Prca 201800087

www.clinical.proteomics-journal.
com Page 1 Proteomics - Clinical Applications
Proteomics in drug development: the dawn of a new era?
Maria Frantzi1*, Agnieszka Latosinska1* and Harald Mischak1,2
1
Mosaiques Diagnostics GmbH, Hannover, Germany
2
BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United
Kingdom
*equally contributing first authors
Corresponding Author
Prof. Harald Mischak
Mosaiques Diagnostics GmbH
Rotenburger Straße 20
D-30659 Hannover, Germany
Email: mischak@mosaiques-diagnostics.com
Phone: +49 (0)511 55 47 44 13
Fax: +49 (0)511 55 47 44 31
Keywords: Drug discovery, Mass Spectrometry, Proteomics
Total number of words: 7.896 (10.288 incl. references and figure legends)
List of Abbreviations (excl. standard abbreviations):
ABPP- activity-based protein profiling; ALK- anaplastic lymphoma kinase; ANGPTL2-
Angiopoietin-related protein 2; ANXA- annexin A BI-TK/GCV- Bifidobacterium infantis
thymidine kinase/nucleoside analogue ganciclovir; BTK- Bruton’s tyrosine kinase; CALU-
Calumenin; CETSA- Cellular thermal shift assay; CDH18- Cadherin-18; CDK- cyclin-
dependent kinases; CMTM6- CKLF-like MARVEL transmembrane domain containing protein
6; CNPY3- Protein canopy homolog 3; CPSN1- calpain small subunit 1; CRSwNP- Chronic
Received: 10 18, 2018; Revised: 01 13, 2019; Accepted: 02 04, 2019
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/prca.201800087.
This article is protected by copyright. All rights reserved.

www.clinical.proteomics-journal.com Page 2 Proteomics - Clinical Applications
rhinosinusitis with nasal polyps; EGFR- Epidermal growth factor receptor; EIF3D- Eukaryotic
translation initiation factor 3 subunit D; EIF5A- Eukaryotic translation initiation factor 5A;
EPHA2- Ephrin type-A receptor 2; ESCC- Esophageal squamous cell carcinoma; FGB-
fibrinogen B; FN1- Fibronectin 1; HSP- Heat shock protein; ICAM1- Adhesion Molecule 1;
IL1RAP- Interleukin 1 Receptor Accessory Protein; IL13RA2- Interleukin-13 receptor subunit
alpha-2; IL18- Interleukin 18; ITD- Internal tandem duplication; ITGB8- Integrin Subunit Beta
8; KRT17- Keratin 17; LGALS3BP- Galectin-3-binding protein; MCAM- Cell surface
glycoprotein MUC18; MCM2- minichromosome maintenance protein 2; MFAP5-
Microfibrillar-associated protein 5; MIBC- muscle invasive bladder cancer; NAP1L1-
Nucleosome assembly protein 1-like 1; NID-1- Nidogen-1; NKCC1- Sodium-potassium-
chloride cotransporter 1; NMIBC- non-muscle invasive bladder cancer; NSCLC- Non–small
cell lung cancer; PAL- photoaffinity labeling; PD-1- Programmed cell death protein 1;
PDLIM2- PDZ and LIM domain protein 2; PGRMC1- Progesterone receptor membrane
component 1; PHAP1- Putative HLA-DR-associated protein 1; PHB- prohibitin; PKM2 -
Pyruvate kinase M2; Plk1- Polo-like kinase 1; POSTN- Periostin; Prx- Peroxiredoxin;
PSMA3- proteasome subunit a3; PTPRZ1- Receptor-type tyrosine-protein phosphatase
zeta; TKI- tyrosine kinase inhibitors; RanBP1- GTPase RAN binding protein 1; RIOK-
Serine/threonine-protein kinase RIO1; S100A2- S100 calcium binding protein A2; SAFB-
Scaffold attachment factor B; SAP- Signalling lymphocytic activation molecule associated
protein; STMN1-Stathmin; TAGLN2- Transgelin-2; TGF‐β1- Transforming growth factor beta
1; TGF‐β3- Transforming growth factor beta 1; UBA5- Ubiquitin-like modifier activating
enzyme 5; UQCRC2- Cytochrome b-c1 complex subunit 2, mitochondrial
Abstract
Mass spectrometry offers the potential of acquiring high resolution data depicting the
functional status of a group of healthy or disease individuals, according to the different
conditions. As most of the drugs are currently targeting proteins, proteomics has a dual
value, both in the discovery of new molecules as therapeutic targets, but also as a

