Precision Medicine

the approximately three million people in the United States who present with a lung nodule on CT each year, about 200,000 will have lung cancer; the rest will have benign lung nodules. A test result indicative of a high probability of being benign allowed patients to enter a period of watchful wait- ing without the need for more invasive diagnostic studies or biopsy. Indi was acquired by Biodesix in 201824. More recently, German-based Uroquant is developing a diagnostic and prognostic assay for bladder cancer that relies upon quantitative MS analysis of biomarkers from urine samples25.

Mining biomarkers in the modified proteome Although techniques for the large-scale assay of proteomic biomarkers have largely failed to trans- late to the clinic and the proteomic biomarkers analysed via standard methodologies that have been integrated into healthcare practices arguably offer minimal diagnostic value20, the promise of clinical proteomics may be realised given increas- ing evidence for the role of the modified proteome in disease. Post-translational modification (PTM) of pro-

teins offers a rapid, elegant and energetically effi- cient method to modulate and maximise the func- tionality of a single gene product, which may occur in a reversible manner. Throughout biology, PTMs are essential to regulate fundamental cellular pro- cesses. Every human protein has the potential to be post-translationally modified at least once during its life span and these cellular homeostatic switches represent critical and sensitive nodes for pathogen- esis, both as drivers and markers. Aberrant PTMs have been associated with many diseases, ranging from the development of cancer to autoimmune disease. One identified reason underlying the fail- ure of immuno-oncological strategies to deliver across large patient populations is due to the vari- ation exhibited in individual patients’ modified protein signatures26,27. Indeed, recent research has demonstrated that glycosylation, phosphorylation, ubiquitination, sumoylation and acetylation of PD- L1 all play a role in the stability of this tumour immune checkpoint ligand, with aberrant modifi- cations modulating PD-L1 mediated immune resis- tance28. Consequently, each of these PTMs could act as novel, alternative biomarkers for diagnostics or as novel drug development targets. Each protein sequence may have many different

modifiable amino acids, but not all will be modi- fied at the same time or within the same copy of the protein. Even for a specific residue competi- tion may exist between different modifications,

Drug Discovery World Winter 2019/20

between acetylation and ubiquitination of lysine residues or between O-phosphorylation and O- GlcNac modification (Figure 2). Thus, each pro- tein exists in an equilibrium of modified states, the balance of which can be shifted to drive different biological responses and it is often the state of the whole protein with all modifications included that is recognised by cell signalling systems. The known cancer driver p53 is an example of this whole protein PTM signature, where function and activity depend on the complete PTM pattern operating synergistically to modulate the protein structure, rather than individual PTMs29-31. This regulation via the whole protein signature is important for distinguishing modified protein biomarkers and in consideration of the methods used to detect them. MS has been considered an unrivalled research

tool for detecting and quantifying PTMs and their changes in a broad variety of samples. Consequently, more than 95% of current data on PTMs is derived from MS-based proteomic stud- ies32. Methuselah Health is attempting to exploit MS analyses of PTMs to identify drug discovery biomarkers for the diseases of ageing, such as dia- betes, neurodegeneration and autoimmunity33. However, analysing complete protein PTM pat- terns is a major challenge for MS. Current PTM research is restricted to the identification of pep- tides after proteolytic digestion (bottom-up pro- teomics), meaning information about the full PTM pattern across the protein is lost and often varia- tion in PTMs are difficult to capture using this approach34. MS-analysis of the full PTM comple- ment of the intact protein (top-down proteomics) is unfortunately unlikely to be applicable to clinical samples in the near future, as it is slow, expensive and limited to proteins <60kDa35,36. An alternative method utilised by Biosignatures

to capitalise on the potential of PTM biomarkers within clinical samples is DIGE. This traditional technique offers sensitive analysis of whole protein PTM patterns37,38, while offering the ability to multiplex assays for simultaneous detection of multiple biomarkers from a single patient sample. Using a next generation DIGE platform that deliv-

ers highly reproducible and accurate results37,39- 41, it has identified a biomarker for prostate cancer that it is currently progressing through clinical val- idation trials and have highlighted a number of other potential biomarkers, for diseases such as cancers and Alzheimer’s disease, that may have failed to translate in previous studies due to alter- nate analytical approaches. While focus in pro- teomics has centred around MS, the potential to


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