MANF Produced by MRL Mouse-Derived Mesenchymal Stem Cells Is Pro-regenerative and Protects From Osteoarthritis

Tejedor, G., Luz-Crawford, P., Barthelaix, A., Toupet, K., Roudières, S., Autelitano, F., Jorgensen, C. and Djouad, F.

The super healer Murphy Roths Large (MRL) mouse represents the “holy grail” of mammalian regenerative model to decipher the key mechanisms that underlies regeneration in mammals. At a time when mesenchymal stem cell (MSC)-based therapy represents the most promising approach to treat degenerative diseases such as osteoarthritis (OA), identification of key factors responsible for the regenerative potential of MSC derived from MRL mouse would be a major step forward for regenerative medicine. In the present study, we assessed and compared MSC derived from MRL (MRL MSC) and C57BL/6 (BL6 MSC) mice. First, we compare the phenotype and the differentiation potential of MRL and BL6 MSC and did not observe any difference. Then, we evaluated the proliferation and migration potential of the cells and found that while MRL MSC proliferate at a slower rate than BL6 MSC, they migrate at a significantly higher rate. This higher migration potential is mediated, in part, by MRL MSC-secreted products since MRL MSC conditioned medium that contains a complex of released factors significantly increased the migration potential of BL6 MSC. A comparative analysis of the secretome by quantitative shotgun proteomics and Western blotting revealed that MRL MSC produce and release higher levels of mesencephalic astrocyte-derived neurotrophic factor (MANF) as compared to MSC derived from BL6, BALB/c, and DBA1 mice. MANF knockdown in MRL MSC using a specific small interfering RNA (siRNA) reduced both MRL MSC migration potential in scratch wound assay and their regenerative potential in the ear punch model in BL6 mice. Finally, injection of MRL MSC silenced for MANF did not protect mice from OA development. In conclusion, our results evidence that the enhanced regenerative potential and protection from OA of MRL mice might be, in part, attributed to their MSC, an effective reservoir of MANF.

Frontiers in Cell and Developmental Biology

ENDPOINTS AND DESIGN OF CLINICAL TRIALS IN PATIENTS WITH DECOMPENSATED CIRRHOSIS: POSITION PAPER OF THE LIVERHOPE CONSORTIUM

Solà E, Pose E, Campion D, Piano S, Roux O, Simon-Talero M, Uschner F, de Wit K, Zaccherini G, Alessandria C, Beuers U, Caraceni P, Francoz C, Mookerjee R, Trebicka J, Vargas V, Serra M, Torres F, Montagnese S, Krag A, Hernáez R, Korenjak M, Watson H, Abraldes J, Kamath P, Ginès P, for the LiverHope Consortium Investigators

Management of decompensated cirrhosis is currently geared towards the treatment of complications once they occur. To date there is no established disease-modifying therapy aimed at halting progression of the disease and preventing the development of complications that can be used for patients with decompensated cirrhosis. The design of clinical trials to investigate new therapies for patients with decompensated cirrhosis is complex. The population of patients with decompensated cirrhosis is heterogeneous (i.e., different etiologies, comorbidities, severity of the disease), leading to the inclusion of diverse populations in clinical trials. In addition, primary endpoints selected for trials that include patients with decompensated cirrhosis are not homogeneous and at times may not be appropriate endpoints. This leads to difficulties in comparing of results obtained from different trials. Against this background, the LiverHope Consortium organized a meeting of experts with the goal of making recommendations for the design of clinical trials and defining appropriate endpoints both for trials aimed at modifying the natural history and preventing progression of decompensated cirrhosis and trials aimed at investigating new therapies for the management of each complication of cirrhosis.

Journal of Hepatology

Accelerated preclinical paths to support rapid development of COVID-19 therapeutics

Jay A. Grobler, Annaliesa S. Anderson, Prabhavathi Fernandes, Michael S. Diamond, Christine M. Colvis, Joseph P. Menetski, Rosa M. Alvarez, John AT. Young, Kara L. Carter, on behalf of the ACTIV Preclinical Working Group

Cell Host and Microbe (2020)

ABHD11, a new diacylglycerol lipase involved in weight gain regulation

Escoubet, J., Kenigsberg, M., Derock, M., Yaligara, V., Bock, M.-D., Roche, S., Massey, F., de Foucauld, H., Bettembourg, C., Olivier, A., Berthemy, A., Capdevielle, J., Legoux, R., et al.

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.

PLOS ONE

IDENTIFICATION OF PHENOTYPES SUITABLE FOR DRUG TESTING IN A GENETIC OR TOXIN-INDUCED MODEL OF PARKINSON'S DISEASE USING HUMAN IPSC-DERIVED DOPAMINERGIC NEURONS

Erika Torchio, Teresa Ferraro, Elisa Bianchini, Alessandra Toti, Andrea Kuan Cie Wong, Cristiana Griffante and Rosaria Remelli

Evotec

A Whole Genome-Wide Arrayed CRISPR Screen in Primary Organ Fibroblasts to Identify Regulators of Kidney Fibrosis

Robert J. Turner, Stefan Golz, Carina Wollnik, Nils Burkhardt, Ina Sternberger, Uwe Andag, Hauke Cornils

Kidney fibrosis presents a hallmark of chronic kidney disease. With ever-increasing patient numbers and limited treatment options available, novel strategies for therapeutic intervention in kidney disease are warranted. Fibrosis commonly results from a wound healing response to repeated or chronic tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue. In order to identify targets relevant for kidney fibrosis, we aimed to employ CRISPR screening in primary human kidney fibroblasts. We demonstrate that CRISPR technology can be applied in primary kidney fibroblasts and can furthermore be used to conduct arrayed CRISPR screening using a high-content imaging readout in a whole genome-wide manner. Hits coming out of this screen were validated using orthogonal approaches and present starting points for validation of novel targets relevant to kidney disease.

SLAS Discovery

Orphan drugs: Overcoming the challenges associated with developing innovative therapeutics for rare diseases

Recent years have witnessed significant growth in the number of sponsors developing drugs for rare or so-called ‘orphan’ diseases. Defined by regulatory authorities as diseases that affect fewer than 200,000 patients in the US, or no more than five in 10,000 people in the EU, orphan diseases are often genetic and therefore lifelong conditions, typically affecting patients from a very early age. While each rare disease individually impacts on a relatively small number of people, collectively, it’s thought that there are more than 350 million sufferers of orphan diseases worldwide. Given the need to find effective treatments for these individuals, many countries have adopted legislation to give sponsors incentives to develop drugs for orphan diseases. With increased regulatory flexibility to encourage innovation, financial incentives to offset the cost of developing drugs for small patient markets, as well as the benefits associated with significant intellectual property value of rare disease therapeutics, the industry has responded with interest and commitment. Prior to the introduction of the US Orphan Drug Act in 1983, just 38 drugs designed for rare diseases were available on the market. Since then, more than 3,600 orphan drugs have been designated by the FDA, and over 600 have been approved. Global sales of orphan drugs are expected to exceed $209 bn by the end of 2022, approximately 21 percent of the whole prescription market, excluding generics. While financial incentives and flexible regulatory framework present a wealth of opportunities for developers, the complexities around developing drugs for small patient populations can present significant additional challenges for sponsors. Here, we look at some of the challenges facing developers of drugs for orphan diseases, and how these can be overcome.

Evotec SE

Quantum Mechanics in Drug Discovery

Alexander Heifetz (Ed.)

This volume looks at applications of quantum mechanical (QM) methods in drug discovery. The chapters in this book describe how QM approaches can be applied to address key drug discovery issues, such as characterizing protein-water-ligand and protein-protein interactions, providing estimates of binding affinities, determining ligand energies and bioactive conformations, refinement of molecular geometries, scoring docked protein–ligand poses, describing molecular similarity, structure–activity-relationship (SAR) analysis, and ADMET prediction. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary software and tools, step-by-step, readily reproducible modeling protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and unique, Quantum Mechanics in Drug Discovery is a valuable resource for structural and molecular biologists, computational and medicinal chemists, pharmacologists, and drug designers.

Springer Protocols

Structure Based Design of Potent Selective Inhibitors of Protein Kinase D1 (PKD1)

Feng, J.A., Lee, P., Alaoui, M.H., Barrett, K., Castanedo, G., Godemann, R., McEwan, P., Wang, X., Wu, P., Zhang, Y., Harris, S.F., Staben, S.T.

We previously disclosed a series of type I 1/2 inhibitors of NF-κB inducing kinase (NIK). Inhibition of NIK by these compounds was found to be strongly dependent on the inclusion and absolute stereochemistry of a propargyl tertiary alcohol as it forms critical hydrogen bonds (H-bonds) with NIK. We report that inhibition of protein kinase D1 (PKD1) by this class of compounds is not dependent on H-bond interactions of this tertiary alcohol. This feature was leveraged in the design of highly selective inhibitors of PKD1 that no longer inhibit NIK. A structure-based hypothesis based on the position and flexibility of the α-C-helix of PKD1 vs NIK is presented.

ACS Med. Chem. Lett.

Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS–SOS1 interaction

Hillig, R.C., Sautier, B., Schroeder, J., Moosmayer, D., Hilpmann, A., Stegmann, C.M., Werbeck, N.D., Briem, H., Boemer, U., Weiske, J., Badock, V., Mastouri, J., Petersen, K., Siemeister, G., Kahmann, J.D., Wegener, D., Böhnke, N., Eis, K., Graham, K., Wortmann, L., von Nussbaum, F., Bader, B.

Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques.

PNAS

The novel fungal CYP51 inhibitor VT-1598 displays classic dose-dependent antifungal activity in murine models of invasive aspergillosis

Garvey, E.P., Sharp, A.D., Warn, P.A., Yates, C.M, Atari, M., Thomas, S.

Aspergillus spp. infections remain a global concern, with ∼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 μg∗h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 μg∗h/ml).

Medical Mycology

Essential ingredients for rational drug design

Jarvis, A., Ouvry, G.

This short review focuses on three aspects of rational drug design that we consider of utmost importance: the conformation of small molecules in solid form, the conformation of small molecules in solution and lesser studied interactions in protein-ligand complexes. Using examples from recent literature, we will illustrate these different aspects and how they have contributed to the discovery of potent modulators.

Bioorganic & Medicinal Chemistry Letters

Liquid chromatography–tandem mass spectrometry for the simultaneous quantitation of enmetazobactam and cefepime in human plasma

Mamelia, M., Vezzellia, A., Verze’, S., Biondi, S., Motta, P., Greco, A., Michi, M., Breda, A.

A simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the simultaneous analysis enmetazobactam (also known as AAI101) and cefepime in human plasma. Sample preparation was based on protein precipitation with acetonitrile. Separation was performed on Acquity BEH HILIC column (50 mm × 2.1 mm, 1.7 μm) with a mobile phase containing ammonium formate in water and acetonitrile. The analytes were analyzed with the corresponding isotopically labeled internal standards and were detected in multiple reactions monitoring (MRM) using API 5000 triple-quadrupole mass spectrometer with electrospray (ESI) source operating in positive ion mode. The calibration curves were linear over the selected ranges (r > 0.9970 for both analytes). The intra and inter-assay precision of the Quality Control samples showed CV ≤ 15% and the accuracy was within 85 and 115% in all cases for both compounds. The lower limit of quantification was 0.05 μg/mL for enmetazobactam and 0.5 μg/mL for cefepime.

Journal of Pharmaceutical and Biomedical Analysis

GNS561, a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma

Brun, S., Bassissi, F., Serdjebi, C., Novello, M., Tracz, J., Autelitano, F., Guillemot, M., Fabre, P., Courcambeck, J., Ansaldi, C., Raymond, E., Halfon, P.

Among the acquired modifications in cancer cells, changes in lysosomal phenotype and functions are well described, making lysosomes a potential target for novel therapies. Some weak base lipophilic drugs have a particular affinity towards lysosomes, taking benefits from lysosomal trapping to exert anticancer activity. Here, we have developed a new lysosomotropic small molecule, GNS561, and assessed its activity in multiple in vitro intrahepatic cholangiocarcinoma models (HuCCT1 and RBE cell lines and patient-derived cells) and in a chicken chorioallantoic membrane xenograft model. GNS561 significantly reduced cell viability in two intrahepatic cholangiocarcinoma cell lines (IC50 of 1.5 ± 0.2 μM in HuCCT1 and IC50 of 1.7 ± 0.1 μM in RBE cells) and induced apoptosis as measured by caspases activation. We confirmed that GNS561-mediated cell death was related to its lysosomotropic properties. GNS561 induced lysosomal dysregulation as proven by inhibition of late-stage autophagy and induction of a dose-dependent build-up of enlarged lysosomes.

Investigational New Drugs

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition

Ballinger, E., Mosior, J., Hartman, T., Burns-Huang, K., Gold, B., Morris, R., Goullieux, L., Blanc, I., Vaubourgeix, J., Lagrange, S., Fraisse, L., Sans, S., Couturier, C., Bacqué, E., Rhee, K., Scarry, S. M., Aubé, J., Yang, G., Ouerfelli, O., Schnappinger, D., Ioerger, T. R., Engelhart, C. A., McConnell, J. A., McAulay, K., Fay, A., Roubert, C., Sacchettini, J., Nathan, C.

Tuberculosis is a global health crisis that threatens to become worse as resistance to existing drugs emerges. Identifying ideal targets for drug development requires knowledge of weak points in biochemical pathways that are specific for the pathogen but are absent in hosts. Ballinger et al. identified a small molecule that inhibits the enzyme phosphopantetheinyl transferase (PptT), which is crucial for biosynthesis of mycobacterial structural and virulence lipids (see the Perspective by Mizrahi and Warner). Treatment resulted in selective killing of the bacteria in vitro and in a mouse model. The target pathways were made sensitive to PptT inhibition by a second enzyme, phosphopantetheine hydrolase, whose activity opposes that of the transferase.

Science

Oral vs. Intraperitoneal Dosing in the Rat Drug Discrimination Model

Tessari, M., Compagnucci, B., Spurio, B., Dacome, L., Pilla, M., Miller, S., Dinklo, T.

The regulatory agencies guidance for the assessment of abuse liability in preclinical models recommend using preferably the clinical route of administration although different routes may be considered depending on the model used and the context of non-medical use. For the drug discrimination the routes of administration with fast onset of action, such as intraperitoneal (IP) or subcutaneous (SC) injection, are largely used. The present study aimed at investigating the effects of some drugs of abuse given through different routes of administration in a two choice drug discrimination operant task.

Safety Pharmacology Society Annual Meeting 2018

Steroid-resistant inflammation in a mouse model of severe asthma is not inhibited by the combination of Theophylline and Budesonide

Russell, V. P., Brana, M., Pompilio, D., Ambrosi, G., Andreetta, F., Venturini, E., Chalidou, A., Manni, M. E.

Severe asthma remains a major unmet medical need and results in a significant demand on healthcare costs. One of the key characteristics of the severe phenotype is a neutrophilic inflammation that is resistant to steroid treatment usually effective in the mild/moderate disease. Steroid-resistant neutrophilic inflammation is also a key characteristic of Chronic Obstructive Pulmonary Disease (COPD) where it has been demonstrated to be due to reduced HDAC activity which can be reversed by the combination of low dose Theophylline and steroid.

European Respiratory Society Congress 2018

Purkinje cells firing recorded by a high density multi-electrode array: a new tool for compounds validation

Ugolini, A., Sessolo, M., Maccione, A., Bettini, E., Federico, D. Gandolfo, M., Corsi, M., Virginio, C.

Restoring altered firing pattern of cerebellar Purkinje cells has been proposed as a promising approach for the treatment of movement disorders such as ataxia (Kasumu et al., 2012; Alviña and Khodakhah, 2010). However new therapeutic molecules would require a full characterization of their effect on Purkinje cells firing in normal and pathological conditions. Here, high-density multi electrode array (HD-MEA) was used to monitor and characterize spiking activity of hundreds Purkinje cells simultaneously in 90 minute long recordings. Since Ca2+-activated K+ channels have been demonstrated to modulate Purkinje cells firing, we used different positive and negative modulators in order to assess their effect on this neuronal population.

FENS Forum 2018

Rotenone-induced toxicity in human iPSC derived dopaminergic neurons (iCell®DopaNeurons): a cellular model to discover novel neuroprotective drugs

Bianchini, E., Remelli, R., Ferraro, T., Griffante, C.

iCell®DopaNeurons are a fully differentiated, highly pure population of human midbrain dopaminergic neuron derived using FCDI’s proprietary differentiation and purification protocols.

FENS Forum 2018

NMDA Receptor Modulators in QPatch

Bettini, E., Aldegheri, L., Castelletti, L., Corsi, M., Virginio, C.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors permeable to Ca2+, Na+ and K+. To be activated, they need to bind to glutamate (via GluN2 subunits), glycine (via GluN1) and release the Mg2+ blockade by membrane depolarization. The majority of NMDARs are tetrameric complexes, consisting of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. GluN1 is coded by a single gene with at least eight different splice variants; four different GluN2 genes originate GluN2A, GluN2B, GluN2C, and GluN2D subunits. NMDARs containing different GluN2 subunits have different pharmacological and kinetic properties.

Ion Channel Modulation Symposium 2018

High Density Multi Electrode Array: a new tool to monitor seizure-like activity evoked by different convulsant drugs

Sessolo, M., Ugolini, A., Maccione, A., Gandolfo, M., Corsi, M., Virginio, C.

Epilepsy is a neurological disorder characterized by recurrent and sustained neuronal discharges. Different approaches have been used to evoke seizure-like activity but chemoconvulsants are the most used. In this work, we challenged mouse hippo-cortical slices with different compounds. Activity evoked by acute application of these compounds was monitored through high-density multi electrode array (HD-MEA) in order to characterize in time and space seizure like activity.

Society for Neuroscience Annual Meeting

In vitro characterization of novel P. aeruginosa QS Inhibitors identified by In silico screening

Garrido, V., Negri, M., Dal Lago, M., Fontana, S., Marinelli, D., Napolitano, C., Andreotti, D., Feriani, A., Felici, A.

The Multiple Virulence Factor Regulator (MvfR) is a Pseudomonas aeruginosa (Pa) Quorum Sensing (QS) transcriptional factor that regulates virulence functions critical for both acute and chronic infections in Cystic Fibrosis patients, making it an interesting drug target.

ESCMID/ASM Conference on Drug Development to Meet the Challenge of Antimicrobial Resistance

Incurred sample reproducibility: 10 years of experiences: views and recommendations from the European Bioanalysis Forum

Kall, M. A., Michi, M., van der Strate, B., Freisleben, A., Stoellner, D., Timmerman, P.

With 10 years of experiences on incurred sample reanalysis (ISR) as an integrated part of regulated bioanalysis, the European Bioanalysis Forum has reflected on the implementation and the use of ISR. Three surveys were conducted in 2016 and 2017 as a revisit of the ISR experiences within European pharmaceutical industry and contract research organizations: has ISR become a tool for postvalidation and process check of a bioanalytical method performance and has ISR become a routine in our laboratories? Do we agree on the interpretation of guidelines/guidance and are we aligned in our approach – among others?

Bioanalysis

Mechanistic in vitro studies indicate that the clinical drug-drug interaction between telithromycin and simvastatin acid is driven by time-dependent inhibition of CYP3A4 with minimal effect on OATP1B1

Elsby, R., Hare, V., Neal, H., Outteridge, S., Pearson, C., Plant, K., Upcott Gill, R., Butler, P., Riley, R.J.

A previous attempt to accurately quantify the increased simvastatin acid exposure due to drug-drug interaction (DDI) with coadministered telithromycin, using a mechanistic static model, substantially underpredicted the magnitude of area under the plasma concentration-time curve ratio (AUCR) based on reversible inhibition of cytochrome P450 (CYP) 3A4 and organic anion transporting polypeptide (OATP) 1B1 (Elsby et al., 2012)...

Drug Metabolism and Disposition

Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones

de Boer, B., Prick, J., Pruis, M. G., Keane, P., Imperato, M. R., Jaques, J., Brouwers-Vos, A. Z., Hogeling, S. M., Woolthuis, C. M., Nijk, M. T., Diepstra, A., Wandinger, S., Versele, M., Attar, R. M., Cockerill, P. N., Huls, G., Vellenga, E., Mulder, A. B., Bonifer, C., Schuringa, J. J.

Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.

Cancer Cell

Time-kill kinetics of cadazolid and comparator antibacterial agents against different ribotypes of Clostridium difficile

Skinner, K., Birchall, S., Corbett, D., Thommes, P., Locher, H.

Clostridium difficile infection (CDI) is an increasing cause of nosocomial diarrhoea worldwide, which has been partly attributed to the emergence of hypervirulent strains including C. difficile BI/NAP1/ribotype 027 and BK/NAP7/ribotype 078. Cadazolid is a new antibiotic currently in late-stage clinical studies for the treatment of CDI. The present study evaluated the in vitro bactericidal effect of cadazolid and comparator antibiotics against four C. difficile strains. The data demonstrate the potent and bactericidal activity of cadazolid against different ribotypes of C. difficile.

Journal of Medical Microbiology

Development and Characterisation of a Human Hepatocyte Low Intrinsic Clearance Assay for Use in Drug Discovery

Lancett, P., Williamson, B., Barton, P., Riley, R.J.

Progression of new chemical entities is a multi-parametric process involving a balance of potency, ADME and safety properties. To accurately predict human pharmacokinetics and estimate human efficacious dose, the use of in vitro measures of clearance is often essential. Low metabolic clearance is often targeted to facilitate in vivo exposure and achieve appropriate half-life. Suspension primary human hepatocytes (PHH) have been successfully utilised in predictions of clearance. However, incubation times are limited, hindering the limit of quantification...

Drug Metabolism and Disposition

Novel approaches to automated utrophin imaging in cells and tissue for Duchenne muscular dystrophy drug discovery

Elsey, D., Guiraud, S., Monecke, S., Geese, M., Edward, B., Squire, S., Guhl, A., Lomow, A., Tinsley, J., Harriman, S., Davies, K., Powell, D., Burch, P.

Utrophin is an autosomal paralogue of dystrophin shown to functionally substitute for the loss of dystrophin in preclinical models of DMD. Ezutromid is a first-in-class class small molecule that that modulates the expression of utrophin in both DMD patient derived muscle cells and the dystrophin deficient mdx mouse model. Here we report the development and validation of cell and tissue based imaging approaches to help determine ezutromid’s mechanism of action and facilitate the identification of additional utrophin modulators...

New Directions in Biology and Disease of Skeletal Muscle Conference, 25-28 June, 2018, New Orleans, LA

Identification of a novel non-brain penetrant A2AR inhibitor and proof-of-concept of CD73 and A2AR/CD73 small-molecule inhibitors for cancer immunotherapy

Fons, P., Bell, A., Versluys, S. et al.

Partnership to discover adenosinergic molecules for immuno-oncology therapeutics established between Exscientia and Evotec

AACR Annual Meeting 2018, April 14-18, 2018, Chicago, Illinois

Translation to the clinic of EVT801: A novel immune-oncology agent for expanding patient population responding to immune checkpoint therapies

Fons, P., Esquerré, M., Mazières, J. et al.

Selective inhibitor of VEGFR3 for patients resistant to immune checkpoint inhibitors: Clinical translation activities

AACR Annual Meeting 2018, April 14-18, 2018, Chicago, Illinois

Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function

Line V. Kristensen, Felix S. Oppermann, Matthias J. Rauen, Karina Fog, Thorsten Schmidt, Jana Schmidt, Tina Harmuth, Rasmus Hartmann-Petersen, Kenneth Thirstrup

Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3 contains an expanded polyglutamine (polyQ) stretch that leads to aggregation of the protein and neuronal dysfunction. Increasing the understanding of ataxin-3 protein interaction partners could help to elucidate disease mechanisms...

Neurochemistry International

NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus

Bents, K., Godemann, R., Linge, S. et al

Abstract NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.

Published online Nature Communications 9, Article number: 179

Computational Methods for GPCR Drug Discovery

Heifetz, A. (Ed.)

Computational Methods for GPCR Drug Discovery

Springer, ISBN 978-1-4939-7465-8, 1st ed. 2018

The Ste20 family kinases MAP4K4, MINK1 and TNIK, converge to regulate stress induced JNK signaling in neurons

Martin Larhammar, Sarah Huntwork-Rodriguez, York Rudhard, Arundhati Sengupta-Ghosh, Joseph W. Lewcock

The c-Jun-N-terminal Kinase (JNK) signaling pathway regulates nervous system development, axon regeneration, and neuronal degeneration following acute injury or in chronic neurodegenerative disease. Dual Leucine Zipper Kinase (DLK) is required for stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway activity remain poorly defined. In the present study, we identify the Ste20 kinases MAP4K4, TNIK, and MINK1 as upstream regulators of DLK/JNK signaling in neurons. Using a trophic factor withdrawal-based model of neurodegeneration in both male and female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, TNIK, and MINK1 act redundantly to regulate DLK activation and downstream JNK dependent phosphorylation of c-Jun in response to stress...

Journal of Neuroscience

Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease

Le Pichon CE, Meilandt WJ, Dominguez S, Solanoy H, Lin H, Ngu H, Gogineni A, Sengupta Ghosh A, Jiang Z, Lee SH, Maloney J, Gandham VD, Pozniak CD, Wang B, Lee S, Siu M, Patel S, Modrusan Z, Liu X, Rudhard Y, Baca M, Gustafson A, Kaminker J, Carano RAD, Huang EJ, Foreman O, Weimer R, Scearce-Levie K, Lewcock JW

Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer’s disease...

Science Translational Medicine

Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells

Kenneth Thirstrup, Justus C. Dächsel, Felix S. Oppermann, Douglas S. Williamson, Garrick P. Smith, Karina Fog, Kenneth V. Christensen

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson’s disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified...

Nature

Polyglutamine expansion of ataxin-3 alters its degree of ubiquitination and phosphorylation at specific sites

Line V. Kristensen, Felix S. Oppermann, Matthias J. Rauen, Rasmus Hartmann-Petersen, Kenneth Thirstrup

Ubiquitination and phosphorylation of proteins represent post translational modifications (PTMs) capable of regulating a variety of cellular processes. In the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3), the disease causing protein ataxin-3 carries an expanded polyglutamine (polyQ) stretch causing it to aggregate in nuclear inclusions. These inclusions are decorated with ubiquitin suggestive of a malfunction in the clearance of the mutant protein. Differences in ubiquitin chain topology between normal and polyQ expanded ataxin-3 could be involved in the differential clearance of the two proteins and play a role in SCA3 pathogenesis. Likewise, changes in phosphorylation patterns between the two variants could contribute to pathogenic processes involved in SCA3. We therefore determined the ubiquitination and phosphorylation patterns, together with the ubiquitin-linkage types associated with normal and polyQ expanded ataxin-3 by mass spectrometry (MS). This analysis revealed a similar ubiquitin linkage pattern on normal and expanded ataxin-3...

Neurochemistry International

Therapeutic potential of NADPH oxidase 1/4 inhibitors

Teixeira G, Szyndralewiez C, Molango S, Carnesecchi S, Heitz F, Wiesel P, Wood JM

The NADPH oxidase (NOX) family of enzymes produces ROS as their sole function and is becoming recognized as key modulators of signal transduction pathways with a physiological role under acute stress and a pathological role after excessive activation under chronic stress. The seven isoforms differ in their regulation, tissue and subcellular localization and ROS products.

Br J Pharmacol. 2017 Jun; 174 (12): 1647-1669. doi: 10.1111/bph.13532. Epub 2016 Jul 14.

Time-Kill Kinetics and Post-Antibiotic Effect of Cdazolid against Clostridium difficile

Birchall, S., Corbett, D., Skinner, K., Thommes, P. et. al.

ECCMID, 22 - 25 April 2017, Vienna, Austria

Steroidal Mineralocorticoid Receptor Antagonists: Synthesis and Biology

Yarnold, C.; Bainbridge, J.M.;Boehm, H.S. et al.