2
methodology to perform high throughput drug profiling. As there is an evident need for drugs
to be improved in terms of efficacy, a mechanistic insight for downstream effectors can be
valuable in order to predict side effects and resistance mechanisms. Recently developed
assays, like thermal proteome profiling enables comprehensive drug target profiling and is,
therefore, of high value in drug discovery. In this review, we have conducted a systematic
literature search and present the most prominent proteomics studies as implicated in
assisting drug discovery and development. Focus is placed on investigations that are closer
to implementation, therefore particular emphasis is given in studies conducted in human
diseased population and further verified in vitro or in vivo.
A. Concept of improving drug discovery and development by proteomics
Proteomic methodologies have proven to be valuable tools to provide novel insights into
disease processes. Proteins are the principal targets of drug discovery[1] and as such,
proteomics is a key technology to support development of specific drugs to stop or reverse
effects of the disease.[2]
In principle, proteomic analysis by mass spectrometry is based on the identification of
ionised compounds in a sample mixture by recording their mass-to-charge ratio.[3] The
relative abundance of the ionised compounds is measured and information on the amino
acid composition of the compounds is acquired, mainly via tandem MS or peptide mass
fingerprinting. Hence, depending on the focus, proteomics analysis can provide insights not
only for the peptide sequence, but also quantification of protein abundance, post
translational modifications (PTMs) and protein interactions. Compared to genome, which is
constant and depicts more the potential of translation, proteome is highly dynamic,
fundamentally the result of the translation process and its regulation. As a result, proteomics
represents a functional snapshot of the cell, in a much more dynamic perspective. Yet,
considering the complexity of the proteome,[4] interpretation of proteomics data is certainly

3
more challenging (for example, the definition of ion signal threshold over instrument noise is
crucial).[5] Moreover, the analytical sensitivity is lower, mainly because of the inability to
amplify the signal as in the case of nucleic acid amplification.[6] Still, proteomics has a great
potential in drug discovery, as can reflect protein activity, structure and kinetics.
Drug development is a long and costly process, frequently hampered by the fact that the
developed drugs have low response rates due to resistance mechanisms and/or
downstream pathways that are subsequently altered. Mass spectrometry techniques have
been advanced by now and are combined with new assays (e.g. thermal shift assay for drug
screening)[7, 8]
that enable high-throughput profiling. Due to the high-throughput potential,
proteins can be measured simultaneously in complex samples, in an unbiased, untargeted
manner. The approach finds applicability in practically every step of the drug development,
covering from drug target discovery to the clinical trial phases, with common applications of
proteomics in the pharmaceutical industry, which can be categorised to: a) discovery of
potential drug targets, b) investigations into mechanisms of drug action and protein-protein
interactions, and c) biomarkers from readily accessible biological fluids (Figure 1).
The challenge in the drug discovery process is to identify the underlying causes of disease
and to reverse them to a normal condition. Therefore, gaining a holistic understanding of
disease’s underlying pathology at the molecular level is essential for moving towards
personalised medicine. While the pathophysiology and causes of many diseases, like cancer
and other chronic conditions vary greatly in their nature and origin, in some cases, the cause
is found at the protein level, involving protein function, protein regulation, or protein-protein
interactions. Discovery investigations involve the identification of proteins whose expression
levels or activities change in disease state. In parallel, profiling of disease phenotype and
correlation with the drug mechanism of action and/or potential pathways controlling
resistance, provides with a mechanistic link which can be further explored towards
identification of systemic biomarkers.[9] As a result of our increased understanding at the
molecular level proteomics biomarkers have been additionally introduced to stratify the

