Comparison of the modes of action of Apremilast and Roflumilast

The phosphodiesterase 4 (PDE4) family are cAMP specific phosphodiesterases that play an important role in the inflammatory response and is the major PDE type found in inflammatory cells. A significant number of PDE4 specific inhibitors have been developed and are currently being investigated for use as therapeutic agents. Apremilast, a small molecule inhibitor of PDE 4 is in development for chronic inflammatory disorders and has shown promise for the treatment of psoriasis, psoriatic arthritis as well as other inflammatory diseases. It has been found to be safe and well tolerated in humans and in March 2014 it was approved by the US food and drug administration for the treatment of adult patients with active psoriatic arthritis. The only other PDE4 inhibitor on the market is Roflumilast and it is used for treatment of respiratory disease. Roflumilast is approved in the EU for the treatment of COPD and was recently approved in the US for treatment to reduce the risk of COPD exacerbations. Roflumilast is also a selective PDE4 inhibitor, administered as an oral tablet once daily, and is thought to act by increasing cAMP within lung cells. As both (Apremilast and Roflumilast) compounds selectively inhibit PDE4 but are targeted at different diseases, there is a need for a clear understanding of their mechanism of action (MOA). Differences and similarity of MOA should be defined for the purposes of labelling, for communication to the scientific community, physicians, and patients, and for an extension of utility to other diseases and therapeutic areas. In order to obtain a complete comparative picture of the MOA of both inhibitors, additional molecular and cellular biology studies are required to more fully elucidate the signalling mediators downstream of PDE4 inhibition which result in alterations in pro- and anti-inflammatory gene expression. My studies were conducted to directly compare Apremilast with Roflumilast, in order to substantiate the differences observed in the molecular and cellular effects of these compounds, and to search for other possible differentiating effects. Therefore the main aim of this thesis was to utilise cutting-edge biochemical techniques to discover whether Apremilast and Roflumilast work with different modes of action. In the first part of my thesis I used novel genetically encoded FRET based cAMP sensors targeted to different intracellular compartments, in order to monitor cAMP levels within specific microdomains of cells as a consequence of challenge with Apremilast and Roflumilast, which revealed that Apremilast and Roflumilast do regulate different pools of cAMP in cells. In the second part of my thesis I focussed on assessing whether Apremilast and Roflumilast cause differential effects on the PKA phosphorylation state of proteins in cells. I used various biochemical techniques (Western blotting, Substrate kinase arrays and Reverse Phase Protein array and found that Apremilast and Roflumilast do lead to differential PKA substrate phosphorylation. For example I found that Apremilast increases the phosphorylation of Ribosomal Protein S6 at Ser240/244 and Fyn Y530 in the S6 Ribosomal pathway of Rheumatoid Arthritis Synovial fibroblast and HEK293 cells, whereas Roflumilast does not. This data suggests that Apremilast has distinct biological effects from that of Roflumilast and could represent a new therapeutic role for Apremilast in other diseases. In the final part of my thesis, Phage display technology was employed in order to identify any novel binding motifs that associate with PDE4 and to identify sequences that were differentially regulated by the inhibitors in an attempt to find binding motifs that may exist in previously characterised signalling proteins. Petide array technology was then used to confirm binding of specific peptide sequences or motifs. Results showed that Apremilast and Roflumilast can either enhance or decrease the binding of PDE4A4 to specific peptide sequences or motifs that are found in a variety of proteins in the human proteome, most interestingly Ubiquitin-related proteins. The data from this chapter is preliminary but may be used in the discovery of novel binding partners for PDE4 or to provide a new role for PDE inhibition in disease. Therefore the work in this thesis provides a unique snapshot of the complexity of the cAMP signalling system and is the first to directly compare action of the two approved PDE4 inhibitors in a detailed way.