Abstract The development of selective steroidal mineralocorticoid receptor antagonists with improved pharmacological profiles over existing marketed drugs is an attractive goal. Such compounds offer potential for the treatment of hypertension, heart failure and renal disease. With this aim, new spirolactones were prepared exploring substitutions at carbons 6, 7, 9–11, 15–16 and 21. Spirolactones 11 a and 20 were identified with promising biological profiles. Both compounds restored Na+/K+ ratios to physiological levels in an in vivo model

Chemistry Select, 2017, Vol 2,(1): 175–189

Stereoselective synthesis of orthogonally protected 2,3-disubstituted morpholines using a base-catalysed cascade reaction

Marlin, F. J.

Abstract The stereoselective synthesis of differentially protected [3-(hydroxymethyl)morpholin-2-yl]methanols is described, starting from chiral epoxides. The key step involves a one-pot oxazolidinone formation via intramolecular epoxide opening and concomitant cyclisation to form the morpholine ring. Selective deprotection reveals the free hydroxymethyl group at either the 2- or 3-position of the morpholine.

ELSEVIER, Volume 58, Issue 31, 2 August 2017, Pages 3078–3080

Potentiation of Antibiotic Activity by a Novel Cationic Peptide: Potency and Spectrum of Activity of SPR741

Corbett, D., Langley, T., Wise, A. et.al.

ABSTRACT Novel approaches for the treatment of multidrug-resistant Gram-negative bacterial infections are urgently required. One approach is to potentiate the efficacy of existing antibiotics whose spectrum of activity is limited by the permeability barrier presented by the Gram-negative outer membrane. Cationic peptides derived from polymyxin B have been used to permeabilize the outer membrane, granting antibiotics that would otherwise be excluded access to their targets. We assessed the in vitro efficacies of combinations of SPR741 with conventional antibiotics against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Of 35 antibiotics tested, the MICs of 8 of them were reduced 32- to 8,000-fold against E. coli and K. pneumoniae in the presence of SPR741. The eight antibiotics, azithromycin, clarithromycin, erythromycin, fusidic acid, mupirocin, retapamulin, rifampin, and telithromycin, had diverse targets and mechanisms of action. Against A. baumannii, similar potentiation was achieved with clarithromycin, erythromycin, fusidic acid, retapamulin, and rifampin. Susceptibility testing of the most effective antibiotic-SPR741 combinations was extended to 25 additional multidrug-resistant or clinical isolates of E. coli and K. pneumoniae and 17 additional A. baumannii isolates in order to rank the potentiated antibiotics. SPR741 was also able to potentiate antibiotics that are substrates of the AcrAB-TolC efflux pump in E. coli, effectively circumventing the contribution of this pump to intrinsic antibiotic resistance. These studies support the further development of SPR741 in combination with conventional antibiotics for the treatment of Gram-negative bacterial infections.

AAC, posted online 22 May 2017

Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells

Roolf C, Dybowski N, Sekora A, Schaab C et al.

Abstract Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 μm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD- cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD- and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Mol Cell Proteomics. 2017 Jul;16(7):1365-1376

NF-L in cerebrospinal fluid and serum is a biomarker of neuronal damage in an inducible mouse of neurodegeneration

Pech, C. et. al.

Abstract Accumulation of neurofilaments (NFs), the major constituents of the neuronal cytoskeleton, is a distinctive feature of neurological diseases and several studies have shown that soluble NFs can be detected in the cerebrospinal fluid (CSF) of patients with neurological diseases, such as multiple sclerosis and frontotemporal dementia. Here we have used an inducible transgenic mouse model of neurodegeneration, CamKII-TetOp25 mice, to evaluate whether NF-L levels in CSF or blood can be used as a biochemical biomarker of neurodegeneration. Induction of p25 transgene brain expression led to increase in CSF and serum NF-L levels that correlated with ongoing neurodegeneration. Switching off p25 prevented further increases in both CSF and serum NF-L levels and concomitantly stopped the progression of neurodegeneration. The levels of CSF NF-L detected in p25 mice are about 4–fold higher than the CSF levels detected in patients with chronic neurodegenerative diseases, such as symptomatic FTD (bvFTD). In addition, our data indicate that the NF-L detected in CSF is most likely a cleaved form of NF-L. These results suggest that CSF and serum NF-L are of interest to be further explored as potential translational dynamic biomarkers of neurodegeneration or as pharmacodynamics biomarkers at least in preclinical animal studies.

ELSEVIER, 2017, published online

NF-L in cerebrospinal fluid and serum is a biomarker of neuronal damage in an inducible mouse model of neurodegeneration

Pech, C. et. al.

ELSEVIER, 2017, published online

In Vivo Efficacy of Combinations of Novel Antimicrobial Peptide Spr741 and Rifampicin in Short-Duration Murine Thigh Infection Models of Gram-Negative Bacterial Infection

Thommes, P., Vaddi, S., Warn, P. et. al.

ABSTRACT Objectives: There is an acute shortage of effective antimicrobial agents to treat multi-drug-resistant Gram-negative infection. An attractive approach to addressing the dearth of treatment options is potentiation of antimicrobial agents to either increase the spectrum of activity or enhance activity. In these studies we assessed the efficacy of combinations of a novel antimicrobial cationic peptide (SPR741) with rifampicin (Rif) in murine models of thigh muscle infection. Methods: Male ICR mice were rendered neutropenic using 2 doses of cyclophosphamide on days -4 & -1. Mice were infected by IM injection into the lateral thigh muscle on day 0 with either E. coli (ATCC 25922) Klebsiella pneumoniae (IR60 [blaNDM-1)) or ATCC BAA 2146 [blaNDM-1]), Enterobacter cloacae (Kp114 [blaKPC] or Acinetobacter baumannii (ATCC BAA 747). Treatment was initiated 1h post infection with SPR741 administered at 1, 3.5 and 7h post infection and Rif administered at 1 and 5h post infection). SPR741 was administered at 10, 20 and 40mg/kg/dose, the doses of Rif were based on preliminary dose response experiments (range 0.376-64mg/kg/dose). Mice were euthanized 9h post infection and the thigh muscle quantitatively cultured. Results: SPR741 and Rif were well tolerated and all animals continued to the study end. All isolates demonstrated robust in vivo growth of 1.55-3.4Log10cfu/g thigh tissue between pre-treatment and harvest samples. Monotherapy with SPR741 at 40mg/kg/dose or Rif (at the doses used) had little effect on the burdens and did not achieve stasis against any isolate. In contrast in all models combinations with ≤20mg/kg/dose SPR741 with Rif led to highly significant reductions in burden below stasis (2.2, 3.7, 4.7, 1.6 and 2.9Log10cfu/g below stasis for ATCC 25922, IR60, Kp114, ATCC BAA 2146 and ATCC BAA 747 respectively). Conclusions: The combination of SPR741 with Rif was highly effective at reducing the thigh burden of mice infected with E. coli, K. pneumonia, E. cloacae and A. baumannii including strains expressing blaKPC and blaNDM-1. These studies support continued development of novel antimicrobial cationic peptide for the treatment of multi-drug-resistant Gram-negative infections

ASM-Microbe, 1 -5 June 2017, New Orleans, USA

In vivo Efficacy of Combinations of Novel Antimicrobial Peptide SPR741 and Rifampicin in Neutropenic Murine Pneumonia Models of Gram-Negative Bacterial Infection

Corbett, S., Teague, J., Warn, P. et. al.

ASM-Microbe, 1 -5 June 2017, New Orleans, USA

In Vitro and In Vivo Characterization of Non-Hydroxamate LpxC Inhibitor FG-630

Corbett, D., Parkes, A., Williamson, B. et. al.

ASM-Microbe, 1 -5 June 2017, New Orleans, USA

Anti-fungal activity of PC1244, a novel azole, on azole sensitive and resistant Aspergillus fumigatus strains and other fungi

Corbett, D., Birchall, S., Wise, A. et. al.

Anti-fungal activity of PC1244

Small molecule in combination with Immune Checkpoint therapies

Fons, P., Esquerré, M., Badet, G. et. al.

Small molecule in combination with Immune Checkpoint therapies

AACR Annual Meeting, 1 - 5 April 2017, Washington, DC, USA

Targeting the adenosine immunosuppressive pathway for cancer immunotherapy with small molecule agents

Fons, P., Bell, P., Versluys, S. et. al.

Targeting the adenosine immunosuppressive pathway for cancer immunotherapy with small molecule agents

AACR Annual Meeting, 1 - 5 April 2017, Washington, DC, USA

Neurturin and a Glp-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats

Reers, C., Schreiter, K., Austen, M., Andag, U. et. al.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor (GDNF) family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. Here, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a GLP-1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and β-cell mass, and prevented deterioration of islet organization.

ACS Publications, 2017, published online

The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice

Dickie, A.P., Schreiter K., Wehr, R. et. al.

Abstract The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10 mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10 ± 0.08 μg/mL at 0.25–0.5 h post-dose with an AUCinf of 1.74 ± 0.52 μg h/mL and an effective half-life for the drug of 1.42 ± 0.72 h (n = 3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15 ± 0.08 μg/mL at 0.25 h post-dose with an AUCinf of 1.94 ± 0.22 μg h/mL and an effective half-life of 1.28 ± 0.02 h (n = 3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans. Graphical abstract Unlabelled figure Download high-res image (163KB)Download full-size image

ELSEVIER, 2017, published online

Thermodynamic profiling of inhibitors of Nrf2:Keap1 interactions

Nasiri, H., Linge, S., Ullmann, D.

