Development of mass spectrometric approaches to advance drug discovery

PhD ThesisThe work in this thesis concerns the development of advanced mass spectrometric techniques to study model systems for drug discovery and accelerate the process of high-throughput screening (HTS) in the field of inflammation. This has primarily been achieved using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) to develop screening assays for anti-inflammatory compounds. MALDI-TOF-MS utilises a laser to ablate and consequently ionise biomolecules that are co-crystallised with a matrix. The absorbed laser energy enables the transfer of charge to the analyte molecule from the excited matrix molecules before acceleration into the TOF tube where analytes are then separated based on their mass to charge ratio (m/z) and detected to generate a mass spectrum. With the development of advanced mass spectrometers that have unmatched laser and digitiser speeds these instruments now rival established fluorescent based drug discovery assays. The first study presented in this thesis is the development of a HTS assay to identify inhibitors of salt inducible kinases (SIKs) in vitro. These kinases are an attractive drug target as they mediate the production and secretion of the cytokine interleukin 10 (IL-10) in immune cells. When secreted, IL-10 dampens the pro-inflammatory response in surrounding macrophages and suppresses a further inflammatory response. Using a peptide-based assay, a proof of concept screen generated a positive correlation with an established fluorescent based assay, thus validating MALDI-TOF-MS as a viable alternative for HTS. Interestingly, several compounds were identified as kinase activators. Inhibitors of SIKs could be particularly beneficial for patients with autoimmune diseases who require immunosuppressive drugs as they may increase the production of IL-10, thus preventing excessive inflammation and subsequent onset of pathogenesis. 3 MALDI-TOF-MS was then applied to the development of cellular based assays to screen compounds against cell types that transition into different phenotypes. Rigorous development of a technically reproducible sample preparation technique enabled robust phenotyping of different cell lines. This method was then successfully applied to study mouse embryonic stem cells (mESCs), which transition between naïve and pluripotent phenotypes under pharmacologically controlled conditions. Moreover, a cellular assay to identify inhibitors of inflammation in the presence of a bacterial ligand lipopolysaccharide (LPS) in monocytes was developed. Here, the BCR-ABL inhibitor nilotinib was identified as having anti-inflammatory properties. Interestingly, its first-generation counterpart imatinib was not identified as having this effect, suggesting novel off-target effects for nilotinib. Its anti-inflammatory properties were then validated by cell biology techniques that demonstrated the reduction of the pro-inflammatory cytokine TNF-α in nilotinib-treated cells. Finally, I utilised advanced high-resolution mass spectrometry to undertake secretomic analysis of L929 fibroblasts, which are commonly used to generate supplemented media that aids the differentiation of bone marrow-derived macrophages (BMDMs). These cells are promising drug targets and the understanding of their differentiation state is paramount to potential drug screening campaigns. A detailed proteomic profile of L929 supplemented media, as well an in-depth proteomic profile of BMDMs differentiated under various conditions were generated.Taken together the work of this thesis contributes valuable knowledge in the field of how mass spectrometry can be utilised to advance HTS campaigns as well as provide valuable insight to the biology of promising drug models

HLA-DQA1*05 carriage associated with development of anti-drug antibodies to infliximab and adalimumab in patients with Crohn's Disease

Anti-tumor necrosis factor (anti-TNF) therapies are the most widely used biologic drugs for treating immune-mediated diseases, but repeated administration can induce the formation of anti-drug antibodies. The ability to identify patients at increased risk for development of anti-drug antibodies would facilitate selection of therapy and use of preventative strategies.This article is freely available via Open Access. Click on Publisher URL to access the full-text

A Direct-to-Biology High-Throughput Chemistry Approach to Reactive Fragment Screening

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive covalent fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.</p

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive covalent fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele