed with the University of Maryland, Baltimore and Johns Hopkins University makes this facility best of its kind.


News Article | February 15, 2017
Site: www.nature.com

No statistical methods were used to predetermine sample size. Affinity-purified antibodies against total and Ser886 and Ser999 phospho-sites of EPRS were generated as described7, 31. Antibody against phospho-Ser was from Meridian Life Science. Antibodies specific for the C terminus of S6K1 and N terminus of S6K2 were purchased from Abcam and LifeSpan, respectively. Antibodies against PKA, DMPK, PKN, GAPDH, caveolin1, CD36/FAT, GLUT4, His-tag, β-actin and FATP1, FATP3, and FATP4 were from Santa Cruz. Antibody specific for FABP4 and FABP5 were from R&D and for FABPpm/GOT2 was from GeneTex. All other antibodies and rapamycin were from Cell Signaling. SignalSilence siRNAs targeting RSK1, AKT and S6K1 were from Cell Signaling, and those targeting raptor and rictor were from Santa Cruz. The 3′-UTR-specific duplex siRNAs, 5′-UGAUACGAAGAUCUUCUCAG-3′ and 5′-GCCUAAAUUAACAGUGGAA-3′, targeting mouse EPRS were from Origene. Smart pool siRNA targeting the coding sequence of mouse FATP1 (SLC27a1) was from Dharmacon and 3′-UTR-specific trilencer siRNA targeting human S6K1 was from Origene. Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7, 8. Recombinant active S6K1 (ref. 32) and RSK1–3 were from Cell Signaling; Akt1 and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33. Full-length human S6K1 cDNA in pCMV6-entry vector was purchased from Origene and recloned, deleting the 23-amino acid N terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen). Human U937 monocytic cells (CRL 1593.2; ATCC authenticated by STR DNA profiling) were cultured in RPMI 1640 medium and 10% fetal bovine serum (FBS) with penicillin and streptomycin at 37 °C in 5% CO . Bone-marrow-derived macrophages (BMDM) were flushed from femur and tibia marrows of S6K1−/−S6K2−/−, and double-knockout S6K1−/−S6K2−/− mice (from G. Thomas and S. Kozma), and then cultured for one week in RPMI 1640 medium containing 10% FBS and 20% L929 cell-conditioned medium at 37 °C in 5% CO . 1 × 107 cells were treated with 500 U ml−1 IFNγ (R&D) for up to 24 h, as described previously34, 35. 3T3-L1 fibroblasts (CL-173; ATCC-certified) were cultured in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM), 10% FBS and antibiotics/antimycotic at 37 °C in 10% CO to near 75% confluence. Confluent fibroblasts were induced to differentiate in medium containing DMEM and 10% FBS supplemented with 1× solutions of insulin:dexamethasone:3-isobutyl-1-methylxanthine (Cayman). After 72 h, the medium was replaced with 10% FBS and DMEM containing only insulin and maintained for a week with 3 changes in the same medium. Adipocytes were maintained in DMEM medium with 10% calf serum and antibiotics/antimycotic for at least 3 d before utilization. Differentiated adipocytes were serum-deprived for 4 h followed by treatment with 100 nM insulin (Sigma-Aldrich) for 4 h, or as indicated. Cell lysates were prepared using Phosphosafe Extraction buffer (Novagen) supplemented with protease inhibitors. As certified U937 monocytes and 3T3-L1 fibroblasts were directly procured from ATCC, they were not subjected to any further testing for contamination. Primary adipocytes from white adipose tissue (WAT) were prepared as described14, 36. Briefly, after mouse sacrifice, fat pads were removed and minced in Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer (pH 7.4) containing 10 mM sodium bicarbonate, 30 mM HEPES, 200 nM adenosine, and 1% fatty acid-free bovine serum albumin (BSA, Sigma). WATs were digested with collagenase (2 mg g−1) in KRBH buffer at 37 °C for 1 h. Digested WATs were suspended in DMEM supplemented with 10% FBS, and filtered through 100-μm mesh cell strainer (BD Falcon) to remove undigested material. The cell suspension was incubated for 10 min at room temperature, and adipocytes collected from the floating layer after centrifugation. Adipocytes were incubated for 1 h at room temperature with gentle shaking and washed three times with DMEM. Differentiated human adipocytes in adipocyte maintenance medium were obtained from Cell Applications. Adipocytes were maintained in DMEM medium with 10% calf serum and antibiotics/antimycotic for 2 d before utilization, and 5 × 106 cells were serum-deprived for 4 h followed by treatment with 100 nM insulin for 4 h. Hepatocytes were isolated by collagenase perfusion of mouse livers and cells seeded for 4 h on collagen-coated 6-well plates (1 × 106 cells per well) in Williams’ medium E with 10% FBS, 25 mM HEPES, 100 nM insulin, and 100 nM triiodothyronine37, 38, 39. Cells were cultured for 48 h in serum-free Williams medium E with two medium changes. Before experiments, hepatocytes were pre-incubated overnight in serum-, insulin-, and triiodothyronine-free DMEM, and then with 100 nM insulin. Adult mouse cardiac cells were isolated by sequential plating using non-perfusion adult cardiomyocyte isolation kit (Cellutron)40. After isolation, 1 × 106 cardiac cells were incubated for 24 h in serum containing AS medium, and then with serum-free AW medium for another 24 h. Before experiments, cells were incubated for 4 h in serum-free DMEM, and then with 100 nM insulin. All studies using cultured cells were repeated at least three times. The number of replicates was estimated from comparable published studies that gave statistically significant results. U937 cells (1 × 107), PBMs and differentiated 3T3-L1 adipocytes (5 × 106 cells for both) were transfected with endotoxin-free plasmid DNAs or siRNAs (target-specific and scrambled control) using nucleofector (100 μl solution V for U937 cells and PBMs and 100 μl solution L for 3T3-L1 adipocytes) from Amaxa nucleofection kit (Lonza) following the manufacturer’s protocol. Transfected cells were immediately transferred to pre-warmed Opti-MEM media for 6 h and then to RPMI 1640 (for U937 cells and PBMs) and DMEM (for 3T3-L1 adipocytes) containing 10% FBS supplemented with penicillin, streptomycin, and geneticin (G418; 20 μg ml−1) for 18 to 24 h before treatment with insulin and inhibitors. Cell lysates or purified active kinases were pre-incubated with recombinant EPRS linker (wild-type and mutant) for 5 min in kinase assay buffer (50 mM Tris-HCl (pH 7.6), 1 mM dithiotheitol, 10 mM MgCl , 1 mM CaCl , and phosphatase inhibitor cocktail)7, 8, 33. Phosphorylation was initiated by addition of 5 μCi [γ-32P]ATP (Perkin-Elmer) for 15 min, and terminated using SDS gel-loading buffer and heat denaturation. Phosphorylated proteins were detected after resolution on Tris-glycine SDS–PAGE, fixation in 40% methanol and 10% acetic acid, and autoradiography. Immunoblot with anti-His tag antibody to detect EPRS linker served as control. To assay kinase activity using peptide substrates, 50 μM of synthetic peptides were phosphorylated with 1 μCi [γ-32P]ATP in kinase assay buffer. Equal volumes were spotted onto P81-phosphocellulose squares, washed in 0.5% H PO , and 32P incorporation determined by scintillation counting. U937 cell lysates were pre-cleared using protein A-sepharose, and target AGC kinase members and a non-member, MK2 were immunoprecipitated by incubation with specific antibodies for 4 h. The immunocomplex was captured by incubating with protein A-sepharose beads for 4 h, and washed three times with kinase assay buffer supplemented with 0.1% Triton X-100. The immunocomplex was resuspended in kinase assay buffer and used to phosphorylate EPRS linker as above, and 32P incorporation into peptide substrates was determined by scintillation counting41. Target peptides for S6K1, RSK1, MSK1, SGK494, NDR1, MRCKα, CRIK, RSKL1, ROCK1 and 2 (RRRLSSLRA), GRK2 (CKKLGEDQAEEISDDLLEDSLSDEDE), LATS1 (CKKRNRRLSVA), MAST1 (KKSRGDYMTMQIG), PRKX (RRRLSFAEPG), DMPK (KKSRGDYMTMQIG), and PDK1 (KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC) were from SignalChem; for MK2 (KKLNRTLSVA) from Enzo Life Sciences; for PKA (RRKASGP), SGK1/AKT (RPRAATF), PKC/PKN (HPLSRTLSVAAKK), PKG, (RKISASEFDRPLR), and Cdk5 (PKTPKKAKKL) were from Santa Cruz. All mice were housed in microisolator cages (maximum 5 per cage of same-sex littermates) and maintained in climate/temperature- and photoperiod-controlled barrier rooms (22 ± 0.5 °C, 12–12 h dark–light cycle) with unrestricted access to water and standard rodent diet (Harlan Teklad 2918) deriving 24, 18 and 58 kcal% from protein, fat and carbohydrate, respectively. Mice were fed standard rodent diet unless otherwise indicated. The number of animals used in each experiment was estimated from examination of comparable published studies that gave statistically significant results. All mouse studies were performed in compliance with procedures approved by the Cleveland Clinic Lerner Research Institute Institutional Animal Care and Use Committee. Genetically-modified EPRS phospho-deficient S999A and phospho-mimetic S999D knock-in mice were generated (Xenogen Biosciences, Taconic). The RP23-86H18 BAC clone from mouse chromosome 1 containing full-length mouse Eprs gene was used to generate 5′ and 3′ homology arms, the knock-in region for the gene targeting vector, and Southern blot probes for screening targeted events. The homology arms and the knock-in region were generated by high-fidelity PCR, and cloned into the pCR4.0 vector. The S999A and S999D mutations (TCA to GCA or GAT, respectively) in exon 20 were introduced by PCR-based site-directed mutagenesis. The final vector also contained Frt sequences flanking the Neo expression cassette for positive embryonic stem cell selection, and a DTA expression cassette for negative selection. The targeting vector was electroporated into C57BL/6 embryonic stem cells and screened with G418. Positive expanded clones with confirmed mutation were selected. Neo was deleted by Flp electroporation, and blastocysts injected. Male chimaeras were bred with C57BL/6 wild-type females, and resulting F1 heterozygotes interbred to generate homozygotes in C57BL/6 background. Genotyping was done using forward primer 5′-CAGCATAAGAACAGTTGCCAAATAAAGG-3′ and reverse primer 5′-TTCTTGAACACACACATGCACAGACTC-3′. For all experiments the wild-type (EprsS/S), EprsA/A and EprsD/D were generated exclusively by breeding heterozygotes (EprsS/A and EprsS/D), and most experiments shown use male mice unless otherwise indicated. Mice were not randomized and studies were performed unblinded with respect to mouse genotype. S6K1−/− mice in C57BL/6 background were generated at the National Jewish Medical and Research Centre (Denver, Colorado) by blastocyst injection of embryonic stem cells with targeted disruption of the S6K1 gene as described previously19, 42. Briefly, neomycin (Neo) selection cassette was inserted to disrupt the exon corresponding to amino acids 207–237 in the catalytic domain of S6K1, thereby frame-shifting the downstream coding region. S6K1−/− mice exhibited phenotypes consistent with the previously reported mice that were generated by similar approach that is, replacing the catalytic domains of S6K1 with a Neo selection cassette9, 20. EprsD/DS6K1−/− and EprsS/SS6K1−/− were generated by EprsS/DS6K1−/− × EprsS/DS6K1−/− crosses. Mice wild-type for both Eprs and S6K1 genes (EprsS/SS6K1+/+) were generated from crosses of S6K1+/− heterozygotes. Male and female mice of EprsS/S and EprsA/A genotypes were recruited (n = 212 total mice) exclusively from crosses of heterozygotes (EprsS/A). All mice were housed in microisolator cages (maximum 5 per cage of same-sex littermates) with routine cage maintenance as above. Weaned mice (>21 days), born between June 2010 and December 2012 from 40 heterozygous parents, were monitored daily and weighed biweekly for the entire duration of their life. Mice that spontaneously developed conditions common in the C57BL/6 strain, such as malocclusion and hydrocephalus, were sacrificed and excluded from the study43. Assessments of deterioration in general health and quality of individual life were made in consultation with veterinary services of the Biological Resources Unit (BRU) of the Cleveland Clinic Lerner Research Institute. Severely sick and moribund mice that were judged to not survive another 48 h were euthanized with this date considered date of death, and included in the longevity analysis. Mice euthanized owing to imminent death include 11.5% (6 out of 52) male and 11.1% (6 out of 54) female of EprsS/S genotype, and 7.7% (4 out of 52) male and 9.3% (5 out of 54) female of EprsA/A genotype. Longevity was analysed by Kaplan–Meier survival curves from 212 mice (52 male and 54 female of each genotype, EprsS/S and EprsA/A) using known birth and death dates. Statistical differences were evaluated by log-rank Mantel–Cox and Gehan–Breslow–Wilcoxon tests using GraphPad Prism 5. Male and female mice of EprsS/S and EprsD/D genotypes were recruited (n = 89 total mice) exclusively from crosses of heterozygotes (EprsS/D). All weaned mice (>21 days born between February, 2011 and September, 2014 from 23 EprsS/D parents) were housed in microisolator cages (maximum 5 per cage of same-sex littermates) with routine cage maintenance and health monitoring as above. Mice killed owing to imminent death (as described above) include 8.7% (2 out of 23) male and 9.5% (2 out of 21) female of EprsS/S genotype, and 8.3% (2 out of 24) male and 4.8% (1 out of 21) female of EprsA/A genotype. Longevity was analysed by Kaplan–Meier survival curves from 89 mice (23, 21 male and 24, 21 male of genotype, EprsS/S and EprsA/A, respectively) using known birth and death dates and statistical analysis, as above. Male and female mice of S6K1+/+ and S6K1−/− genotypes were recruited (n = 112 total mice) exclusively from crosses of heterozygotes (S6K1+/−). All weaned mice (>21 days born between February 2011 and December 2013 from 23 S6K1+/− parents) were housed in microisolator cages (maximum 5 per cage of same-sex littermates) with routine cage maintenance and health monitoring as above. Mice killed owing to imminent death (as described above) include 13.8% (4 out of 29) male and 10.3% (3 out of 29) female of S6K1+/+ genotype, and 14.3% (4 out of 28) male and 14.3% (3 out of 21) female of S6K1−/− genotype. Longevity estimation was analysed by Kaplan–Meier survival curves from 112 mice (29, 29 male and 28, 26 female of genotype, S6K1+/+ and S6K1−/−, respectively) using known birth and death dates and statistical analysis as above. Univariate and multivariate CPH regression models were performed to analyse the effects of 4 variables; genotype, date of birth (DOB), gender, and parental identity (PID), on longevity of mice recruited for the study. The independent variables were fitted as categorical variables in the model. Genotype and gender were coded as binary variables. DOB and PID were coded as multiple categories. For CPH regression analysis of EprsS/S and EprsA/A mice (n = 212), the data were coded as follows: genotype, EprsS/S (1) and EprsA/A (0); gender, male (0) and female (1). On the basis of unique occurrences, DOB and PID were categorized into 79 (0–78, 0 being the DOB for oldest mice in the study) and 40 (1–40) categories, respectively. Oldest DOB category represents the reference for DOB. PID-1 was considered reference for PID variable. Models were fit using Cox proportional hazards regression in R package ‘survival’ using coxph function. Univariate model was built fitting each of the four variables individually and multivariate model was built fitting all four variables simultaneously. For CPH regression analysis of EprsS/S and EprsD/D mice (n = 89), the data were coded as follows: genotype, EprsS/S (1) and EprsD/D (0); gender, male (0) and female (1). On the basis of unique occurrences, DOB and PID were categorized into 38 (0–37, 0 being the DOB for oldest mice in the study) and 23 (1–23) categories, respectively. For CPH regression analysis of S6K1+/+ and S6K1−/− mice (n = 112), the data were coded as follows: genotype, S6K1+/+ (1) and S6K1−/− (0); gender, male (0) and female (1). On the basis of unique occurrences, DOB and PID were categorized into 36 (0–35, 0 being the DOB for oldest mice in the study) and 23 (1–23) categories, respectively. Scanning electron microscopy was performed by the Cleveland Clinic Imaging Core. WAT from 20-week-old male mice was fixed using 2.5% glutaraldehyde and 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C. Tissues were washed three times in PBS followed by post-fixation with 1% osmium tetroxide in PBS for 1 h at 4 °C. Finally, the tissues were dehydrated through graded alcohol (50, 70, 90, and 100%), twice in ethanol:hexamethyldisilizane (HMDS; 1:1), and three times in 100% HMDS for 10 min each, and dried at room temperature. Samples were mounted on aluminium stubs and coated with palladium-gold using a sputter-coater, and viewed at X500 magnification with a Jeol JSM 5310 Electron Microscope (EOL). Adipose tissues from 20-week-old male mice were fixed in formalin, dehydrated in ethanol, embedded in paraffin, and cut at 5-μm thickness. Sections were deparaffinized, rehydrated, and stained with haematoxylin and eosin by the Cleveland Clinic Histology Core. Stained tissues were visualized with Leica DM2500 microscope, captured with Micropublisher 5.0 RTV digital camera (QImaging) using a 5X objective lens for magnification, and QCapture Pro 6.0 (QImaging) software for image acquisition. Adipocytes from 100 mg EWAT of 20-week male mice were isolated as described above and suspended in DMEM. Cells were counted in a haemocytometer. Basal lipolysis in primary adipocytes from EprsS/S, EprsA/A, and EprsD/D EWAT was measured by glycerol release using adipolysis assay kit (Cayman). Fatty acid oxidation in EWAT of 20-week-old male mice was performed as described13, 44. Explants were placed in an Erlenmeyer flask (Kimble-chase Kontes) containing the reaction mixture (DMEM with 0.1 μCi of [14C]oleic acid, 100 mM l-carnitine, and 0.2% fat-free BSA), and conditioned for 5 min in a 37 °C CO incubator. The flask was sealed with a rubber stopper containing a centre-well (Kimble-chase Kontes) fitted with a loosely folded filter paper moistened with 0.2 ml of 1 N NaOH, and incubated for 5 h at 37 °C. 14CO in the filter paper was trapped by addition of 200 μl of perchloric acid to the reaction mixture followed by incubation at 55 °C for 1 h. Radioactivity in the filter paper was determined by scintillation counting. At 16 weeks, mice were individually housed and given standard rodent diet and water ad libitum. Cumulative food intake was measured by weighing the mouse and food every second day for 30 consecutive days. Intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) in EprsS/S, EprsA/A, and EprsD/D mice were determined as described22, 39. Briefly, GTT was done after an overnight (12 h) fast followed by peritoneal injection of glucose (2 mg g−1 body weight, Sigma). ITT was performed in 6-h fasted mice by injection of 0.75 U kg−1 body weight of insulin (Sigma). Blood glucose was determined using a commercial glucometer (Contour, Bayer). Serum triglycerides, free fatty acids, glucose, and insulin in 12-h fasted and in 1-h post-prandial (fed) mice were determined using commercially available kits. Serum triglycerides, free fatty acids, and glucose kits were from Wako. Insulin was determined using enzyme-linked immunoassay-based, ultra-sensitive mouse insulin kit (Crystal). Determination of serum β-hydroxybutyrate (for ketone body analysis) from 6-h fasted mice was done using colorimetric assay kit from Cayman. White blood cell counts in blood freshly collected by cardiac puncture in the presence of 10 mM EDTA were determined using Advia hematology system. Lipid content in mouse faeces was determined after extraction with chloroform:methanol (2:1)45, 46. GAIT system activity in insulin-treated adipocytes was determined by in vitro translation of capped poly(A)-tailed Luc-Cp GAIT and T7 gene 10 reporter RNAs as described35, 47. Gel-purified RNAs were incubated with lysates from U937 monocytes and differentiated 3T3-L1 adipocytes in the presence of rabbit reticulocyte lysate and [35S]methionine. Translation of the two transcripts was determined following resolution on 10% SDS–PAGE and autoradiography. Cytokine levels in mouse serum (100 μg protein) were determined using mouse cytokine antibody array C3 kit (RayBiotech). Mouse liver triglyceride content was determined by measurement of glycerol following saponification in ethanolic KOH (2:1, ethanol: 30% KOH)48. For assessment of total neutral lipid, freshly isolated liver slices were frozen in OCT, 5-μm sections stained with Oil Red O, and analysed by densitometry using NIH image J as described49. Mouse energy metabolism was determined by indirect calorimetry using the Oxymax CLAMS system (Columbus Instruments) in the Rodent Behavioural Core of the Cleveland Clinic Lerner Research Institute. Mice were housed individually in CLAMS cages and allowed to acclimate for 48 h with unrestricted excess to food and water. Thereafter, O consumption (VO ), CO release, RER and heat generation were recorded for 24 h spanning a single light–dark cycle. Adipocytes from 500 mg WAT from wild-type and EprsA/A mice were labelled with 150 μCi of 32P-orthophosphate (MP Biomedicals) in phosphate-free DMEM medium in absence or presence of insulin (100 nM) for 4 h. EPRS was immunoprecipitated with antibodies cross-linked to protein A-sepharose beads (Sigma) in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease/phosphatase inhibitor cocktail. Immunoprecipitated beads were washed with 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100, and then in 50 mM Tris-HCl (pH 7.6) and 150 mM NaCl. 32P incorporation in immunoprecipitated proteins was determined by Tris-glycine SDS–PAGE, fixation and autoradiography. Adipocytes (0.25 × 106 cells) were pre-incubated in serum-free DMEM for 4 h. Subsequently, the medium was supplemented with 2.5 μCi [14C]Glu or [14C]Pro (Perkin-Elmer), and cells incubated for additional 6 h. Adipocytes were lysed and 14C incorporation determined by trichloroacetic acid-precipitation and scintillation counting. Mouse adipocytes (0.25 × 106 cells) were pre-incubated in methionine-free RPMI medium (Invitrogen) with 10% FBS for 30 min. [35S]Met/Cys (250 μCi, Perkin-Elmer) was added and incubated at 37 °C with 5% CO for 15 min. Labelled cells were lysed in RIPA buffer (Thermo Fisher) and analysed by Tris-glycine SDS–PAGE, fixation and autoradiography. Cell lysates or immunoprecipitates were denatured in Laemmli sample buffer (Bio-Rad) and resolved on Tris-glycine SDS–PAGE (10, 12, or 15% polyacrylamide) prepared using 37.5:1 acrylamide:bis-acrylamide stock solution (National Diagnostics). After transfer to polyvinyl difluoride membrane, the membranes were probed with target-specific antibody, followed by incubation with horseradish peroxidase conjugated secondary antibody and detection with Amersham ECL prime western blotting detection reagent (GE Healthcare). Immunoblots shown are typical of experiments independently done at least three times. Pre-cleared cell lysates (1 mg) were incubated with antibody cross-linked to protein A-sepharose beads in detergent-free buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and EDTA-free protease/phosphatase inhibitor cocktail. Immunoprecipitates were analysed by Tris-glycine SDS–PAGE and immunoblotting either after washing the beads three times in the same buffer or after elution, followed by neutralization with 0.2 M glycine-HCl (pH 2.6) or 50 mM Tris-HCl (pH 8.5), respectively. Fatty acid uptake assay kit (QBT, Molecular Devices) that utilizes fluorescent bodipy-C , a LCFA analogue, was used to determine fatty acid uptake50. Differentiated 3T3-L1 adipocytes were plated at 5 × 104 cells per well in a 96-well plate. Adipocytes were first incubated in serum-free Hank’s balanced salt (HBS) solution for 4 h, and then with 100 nM insulin and bodipy-C for an additional 4 h. After 30 min, relative fluorescence was read at 485 nm excitation and 515 nm emission wavelength in bottom-read mode (SpectraMax GeminiEM, Molecular Devices). LCFA uptake was also determined in differentiated 3T3-L1 adipocytes as cellular accumulation of [14C]oleate (Perkin-Elmer). Adipocytes (10,000 cells) were seeded in a 24-well plate in DMEM with 10% calf serum overnight. Cells were serum-deprived for 4 h, treated with 100 nM insulin for 3.5 h, and then with 50 μM of [14C]oleate in HBS containing 0.1% fatty acid-free BSA for 30 min51, 52. Cells were washed extensively in cold HBS with 0.1% fatty acid-free BSA to remove unincorporated [14C]oleate, lysed in RIPA buffer (Thermo Fisher), and centrifuged at 2000 rpm for 5 min. Supernatant radioactivity was determined by scintillation counting and normalized to protein. LCFA uptake by mouse WAT, hepatocytes, cardiac cells, BMDM, and soleus muscle strips were measured using essentially the same method13. Adipocytes from wild-type and mutant mice were pre-incubated for 4 h in serum- and glucose-free DMEM and then rinsed with Krebs-Ringer buffer containing 20 mM HEPES (pH 7.4), 5 mM sodium phosphate, 1 mM MgSO , 1 mM CaCl , 136 mM NaCl, and 4.7 mM KCl53, 54. Adipocytes were incubated for 4 h in the presence of 1 μCi of [14C]2-deoxy-d-glucose (DG; Perkin-Elmer) and 100 nM insulin in the same buffer supplemented with 100 mM unlabelled 2-DG (Sigma). Uptake was stopped using ice-cold PBS containing 50 μM cytochalasin, followed by four washes with PBS. Lysate radioactivity was determined by scintillation counting. Membrane fraction from differentiated 3T3-L1 adipocytes was isolated by phase partitioning using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo-Scientific). Plasma membrane fractions from 3T3-L1 adipocytes were prepared as described14. Differentiated 3T3-L1 adipocytes were washed in buffer containing 250 mM sucrose, 10 mM Tris (pH 7.4), and 0.5 mM EDTA. Lysates were prepared by homogenization in the same buffer supplemented with protease and phosphatase inhibitor cocktail, and centrifuged at 16,000g for 20 min at 4 °C. The re-suspended pellet was layered onto a solution containing 1.12 M sucrose, 10 mM Tris (pH 7.4), and 0.5 mM EDTA, and centrifuged at 150,000g for 20 min at 4 °C. The resulting pellet was suspended in RIPA buffer (Sigma) and plasma membrane was obtained by centrifugation at 74,000g for 20 min at 4 °C. All data generated are included in the published article and in the supplementary information files. Additional statistical data sets generated are available from the corresponding author upon request.