Abstract Keap1 binds to the transcription factor Nrf2 and negatively modulates the expression of genes involved in cellular protection against oxidative stress. Small molecules have been discovered to inhibit the Nrf2:Keap1 interactions and act as antagonists of Keap1. The affinities of these small molecules are not very high and need further improvement in follow up hit-to-lead programs. In addition to the affinity parameters Ki, Kd, and IC50 thermodynamic parameters provide useful information for the selection and optimization of these hit molecules at the early stage of the lead discovery process. In this letter a tracer displacement assay was used to determine the thermodynamic signature of some of the known inhibitors of the Nrf2:Keap1 interaction. An optimized assay protocol is presented, which can be applied to other small molecules in hit-to-lead programs in a medium throughput manner.

Science Direct, 2016, published online

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease

Neagoe, I., Deisemann, H., Winkler, D. et al.

Abstract Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntington's disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.

Experimental Neurology 282 (2016) 99–118

The KNIME Cookbook

Matzanetz, M.P., Silipo, R.

Matzanetz, M.P., Silipo, R.

KNIME Press, 2016, published online

Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

Wandinger, S. K., Lahortiga, I., Jacobs, K. et al.

Abstract The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.

PLoS ONE, 2016, 11(1)

Predictions of Ligand Selectivity from Absolute Binding Free Energy Calculations

Bodkin, M.J., Heifetz, A., et al.

Bodkin, M.J., Heifetz, A., et al.

ACS Publications, 2016, published online

Potentiation of Antibiotic Activity by a Novel Cationic Peptide, SPR741

Corbett, D., Birchall, S., Wise, A. et.al.

ASM-Microbe, 16 - 20 June 2016, Boston, USA

Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington’s Disease Models

Beaumont, V., Elschenbroich, S., Schaab, C. et al.

Beaumont, V., Elschenbroich, S., Schaab, C. et al.

Cell Press, 2016, published online

KNIME-ing through the EVOSpace of FTrees

Bodkin, M., Dawson, G., Mazanetz, M.P.

UGM & Conference 2016 Europe May 17-20, 2016 , Vienna, Austria

Integrated genomics and proteomics define huntingtin CAG length–dependent networks in mice

Schaab, C., Langfelder, P., Cantle, J.P. et al.

Abstract To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length–dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo.

Nature neuroscience, 2016, published online

Fragment Molecular Orbital Method Applied to Lead Optimization of Novel Interleukin-2 Inducible T-Cell Kinase (ITK) Inhibitors

Heifetz, A., Trani, G., MacKinnon, C. H. et al.

Abstract Abstract Image Inhibition of inducible T-cell kinase (ITK), a nonreceptor tyrosine kinase, may represent a novel treatment for allergic asthma. In our previous reports, we described the discovery of sulfonylpyridine (SAP), benzothiazole (BZT), indazole (IND), and tetrahydroindazole (THI) series as novel ITK inhibitors and how computational tools such as dihedral scans and docking were used to support this process. X-ray crystallography and modeling were applied to provide essential insight into ITK–ligand interactions. However, “visual inspection” traditionally used for the rationalization of protein–ligand affinity cannot always explain the full complexity of the molecular interactions. The fragment molecular orbital (FMO) quantum-mechanical (QM) method provides a complete list of the interactions formed between the ligand and protein that are often omitted from traditional structure-based descriptions. FMO methodology was successfully used as part of a rational structure-based drug design effort to improve the ITK potency of high-throughput screening hits, ultimately delivering ligands with potency in the subnanomolar range.

J. Med. Chem., 2016, 59 (9), 4352-4363

Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design

Dietz, M., Dirksen, A., Volgraf, M. et al.

Abstract Abstract Image The N-methyl-d-aspartate receptor (NMDAR) is a Na+ and Ca2+ permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer’s disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure–activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

ACS Publications, 2016, published online

Applications of the Fragment Molecular Orbital Method to Drug Research

Matzanetz, M.P., Chudyk, E., Fedorov et al.

Abstract The study of molecular behavior at high levels of theoretical accuracy has entered into a new age in computational drug discovery where quantum mechanical (QM) methods are becoming increasingly popular. Theoretically rigorous calculations can be prohibitively computationally expensive and time consuming. These two factors have necessitated the development of faster methods, and the fragment molecular orbital method (FMO) is one such method that has been used for efficient and accurate QM calculations in drug design. In this chapter, the use of FMO is described in detail for predicting geometry, estimating the binding energy of the ligands, conformational sampling, analysis of molecular interactions, deriving partial charges, and generating quantitative structure-activity relationship (QSAR) models.

Springer Science 2016, published online

Structure-Based Design of Tricyclic NF-κB Inducing Kinase (NIK) Inhibitors That Have High Selectivity over Phosphoinositide-3-kinase (PI3K)

Godemann, R., Knüppel, K., Krämer, J., Krüger, S., McEwan, P., Wang, X. et al.

Structure-Based Design of Tricyclic NF-κB Inducing Kinase (NIK) Inhibitors That Have High Selectivity over Phosphoinositide-3-kinase (PI3K)

ACS Publications, 2016, published online

Targeting the podocyte to treat glomerular kidney disease

Lal, M. A., Young, K. W., Andag, U.

Abstract The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics.

Drug Discovery Today, 2015, Jun 19

Targeting deubiquitinase activity with a novel small-molecule inhibitor as therapy for B-cell malignancies

Peterson, L. F.; Ermann, M.; Courtney, S. M. et al.

Abstract Usp9x was recently shown to be highly expressed in myeloma patients with short progression-free survival and is proposed to enhance stability of the survival protein Mcl-1. In this study, we found that the partially selective Usp9x deubiquitinase inhibitor WP1130 induced apoptosis and reduced Mcl-1 protein levels. However, short hairpin RNA–mediated knockdown (KD) of Usp9x in myeloma cells resulted in transient induction of apoptosis, followed by a sustained reduction in cell growth. A compensatory upregulation of Usp24, a deubiquitinase closely related to Usp9x, in Usp9x KD cells was noted. Direct Usp24 KD resulted in marked induction of myeloma cell death that was associated with a reduction of Mcl-1. Usp24 was found to sustain myeloma cell survival and Mcl-1 regulation in the absence of Usp9x. Both Usp9x and Usp24 were expressed and activated in primary myeloma cells whereas Usp24 protein overexpression was noted in some patients with drug-refractory myeloma and other B-cell malignancies. Furthermore, we improved the drug-like properties of WP1130 and demonstrated that the novel compound EOAI3402143 dose-dependently inhibited Usp9x and Usp24 activity, increased tumor cell apoptosis, and fully blocked or regressed myeloma tumors in mice. We conclude that small-molecule Usp9x/Usp24 inhibitors may have therapeutic activity in myeloma.

Blood 2015, 125, 3588-3597

Systematic evaluation of label-free and super-SILAC quantification for proteome expression analysis

Tebbe, A.; Klammer, M.; Sighart, S. et al.

Abstract Rationale Advanced implementations of mass spectrometry (MS)-based proteomics allow for comprehensive proteome expression profiling across many biological samples. The outcome of such studies critically depends on accurate and precise quantification, which has to be ensured for high-coverage proteome analysis possible on fast and sensitive mass spectrometers such as quadrupole orbitrap instruments. Methods We conducted ultra-high-performance liquid chromatography (UHPLC)/MS experiments on a Q Exactive to systematically compare label-free proteome quantification across six human cancer cell lines with quantification against a shared reference mix generated by stable isotope labeling with amino acids in cell culture (super-SILAC). Results Single-shot experiments identified on average about 5000 proteins in the label-free compared to about 3500 in super-SILAC experiments. Label-free quantification was slightly less precise than super-SILAC in replicate measurements, verifying previous results obtained for lower proteome coverage. Due to the higher number of quantified proteins, more significant differences were detected in label-free cell line comparisons, whereas a higher percentage of quantified proteins was identified as differentially expressed in super-SILAC experiments. Additional label-free replicate analyses effectively compensated for lower precision of quantification. Finally, peptide fractionation by high pH reversed-phase chromatography prior to LC/MS analysis further increased the robustness and precision of label-free quantification in conjunction with higher proteome coverage. Conclusions Our results benchmark and highlight the utility of label-free proteome quantification for applications such as target and biomarker discovery on state-of-the-art UHPLC/MS workflows. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid Communications in Mass Spectrometry, 2015, 29, 9, 795-801

Synthesis, SAR, and Series Evolution of Novel Oxadiazole-Containing 5-Lipoxygenase Activating Protein Inhibitors: Discovery of 2-[4-(3-{(R)-1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915)

Takahashi, H.; Crux, R.; Dines, J. A. et al.

Abstract Abstract Image The synthesis, structure–activity relationship (SAR), and evolution of a novel series of oxadiazole-containing 5-lipoxygenase-activating protein (FLAP) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (IC50 < 100 nM). Optimization of binding and functional potencies, as well as physicochemical properties resulted in the identification of compound 69 (BI 665915) that demonstrated an excellent cross-species drug metabolism and pharmacokinetics (DMPK) profile and was predicted to have low human clearance. In addition, 69 was predicted to have a low risk for potential drug–drug interactions due to its cytochrome P450 3A4 profile. In a murine ex vivo whole blood study, 69 demonstrated a linear dose–exposure relationship and a dose-dependent inhibition of LTB4 production.

J. Med. Chem., 2015, 58 (4), 1669–1690

SMT19969 for Clostridium difficile Infection: Comparative Efficacy Compared to Fidaxomicin and Vancomycin in the Hamster Model of CDI

Teague, J., Thommes, P., Warn, P. et.al.

