Alain Schilb

Development and Implementation of a Highly Miniaturized Confocal 2D-FIDA–Based High-Throughput Screening Assay to Search for Active Site Modulators of the Human Heat Shock Protein 90β

The beta isoform of the heat shock protein 90 (Hsp90β) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90β are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90β. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90β inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90β. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II™ from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

Blockade of activin type II receptors with a dual anti-ActRIIA/IIB antibody is critical to promote maximal skeletal muscle hypertrophy

Significance We recently reported that activin type II receptors (ActRIIs) blockade using bimagrumab could positively impact muscle wasting in mice and humans. However, the specific role of each individual ActRII at regulating adult muscle mass had not been clarified. Here, we highlight the importance of concomitant neutralization of both ActRIIs in controlling muscle mass. Through comparison with single specificity antibodies, we uncover unique features related to bimagrumab and its neutralizing interactions with both ActRIIA and ActRIIB at the structural and cellular levels and in vivo in adult mice. The need for simultaneous engagement and neutralization of both ActRIIs to generate a strong skeletal muscle response confers unique therapeutic potential to bimagrumab, in the context of muscle wasting conditions. The TGF-β family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc “trap,” to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit.

Comprehensive proteome analysis of human skeletal muscle in cachexia and sarcopenia: a pilot study

Cancer cachexia (cancer‐induced muscle wasting) is found in a subgroup of cancer patients leaving the patients with a poor prognosis for survival due to a lower tolerance of the chemotherapeutic drug. The cause of the muscle wasting in these patients is not fully understood, and no predictive biomarker exists to identify these patients early on. Skeletal muscle loss is an inevitable consequence of advancing age. As cancer frequently occurs in old age, identifying and differentiating the molecular mechanisms mediating muscle wasting in cancer cachexia vs. age‐related sarcopenia are a challenge. However, the ability to distinguish between them is critical for early intervention, and simple measures of body weight may not be sufficiently sensitive to detect cachexia early.

Comprehensive proteome analysis of human skeletal muscle in cachexia and sarcopenia

Cancer cachexia (cancer‐induced muscle wasting) is found in a subgroup of cancer patients leaving the patients with a poor prognosis for survival due to a lower tolerance of the chemotherapeutic drug. The cause of the muscle wasting in these patients is not fully understood, and no predictive biomarker exists to identify these patients early on. Skeletal muscle loss is an inevitable consequence of advancing age. As cancer frequently occurs in old age, identifying and differentiating the molecular mechanisms mediating muscle wasting in cancer cachexia vs. age‐related sarcopenia are a challenge. However, the ability to distinguish between them is critical for early intervention, and simple measures of body weight may not be sufficiently sensitive to detect cachexia early.

Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly

Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p<0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p<0.0001). Bax was significantly increased in CWS, CWL and HE (P<0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p<0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained.

Alterations in the in vitro and in vivo regulation of muscle regeneration in healthy ageing and the influence of sarcopenia

Sarcopenia is defined as the age‐related loss of skeletal muscle mass and function. While all humans lose muscle with age, 2–5% of elderly adults develop functional consequences (disabilities). The aim of this study was to investigate muscle myogenesis in healthy elderly adults, with or without sarcopenia, compared with middle‐aged controls using both in vivo and in vitro approaches to explore potential biomarker or causative molecular pathways associated with sarcopenic versus non‐sarcopenic skeletal muscle phenotypes during ageing.

Abstract 4541: Highly sensitive kinase assays combining Carna Biosciences QSS Assist ELISA reagents with the Meso Scale Discovery MULTI-ARRAY platform.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We developed a sensitive kinase asay using a QSS Assist ELISA kinase assay kit from Carna Biosciences, Inc. and the detection method from Meso Scale Discovery. The QSS Assist ELISA kit is optimized for screening of compounds. Comparing to most other kinase assay kits, QSS Assist ELISA kits are available for both peptide and protein substrates and several ELISA kits developed by Carna Biosciences employ a naturally occurring protein substrate, e.g. MAP kinases for MAPK kinases. Detection of kinase activity is achieved with a primary anti-phosphoserine/threonine or tyrosine antibody and a secondary anti-Ig-antibody. Commonly, such secondary antibodies are coupled to horseradish peroxidase (HRP), which allows for signal detection by adding a color reagent, i.e. an HRP substrate which can be detected upon reaction by measuring optical density. Here we applied an MSD SULFO-TAG labeled secondary antibody. These antibodies emit light upon electrochemical stimulation initiated at the electrode surfaces of MULTI-ARRAY microplates, and thus detect the primary antibody with ultimate sensitivity without direct labeling it. We have sucessfully miniaturized the highly sensitive assay and this new assay technology has been validated with published kinase inhibitors from AstraZeneca and Tocris Bioscience. Citation Format: Carsten Jacobi, Alain Schilb, Yusuke Kawase, Yasuyuki Kirii, Brigitte Boldyreff. Highly sensitive kinase assays combining Carna Biosciences QSS Assist ELISA reagents with the Meso Scale Discovery MULTI-ARRAY platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2013-4541