News Article | December 15, 2016
Site: www.businesswire.com

HAMBURG, Germany--(BUSINESS WIRE)--Evotec AG (Frankfurt Stock Exchange: EVT, TecDAX, ISIN: DE0005664809) today announced the successful closing of the acquisition of 100% shares in Cyprotex PLC ("Cyprotex", AIM: CRX-GB), a specialist pre-clinical contract research organisation in ADME-Tox and DMPK headquartered in the UK. The proposed acquisition was announced in detail on 26 October 2016. Following a scheme of arrangement regulated by the UK takeover code, all shares of Cyprotex have been acquired by and transferred to Evotec AG effective 14 December 2016 and the shares will this morning be cancelled from AIM. Evotec is paying £ 55.7 m (EUR 66.3 m; at an assumed £/EUR exchange rate of 1.19) in cash for the acquisition of all 26.1 million issued and to be issued Cyprotex shares and the funding of company debt mainly in the context of loan notes. The offer of 1.60 £ per Cyprotex share reflects a 9.4% premium to the VWAP of the past 30 trading days at AIM prior to the offer on 26 October 2016. MCF Corporate Finance, led by Ian Henderson, acted as Evotec's exclusive financial adviser throughout the acquisition process. Cyprotex, headquartered in the UK, was founded in 1999 and is publicly traded on AIM (CRX). The company currently has 136 employees working from sites at Macclesfield and Alderley Park, both of which are located near Manchester in the UK, and at Watertown, MA, and Kalamazoo, MI, in the USA. Cyprotex will continue to operate and serve its loyal client base in all currently existing segments under its brand name "Cyprotex" whilst employees and capabilities will be integrated into Evotec's global drug discovery group, thereby leveraging both companies' extensive partner networks and identifying further commercial synergies. Dr Mario Polywka, Chief Operating Officer of Evotec, commented: "We are pleased the acquisition has closed and we can now approach the exciting phase of welcoming Cyprotex' employees and clients to our global drug discovery services platform. The addition of the market's most industrialised ADME-Tox platform and proven expertise in in vitro ADME screening, mechanistic and high-content toxicology screening and predictive modelling to our offering substantially improves our ability to provide our alliance partners with access to the most comprehensive drug discovery platform. Cyprotex' proven technology platform and its expert and dedicated employees perfectly augment Evotec's business strategy and offering." Dr Werner Lanthaler, Chief Executive Officer of Evotec, added: "The highest quality and completeness of our drug discovery platform is key to improve the efficiency in the process for our partners. With Cyprotex we make here an important next step. We warmly welcome the Cyprotex employees to the Evotec Group and look forward to working with them." Evotec confirms its liquidity guidance for 2016. The Company expects liquidity to be at a similar level to the prior year, excluding any potential cash outflow for M&A or similar transactions. Based on current estimates, it is expected that the Cyprotex business will add approx. EUR 18-20 m in revenues in 2017 and will be accretive to Evotec's 2017 EBITDA. Cyprotex is listed on the AIM market of the London Stock Exchange (CRX). It has sites at Macclesfield and Alderley Park, both of which are near Manchester in the UK, and at Watertown, MA and Kalamazoo, MI in the US. The Company was established in 1999 and works with more than 1500 partners within the pharmaceutical and biotech industry, cosmetics and personal care industry and the chemical industry. Cyprotex acquired Apredica and the assets of Cellumen Inc. in August 2010 and the combined business provides support for a wide range of experimental and computational ADME-Tox and PK services. The acquisition of the assets and business of CeeTox in January 2014 has enabled Cyprotex to expand its range of services to target the personal care, cosmetics and chemical industries. In 2015, Cyprotex launched its new bioscience division to expand its capabilities into phenotypic and target based screening. The Company's core capabilities include high quality in vitro ADME services, mechanistic toxicology and high content toxicology screening services, including its proprietary CellCiphr(R) toxicity prediction technology, bioscience services, predictive modelling solutions including Cloe(R) PK, chemPK(TM), chemTarget, chemTox and DDI-Fusion and a range of skin, ocular and endocrine disruption services. For more information, please visit www.cyprotex.com. Evotec is a drug discovery alliance and development partnership company focused on rapidly progressing innovative product approaches with leading pharmaceutical and biotechnology companies, academics, patient advocacy groups and venture capitalists. We operate worldwide providing the highest quality stand-alone and integrated drug discovery solutions, covering all activities from target-to-clinic to meet the industry's need for innovation and efficiency in drug discovery (EVT Execute). The Company has established a unique position by assembling top-class scientific experts and integrating state-of-the-art technologies as well as substantial experience and expertise in key therapeutic areas including neuroscience, diabetes and complications of diabetes, pain and inflammation, oncology and infectious diseases. On this basis, Evotec has built a broad and deep pipeline of more than 70 partnered product opportunities at clinical, pre-clinical and discovery stages (EVT Innovate). Evotec has established multiple long-term discovery alliances with partners including Bayer, CHDI, Sanofi or UCB and development partnerships with e.g. Janssen Pharmaceuticals in the field of Alzheimer's disease, with Sanofi in the field of diabetes and with Pfizer in the field of tissue fibrosis. For additional information please go to www.evotec.com. MCF Corporate Finance ("MCF") is a leading independent and international corporate finance advisory firm with offices in Hamburg, Helsinki, London and Stockholm. The company was established in 1987 and is run as an independent partnership. MCF specialises in cross-border and domestic M&A transactions in the European markets. Its multinational team consists of more than 40 corporate finance specialists with extensive experience in industry, banking, finance, accounting and law. MCF has previously advised Evotec on several transactions in both Germany and the UK. For further information, please go to www.mcfcorpfin.com/en. Information set forth in this press release contains forward-looking statements, which involve a number of risks and uncertainties. The forward-looking statements contained herein represent the judgement of Evotec as of the date of this press release. Such forward-looking statements are neither promises nor guarantees, but are subject to a variety of risks and uncertainties, many of which are beyond our control, and which could cause actual results to differ materially from those contemplated in these forward-looking statements. We expressly disclaim any obligation or undertaking to release publicly any updates or revisions to any such statements to reflect any change in our expectations or any change in events, conditions or circumstances on which any such statement is based.