ECCMID, 25 - 28 April 2015, Copenhagen, Denmark

Selective CB2 receptor agonists. Part 3: The optimization of a piperidine-based series that demonstrated efficacy in an in vivo neuropathic pain model

Bartolozzi, A., Albrecht, C., Gemkow, M. et al.

Abstract A novel class of potent cannabinoid receptor 2 (CB2) agonists based on a (S)-piperidine scaffold was identified using ligand-based pharmacophore models. Optimization of solubility and metabolic stability led to the identification of several potent CB2 agonists (e.g., 30) that displayed selectivity over cannabinoid receptor 1 (CB1) and acceptable drug like properties. In rats, compound 30 demonstrated a favorable pharmacokinetic profile and efficacy in a Streptozotocin-induced diabetic neuropathy model, with full reversal of mechanical hyperalgesia. Graphical abstract

Bioorganic & Medicinal Chemistry Letters, 2015, 25, 587-592

Selective CB2 receptor agonist. Part 2: Structure-acitivity relationship studies and optimization or proline-based compounds

Riether, D., Jenkins, J.E., Albrecht, C. et al.

Abstract Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure–activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia.

Bioorganic & Medicinal Chemistry Letters, 2015, 25, 581-586

Selective CB2 receptor agonist. Part 1: The identification of novel ligands through computer-aided drug design (CADD) approaches

Hickey, E.R., Ermann, M., Albrecht, C. et al.

Abstract Computer-aided drug design scaffold hopping strategies were utilized to identify new classes of CB2 agonists when compounds of an established series with low nanomolar potency were challenging to optimize for good drug-like properties. Use of ligand-based design strategies through BI Builder (a tool for de novo design) and PharmShape (a virtual screening software package) approaches led to the discovery of new chemotypes. Specifically, compounds containing azetidine-, proline-, and piperidine-based cores were found to have low nanomolar and picomolar CB2 agonist activities with drug-like properties considered appropriate for early profiling.

Bioorganic & Medicinal Chemistry Letters, 2015, 25, 575-580

Phenotypic discovery and characterization of neuroprotective compounds relevant to ALS

Rudhard, Y.; Höing, S.; Reinhardt, P. et al

ALS/MND Symposium 2013, 05-08 December 2013, Milan, Italy

Modeling of ribonucleic acid–ligand interactions

Stefaniak, F., Chudyk, E. I., Bodkin, M. et al.

Stefaniak, F., Chudyk, E. I., Bodkin, M. et al.

WIREs Comput Mol Sci 2015

Large scale storage stability analysis of molecules in the NCATS SMR (formerly MLSMR)

Laggner, C.; Shayo, Y.; Hendarto, C. et al.

249th ACS National Meeting & Exposition, Spring 2015 22-26 March 2015, Denver, USA

In vitro susceptibility of Clostridium difficile to SMT19969 and comparators, as well as the killing kinetics and post-antibiotic effects of SMT19969 and comparators against C. difficile

Birchall, S., Corbett, D., Wise, A., et. al.

Abstract Objectives SMT19969 is a novel antimicrobial under clinical development for the treatment of Clostridium difficile infection (CDI). The objective was to determine the comparative susceptibility of 82 C. difficile clinical isolates (which included ribotype 027 isolates and isolates with reduced metronidazole susceptibility) to SMT19969, fidaxomicin, vancomycin and metronidazole and to determine the killing kinetics and post-antibiotic effects of SMT19969, fidaxomicin and vancomycin against C. difficile. Methods MICs were determined by agar incorporation. Killing kinetics and post-antibiotic effects were determined against C. difficile BI1, 630 and 5325 (ribotypes 027, 012 and 078, respectively). Results SMT19969 showed potent inhibition of C. difficile (MIC90=0.125 mg/L) and was markedly more active than either metronidazole (MIC90 = 8 mg/L) or vancomycin (MIC90 = 2 mg/L). There were no differences in susceptibility to SMT19969 between different ribotypes. Fidaxomicin was typically one doubling dilution more active than SMT19969 and both agents maintained activity against isolates with reduced susceptibility to metronidazole. In addition, SMT19969 was bactericidal against the C. difficile strains tested, with reductions in viable counts to below the limit of detection by 24 h post-inoculation. Vancomycin was bacteriostatic against all three strains. Fidaxomicin was bactericidal although reduced killing was observed at concentrations <20 × MIC against C. difficile BI1 (ribotype 027) compared with other strains tested. Conclusions These data demonstrate that SMT19969 is associated with potent and bactericidal activity against the strains tested and support further investigation of SMT19969 as potential therapy for CDI.

Journal of Antimicrobial Chemotherapy, 2015, Volume 70, Issue 6, 1, 1751–1756

Identification of 12/15-lipoxygenase as a regulator of axon degeneration through high content screening

Rudhard, Y.; Ghosh,A. S.; Lippert, B.

ABSTRACT: Small molecule inhibitors of protein kinases are key tools for signal transduction research and represent a major class of targeted drugs. Recent developments in quantitative proteomics enable an unbiased view on kinase inhibitor selectivity and modes of action in the biological context. While chemical proteomics techniques utilizing quantitative mass spectrometry interrogate both target specificity and affinity in cellular extracts, proteome-wide phosphorylation analyses upon kinase inhibitor treatment identify signal transduction pathway and network regulation in an unbiased manner. Thus, critical information is provided to promote new insights into mechanisms of kinase signaling and their relevance for kinase inhibitor drug discovery.

Journal of Neuroscience

Evaluation of water displacement energetics in protein binding sites with grid cell theory

Gerogiokas, G.; Southey, M.W.Y.; Mazanetz, M.P. et al.

Excess free energies, enthalpies and entropies of water in protein binding sites were computed via classical simulations and Grid Cell Theory (GCT) analyses for three pairs of congeneric ligands in complex with the proteins scytalone dehydratase, p38a MAP kinase and EGFR kinase respectively. Comparative analysis is of interest since the binding modes for each ligand pair differ in the displacement of one binding site water molecule, but significant variations in relative binding affinities are observed. Protocols that vary in their use of restraints on protein and ligand atoms were compared to determine the influence of protein–ligand flexibility on computed water structure and energetics, and to assess protocols for routine analyses of protein–ligand complexes. The GCT-derived binding affinities correctly reproduce experimental trends, but the magnitude of the predicted changes in binding affinities is exaggerated with respect to results from a previous Monte Carlo Free Energy Perturbation study. Breakdown of the GCT water free energies into enthalpic and entropic components indicates that enthalpy changes dominate the observed variations in energetics. In EGFR kinase GCT analyses revealed that replacement of a pyrimidine by a cyanopyridine perturbs water energetics up three hydration shells away from the ligand

Phys. Chem. Chem. Phys., 2015, 17, 8416-8426

Efficacy of BAL30072 in Murine Lung Infection Models of Multi-Resistant Gram-Negative Bacteria

Sattar, A., Thommes, P., Vaddi, S.

ECCMID, 25 - 28 April 2015, Copenhagen, Denmark

Efficacy of BAL30072 in Combination with Meropenem in Murine Thigh Infection Models of Multi-Resistant Gram-Negative Bacteria

Gould, J., Thommes, P., Sattar, A. et. al.

ECCMID, 25 - 28 April 2015, Copenhagen, Denmark

Discovery of novel amino-pyrimidine inhibitors of the insulin-like growth factor 1 (IGF1R) and insulin receptor (INSR) kinases; parallel optimization of cell potency and hERG inhibition

Klammer, M.; Dybowski, J. N.; Schaab, C. et al.

Abstract Multivariate biomarkers that can predict the effectiveness of targeted therapy in individual patients are highly desired. Previous biomarker discovery studies have largely focused on the identification of single biomarker signatures, aimed at maximizing prediction accuracy. Here, we present a different approach that identifies multiple biomarkers by simultaneously optimizing their predictive power, number of features, and proximity to the drug target in a protein-protein interaction network. To this end, we incorporated NSGA-II, a fast and elitist multi-objective optimization algorithm that is based on the principle of Pareto optimality, into the biomarker discovery workflow. The method was applied to quantitative phosphoproteome data of 19 non-small cell lung cancer (NSCLC) cell lines from a previous biomarker study. The algorithm successfully identified a total of 77 candidate biomarker signatures predicting response to treatment with dasatinib. Through filtering and similarity clustering, this set was trimmed to four final biomarker signatures, which then were validated on an independent set of breast cancer cell lines. All four candidates reached the same good prediction accuracy (83%) as the originally published biomarker. Although the newly discovered signatures were diverse in their composition and in their size, the central protein of the originally published signature — integrin β4 (ITGB4) — was also present in all four Pareto signatures, confirming its pivotal role in predicting dasatinib response in NSCLC cell lines. In summary, the method presented here allows for a robust and simultaneous identification of multiple multivariate biomarkers that are optimized for prediction performance, size, and relevance.

PLoS One 2015, 10 (6), e0128542

Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model of Huntington´s Disease: An In Vivo High-Content Imaging Study

Carty, N.; Berson, N.; Tillack, K. et al.

Abstract Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2–12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.

PLoS One 2015, 10 (4), e0123527

Binding mode and structure-activity relationships around direct inhibitors of the Nrf2-Keap1 complex

Jnoff, E., Albrecht, C., Barker, J.J. et al.

Abstract An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.

