Controversial Aspects of Diagnostics and Therapy of Arthritis of the Temporomandibular Joint in Rheumatoid and Juvenile Idiopathic Arthritis: An Analysis of Evidence- and Consensus-Based Recommendations Based on an Interdisciplinary Guideline Project

Introduction: Due to potentially severe sequelae (impaired growth, condylar resorption, and ankylosis) early diagnosis of chronic rheumatic arthritis of the temporomandibular joint (TMJ) and timely onset of therapy are essential. Aim: Owing to very limited evidence the aim of the study was to identify and discuss controversial topics in the guideline development to promote further focused research. Methods: Through a systematic literature search, 394 out of 3771 publications were included in a German interdisciplinary guideline draft. Two workgroups (1: oral and maxillofacial surgery, 2: interdisciplinary) voted on 77 recommendations/statements, in 2 independent anonymized and blinded consensus phases (Delphi process). Results: The voting results were relatively homogenous, except for a greater proportion of abstentions amongst the interdisciplinary group (p < 0.001). Eighty four percent of recommendations/statements were approved in the first round, 89% with strong consensus. Fourteen recommendations/statements (18.2%) required a prolonged consensus phase and further discussion. Discussion: Contrast-enhanced MRI was confirmed as the method of choice for the diagnosis of TMJ arthritis. Intraarticular corticosteroid injection is to be limited to therapy refractory cases and single injection only. In adults, alloplastic joint replacement is preferable to autologous replacement. In children/adolescents, autologous reconstruction may be performed lacking viable alternatives. Alloplastic options are currently still considered experimental

Affinity-based screening techniques: Their impact and benefit to increase the number of high quality leads

The generation of new chemical leads as a starting point for drug development is the first critical step in the drug discovery process after target identification and validation. Since the beginning of 90s high-throughput screening (HTS) has rapidly evolved to one of the main sources for new chemical entities by testing large compound libraries for activity against a target of interest in biochemical in vitro tests using the recombinant protein or cell-based assays. Since the last recent years traditional functional assay read-out technologies using labeled detection reagents are complemented by biophysical methods which directly measure the physical interaction (affinity) between a low molecular weight compound and a target protein in a completely label-free mode. Affinity-based methods offer the opportunities (i) to address non-tractable targets e.g. orphan receptors or functionally inactive protein forms, (ii) to screen extremely large compound files (> 10 mil.) e.g. DNA-encoded libraries and (iii) to characterize HTS hits with an orthogonal assay read out enabling to sort out HTS assay artefacts and to prioritize compounds. This review will focus on the principle, application and impact of selected affinity-based technologies in (i) primary screening for hit identification and (ii) hit validation and qualification to select the most promising chemotypes for further investigation. We will highlight how throughput, robustness and information content of affinity-based methods guide and determine their efficient best value use in the different phases of lead finding

Applications of Biophysics in High Throughput Screening Hit Validation.

For approximately a decade, biophysical methods have been utilized to validate positive hits selected from high throughput screening campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high content information is required in order to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include DLS (Dynamic Light Scattering), Turbidometry, RWG (Resonance Waveguide Grating), SPR (Surface Plasmon Resonance), DSF (Differential Scanning Fluorimetry), MS (Mass Spectrometry), and others. Each technology can provide different types of information of the binding interaction of interest. Thus, these technologies can be incorporated in a hit validation strategy according to the profile of chemical matter that is desired by the medicinal chemists and naturally to the amenability of the target protein to the technology’s screening format. Here we present the results of several screening strategies using biophysics with the objective to compare their approaches, discuss their advantages and challenges, and summarize their benefits in reference to lead discovery

Conformational Changes of Calmodulin on Calcium and Peptide Binding Monitored by Film Bulk Acoustic Resonators

Film bulk acoustic resonators (FBAR) are mass sensitive, label-free biosensors that allow monitoring of the interaction between biomolecules. In this paper we use the FBAR to measure the binding of calcium and the CaMKII peptide to calmodulin. Because the mass of the calcium is too small to be detected, the conformational change caused by the binding process is measured by monitoring the resonant frequency and the motional resistance of the FBAR. The resonant frequency is a measure for the amount of mass coupled to the sensor while the motional resistance is influenced by the viscoelastic properties of the adsorbent. The measured frequency shift during the calcium adsorptions was found to be strongly dependent on the surface concentration of the immobilized calmodulin, which indicates that the measured signal is significantly influenced by the amount of water inside the calmodulin layer. By plotting the measured motional resistance against the frequency shift, a mass adsorption can be distinguished from processes involving measurable conformational changes. With this method three serial processes were identified during the peptide binding. The results show that the FBAR is a promising technology for the label-free measurement of conformational changes.ISSN:2079-637

Applications of Biophysics in High Throughput Screening Hit Validation.

For approximately a decade, biophysical methods have been utilized to validate positive hits selected from high throughput screening campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high content information is required in order to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include DLS (Dynamic Light Scattering), Turbidometry, RWG (Resonance Waveguide Grating), SPR (Surface Plasmon Resonance), DSF (Differential Scanning Fluorimetry), MS (Mass Spectrometry), and others. Each technology can provide different types of information of the binding interaction of interest. Thus, these technologies can be incorporated in a hit validation strategy according to the profile of chemical matter that is desired by the medicinal chemists and naturally to the amenability of the target protein to the technology’s screening format. Here we present the results of several screening strategies using biophysics with the objective to compare their approaches, discuss their advantages and challenges, and summarize their benefits in reference to lead discovery

Non-stoichiometric Inhibition in High-Throughput Screening

Abstract Introduction: Over the last two decades, high-throughput screening (HTS) has become one of the key strategies for the generation of new leads. Non-stoichiometric inhibition is one of the most extensively studied mechanisms responsible for the large percentage of hit compounds from biochemical screens that cannot be developed into leads and therefore, HTS hit lists need to be sorted rapidly and efficiently into stoichiometrically binding inhibitors and compounds that affect enzyme activity non-stoichiometrically. Areas to be covered: In this review, we first explore non-stoichiometric inhibition of enzymatic activity in biochemical screens, particularly by compound aggregation and explain the terminology we use to describe such compound behavior. We then provide a short historical overview of both academic and industrial research on compound aggregation specifically. Finally, we discuss the implications for industrial drug discovery and the measures that can be taken to identify non-stoichiometric and aggregating inhibitors early in this process. Expert Opinion: The most pragmatic approach in a lead finding campaign is to focus on the early identification of selective and stoichiometric inhibitors. The combination of multiple approaches (assessing both activity and binding) allows the enrichment of stoichiometric inhibitors at each stage of the flowchart

Encoded Library Technologies as Integrated Lead Finding Platforms for Drug Discovery

The scope of targets investigated in pharmaceutical research is continuously moving into uncharted territory. Consequently, finding suitable chemical matter with current compound collections is proving increasingly difficult. Encoded library technologies enable the rapid exploration of large chemical space for the identification of ligands for such targets. These binders facilitate drug discovery projects both as tools for target validation, structural elucidation and assay development as well as starting points for medicinal chemistry. Novartis internalized two complementing encoded library platforms to accelerate the initiation of its drug discovery programs. For the identification of low-molecular weight ligands, we apply DNA-encoded libraries. In addition, encoded peptide libraries are employed to identify cyclic peptides. This review discusses how we apply these two platforms in our research and why we consider it beneficial to run both pipelines in-house