News Article | December 15, 2016
Site: www.businesswire.com

HAMBURG, Deutschland--(BUSINESS WIRE)--Evotec AG (Frankfurter Wertpapierbörse: EVT, TecDAX, ISIN: DE0005664809) gab heute den erfolgreichen Abschluss der Akquisition von 100% aller Anteile an der Cyprotex PLC ("Cyprotex", AIM: CRX-GB) bekannt. Cyprotex ist ein auf präklinische ADME-Tox- und DMPK-Leistungen spezialisiertes Auftragsforschungsunternehmen mit Hauptsitz in UK. Die beabsichtigte Akquisition wurde detailliert am 26. Oktober 2016 bekannt gegeben. Mittels eines detaillierten Übernahmeplans, des sogenannten Scheme of Arrangements, der durch den UK Takeover Code geregelt wird, wurden alle Anteile an der Cyprotex mit Wirkung zum 14. Dezember 2016 durch die Evotec AG erworben und transferiert. Im Verlauf des heutigen Morgens erfolgt ein Delisting der Cyprotex-Aktien von der AIM. Evotec entrichtet 55,7 Mio. £ (66,3 Mio. EUR bei einem angenommenen £/EUR-Kurs von 1,19) in bar für die Übernahme aller 26,1 Mio. ausgegebenen und noch auszugebenden Cyprotex-Aktien und die Übernahme der Verbindlichkeiten des Unternehmens, die hauptsächlich aus ausgegebenen Anleihen bestehen. Das Angebot von 1,60 £ pro Cyprotex-Aktie entspricht einem 9,4%-Aufschlag auf den VWAP (volumengewichteter Durchschnittskurs) der letzten 30 Handelstage an der AIM vor Unterbreitung des Angebots am 26. Oktober 2016. MCF Corporate Finance unter der Leitung von Ian Henderson fungierte während des Übernahmeprozesses als exklusiver Finanzberater von Evotec. Cyprotex hat seinen Hauptsitz in UK, wurde im Jahr 1999 gegründet und ist an der AIM (CRX) notiert. Das Unternehmen hat derzeit 136 Mitarbeiter, die an den Standorten Macclesfield and Alderley Park (beide in der Nähe von Manchester, UK) sowie Watertown, MA, und Kalamazoo, MI, in den USA beschäftigt sind. Cyprotex wird weiterhin seinen loyalen Kundenstamm in allen derzeitig bestehenden Segmenten unter der Marke Cyprotex bedienen. Die Mitarbeiter und Kapazitäten sowie Fähigkeiten werden gleichzeitig in Evotecs globale Wirkstoffforschungsplattform integriert, um die umfangreichen Partnernetzwerke beider Unternehmen wirksam einzusetzen und weitere kommerzielle Synergien zu identifizieren. Dr. Mario Polywka, Chief Operating Officer von Evotec, kommentierte: "Wir freuen uns sehr, dass die Übernahme nun abgeschlossen ist und wir in die nächste spannende Phase eintreten können, in der wir die Cyprotex-Mitarbeiter und -Kunden auf unserer globalen Wirkstoffforschungsplattform begrüßen dürfen. Die Akquisition dieser industrialisierten ADME-Tox-Plattform und nachgewiesener Expertise in in vitro-ADME-Screening, mechanistischem sowie High-Content Toxicology-Screening und Vorhersagemodellen bedeutet eine Stärkung unserer hochwertigen Wirkstoffforschungsplattform und Expertise in diesem Bereich und ermöglicht unseren Partnern Zugang zu einer äußerst umfangreichen Wirkstoffforschungsplattform. Die bewährte Technologieplattform von Cyprotex und die erfahrenen und engagierten Mitarbeiter stellen eine sehr gute Ergänzung unserer Strategie und unseres Angebots dar." Cyprotex ist im AIM-Segment der London Stock Exchange notiert (CRX). Das Unternehmen verfügt über Standorte in Macclesfield und Alderley Park, beide in der Nähe von Manchester, UK, sowie in Watertown, MA, und Kalamazoo, MI, in den USA. Cyprotex wurde im Jahr 1999 gegründet und arbeitet mit mehr als 1.500 Partnern aus der Pharma- und Biotechbranche, Kosmetik-, Personal Care- und Chemiebranche zusammen. Im Jahr 2010 hat Cyprotex Apredica und die Vermögenswerte von Cellumen Inc. erworben. Das daraus resultierende gemeinsame Geschäft bietet Unterstützung in einer Vielzahl von experimentellen und computerbasierten ADME-Tox- und PK-Leistungen an. Infolge der Übernahme von CeeTox im Januar 2014 konnte Cyprotex seine Bandbreite von Leistungen auf die Bereiche Personal Care, Kosmetik und Chemie ausweiten. Im Jahr 2015 erweiterte Cyprotex mit dem neuen Bioscience-Bereich seine Fähigkeiten in den Bereichen phänotypisches und targetbasiertes Screening. Zu den Kernkompetenzen des Unternehmens zählen hochwertige in vitro-ADME-Leistungen, mechanistische Toxikologie und High Content Toxicology Screening Services, darunter die proprietären Technologien CellCiphr(R) (toxicity prediction technology), Bioscience-Leistungen, Vorhersagemodelle wie Cloe(R) PK, chemPKTM, chemTarget, chemTox und DDI-Fusion, sowie eine Vielzahl von Leistungen in Bezug auf Haut-, okulare und endokrine Disruptoren. Weitere Informationen finden Sie unter www.cyprotex.com. Evotec ist ein Wirkstoffforschungs- und -entwicklungsunternehmen, das in Forschungsallianzen und Entwicklungspartnerschaften mit führenden Pharma- und Biotechnologieunternehmen, akademischen Einrichtungen, Patientenorganisationen und Risikokapitalgesellschaften innovative Ansätze zur Entwicklung neuer pharmazeutischer Produkte zügig vorantreibt. Wir sind weltweit tätig und bieten unseren Kunden qualitativ hochwertige, unabhängige und integrierte Lösungen im Bereich der Wirkstoffforschung an. Dabei decken wir alle Aktivitäten vom Target bis zur klinischen Entwicklung ab, um dem Bedarf der Branche an Innovation und Effizienz in der Wirkstoffforschung begegnen zu können (EVT Execute). Durch das Zusammenführen von erstklassigen Wissenschaftlern, modernsten Technologien sowie umfangreicher Erfahrung und Expertise in wichtigen Indikationsgebieten wie zum Beispiel Neurowissenschaften, Diabetes und Diabetesfolgeerkrankungen, Schmerz und Entzündungskrankheiten, Onkologie und Infektionskrankheiten ist Evotec heute einzigartig positioniert. Auf dieser Grundlage hat Evotec ihre Pipeline bestehend aus mehr als 70 verpartnerten Programmen in klinischen, präklinischen und Forschungsphasen aufgebaut (EVT Innovate). Evotec arbeitet in langjährigen Forschungsallianzen mit Partnern wie Bayer, CHDI, Sanofi oder UCB zusammen. Darüber hinaus verfügt das Unternehmen über Entwicklungspartnerschaften u. a. mit Janssen Pharmaceuticals im Bereich der Alzheimer'schen Erkrankung, mit Sanofi im Bereich Diabetes und mit Pfizer auf dem Gebiet Organfibrose. Weitere Informationen finden Sie auf unserer Homepage. www.evotec.com. MCF Corporate Finance ("MCF") ist eine führende unabhängige und international tätige Corporate Finance Beratung mit eigenen Standorten in Hamburg, Helsinki, London und Stockholm. MCF wurde 1987 gegründet und wird als unabhängige Partnerschaft geführt. MCF ist auf inländische und grenzüberschreitende M&A Transaktionen im europäischen Raum spezialisiert. Das multinationale Team von MCF besteht aus mehr als 40 Corporate Finance-Spezialisten mit umfangreicher Expertise in den Bereichen Industrie, Bank, Finanzen, Rechnungswesen und Recht. MCF hat Evotec bereits bei diversen Transaktionen sowohl in Deutschland als auch in UK beraten. Weitere Informationen finden Sie unter www.mcfcorpfin.com/en.