ChemMedChem., 2014 April, 9 (4), 699-705

3D Enriched Fragment Library Design for X-ray Crystallography

Barker, J. Bodkin, M. Mazanetz, M.P.

Fragments 2015: Fifth RSC-BMCS Fragment-based Drug Discovery meeting, 22 - 24 March 2015, Cambridge, UK

Tuning covalent reactivity: A Chemist’s toolbox

Ermann, Monika; Morao, Inaki; Courtney, Stephen M. et al.

The 34th National Medicinal Chemistry Symposium, 18-21 May 2014, Charleston, SC, USA

Tropomyosin receptor kinase inhibitors: a patent update 2009 – 2013

McCarthy, C., Walker, E.

Abstract G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate GPCR drug discovery. Several key points emerged. Firstly, structures for all three major classes of GPCRs have now been solved and there is increasing coverage across the GPCR phylogenetic tree. This is likely to be substantially enhanced with data from x-ray free electron sources as they move beyond proof of concept. Secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many GPCRs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. Thirdly, there will almost certainly be some GPCRs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. Subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. This is particularly important because it has ramifications for how we interpret pre-clinical data. In summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of GPCRs and will be essential for resolving problems associated with the more difficult targets in the future. Keywords: GPCRs, G-protein coupled receptors; β2AR, β2-adrenergic receptor; GLP-1, Glucagon-like peptide-1 receptor; CCK2R, cholecystokinin receptor-2; δ-OR, delta-opioid receptor; CRF1, corticotropin releasing factor receptor 1; CXCR1, CXCR2, CCR4 and CCR5, chemokine receptors; 5-HT2B and 5-HT2C, human 5-hydroxytryptamine receptors 2B and 2C, respectively; H1, histamine receptor 1; hM3R, human muscarinic M3 receptor; Dopamine D2 receptor; α1B Adrenergic receptor; T4L, T4-lysozyme; BRIL, apocytochrome b562RIL; XFELs, x-ray free electron lasers; SDM, site-directed mutagenesis; MD, molecular dynamic simulations; 3D, three-dimensional; 7TM, seven-transmembrane domain; TM, trans-membrane helix; ECL, extracellular loop; HGMP, hierarchical GPCR modelling protocol; GLAS, GPCR-likeness assessment score; ProS, pairwise protein similarity method; PDB, Protein Data Bank

Expert Opinion on Therapeutic Patents, 2014, 24, 7, 731–744

Towards a LEANer platform for parallel protein production

Black, Hara; Hitchman, Richard; Yadav, Rahul et al.

PEGS Summit, 05-09 May 2014, Boston, MA, USA

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

Macdonald, D., Tessari, M., Herrmann, F. et al

Abstract The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

Plos One, 2014, 9 (5), e96854-ff

Membrane protein production & structural biology capabilities at Evotec

Singh, Shweta; Duclo, Stephanie; Smith, Myron et al.

Keystone Symposia on Molecular and Cellular Biology, 30 March - 04 April 2014 , Snowbird, Utah, USA

Investigation of the Flexibility of Protein Kinases Implicated in the Pathology of Alzheimer’s Disease

Mazanetz, M.P., Laughton, C.A., Fischer, P.M.

Abstract The pathological characteristics of Alzheimer’s Disease (AD) have been linked to the activity of three particular kinases—Glycogen Synthase Kinase 3β (GSK3β), Cyclin-Dependent Kinase 5 (CDK5) and Extracellular-signal Regulated Kinase 2 (ERK2). As a consequence, the design of selective, potent and drug-like inhibitors of these kinases is of particular interest. Structure-based design methods are well-established in the development of kinase inhibitors. However, progress in this field is limited by the difficulty in obtaining X-ray crystal structures suitable for drug design and by the inability of this method to resolve highly flexible regions of the protein that are crucial for ligand binding. To address this issue, we have undertaken a study of human protein kinases CDK5/p25, CDK5, ERK2 and GSK3β using both conventional molecular dynamics (MD) and the new Active Site Pressurisation (ASP) methodology, to look for kinase-specific patterns of flexibility that could be leveraged for the design of selective inhibitors. ASP was used to examine the intrinsic flexibility of the ATP-binding pocket for CDK5/p25, CDK5 and GSK3β where it is shown to be capable of inducing significant conformational changes when compared with X-ray crystal structures. The results from these experiments were used to quantify the dynamics of each protein, which supported the observations made from the conventional MD simulations. Additional information was also derived from the ASP simulations, including the shape of the ATP-binding site and the rigidity of the ATP-binding pocket. These observations may be exploited in the design of selective inhibitors of GSK3β, CDK5 and ERK2. View Full-Text

Molecules 2014, 19(7), 9134-9159

Identification of Significant Features by the Global Mean Rank

Klammer, M., Dybowski, J.N., Hoffmann, D., Schaab, C.

Abstract With the introduction of omics-technologies such as transcriptomics and proteomics, numerous methods for the reliable identification of significantly regulated features (genes, proteins, etc.) have been developed. Experimental practice requires these tests to successfully deal with conditions such as small numbers of replicates, missing values, non-normally distributed expression levels, and non-identical distributions of features. With the MeanRank test we aimed at developing a test that performs robustly under these conditions, while favorably scaling with the number of replicates. The test proposed here is a global one-sample location test, which is based on the mean ranks across replicates, and internally estimates and controls the false discovery rate. Furthermore, missing data is accounted for without the need of imputation. In extensive simulations comparing MeanRank to other frequently used methods, we found that it performs well with small and large numbers of replicates, feature dependent variance between replicates, and variable regulation across features on simulation data and a recent two-color microarray spike-in dataset. The tests were then used to identify significant changes in the phosphoproteomes of cancer cells induced by the kinase inhibitors erlotinib and 3-MB-PP1 in two independently published mass spectrometry-based studies. MeanRank outperformed the other global rank-based methods applied in this study. Compared to the popular Significance Analysis of Microarrays and Linear Models for Microarray methods, MeanRank performed similar or better. Furthermore, MeanRank exhibits more consistent behavior regarding the degree of regulation and is robust against the choice of preprocessing methods. MeanRank does not require any imputation of missing values, is easy to understand, and yields results that are easy to interpret. The software implementing the algorithm is freely available for academic and commercial use.

PLoS One 2014, 9 (8), e104504

High-Throughput Screening

Böcker, A., Schaertl, S., Hess, S.

About this book Ion channel drug discovery is a rapidly evolving field fuelled by recent, but significant, advances in our understanding of ion channel function combined with enabling technologies such as automated electrophysiology. The resurgent interest in this target class by both pharmaceutical and academic scientists was clearly highlighted by the over-subscribed RSC/BPS 'Ion Channels as Therapeutic Targets' symposium in February 2009. This book builds on the platform created by that meeting, covering themes including advances in screening technology, ion channel structure and modelling and up-to-date case histories of the discovery of modulators of a range of channels, both voltage-gated and non-voltage-gated channels. The editors have built an extensive network of contacts in the field through their first-hand scientific experience, collaborations and conference participation and the organisation of the meeting at Novartis, Horsham, increased the network enabling the editors to draw on the experience of eminent researchers in the field. Interest and investment in ion channel modulation in both industrial and academic settings continues to grow as new therapeutic opportunities are identified and realised for ion channel modulation. This book provides a reference text by covering a combination of recent advances in the field, from technological and medicinal chemistry perspectives, as well as providing an introduction to the new 'ion channel drug discoverer'. The book has contributions from highly respected academic researchers, industrial researchers at the cutting edge of drug discovery and experts in enabling technology. This combination provides a complete picture of the field of interest to a wide range of readers.

Ion Channel Drug Discovery, 2014, 16-41

GPCR structure, function, drug discovery and crystallography: report from Academia-Industry International Conference (UK Royal Society) Chicheley Hall, 1-2 September 2014

Bodkin MJ, Heifetz A., Law RJ, Singh S. et al.

Abstract G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate GPCR drug discovery. Several key points emerged. Firstly, structures for all three major classes of GPCRs have now been solved and there is increasing coverage across the GPCR phylogenetic tree. This is likely to be substantially enhanced with data from x-ray free electron sources as they move beyond proof of concept. Secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many GPCRs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. Thirdly, there will almost certainly be some GPCRs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. Subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. This is particularly important because it has ramifications for how we interpret pre-clinical data. In summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of GPCRs and will be essential for resolving problems associated with the more difficult targets in the future. Keywords: GPCRs, G-protein coupled receptors; β2AR, β2-adrenergic receptor; GLP-1, Glucagon-like peptide-1 receptor; CCK2R, cholecystokinin receptor-2; δ-OR, delta-opioid receptor; CRF1, corticotropin releasing factor receptor 1; CXCR1, CXCR2, CCR4 and CCR5, chemokine receptors; 5-HT2B and 5-HT2C, human 5-hydroxytryptamine receptors 2B and 2C, respectively; H1, histamine receptor 1; hM3R, human muscarinic M3 receptor; Dopamine D2 receptor; α1B Adrenergic receptor; T4L, T4-lysozyme; BRIL, apocytochrome b562RIL; XFELs, x-ray free electron lasers; SDM, site-directed mutagenesis; MD, molecular dynamic simulations; 3D, three-dimensional; 7TM, seven-transmembrane domain; TM, trans-membrane helix; ECL, extracellular loop; HGMP, hierarchical GPCR modelling protocol; GLAS, GPCR-likeness assessment score; ProS, pairwise protein similarity method; PDB, Protein Data Bank

PMC US National Library of Medicine National Institutes of Health, 2015

Global phosphoproteome analysis of human bone marrow reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib

Schaab, C., Oppermann, F.S., Klammer, M. et al.

Global phosphoproteome analysis of human bone marrow reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib Leukemia (2014) 28, 716–719; doi:10.1038/leu.2013.347 Treatment with inhibitors of the receptor tyrosine kinase FLT3 are currently studied as promising therapies in acute myeloid leukemia (AML). However, only a subset of patients benefit from these treatments and the presence of activating mutations within FLT3 can predict response to a certain extent only. AC220 (quizartinib) is an example of a potent FLT3 inhibitor1 that was studied in a recent phase II open-label study in patients with relapsed/refractory AML. The complete remission rate (including CRp and CRi) in FLT3-ITD-positive patients was 54% (50/92) and the corresponding partial remission rate (PR) was 17% (16/92)2 Thus, although the FLT3-ITD mutation status correlates with response, the error rate in stratification of patients into responders and non-responders is high, as still 29% of the FLT3-ITD-positive patients failed to respond. Exclusion of FLT3-ITD-negative patients from AC220 treatment also seems critical, as the total response rate (CR þ PR) in FLT3-ITD-negative patients is substantially lower (41%, 17/41). As AC220 is a tyrosine kinase inhibitor, we hypothesized that investigating phosphorylation-based signaling on a system-wide scale in AML cells allows for identification of markers enabling more accurate prediction of therapy response as compared to commonly used genetic markers. Hence, we applied quantitative mass spectrometry to decipher a multivariate phosphorylation site marker, which we refer to as phosphosignature, in patient-derived AML blasts that might be useful as predictive biomarkers for AC220 treatment. We first collected bone marrow aspirates of 21 patients enrolled in the phase II clinical trial of AC220 monotherapy in AML (ACE, NCT00989261) with FLT3-ITD before treatment (Supplementary Table 1). We processed the aspirates according to a previously established sample preparation workflow (Figure 1 and Supplementary Methods). Twelve of the twenty-one samples were processed at the beginning of this study (training group) and were used to generate a training data-set for phosphosignature identification. Nine additional samples were processed toward the end of this study and were used for validating the phospho-signature (validation group). All patients with CR or PR were counted as responder in our study (6/12 in the training subgroup and 6/9 in the validation subgroup). To monitor quantitatively the phospho-proteomes of the patient-derived AML blasts, we used super-SILAC in combination with quantitative mass spectrometry (see Figure 1 and Supplementary Methods). Data analysis was finally performed by using the MaxQuant software3 and further bioinformatics tools as outlined below. In total, 13 236 phospho-sites were identified in the training group. Of these, 7831 were confidently assigned to specific serine, threonine or tyrosine residues (class I sites). We first investigated whether we can identify differentially regulated phospho-sites when comparing responder and nonresponder samples (Figure 2a). Only class I sites quantified in at least two thirds of the experiments were used (2119 sites with approximately 10.6% missing values on average). Indeed, application of the mean-rank test4 revealed three significantly different sites at a false-discovery rate of 10% (see Supplementary Table 2). The first regulated site (S160) is located on the endonuclease/ exonuclease/phosphatase family domain-containing protein 1 (EEPD1). The protein carrying the second phosphorylation site (S630) was B-cell lymphoma/leukemia 11A (BCL11A), which functions as a myeloid and B-cell proto-oncogene and may play a role in leukemogenesis and hematopoiesis.5 Furthermore, the expression of BCL11A is associated with a poor outcome of AML patients.6 The third phosphorylation site (S333) is located on Ranbinding protein 3 (RANBP3). RANBP3 mediates nuclear export of Smad2/3 and thereby inhibits TGF-b signaling.7

Leukemia, 2014, 28, 716–719

Evaluation of Host/Guest Binding Thermodynamics of Model Cavities with Grid Cell Theory

Law, R.J., Mazanetz, M.P., Southey, M.W.Y. et al.

Abstract Abstract Image A previously developed cell theory model of liquid water was used to evaluate the excess thermodynamic properties of confined clusters of water molecules. The results are in good agreement with reference thermodynamic integration calculations, suggesting that the model is adequate to probe the thermodynamic properties of water at interfaces or in cavities. Next, the grid cell theory (GCT) method was applied to elucidate the thermodynamic signature of nonpolar association for a range of idealized host–guest systems. Polarity and geometry of the host cavities were systematically varied, and enthalpic and entropic solvent components were spatially resolved for detailed graphical analyses. Perturbations in the thermodynamic properties of water molecules upon guest binding are restricted to the immediate vicinity of the guest in solvent-exposed cavities, whereas longer-ranged perturbations are observed in buried cavities. Depending on the polarity and geometry of the host, water displacement by a nonpolar guest makes a small or large enthalpic or entropic contribution to the free energy of binding. Thus, no assumptions about the thermodynamic signature of the hydrophobic effect can be made in general. Overall the results warrant further applications of GCT to more complex systems such as protein–ligand complexes.

Journal of Chemical Theory and Computation, 2014, 10 (9), pp 4055–4068

Design, synthesis and structure–activity relationships of a novel class of sulfonylpyridine inhibitors of Interleukin-2 inducible T-cell kinase (ITK)

Trani, G., Barker, J. J., Bromidge, S. M. et al.

Abstract Starting from benzylpyrimidine 2, molecular modeling and X-ray crystallography were used to design highly potent inhibitors of Interleukin-2 inducible T-cell kinase (ITK). Sulfonylpyridine 4i showed sub-nanomolar affinity against ITK, was selective versus Lck and its activity in the Jurkat cell-based assay was greatly improved over 2. Graphical abstract Download high-res image (61KB)Download full-size image

Bioorganic & Medicinal Chemistry Letters, 2014, 24, 5818-5823

Dendritic Structural Degeneration Is Functionally Linked to Cellular Hyperexcitability in a Mouse Model of Alzheimer’s Disease

Šišková, Z., Justus, D., von der Kammer, H. et al.

Highlights •Dendritic structural degeneration alters neuronal electrical properties •CA1 pyramidal neurons show increased bursting and higher in vivo firing rates •Neuronal structural pathology and cellular hyperexcitability are functionally linked Summary Dendritic structure critically determines the electrical properties of neurons and, thereby, defines the fundamental process of input-to-output conversion. The diversity of dendritic architectures enables neurons to fulfill their specialized circuit functions during cognitive processes. It is known that this dendritic integrity is impaired in patients with Alzheimer’s disease and in relevant mouse models. It is unknown, however, whether this structural degeneration translates into aberrant neuronal function. Here we use in vivo whole-cell patch-clamp recordings, high-resolution STED imaging, and computational modeling of CA1 pyramidal neurons in a mouse model of Alzheimer’s disease to show that structural degeneration and neuronal hyperexcitability are crucially linked. Our results demonstrate that a structure-dependent amplification of synaptic input to action potential output conversion might constitute a novel cellular pathomechanism underlying network dysfunction with potential relevance for other neurodegenerative diseases with abnormal changes of dendritic morphology. Introduction Dendritic geometry and function are inseparably linked, defining the dendritic integration of synaptic signals, their propagation, and their capability to evoke action potential output (Häusser et al., 2000, Magee, 2000, Poirazi and Mel, 2001, Spruston, 2008). This fundamental relationship between the geometry of dendrites and their cable properties has been explored intensely using computational modeling (Rall, 1962, Rall, 1967, Jaffe and Carnevale, 1999, Vetter et al., 2001). Any physiological or degenerative alteration of the geometry may potentially change the electrical properties of the dendritic cables, which may have functional consequences on the neuronal input-output transformation. Indeed, dendritic degeneration is a common phenomenon observed in many neurodegenerative diseases (Ferrer et al., 1988, Ferrer, 1999, Spires and Hyman, 2004, Zaja-Milatovic et al., 2005, Grutzendler et al., 2007). In Alzheimer’s disease, reduced dendritic branching and length are found consistently in hippocampal and cortical pyramidal neurons of patients (Spires and Hyman, 2004, Grutzendler et al., 2007, Adlard and Vickers, 2002, Falke et al., 2003, Geula, 1998) and in animal models (Grutzendler et al., 2007, Le et al., 2001, Tsai et al., 2004, Moolman et al., 2004). However, despite the omnipresence of structural alterations in neurodegenerative diseases, the specific consequences of dendritic degeneration on their electrical properties and neuronal function are not known. The basic modeling studies performed in wild-type neuronal morphologies predict that a reduction in dendritic branching, length, and surface, as observed in Alzheimer’s disease, would render a given neuron electrically more compact. In a more compact neuron, synaptic currents would be translated more efficiently into postsynaptic and axosomatic depolarization, which then, in turn, could lead to increased action potential output (Johnston et al., 1996). The consequences may not only include neuronal hyperexcitability on the single cell level but also aberrant circuit synchronization, both of which have been found to contribute to cognitive dysfunction in patients and in animal models of Alzheimer’s disease (Palop et al., 2007, Minkeviciene et al., 2009, Busche et al., 2008, Busche et al., 2012, Sanchez et al., 2012, Vossel et al., 2013). The aim of this study was to establish a framework linking the pathological changes of dendritic morphology to neuronal malfunction. In particular, we intended to determine whether the integration of synaptic signals and their transformation into action potential output is altered as a consequence of the pathological changes in dendrite morphology.

Neuron, 2014

Applying QED to antibacterials - Development of QEA, a quantitative estimate of antibacterial drug-likeness

James, T., Parkes, A.

BioInfect 2014, 04 Nov 2014, Alderley Park, Cheshire, UK