4
patients for receiving an intervention and predict response- a topic which is as widely
covered in recent reviews.[10, 11]
B. New technological advancements
Latest technological breakthroughs have revolutionized drug development, particularly
regarding screening of ligands and/or inhibitors. Thermal proteome profiling has been
recently introduced,[12-15] as an evolution to the traditional fluorometric/ calorimetric thermal
shift assay, that now, when combined with mass spectrometry, can provide insights on
drug’s mechanism of action through comprehensive screening in living cells.[16] The
conventional thermal shift assay is based on the investigation of protein stabilization when
interacting with other biomolecules like drugs, DNA and co-factors. Changes in thermal
denaturation temperature of a protein under different conditions is representative of protein-
ligand interactions, thus appropriate for high throughput screening of small molecules/
inhibitors to develop ligand libraries.[16] On this basis, thermal proteome profiling assays were
further introduced in seminal studies, reporting new technologies like cellular thermal shift
assay (CETSA), activity-based protein profiling (ABPP),[17] photoaffinity labeling (PAL),[18]
and are comparatively summarized in recent reviews.[16, 19] Both PAL and ABPP assays are
based on labeling of proteins- in PAL assay through photoaffinity labeling with the ligand to
be covalently linked through UV irradiation[18] and in ABPP methodology through biotinylated
probes and affinity enriched on streptavidin beads.[17] Both approaches are subsequently
linked to downstream MS analysis and can provide insights in the structural proteome (in
case of PAL assay)[18] and enzyme functionality (ABPP assay).[17] Probably, the most recent
greatest breakthrough technology, however, is cellular thermal shift assay (CETSA),[7] as
when coupled with MS, enables untargeted label-free proteomics profiling to monitor drug/
target interactions in living cells. After its first introduction in 2013,[7] several reports have
made use of the technology in cell lines to monitor kinases inhibitors,[13] transmembrane cell

5
surface proteins[12] and even on- and off-target effect of chemotherapeutic agents, like
Methotrexate.[20] The potential of such applications is of high impact in the years to come.
C. From proteomics to personalized medicine: Literature review
Because of the wide area of applications, in this article, we focus on the application of
proteomics, as directly applied for discovery of novel targets and the use of mass
spectrometry (MS) techniques in the profiling of drug effects or mechanisms of action and
downstream effectors. For this purpose, a literature search was conducted using the Web of
Science database (22/08/18), as follows: TOPIC: "drug discover*" OR "drug target" OR
"therapeutic target" AND TOPIC: proteom*. Only manuscripts defined as “ARTICLE” OR
“REVIEW” types published within the last 5 years (2014 -2018) were considered. A total of
1625 papers were retrieved (excluding replicates) and those were further screened
independently by two co-authors (Supplementary Table 1). The manuscripts were further
shortlisted by including only research articles employing mass spectrometry platforms. For
the discovery studies, we considered only those, where association of proteomics results
was further verified in vitro and/or in vivo. Due to the broad spectrum of the field, in this
article we focused on chronic diseases. Therefore, biomarker studies, any investigations in
infectious diseases as well as technical papers on method development were excluded.
Forty-nine studies on chronic diseases were selected and are described in this review,
separated in the four following categories, according to the context of research: 1) discovery
studies for identification of potential drug targets and downstream pathways, 2) profiling
studies for drug effects on proteome, 3) chemical proteomic studies for drug selectivity and
inhibitor binding and 4) structural proteomics studies.
D. Discovery of potential drug targets
i. Tissue proteomics for drug discovery in oncology

6
Cancer includes a group of malignancies that presents high heterogeneity.[21] Cancer at its
advanced stage is still lethal mainly because of the variability that is observed among the
patients, resulting in a very low success rate for the proposed drugs.[21] Nevertheless, one
advantage compared to other chronic diseases, particularly for drug research is the
availability of tissue specimens, which can be rich in information. In this context, mass
spectrometry has been employed for large scale profiling to improve on the discovery, but
also in characterization of drugs’ action.[22] Several cancer discovery studies involving high
resolution proteomics, in which patient-derived tumour specimens were investigated, were
retrieved and are described below.
As many therapeutic targets for high grade glioma include immunotoxins targeting surface
sialylated glycoproteins, Autelitano et al.[23] have attempted to profile sialo-glycoproteins
implicated in glioma, through metabolic labeling with Ac4ManNAz and biorthogonal chemical
reporting with biotin-linked phopshine to bind the labelled glycans.[23] The enrichment was
followed by label-free MS to identify potential drug targets in human tissues from four cancer
patients. Although the number of patients was low, the findings were compared with fetal
astrocytes and human neural progenitor cells and additional verification was performed by
immunohistochemical staining (IHC).[23] Fifty-two proteins were classified as glioblastoma-
specific, including among others Angiopoietin-related protein 2 (ANGPTL2), Intercellular
Adhesion Molecule 1 (ICAM1), Interleukin 1 Receptor Accessory Protein (IL1RAP), IL13RA2
(Interleukin-13 receptor subunit alpha-2), Integrin Subunit Beta 8 (ITGB8), Galectin-3-binding
protein (LGALS3BP), Receptor-type tyrosine-protein phosphatase zeta (PTPRZ1). The idea
of employing an enrichment protocol to target glycoproteins is of particular interest,
considering that alterations of glycosylation are linked to tumour development, progression
as well as formation of metastasis.[24] However, these results are still at a preliminary phase,
as did not yet reach pre-clinical testing. Similarly, Collet et al.[25] analysed 23 primary cell
lines from 16 glioma patients, for which treatment and follow-up information was available.
As primary glioma cultures present different phenotypes (either adherent or as