No statistical methods were used to predetermine sample size. All procedures involving mice were performed in accordance with AAALAC standards or under UK Home Office regulations, and were reviewed and approved in accordance with the Novartis Animal Welfare Policy. Sample size was determined on the basis of the minimum number of animals required for good data distribution and statistics. Blinding was not possible in these experiments but animals were selected randomly for each group. Reported IC / EC / CC values were calculated by averaging IC / EC / CC values obtained from individual technical replicate experiments (n; specified in relevant Figure captions and Methods sub-sections). Each technical replicate experiment was performed on a different day with freshly prepared reagents. Reported standard errors of mean (s.e.m.) were calculated using IC / EC / CC values determined in individual technical replicate experiments. To calculate IC / EC / CC values, measured dose response values were fitted with 4-parameter logistic function (model 201, XLfit, IDBS), where x refers to compound concentration and y corresponds to an assay readout value. RPMI-1640 medium (HyClone) was supplemented with 20% heat-inactivated fetal bovine serum (Omega Scientific), 23 μM folic acid (Sigma-Aldrich), 100 μM adenosine (Sigma-Aldrich), 22 mM d-glucose (Sigma-Aldrich), 4 mM l-glutamine (Hyclone), 25 mM 2-(4-morpholino) ethanesulfonic acid (Sigma-Aldrich) and 100 IU penicillin/ 100 μg/ml streptomycin (HyClone), and adjusted to pH = 5.5 with 6 M hydrochloric acid (Fisher Scientific) at 37 °C. Leishmania donovani MHOM/SD/62/1 S-CL2D axenic amastigotes were cultured in 10 ml of this medium (Axenic Amastigote Medium) in T75 CELL-STAR flasks (Greiner Bio-One) at 37 °C/ 5% CO and passaged once a week. To determine compound growth inhibitory potency on L. donovani axenic amastigotes, 100 nl of serially diluted compounds in DMSO were transferred to the wells of white, solid bottom 384-well plates (Greiner Bio-One) by Echo 555 acoustic liquid handling system (Labcyte). Then, 1 × 103 of L. donovani axenic amastigotes in 40 μl of Axenic Amastigote Medium were added to each well, and plates were incubated for 48 h at 37 °C/ 5% CO . Parasite numbers in individual plate wells were determined through quantification of intracellular ATP. The CellTiter-Glo luminescent cell viability reagent (Promega) was added to plate wells, and ATP-dependent luminescence signal was measured on an EnVision MultiLabel Plate Reader (Perkin Elmer). Luminescence values in wells with compounds were divided by the average luminescence value of the plate DMSO controls, and used for calculation of compound EC values as described above. Axenic amastigote EC values shown in Fig. 4b correspond to means of 2 technical replicates. Female BALB/cJ mice (Envigo) infected with L. donovani MHOM/ET/67/HU3 (ATCC) for 50–80 days were euthanized, and infected spleens were removed and weighed. The weight of an infected spleen ranged from 300 to 600 mg. For comparison, spleens from non-infected age-matched BALB/cJ mice weighed ~100 mg. Infected spleens were washed in Axenic Amastigote Medium (composition described above) and placed into Falcon 50 ml conical centrifuge tubes (Fisher Scientific) containing ice-cold Axenic Amastigote Medium (15 ml per infected spleen). Spleens were homogenized on ice in a Dounce homogenizer and centrifuged at 200g for 15 min at 4 °C to remove tissue debris. Leishmania donovani amastigotes present in the supernatant were pelleted by centrifugation at 1,750g for 15 min at 4 °C and re-suspended either in Axenic Amastigote Medium (when used for in vitro macrophage infections) or in Hanks’ Balanced Salt Solution (when used for mouse infections; Hyclone). Suspensions of splenic amastigotes were kept on ice and used for in vitro or in vivo infections within 2–3 h. To propagate L. donovani amastigotes in vivo, 6–7-week-old female BALB/cJ mice were infected with 8 × 107 purified splenic amastigotes in 200 μl of Hanks’ Balanced Salt Solution by tail vein injection. In vitro compound potencies on intra-macrophage L. donovani MHOM/ET/67/HU3 were determined using primary murine peritoneal macrophages infected with L. donovani splenic amastigotes. Primary macrophages were elicited in female BALB/cJ mice for 72 h following the injection of 500 μl of sterile aqueous 2% starch (J. T. Baker) solution into the mouse peritoneal cavity. The protocol used for isolation of peritoneal macrophages was described in detail previously31. The isolated macrophages were re-suspended in Macrophage Infection Medium (RPMI-1640 medium supplemented with 2 mM l-glutamine, 10% heat-inactivated fetal bovine serum, 10 mM sodium pyruvate (Hyclone), and 100 IU penicillin/ 100 μg/ml streptomycin), and 50 μl of macrophage suspension (4 × 105 macrophages/ml) were added to microscopy-grade, clear-bottom, black 384-well plates (Greiner Bio-One). Following overnight incubation at 37 °C/ 5% CO , plate wells were washed with Macrophage Infection Medium to remove non-adherent cells using ELx405 Select microplate washer (BioTek), and then filled with 40 μl of Macrophage Infection Medium. Leishmania donovani HU3 splenic amastigotes isolated from infected spleens were re-suspended in Macrophage Infection Medium at a concentration of 6 × 107 cells/ml, and 10 μl of the suspension were added to assay plate wells containing adherent macrophages. After a 24-h infection period at 37 °C/ 5% CO , plate wells were washed with Macrophage Infection Medium to remove residual extracellular parasites and re-filled with 50 μl of the medium. Leishmania donovani-infected macrophages were subsequently treated with DMSO-dissolved compounds (0.5% final DMSO concentration in the assay medium) in dose response for 120 h at 37 °C/ 5% CO . Next, treated macrophages were washed with the phosphate-buffered saline buffer (PBS; Sigma-Aldrich) supplemented with 0.5 mM magnesium chloride (Sigma-Aldrich) and 0.5 mM calcium chloride (Sigma-Aldrich), fixed with 0.4% paraformaldehyde (Sigma-Aldrich) in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS, and stained with SYBR Green I nucleic acid stain (Invitrogen, 1:100,000 dilution in PBS) overnight at 4 °C. Image collection and enumeration of macrophage cells and intracellular L. donovani amastigotes was performed using the OPERA QEHS automated confocal microscope system equipped with 20× water immersion objective (Evotec Technologies) and the OPERA Acapella software (Evotec Technologies) as described previously32. All reported intra-macrophage L. donovani EC values were calculated from at least 3 technical replicates (n = 3 or n = 4; specified in relevant figure captions). Bloodstream form Trypanosoma brucei Lister 427 parasites were continuously passaged in HMI-9 medium formulated from IMDM medium (Invitrogen), 10% heat-inactivated fetal bovine serum, 10% Serum Plus medium supplement (SAFC Biosciences), 1 mM hypoxanthine (Sigma-Aldrich), 50 μM bathocuproine disulfonic acid (Sigma-Aldrich), 1.5 mM cysteine (Sigma-Aldrich), 1 mM pyruvic acid (Sigma-Aldrich), 39 μg/ml thymidine (Sigma-Aldrich), and 14 μl/lβ-mercapthoethanol (Sigma-Aldrich); all concentrations of added components refer to those in complete HMI-9 medium. The parasites were cultured in 10 ml of HMI-9 medium in T75 CELL-STAR tissue culture flasks at 37 °C/ 5% CO . To determine compound growth inhibitory potency on T. brucei bloodstream form parasites, 100 nl of serially diluted compounds in DMSO were transferred to the wells of white, solid bottom 384-well plates (Greiner Bio-One) by Echo 555 acoustic liquid handling system. Then, 5 × 103 of T. brucei parasites in 40 μl of HMI-9 medium were added to each well, and the plates were incubated for 48 h at 37 °C/ 5% CO . Parasite numbers in individual plate wells were determined through quantification of intracellular ATP amount. The CellTiter-Glo luminescent cell viability reagent was added to plate wells, and ATP-dependent luminescence signal was measured on an EnVision MultiLabel Plate Reader. Luminescence values in wells with compounds were divided by the average luminescence value of the plate DMSO controls, and used for calculation of compound EC values as described above. Trypanosoma brucei EC values shown in Fig. 1 and Extended Data Fig. 3 correspond to means of 4 technical replicates. NIH 3T3 fibroblast cells (ATCC) were maintained in RPMI-1640 medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum and 100 IU penicillin/ 100 μg/ml streptomycin at 37 °C/ 5% CO . Trypanosoma cruzi Tulahuen parasites constitutively expressing Escherichia coli β-galactosidase33 were maintained in tissue culture as an infection in NIH 3T3 fibroblast cells. Briefly, 2 × 107 T. cruzi trypomastigotes were used to infect 6 × 105 NIH 3T3 cells growing in T75 CELL-STAR tissue culture flasks and cultured at 37 °C/ 5% CO until proliferating intracellular parasites lysed host 3T3 cells and were released into the culture medium (typically 6–7 days). During the infection, the tissue culture medium was changed every two days. Number of T. cruzi trypomastigotes present in 1 ml of medium was determined using a haemocytometer. To determine compound potency on intracellular T. cruzi amastigotes, NIH 3T3 cells were re-suspended in phenol red-free RPMI-1640 medium containing 3% heat-inactivated fetal bovine serum and 100 IU penicillin/ 100 μg/ml streptomycin, seeded at 1,000 cells/ well (40 μl) in white, clear bottom 384-well plates (Greiner Bio-One), and incubated overnight at 37 °C/ 5% CO . The following day, 100 nl of each compound in DMSO were transferred to individual plate wells by Echo 555 acoustic liquid handling system. After one hour incubation, 1 × 106 of tissue culture-derived T.cruzi trypomastigotes, in 10 μl of phenol red-free RPMI-1640 medium supplemented with 3% heat-inactivated fetal bovine serum and 100 IU penicillin/ 100 μg/ml streptomycin were added to each well. Plates were then incubated for 6 days at 37 °C/ 5% CO . Intracellular T. cruzi parasites were quantified by measuring the activity of parasite-expressed β-galactosidase. Ten microlitres of a chromogenic β-galactosidase substrate solution (0.6 mM chlorophenol red-β-D-galactopyranoside/ 0.6% NP-40 in PBS; both reagents from Calbiochem) were added to each well and incubated for 2 h at room temperature. After incubation, absorption was measured at 570 nM on SpectraMax M2 plate reader (Molecular Devices). Measured absorbance values in wells with compounds were divided by the average absorbance value of the plate DMSO controls, and used for calculation of compound EC values as described above. Trypanosoma cruzi amastigote EC values shown in Fig. 1 and Extended Data Fig. 3 correspond to means of 4 technical replicates. Trypanosoma cruzi CL epimastigotes were continuously passaged in LIT medium containing 9 g/l liver infusion broth (Difco), 5 g/l bacto tryptose (Difco), 1 g/l sodium chloride, 8 g/l dibasic sodium phosphate (Sigma-Aldrich), 0.4 g/l potassium chloride (Sigma-Aldrich), 1 g/l d-glucose, 10% heat-inactivated fetal bovine serum and 10 ng/ml of hemin (Sigma-Aldrich). The medium was adjusted to pH = 7.2 with 6 M hydrochloric acid. The parasites were cultured in 10 ml of LIT medium in T75 CELL-STAR tissue culture flasks at 27 °C. To determine compound growth inhibitory potency on T. cruzi epimastigotes, 100 nl of serially diluted compounds in DMSO were transferred to the wells of white, solid bottom 384-well plates (Greiner Bio-One) by an Echo 555 acoustic liquid handling system. Then, 5 × 103 of T. cruzi epimastigotes in 40 μl of LIT medium were added to each well, and the plates were incubated for 7 days at 27 °C. Parasite numbers in individual plate wells were determined through quantification of intracellular ATP amount. The CellTiter-Glo luminescent cell viability reagent was added to plate wells, and ATP-dependent luminescence signal was measured on an EnVision MultiLabel Plate Reader. Luminescence values in wells with compounds were divided by the average luminescence value of the plate DMSO controls, and used for calculation of compound EC values as described above. Trypanosoma cruzi epimastigote EC values shown in Extended Data Fig. 4 correspond to means of 3 technical replicates. NIH 3T3 fibroblast cells were maintained in RPMI-1640 medium with glutamine (Life Technologies) supplemented with 5% heat-inactivated fetal bovine serum and 100 IU penicillin/ 100 μg/ml streptomycin (3T3 medium) at 37 °C/ 5% CO . NIH 3T3 fibroblast cells were purchased from ATCC. We did not perform cell line authentication and did not test the cells for mycoplasma contamination. This cell line is not listed in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample. To determine compound potency, NIH 3T3 cells re-suspended in 3T3 medium were seeded at 1,000 cells/well (50 μl) in white 384-well plates (Greiner Bio-One) and incubated overnight at 37 °C/ 5% CO . The following day, 100 nl of each compound in DMSO were transferred to individual plate wells by Echo 555 acoustic liquid handling system and plates were incubated for five days at 37 °C/ 5% CO . Cell numbers in individual plate wells were determined through quantification of intracellular ATP amount. The CellTiter-Glo luminescent cell viability reagent was added to plate wells, and ATP-dependent luminescence signal was measured on an EnVision MultiLabel Plate Reader. Luminescence values in wells with compounds were divided by the average luminescence value of the plate DMSO controls, and used for calculation of compound CC values as described above. NIH 3T3 CC values shown in Fig. 1 and Extended Data Fig. 3 correspond to means of 4 technical replicates. Primary macrophage cell viability was determined on mouse peritoneal macrophages infected with L. donovani and was expressed as the ratio of the number of macrophage cells in wells treated with a compound to those in wells treated with DMSO. The number of macrophage cells in wells was determined by high content microscopy as described previously32. All reported macrophage CC values were calculated from 4 technical replicates (n = 4; also specified in Fig. 1 and Extended Data Fig. 3 captions). Trypanosoma cruzi epimastigotes cultures resistant to GNF3943 and GNF8000 were generated using a methodology described previously32. Briefly, epimastigotes were initially cultured in the presence of compound concentration equivalent to its EC value (GNF3943 EC  = 1.5 μM and GNF8000 EC  = 0.2 μM in 0.2% DMSO) or 0.2% DMSO (control). Once a week, parasites were counted and growth rates were determined. If the parasite cultures exhibited a reduced growth rate compared to 0.2% DMSO-treated parasites, epimastigotes were cultured at the same compound concentration. Once the growth rates matched that of the control epimastigote culture (0.2% DMSO), parasites were transferred into medium containing twofold higher compound concentration. The process was repeated until substantial resistance was achieved (~10- to 20-fold increase in corresponding EC value). The time required for generation of cultures with such a level of resistance was approximately five months. Resistant clones were isolated via cloning by limiting dilution, and two independent clones were analysed by whole-genome sequencing. Chromosomal DNA isolation from GNF3943- and GNF8000-resistant T. cruzi clones, whole-genome sequencing and sequence analysis were performed as described previously32. Sequencing reads were aligned to the T. cruzi CL Brenner genome34. PSMB4 TcCLB503891.100 was amplified from T. cruzi CL Brenner genomic DNA using KOD Hot Start DNA Polymerase (EMD Millipore), and sense (5′-AAAGCGGCCGCATGTCGGAGACAACCATTG-3′) and antisense (5′-CCATGATCTTGATGTAATATAAGGCATTCAGCCCTGCTG-3′) primers. The PSMB4F24L gene was generated from the wild-type PSMB4 construct by site-directed mutagenesis using mutagenic sense (5′-CAGCAGGGCTGAATGCCTTATATTACATCAAGATCATGG-3′) and antisense (5′-CCATGATCTTGATGTAATATAAGGCATTCAGCCCTGCTG-3′) primers and QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The sequences of the wild-type and mutant PSMB4 genes were verified by sequencing and both gene versions were subcloned into the T. cruzi expression vector pTcIndex1 under control of a T7 promoter35. Trypanosoma cruzi CL Brenner epimastigotes were first transfected as described previously36 with the pLEW13 plasmid37 harbouring a tetracycline-inducible T7 RNA polymerase gene. Transfected epimastigotes were selected in medium supplemented with neomycin (G418) at 500 μg/ml, and then transfected a second time with either pTcIndex1-PSMB4wt or pTcIndex1-PSMB4F24L plasmid. Double-transfected epimastigotes were selected in the presence of 500 μg/ml of G418 (Sigma-Aldrich) and 500 μg/ml of hygromycin (Sigma-Aldrich). Susceptibility of double transfected epimastigote cell lines to compounds was assessed using induced (+5 mg/ml of tetracycline) and non-induced parasite cultures after five days of compound treatment. Parasite viability was determined with AlamarBlue (ThermoFisher Scientific). Reported EC values for T. cruzi epimastigotes ectopically expressing PSMB4 proteins were calculated from 3 technical replicates (n = 3; also specified in the Fig. 3a caption). PSMB4 (Tb927.10.4710) was amplified from T. brucei Lister 427 genomic DNA using PCR SuperMix High Fidelity (Invitrogen), sense (5′-GCAAGCTTATGGCAGAGACGACTATCGG-3′) and antisense (5′-GCGGATCCCTAGCTTACAGATTGCACTC-3′) primers. The PSMB4F24L gene was generated from the wild-type PSMB4 construct by site-directed mutagenesis using mutagenic sense (5′- GCTGCGGGGTTAAATGCGTTATACTACATTAAGATAACGG-3′), antisense (5′-CCGTTATCTTAATGTAGTATAACGCATTTAACCCCGCAGC-3′) primers and QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The sequences of the wild-type and mutant PSMB4 genes were verified by sequencing and both gene versions were cloned into the T. brucei expression vector pHD1034 under control of a ribosomal RNA promoter. Transfected T. brucei Lister 427 cells were selected in medium supplemented with puromycin at 1 μg/ml. Susceptibility of transfected T. brucei cell lines to compounds was assessed after 2 days of compound treatment. Parasite viability was determined with CellTiter-Glo. Reported EC values for T. brucei parasites ectopically expressing PSMB4 proteins were calculated from 3 technical replicates (n = 3; also specified in the Fig. 3b caption). Trypanosoma cruzi CL epimastigotes, L. donovani MHOM/SD/62/1 S-CL2D axenic amastigotes and T. brucei Lister 427 bloodstream form trypomastigotes were grown to log phase and harvested by centrifugation. The corresponding cell pellets were stored at −80 °C until further use. Prior to purification, 10 g of cell pellets were thawed, re-suspended in lysis buffer (50 mM Tris-HCl pH = 7.5, 1 mM TCEP, 5 mM EDTA, and 10 μM E-64), and lysed by passing cell suspension three times through a needle (22 gauge) and by subsequent three freeze/thaw cycles. The lysate was first cleared of cellular debris by two centrifugation steps (15,000g at 4 °C for 15 min followed by 40,000g at 4 °C for 60 min) and then fractionated through ammonium sulphate precipitation. The protein fraction precipitated between 45% and 65% of ammonium sulphate saturation was re-suspended in 25 mM Tris-HCl pH = 7.5, 1 mM TCEP buffer, and dialysed overnight at 4 °C against the same buffer. Proteasomes were further purified by anion exchange chromatography (Resource Q column, GE Healthcare Life Sciences) and size-exclusion chromatography (Superose 6 column, GE Healthcare Life Sciences) as described elsewhere38. Active fractions from the latter purification step were pooled and used in proteasome biochemical assays. Purified T. cruzi proteasome sample was buffer-exchanged and concentrated into 100 mM trimethylamine bicarbonate-HCl pH = 8.0, 150 mM NaCl buffer using a 10 kDa molecular weight cut-off micro-concentrator (Milipore Amicon Ultra). The resulting proteasome sample (200 μl, 1 mg/ml) was mixed with 5 μl of a TMTsixplex reagent (Pierce). After 60 s incubation to label primary amines, the reaction was stopped by adding 25 μl of 5% hydroxylamine. The labelled sample was run on 4–20% Bis-Tris PAGE gel (Invitrogen) to separate polypeptides. The gel was stained with eStain 2.0 (GenScript). Stained protein bands were cut out and in-gel-digested separately with elastase (Promega) and asparaginase (Roche). Peptides generated by the digestions were resolved by HPLC using a vented column setup with a 2 cm Poros 10 R2 (Life Technologies, Carlsbad, CA) self-packed pre-column, and a PepMap Easy-Spray C18 analytical column (15 cm × 75 μm ID, Thermo Scientific). Resin-bound proteolytic fragments were eluted with 2 to 40% acetonitrile / 0.1% formic acid operated at 300 nl/min for 120 min. Spectra of eluted peptide species were determined by a column-coupled Q Exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Scientific). Proteome Discoverer v1.4 software (Thermo Scientific) was used to search the T.cruzi genome28 with identified spectra for presence of 20S proteasome subunits (Supplementary Table 7). Search parameters included fixed carbamidomethyl modification of cysteine, and variable oxidation of methionine, deamidation of asparagine, pyro-glu of N-terminal glutamine, and TMT(6-plex) modification of lysine residues. The activity of purified parasite and human 20S proteasomes was monitored by measuring cleavage of various rhodamine-labelled fluorogenic substrates. Purified 20S proteasomes were diluted in proteasome assay buffer (25 mM Tris-HCl pH 7.5, 1 mM dithiothreitol (Sigma-Aldrich), 10 mM sodium chloride, 25 mM potassium chloride, 1 mM magnesium chloride, 0.05% (w/v) CHAPS (Sigma-Aldrich) and 0.9% DMSO) at a final concentration of 162 nM (parasite proteasomes) or 25 nM (human proteasome), and pre-incubated with compound (40 nl; 0.2% final DMSO concentration) for 1 h. Next, the following substrates (Biosynthan GmbH) were added at 3 μM final concentration to monitor specific proteolytic activities (Suc-LLVY-Rh110-dPro: chymotrypsin-like activity; Ac-RLR-Rh110-dPro: trypsin-like activity; Ac-GPLD-Rh110-dPro: caspase-like activity). The reaction was allowed to proceed for two hours at room temperature and fluorescence as a measure of purified 20S proteasome activity was monitored using the EnVision plate reader (excitation at 485 nm/emission at 535 nm). K and K values were calculated using GraphPad Prism (GraphPad Software) ‘non-competitive enzyme inhibition’ function. Data shown in Fig. 4a, c, d and Extended Data Table 3 represent means of 3 technical replicates (n = 3). Data shown in Fig. 4b and Extended Data Fig. 5 represent means of 2 technical replicates (n = 2). Growing T. cruzi CL epimastigotes were seeded into 24-well tissue culture plate (1 × 107 cells per well) in LIT medium and treated for 2–12 h with DMSO (0.2%) or various concentrations of bortezomib and GNF6702 at 27 °C. Following the treatment, parasites were collected by centrifugation (3,500g for 6 min) and washed twice with phosphate-buffered saline (PBS). Epimastigotes were lysed by resuspending washed cells in a buffer containing 50 mM Tris-HCl pH = 7.4, 150 mM sodium chloride, 1% CHAPS, 20 μM E-64 (Sigma-Aldrich), 10 mM EDTA(Sigma-Aldrich), 5 mM N-ethylmaleimide(Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), 10 μg/ml leupeptin (Sigma-Aldrich), 10 μg/ml aprotinin (Sigma-Aldrich), and incubating the suspension on ice for 20 min. Cell lysates were cleared by centrifugation at 21,000g for 30 min at 4 °C. For 3T3 cells, 2 × 105 cells/well were seeded into 24-well tissue culture plates in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, and incubated overnight at 37 °C to allow cells to attach. Attached cells were treated for 2 h with DMSO (0.25%) or various concentrations of bortezomib and GNF6702. Treated cells were washed twice with PBS and then lysed by incubating cells in modified RIPA buffer (50 mM Tris-HCl pH = 7.4, 1% Triton X-100, 0.2% sodium dodecylsulfate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin) for 30 min at 4 °C. Cell lysates were cleared by centrifugation at 21,000g for 30 min at 4 °C. Protein concentration in cell extracts was determined with BCA assay (ThermoFisher), and 10 μg of cell extracts were loaded on NuPAGE Novex 4–12% Bis-Tris gel (Invitrogen). After electrophoresis, resolved proteins were transferred to nitrocellulose membrane. Ubiquitylated proteins were detected with polyclonal anti-ubiquitin primary antibody (Proteintech, catalogue number 10201-2-AP) and rabbit anti-mouse IgG-peroxidase antibody (Sigma-Aldrich, catalogue number A0545), and then imaged using ECL Prime Western Blotting Detection Reagent (Amersham) on Chemidoc XR+ imaging system (BioRad). Collected western blot images were quantified using Image Lab software (BioRad). Briefly, rectangles of identical size and shape were drawn around each blot lane to include inside the shape all ubiquitylated protein bands within 17–198 kDa molecular mass range. Next, integrated signal intensities within the rectangles (reported by the Image Lab software) were used for calculation of EC values. Three technical replicate experiments (n = 3) for each different dose response experiment (GNF6702 on T. cruzi epimastigotes; GNF6702 on 3T3 cells; bortezomib on T. cruzi epimastigotes; bortezomib on 3T3 cells) were performed. The homology model of T. cruzi 20S proteasome was built using ‘Prime’ protein structure prediction program (Schrödinger) and X-ray structure of bovine 20S proteasome (PDB accession code 1IRU)39 as the template. The model was subjected to restrained minimization to relieve inter-chain clashes. ‘SiteMap’ program (Schrödinger) was used to identify pockets on a protein surface suitable for small molecule binding. Flexible ligand docking was performed using ‘Glide 5.8’ (Schrödinger). The grid box was centred in a middle of the identified pocket and extended by 10 Å, with outer box extending an additional 20 Å. The ligand was docked using the standard precision (SP) algorithm and scored using ‘GlideScore’ (Schrödinger). The GNF6702 GlideScore is equal to −8.5. GNF6702 profiling was performed at 10 μM concentration in a selectivity panel at Eurofins (www.eurofinspanlabs.com/Catalog/AssayCatalog/AssayCatalog.aspx). Listed values correspond to the assay readout values expressed relative to the DMSO control. To determine inhibition of a subset of human tyrosine kinases by GNF6702, the inhibitor was profiled on a panel of Ba/F3 cell lines expressing individual Tel-activated kinases as described previously40. All assays were performed as single technical repeats. The solubility of GNF6702 was assessed in a high throughput thermodynamic solubility assay as described previously41. First, 25 μl of GNF6702 DMSO solutions were transferred to individual wells of a 96-well plate. DMSO was evaporated and 250 μl of 67 mM potassium phosphate buffer pH 6.8 were added to yield projected final compound concentrations from 1 μM to 100 μM. The plate was sealed to prevent solvent loss and shaken for 24 h at room temperature. The plate was then filtered to remove non-dissolved material. Concentration of GNF6702 in individual plate wells was determined by measuring solution UV absorbance with reference to a GNF6702 calibration curve. A 96-Multiwell Insert System (BD Biosciences) was used for the Caco-2 cell culture and permeability assay as described previously42. Caco-2 cells were seeded onto insert wells at a density of 1.48 × 105 cells per ml and allowed to grow for 19–23 days before assays. To measure both absorptive (apical to basolateral (A–B)) and secretory (basolateral to apical (B–A)) compound transport, a solution of GNF6702 at 10 μM concentration in 0.5% DMSO were added to donor wells. The plate was incubated at 37 °C for 2 h, with samples taken at the beginning and end of the incubation from both donor and acceptor wells. The concentration of GNF6702 was determined by LC-MS/MS. Apparent drug permeability (P ) was calculated using the following equation: where dQ/dt is the total amount of a test compound transported to the acceptor chamber per unit of time (nmol/s), A is the surface area of the transport membrane (0.0804 cm2), C is the initial compound concentration in the donor chamber (10 μM), and P is expressed as cm/s. Extent of inhibition of major human CYP450 isoforms 2C9, 2D6 and 3A4 by GNF6702 was determined using pooled human liver microsomes and the known specific substrates of various CYP450 isoforms: diclofenac (5 μM), bufuralol (5 μM), midazolam (5 μM), and testosterone (50 μM). Probe substrate concentrations were used at concentrations equal to their reported K values. The CYP450 inhibition assays with probe substrates diclofenac (2C9) or midazolam (3A4) were incubated at 37 °C for 5 to 10 min using a microsomal protein concentration of 0.05 mg/ml. Probe substrates bufuralol (2D6) and testosterone (3A4) were incubated at 37 °C for 20 min using microsomal concentration 0.5 mg/ml. The test concentrations of GNF6702 ranged from 0.5 to 25 μM in the presence of 1% DMSO. The reactions were initiated by adding NADPH (1 mM final concentration; Sigma-Aldrich) after a 5-min preincubation. Incubations were terminated by the addition of 300 μl of acetonitrile to 100 μl of a sample. No detectable cytochrome P450 inhibition was observed. Extent of CYP450 isoform inhibition was determined by quantifying residual concentrations of individual CYP450 substrate probes at the end of reactions by LC-MS/MS. The intrinsic metabolic stability of GNF6702 was determined in mouse and human liver microsomes using the compound depletion approach and LC-MS/MS quantification. The assay measured the rate and extent of metabolism of GNF6702 by measuring the disappearance of the compound. The assay determined GNF6702 in vitro half-life (T ) and hepatic extraction ratios (ER) as described previously43. GNF6702 was incubated for 30 min at 1.0 μM concentration in a buffer containing 1.0 mg/ml liver microsomes. Samples (50 μl) were collected at 0, 5, 15 and 30 min and immediately quenched by addition of 150 μl of ice-cold acetonitrile/methanol/water mixture (8/1/1). Quantification of GNF6702 in samples was performed by LC-MS/MS, and the in vitro intrinsic clearance was determined using the substrate depletion method. The intrinsic clearance, CL was calculated using the following equation: where T is the in vitro half-life, V (μl) is the reaction volume, and M (mg) is the microsomal protein amount. Finally the hepatic extraction ratio is calculated as: CL was calculated using the following equation: where f  = fraction unbound to protein (assumed to be 1). An outline of various in vitro and in vivo DMPK assays used in this study for compound profiling was summarized previously44. The pharmacokinetic properties of GNF compounds and calculation of pharmacokinetic parameters was performed as described previously23. Mean compound plasma concentrations were calculated from fitted functions approximating compound plasma profile throughout eight days of dosing. Blinding was not possible in these experiments. Plasma concentration of GNF6702 was quantified using a LC-MS/MS assay. Solution of 20 ng/ml of verapamil hydrochloride (Sigma-Aldrich) in acetonitrile/methanol mixture (3/1 by volume), was used as an internal standard. Twenty microlitres of plasma samples were mixed with 200 μl of internal standard solution. The samples were vortexed and then centrifuged in an Eppendorf Centrifuge 5810 R (Eppendorf) at 4,000 r.p.m. for 5 min at 4 °C to remove precipitated plasma proteins. The supernatants (150 μl) were transferred to a 96-well plate and mixed with 150 μl H O. The samples (10 μl) were then injected onto a Zorbax SB-C8 analytical column (2.1 × 30 mm, 3.5 μm; Agilent Technologies) and separated using a three step gradient (1st step: 1.5 ml of 0.05% formic acid in 10% acetonitrile; 2nd step: 0.5 ml of 0.05% formic acid in 100% acetonitrile; 3rd step: 0.5 ml of 0.05% formic acid in 10% acetonitrile) at flow rate of 700 μl/min. GNF6702 and verapamil were eluted at retention time 1.19 and 1.17 min, respectively. The HPLC system, consisting of Agilent 1260 series binary pump (Agilent Technologies), Agilent 1260 series micro vacuum degasser (Agilent Technologies) and CTC PAL-HTC-xt analytics autosampler (LEAP Technologies) was interfaced to a SCIEX API 4000 triple quadrupole mass spectrometer (Sciex). Mass spectrometry analysis was carried out using atmospheric pressure chemical ionization (APCI) in the positive ion mode. GNF6702 (430.07 > 333.20) and verapamil (455.16 > 164.90) peak integrations were performed using AnalystTM 1.5 software (Sciex). The lower limit of quantification (LLOQ) in plasma was 1.0 ng/ml. Samples were quantified using seven calibration standards (dynamic range 1–5,000 ng/ml) prepared in plasma and processed as described above. All compounds administered to mice during efficacy experiments were formulated as suspensions in distilled water containing 0.5% methylcellulose (Sigma-Aldrich) and 0.5% Tween 80 (Sigma-Aldrich). During a treatment course, each mouse received 0.2 ml of drug suspension per dose by oral gavage. Female BALB/cJ mice (Envigo; 6–8 weeks old) were infected by tail vein injection with 4 × 107 L. donovani MHOM/ET/67/HU3 splenic amastigotes (protocol number P11-319). Seven days after infection, animals were orally dosed for eight days with vehicle (0.5% methylcellulose/ 0.5% Tween 80, miltefosine (12 mg/kg once-daily; Sigma-Aldrich), or a GNF compound (twice-daily). On the first day of dosing, three mice were used for collection of blood for PK determination and euthanized afterwards. On the last day of dosing, PK samples were collected from remaining five mice, which were also used for determination of compound efficacy (n = 5 mice per group). Liver samples were collected from these five mice and L. donovani parasite burdens were quantified by qPCR as follows. Total DNA was extracted from drug-treated mice livers using the DNeasy Blood and Tissue Kit (Qiagen). Two types of DNA were quantified in parallel using the TaqMan assay: L. donovani major surface glycoprotein gp63 (Ldon_GP63) and mouse Gapdh. Leishmania donovani gp63 DNA was quantified with the following set of primers: TGCGGTTTATCCTCTAGCGATAT (forward), AGTCCATGAAGGCGGAGATG (reverse), and TGGCAGTACTTCACGGAC (TaqMan MGB probe, 5′-FAM-labelled reporter dye, non-fluorescent quencher). Mouse Gapdh DNA was quantified with the following set of primers: GCCGCCATGTTGCAAAC (forward primer), CGAGAGGAATGAGGTTAGTCACAA (reverse primer), and ATGAATGAACCGCCGTTAT (TaqMan MGB probe, 5′-FAM-labelled reporter dye, non-fluorescent quencher). Each qPCR reaction (10 μl) included 5 μl of TaqMan Gene Expression Master Mix (Life Technologies), 0.5 μl of a 20× primer/probe mix (Life Technologies), and 4.5 μl (50 ng) of total DNA from liver samples. DNA amount was quantified using the Applied Biosystems 7900HT instrument. Leishmania donovani parasite burden (RU: relative units) was expressed as the abundance of L. donovani gp63 DNA relative to the abundance of mouse Gapdh DNA. Leishmania major MHOM/SA/85/JISH118 metacyclic promastigotes were generated and purified by the peanut agglutinin method as described elsewhere45. To establish the L. major footpad infection, female BALB/cJ mice (Envigo; 6–8 weeks old; protocol number P11-319) were injected with a suspension of L. major metacyclic promastigotes (1 × 106 parasites in 50 μl) into their left hind footpads. After eight days of infection, animals were dosed with vehicle, miltefosine (30 mg/kg once-daily), or indicated regimens of GNF6702 for seven days (n = 6 mice per group). The progress of infection was monitored by measuring the size (length and thickness) of hind footpad swelling using digital calipers. At the end of the study, the mice were euthanized, and the footpad tissues were extracted and used for genomic DNA isolation with the DNeasy Blood and Tissue kit (Qiagen). The L. major footpad burden was determined by qPCR quantification of kinetoplastid minicircle DNA (forward primer: 5′-TTTTACACCTCCCCCCAGTTT-3′; reverse primer: 5′-CCCGTTCATAATTTCCCGAAA-3′; Taqman MGB probe: 5′-AGGCCAAAAATGG-3′, 5′-FAM (6-carboxyfluorescein)-labelled reporter dye, non-fluorescent quencher). The amounts of mouse chromosomal DNA in extracted samples were quantified in parallel qPCR using a glyceraldehyde-3-phosphate dehydrogenase (Gapdh) TaqMan assay as described for mouse VL model above. Leishmania major burden in footpad was expressed as the ratio of kinetoplast minicircle DNA to mouse Gapdh. P values for the between-groups differences in efficacies were calculated with a Student’s paired t-test with a two-tailed distribution. Compound efficacy in a mouse model of Chagas disease was determined as described previously23. Female C57BL/6 mice (Envigo; 6–8 weeks old; protocol number P11-316) were infected by intraperitoneal injection with 103 tissue culture-derived T. cruzi CL trypomastigotes. Starting at 35 days after infection, the animals were dosed orally once-daily with 100 mg/kg benznidazole (Sigma-Aldrich) and indicated doses of GNF6702 (1, 3, and 10 mg/kg twice-daily, n = 8 per group) for 20 days. Ten days following the end of drug treatment, the mice underwent four cycles of cyclophosphamide immunosuppression, each cycle lasting one week. During each immunosuppression cycle, mice were dosed by oral gavage once-daily with 200 mg/kg cyclophosphamide (suspension in 0.5% methylcellulose/ 0.5% Tween80 aqueous solution) on day 1 and day 4 of the cycle. After the fourth immunosuppression cycle, blood samples were collected from the orbital venous sinus of each mouse, mice were euthanized and heart and colon samples were collected. Samples from treated mice were used for extraction of total DNA using the High Pure PCR template preparation kit (Roche). The amounts of T. cruzi satellite DNA (195-bp fragment) in extracted DNA samples were quantified by real-time qPCR TaqMan assay (Life Technologies) with the following set of primers: AATTATGAATGGCGGGAGTCA (forward primer), CCAGTGTGTGAACACGCAAAC (reverse primer), and AGACACTCTCTTTCAATGTA (TaqMan MGB probe, 5′-FAM (6-carboxyfluorescein)-labelled reporter dye, non-fluorescent quencher). The amounts of mouse chromosomal DNA in extracted samples were quantified in parallel qPCR reactions using a Gapdh (glyceraldehyde-3-phosphate dehydrogenase) TaqMan assay as described for mouse VL model above. Each qPCR mixture (10 μl) included 5 μl of TaqMan Gene Expression master mix (Life Technologies), 0.5 μl of a 20× primer/ probe mix (Life Technologies), and 4.5 μl (50 ng) of total DNA extracted from blood samples. PCRs were run on the Applied Biosystems 7900HT instrument. Trypanosoma cruzi parasitemia was expressed as the abundance of T. cruzi microsatellite DNA relative to the abundance of mouse Gapdh DNA. Female CD1 (Charles River UK; ~8 weeks old; project license number PPL 60/4442) mice were infected by injection into the peritoneum with 3 × 104 T. brucei (GVR35-VSL2) bloodstream form parasites46. Starting on day 21, mice were dosed by oral gavage once-daily with GNF6702 (n = 6) at 100 mg/kg for 7 days or a single dose of diminazene aceturate (Sigma-Aldrich) at 40 mg/kg in sterile water was administered by i.p. injection (n = 3). A group of untreated mice (n = 3) was included as controls. Mice were monitored weekly for parasitemia from day 21 post-infection. Trypanosoma brucei was quantified in blood samples from the tail vein by microscopy, and in vivo bioluminescence imaging of infected mice was performed before treatment on day 21 post-infection and in weeks following the treatment (day 28, 35, 42, 56, 63, 72, 84, 92 post-infection). Imaging on groups of three mice was performed 10 min after i.p. injection of 150 mg d-luciferin (Promega)/kg body weight (in PBS) using an IVIS Spectrum (Perkin Elmer) as described previously25. A group of uninfected mice (aged-matched for day 0 time point; n = 4) were imaged using the same acquisition settings to show the background bioluminescence (Fig. 2e, grey-filled squares) in the absence of luciferase-expressing T. brucei after day 92 of the experiment. Untreated and diminazene-treated mice were euthanized on days 32 and 35, and day 42, respectively, due to high parasitemia or the development of symptoms related to CNS infection. GNF6702-treated mice were euthanized on day 92. No parasitemia or clinical symptoms were observed at this point. At the specified endpoints mice were sacrificed by cervical dislocation, after which whole brains were removed and imaged ex vivo within 10 min after administration of 100 μl of d-luciferin onto the brain surface. Data analysis for bioluminescence imaging was performed using Living Image Software (Perkin Elmer). The same rectangular region of interest (ROI) covering the mouse body was used for each whole-body image to show the bioluminescence in total flux (photons per second) within that region. Image panels of whole mouse bodies are composites of the original images with areas outside the ROI cropped out to save space. For ex vivo brain images the same oval shaped ROI was used to display the bioluminescence detected for each mouse brain at the respective endpoints. The detailed procedures for chemical synthesis are presented in Supplementary Information.


"We've rationally designed something to target multiple pathways, which is contrary to the traditional thinking in medicinal chemistry, where you have one target, one drug," said University of Illinois chemistry professor Steven Zimmerman, who led the research with graduate students Lien Nguyen and Long Luu. "People are slowly discovering that drugs that hit multiple targets are actually better." The team reports its findings in the Journal of the American Chemical Society. DM1 (but not Duchennes muscular dystrophy) results from a genetic error that causes expansion of a region of a particular gene, called DMPK. This gene includes a repeated, three-letter sequence of nucleotides, the gene's chemical building blocks. Normal cells contain as many as 35 of these repeats, but sometimes mutation pushes the number of repeats beyond 50, which can lead to symptoms of the disease. Mutant DMPK genes often continue to expand, amplifying the health problems that can result. In some people, the gene includes as many as 10,000 repeats. No drugs are available to treat DM1, which afflicts an estimated one in 8,000 people worldwide. Scientists are gradually learning how the disease impairs cells. When mutant DMPK is converted into RNA in a first step of protein production, the repeated sequences in the RNA cause it to bind to another protein, MBNL, which regulates RNA processing. When bound by the mutant RNA, MBNL cannot function properly, causing a cascade of problems in protein production, Zimmerman said. "Dozens of other proteins become dysregulated," he said. "There's a chloride channel that causes heart arrhythmias. There's an insulin receptor that, when it's dysregulated, gives diabetic symptoms." In earlier work, Zimmerman and his colleagues developed a compound that stopped the mutant RNA from binding to MBNL. But the disease has other means of creating havoc in cells, researchers have since found. For example, the cell translates the mutant RNA into proteins that also turn out to be toxic. And the mutant RNA interferes with the function of other proteins besides MBNL. "The disease is like a hydra," Zimmerman said. "We cut off one of its modes of action and we learn about two more that need to be dealt with." Nguyen and Luu tackled this problem by tethering new biologically active appendages to the lab's original compound, creating multitarget drugs that are small enough to get easily into cells. In tests, they found that the new compounds have three modes of action. One, they stop the process by which the mutant DNA is converted into RNA. Two, they bind to the mutant RNA and prevent it from attaching to the regulatory protein, MBNL. And three, they chop up the mutant RNA, a process that is slow but appears to be effective in in vitro experiments. The most potent compounds the researchers developed reduce levels of the mutant RNA in cells that replicate the pathology of DM1. The new compounds also reversed two symptoms of the disease in a fruit fly model of DM1. "The new compounds would need to work effectively in mice and pass preclinical benchmarks before they can be tried in humans," Zimmerman said. "It is encouraging that a different approach using a DNA analog is already in clinical trials in human patients." The advantage of the new agents under development in Zimmerman's lab is their small size, he said. "Small molecules are much easier to make than larger compounds, they are easier to get into cells and their potential for getting into the brain is higher," he said. Explore further: Small molecule inhibits pathology associated with myotonic dystrophy type one More information: Lien Nguyen et al. Rationally Designed Small Molecules That Target Both the DNA and RNA Causing Myotonic Dystrophy Type 1, Journal of the American Chemical Society (2015). DOI: 10.1021/jacs.5b09266