Quantitative measurement using scanning thermal microscopy

This thesis reports on the development of quantitative measurement using micromachined scanning thermal microscopy (SThM) probes. These thermal probes employ a resistive element at their end, which can be used in passive or active modes. With the help of a review of SThM, the current issues and potentials associated with this technique are revealed. As a consequence of this understanding, several experimental and theoretical methods are discussed, which expand our understanding of these probes. The whole thesis can be summarized into three parts, one focusing on the thermal probe, one on probe-sample thermal interactions, and the third on heat transfer within the sample. In the first part, a series of experiments are demonstrated, aimed at characterizing the probe in its electrical and thermal properties, benefiting advanced probe design, and laying a fundamental base for quantifying the temperature of the probe. The second part focuses on two artifacts observed during the thermal scans – one induced by topography and the other by air conduction. Correspondingly, two devices, probing these artifacts, are developed. A topography-free sample, utilizing a pattern transfer technique, minimises topography-related artifacts that limited the reliability of SThM data; a controlled temperature ‘Johnson noise device’, with multiple-heater design, offers a uniform, accurate, temperature distribution. Analyzing results of scan from these samples provides data for studying the thermal interactions within the probe and the tip-sample interface. In the final part, the observation is presented that quantification of measurements depends not only on an accurate measurement tool, but also on a deep understanding of the heat transfer within the sample resulting from the nanoscopic contact. It is believed that work in this thesis contributes to SThM gaining wider application in the scientific community.

Engineering the transition from non-living to living matter

Re-creating and understanding the origin of life represents one of the major challenges facing the scientific community. We will never know exactly how life started on planet Earth, however, we can reconstruct the most likely chemical pathways that could have contributed to the formation of the first living systems. Traditionally, prebiotic chemistry has investigated the formation of modern life’s precursors and their self-organisation under very specific conditions thought to be ‘plausible’. So far, this approach has failed to produce a living system from the bottom-up. In the work presented herein, two different approaches are employed to explore the transition from inanimate to living matter. The development of microfluidic technology during the last decades has changed the way traditional chemical and biological experiments are performed. Microfluidics allows the handling of low volumes of reagents with very precise control. The use of micro-droplets generated within microfluidic devices is of particular interest to the field of Origins of Life and Artificial Life. Whilst many efforts have been made aiming to construct cell-like compartments from modern biological constituents, these are usually very difficult to handle. However, microdroplets can be easily generated and manipulated at kHz rates, making it suitable for high-throughput experimentation and analysis of compartmentalised chemical reactions. Therefore, we decided to develop a microfluidic device capable of manipulating microdroplets in such a way that they could be efficiently mixed, split and sorted within iterative cycles. Since no microfluidic technology had been developed before in the Cronin Group, the first chapter of this thesis describes the soft lithographic methods and techniques developed to fabricate microfluidic devices. Also, special attention is placed on the generation of water-in-oil microdroplets, and the subsequent modules required for the manipulation of the droplets such as: droplet fusers, splitters, sorters and single/multi-layer micromechanical valves. Whilst the first part of this thesis describes the development of a microfluidic platform to assist chemical evolution, finding a compatible set of chemical building blocks capable of reacting to form complex molecules with endowed replicating or catalytic activity was challenging. Abstract 10 Hence, the second part of this thesis focuses on potential chemistry that will ultimately possess the properties mentioned above. A special focus is placed on the formation of peptide bonds from unactivated amino acids, despite being one of the greatest challenges in prebiotic chemistry. As opposed to classic prebiotic experiments, in which a specific set of conditions is studied to fit a particular hypothesis, we took a different approach: we explored the effects of several parameters at once on a model polymerisation reaction, without constraints on hypotheses on the nature of optimum conditions or plausibility. This was facilitated by development of a new high-throughput automated platform, allowing the exploration of a much larger number of parameters. This led us to discover that peptide bond formation is less challenging than previously imagined. Having established the right set of conditions under which peptide bond formation was enhanced, we then explored the co-oligomerisation between different amino acids, aiming for the formation of heteropeptides with different structure or function. Finally, we studied the effect of various environmental conditions (rate of evaporation, presence of salts or minerals) in the final product distribution of our oligomeric products.