7
neurospheres), the aim of this study was to profile both and identify common glioma specific
proteins as potential therapeutic targets.[25] Proteomic analysis by nano-LC/Q-TOF MS, was
performed in 11 adherent and 12 neurosphere- cell lines. 342 proteins indicative of cell cycle
processes were specifically identified in neurosphere lines, while 112 proteins indicative of
cell adhesion were solely found in adherent cultures. This is an interesting observation
considering the different adherent properties of the two types of cell lines. Forty-nine proteins
were significantly correlated with patients’ survival, among those, Calumenin (CALU),
Eukaryotic translation initiation factor 5A (EIF5A) and Transgelin-2 (TAGLN2) with increased
expression, while Nucleosome assembly protein 1-like 1 (NAP1L1) with decreased.[25] As in
the previous evaluation, the above proteins need to be further tested in vivo. Similarly, in a
study for proteomics characterization of melanoma,[26] isobaric tags for relative and absolute
quantitation (iTRAQ) was applied in melanoma tissues and matched non-malignant tissue.
Thirty differentially expressed proteins with more than 15‐fold were identified, while 67 were
decreased with more than 0.25‐fold. Among those, Periostin (POSTN) was highly
upregulated and selected to be further evaluated in melanoma cell lines. Co-cultures of
melanoma with human dermal fibroblasts revealed that POSTN expression was induced in
normal cells through induced cytokines, particularly Transforming growth factor beta 1 (TGF‐
β1) and TGF‐β3.[26]
Along these lines, Latosinska et al.[27] performed a comparative proteomic analysis of muscle
invasive (MIBC, n=6) and non-muscle invasive bladder cancer (NMIBC, n=5) tissue samples
using LC-MS/MS. Interestingly, the proteomic profiles were integrated in a pathway and
interactome analysis, which revealed involvement and functional significance of eukaryotic
translation initiation factor 3 subunit D (EIF3D) in MIBC. The validity of the EIF3D as a
potential target was further investigated by silencing of EIF3D which decreased cell
proliferation, colony formation as well as migration capacity in vitro. Additionally, knock-down
of EIF3D decreased tumor growth in xenograft models, highlighting this study as a pure
proteomics-driven investigation of a potential drug target that reached pre-clinical testing in

8
animal models.[27] In another bladder cancer investigation, iTRAQ-based quantitative
proteomics analysis on tissue samples and adjacent non-malignant tissue was conducted by
[28]
Zhang et al. Thirty five proteins were significantly altered (p<0.05), with up-regulation of
Scaffold attachment factor B (SAFB) and GTPase RAN binding protein 1 (RanBP1) further
confirmed by IHC. However, further assessment of these findings in disease models is
required. Additionally, Zha et al.[29] applied iTRAQ labeling coupled to MS analysis for the
comparative assessment of 15 paired cancer and adjacent non-cancerous tissue from
patients with laryngeal carcinoma, revealing 100 differentially expressed proteins. Among
them, S100 calcium binding protein A2 (S100A2), Keratin 17 (KRT17), Fibrinogen B (FGB),
Fibronectin 1 (FN1) and POSTN1 were verified by IHC in 60 laryngeal carcinoma and 20
non-malignant tissues. Although the identified proteins seem to be more related to
inflammation, interference in vitro assays for S100A2, suggested this protein as potential
therapeutic target.[29]
In another study, investigating osteosarcoma, Hypoxia-induced E3 ubiquitin ligase subunit
(WSB1) was highly expressed in tumor tissues, associated with progression and
metastasis, as revealed by IHC analysis in 40 primary osteosarcoma tissues.[30] The invasive
and metastatic potential increased in animal models, where WSB1 was overexpressed to
induce hypoxia. Downstream effectors were investigated by stable isotope labeling with
amino acids in cell culture (SILAC)/ MS. A total of 1,078 proteins were significantly altered
upon WSB1 overexpression (560 upregulated and 518 downregulated; 3.0-fold).[30] RhoGDI2
(inhibitor of Rho proteins) was identified downstream of WSB1 signalling, in decreased
levels. A negative correlation between WSB1 and RhoGDI2 was also confirmed in the
patients’ tissue, suggesting RhoGDI2 as downstream target of WSB1.[30] Zhang et al.[31] also
applied SILAC-based quantitative proteomic approach to investigate changes in human
renal carcinoma tissues and autologous para-cancerous kidney tissues. Eighty-two proteins
were defined as differentially regulated (>1.34 or <0.66-fold), including proteins previously
described in the context of renal cell carcinoma (e.g. nexin family proteins, vimentin, heat