News Article | October 27, 2016
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HAMBURG, Germany--(BUSINESS WIRE)--Evotec AG (Frankfurt Stock Exchange: EVT, TecDAX, ISIN: DE0005664809) today announced that it has made an offer to acquire Cyprotex PLC ("Cyprotex", AIM: CRX-GB), a specialist pre-clinical contract research organisation in ADME-Tox and DMPK. Cyprotex serves the industry's increasing requirement for earlier drug screening, regulatory requirements and reducing the reliance on animal testing. The proposed acquisition, which has been unanimously recommended by the


News Article | October 27, 2016
Site: www.businesswire.com

HAMBURG, Germany--(BUSINESS WIRE)--Evotec AG (Frankfurt Stock Exchange, Prime Standard, ISIN: DE 000 566480 9, WKN 566480) today announced that it has made an offer to acquire Cyprotex PLC (AIM Listing: CRX-GB), a specialist pre-clinical contract research organisation in ADME-Tox and DMPK headquartered in UK. The proposed acquisition, which has been unanimously recommended by the board of Cyprotex, is expected to close before year-end 2016. Evotec will pay approximately £ 55.36 m (EUR 62.00 m;


News Article | October 27, 2016
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HAMBURG, Deutschland--(BUSINESS WIRE)--Evotec AG (Frankfurter Wertpapierbörse, Prime Standard, ISIN: DE 000 566480 9, WKN 566480) gab heute die Unterbreitung eines Angebots zur Akquisition der Cyprotex PLC (AIM-Notierung: CRX-GB) bekannt. Cyprotex ist ein auf präklinische ADME-Tox- und DMPK-Leistungen spezialisiertes Auftragsforschungsunternehmen mit Hauptsitz in UK. Es wird erwartet, dass die beabsichtigte Übernahme, die einstimmig von dem Cyprotex-Vorstand empfohlen wurde, vor Jahresende 2016


News Article | October 27, 2016
Site: www.businesswire.com

HAMBURG, Deutschland--(BUSINESS WIRE)--Evotec AG (Frankfurter Wertpapierbörse: EVT, TecDAX, ISIN: DE0005664809) gab heute die Unterbreitung eines Angebots zur Akquisition der Cyprotex PLC ("Cyprotex", AIM: CRX-GB) bekannt. Cyprotex ist ein auf präklinische ADME-Tox- und DMPK-Leistungen spezialisiertes Auftragsforschungsunternehmen. Damit begegnet das Unternehmen dem steigenden Bedarf der Branche an früheren Wirkstoffscreenings sowie regulatorischen Anforderungen und der Reduzierung der Abhängig

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