Approaches to Study Small Molecule Inhibitors and Their Targets

Bioactive small molecules are valuable tools to understand and manipulate biological pathways. In order to be effective as either probes for understanding cell biology or as clinical drugs, the small molecule’s mechanism of action must be characterized. However, identifying a small molecule’s target and characterizing its interaction with that target remain major challenges in the chemical biology field. In this thesis, I describe methods to improve the process of drug target identification and binding site characterization. In order to identify the target of a small molecule, I have developed an approach in which multiple drug resistant clones are isolated and transcriptome sequencing is used to find mutations in each clone. Further analysis of mutations common to more than one drug-resistant clone can identify a drug’s physiological target and indirect resistance mechanisms. In proof-of-concept studies I analyze clones resistant to two cytotoxic anti-cancer drugs, BI 2536 and bortezomib. For both compounds I detect mutations in the known target that confer resistance to the drug. Unlike other target identification methods, this approach can establish a genetic proof of the target in human cells. I have also developed a method to characterize a small molecule’s binding site after its target is known. In this method, called Stable Isotope Labeled Inhibitors for Crosslinking (SILIC), structure-activity relationship data is used to design inhibitor analogs that incorporate a photo-crosslinking group along with either natural or heavy stable isotopes. An equimolar mixture of these inhibitor analogs is crosslinked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. I applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein

Anion exchange resin and slow precipitation preclude the need for pretreatments in silver phosphate preparation for oxygen isotope analysis of bioapatites

The authors would like to thank Wolfram Meier-Augenstein (Robert Gordon University) for advice on TC/EA677 IRMS and to Raquel Maria (Kimmel Center for Archaeological Science, Weizmann Institute of Science) for advice on FTIR-ATR. Thanks to Birke Brumme (MPI EVA) for practical support with sample preparation. Thanks are also due to Sahra Talamo (MPI EVA/University of Bologna) for providing aliquots of the S-EVA-2000 and S-EVA-2001 in-house bone standards and to Klervia Jaouen (MPI EVA/Géosciences Environnment Toulouse) for providing extracted collagen used in the preparation of synthetic bones. This research was funded by the Max-Planck-Society as part of SP’s doctoral research. The authors would also like to thank the Max-Planck-Society, the University of Aberdeen and the Vreije Universiteit Brussels for professional and financial support during the production of this manuscript. CS thanks the Research Foundation - Flanders for his post-doctoral fellowship. We also thank Christophe Lécuyer and an anonymous reviewer for their valuable comments and suggestions.Peer reviewedPostprin

Mini-Faustball

Faustball auf kleinem Feld mit kleinen Teams. Dynamik, Schnelligkeit, Action und Teamgedanke. Das sind die wesentlichsten Merkmale des attraktiven Faustballsports. Es gibt kaum ein Rückschlagspiel, das so schnell ein Spiel ermöglicht. Dieses Monatsthema basiert auf dem Grundgedanken «Spielen wird im Spiel gelernt und verbessert» und liefert zahlreiche Bausteine für eine Einheit Mini-Faustball

High resolution flat-panel CT arthrography vs. MR arthrography of artificially created osteochondral defects in ex vivo upper ankle joints

Purpose High resolution flat-panel computed tomography arthrography (FPCT-A) and magnetic resonance arthrography (MR-A) are well suited to evaluate osteochondral lesions. The current study compares the performance of FPCT-A versus MR-A in an experimental setting. Methods Fourteen cadaveric ankles were prepared with artificial osteochondral defects of various sizes in four separate talar locations. After intra-articular contrast injection, FPCT-A and 3-T MR-A were acquired. Each defect was then filled with synthetic pallets. The resulting cast was used as reference. Two independent radiologists measured the dimensions of all defects with FPCT-A and MR-A. Intra-class correlation coefficients (ICC) were calculated. Data were compared using t-tests and Bland-Altman plots. Results The correlation for FPCT-A and cast was higher compared to MR-A and cast (ICC 0.876 vs. 0.799 for surface [length x width]; ICC 0.887 vs. 0.866 for depth, p0.05). Depth measurements were significantly smaller by MR-A (mean difference -1.1 mm, p<0.001). There was no bias between the different modalities. Conclusions Ex vivo FPCT-A and MR-A both deliver high diagnostic accuracy for the evaluation of osteochondral defects. FPCT-A was slightly more accurate than MR-A, which was most significant when measuring lesion depth

Can understanding reward help illuminate anhedonia?

Purpose of review: The goal of this paper is to examine how reward processing might help us understand the symptom of anhedonia. Recent findings: There are extensive reviews exploring the relationship between responses to rewarding stimuli and depression. These often include a discussion on anhedonia and how this might be underpinned in particular by dysfunctional reward processing. However, there is no specific consensus on whether studies to date have adequately examined the various sub-components of reward processing or how these might relate in turn to various aspects of anhedonia symptoms. Summary: The approach to understanding the symptom of anhedonia should be to examine all the sub-components of reward processing at the subjective and objective behavioural and neural level, with well validated tasks that can be replicated. Investigating real life experiences of anhedonia and how theses might be predicted by objective lab measures is also needed in future research

Fibroblast Growth Factor 23 Does Not Directly Influence Skeletal Muscle Cell Proliferation and Differentiation or Ex Vivo Muscle Contractility

Skeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C2C12 myoblasts and myotubes. C2C12 proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca2+ ([Ca2+]i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C2C12 proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C2C12 [Ca2+]i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD

Assessing health-related quality of life in COPD: comparing generic and disease-specific instruments with focus on comorbidities

Background: Chronic Obstructive Pulmonary Disease (COPD) influences different aspects of patient's health-related quality of life (HRQL). While disease-specific HRQL instruments focus on symptoms and functional impairments, generic instruments cover a broader view on health. This study compares the generic EQ-5D-3 L and two disease-specific questionnaires (St.-George's Respiratory Questionnaire (SGRQ-C), COPD Assessment Test (CAT)) in a comprehensive spectrum of COPD disease grades with particular attention on comorbidities and assesses the discriminative abilities of these instruments. Methods: Using data from the baseline visit of the German COPD cohort COSYCONET, mean HRQL scores in different COPD grades were compared by linear regression models adjusting for age, sex, education, smoking status, BMI, and low vs. high number of comorbidities or a list of several self-reported comorbid conditions. Discriminative abilities of HRQL instruments to differentiate between COPD grades were assessed by standardized mean differences. Results: In 2,291 subjects in COPD GOLD grades 1-4 EQ-5D-3 L utility, EQ-5D VAS, SGRQ, and CAT were found able to discriminate between COPD grades, with some limitations for the EQ-5D utility in mild disease. Both generic and disease-specific HRQL instruments reflected the burden of comorbid conditions. The SGRQ showed the best discrimination between COPD grades and was less influenced by comorbidities, while EQ-5D utility put a higher weight on comorbid conditions. For all instruments, psychiatric disorders and peripheral artery disease showed the strongest negative associations with HRQL. Conclusion: All HRQL instruments considered reflect considerable impairment of HRQL in COPD patients, worsening with increasing COPD grade and number of comorbidities. Findings may support clinical assessment, choice of HRQL instrument in future studies, and parameterization of decision-analytic models

A Robust Quality Infrastructure Is Key to Safe and Effective Delivery of Immune Effector Cells: How Fact-Finding Can Help

Immune effector cells (IECs) include a broad range of immune cells capable of modulating several disease states, including malignant and nonmalignant conditions. The growth in the use of IECs as both investigational and commercially available products requires medical institutions to develop workflows/processes to safely implement and deliver transformative therapy. Adding to the complexity of this therapy are the variety of targets, diseases, sources, and unique toxicities that a patient experiences following IEC therapy. For over 25 years, the Foundation for the Accreditation of Cellular Therapy (FACT) has established a standard for the use of cellular therapy, initially with hematopoietic cell transplantation (HCT), and more recently, with the development of standards to encompass IEC products such as chimeric antigen receptor (CAR)-T cells. To date, IEC therapy has challenged the bandwidth and infrastructure of the institutions offering this therapy. To address these challenges, FACT has established a programmatic framework to improve the delivery of IEC therapy. In this study, we outline the current state of IEC program development, accreditation, and solutions to the challenges that programs face as they expand their application to novel IEC therapy