9
shock proteins) and novel findings. Progesterone receptor membrane components 1
(PGRMC1), of which upregulation was reported in other types of cancer, was further verified
by immunohistochemistry (p<0.05, n=135 paired tissues). Its overexpression significantly
enhanced proliferation of renal cancer cells (p<0.05). Opposite effect was observed after
PGRMC1 silencing.
As evident by the retrieved studies in human cancerous specimens, patient excised tissue is
a valuable source for identification of a large number of cancer-associated proteins, several
of those can be positively verified either by IHC and/or correlated with disease outcome, and
of which functional relevance in cancer is supported by in vitro investigation. Yet, none of the
studies, except for the study by Latosinska et al. has introduced a proteomics- driven target
that has been initially investigated in a pre-clinical setting including animal models.
ii. Cell line proteomics for drug discovery in oncology
A considerable number of studies involve cell-line based investigations to identify potential
drug targets, of which validity/ relevance was supported by in vitro/ in vivo experiments. In
the study by Hue et al.[32] SILAC was employed to assess changes between HCT116 cell
line and its highly invasive subline (HCT116-I8). 3,161 proteins were commonly identified in
three experiments (FDR of 1%, ≥2 peptides)[32] including 128 differentially abundant proteins
(fold ≥1.5).[32] Cdc42BPA (up-regulated, downstream regulator of Cdc42 signalling pathway)
was selected for subsequent analysis in vitro and in human samples. Knock-down of
Cdc42BPA in two highly invasive cell lines resulted in a reduction of invasive potential. IHC
analysis of Cdc42BPA (n=100 primary colon cancer tissues and n=80 adjacent normal
tissues) revealed stronger staining in tumor vs adjacent normal tissues. High Cdc42BPA
expression was associated with lymph node (p=0.002) and distant metastasis (p=0.026), as
well as shorter survival time (p<0.05). Nevertheless, further in vivo experiments are required
to demonstrate its therapeutic potential. In another study, Zhou et al.[33] analysed by SILAC
the membrane proteome of hepatocellular carcinoma cell lines that differs in metastatic

10
potential. A total of 1,181 proteins were quantified, including 190 differentially abundant
proteins. From the latter, 90 proteins were annotated to plasma membrane. Subsequent
investigation was focused on sodium-potassium-chloride cotransporter 1 (NKCC1), of which
upregulation in cell lines with higher metastatic potential was confirmed by Western blot.
Immunohistochemistry analysis (n=67) showed that NKCC1 is significantly associated
(p<0.05) with tumor differentiation and microvascular invasion. In this study both in vitro and
in vivo experiments were conducted, demonstrating reduction of cell proliferation and
invasion after NKCC1 knockdown, while an opposite effect was observed after NKCC1 over-
expression.[33]
Bassiri et al.[34] aimed at identification of novel drug targets for Merlin-deficient meningioma
and schwannoma through parallel analysis of proteome and phosphoproteome in
schwannoma/ Schwann cells and meningioma/ Meningeal cells. This is a particularly
interesting approach, considering that PTMs play a major role in protein functionality, and
phosphorylation is one of the most intensively studied modification.[35] Importantly, aberrant
phosphorylation status have been implicated in cancer, and therapeutics targeting
phosphorylation pathways are emerging in drug discovery.[35] Upon characterisation of
individual types of cancer, the analysis was focused on 11 commonly deregulated
phosphoproteins between schwannoma and meningioma. Among those, overexpression of
PDZ and LIM domain protein 2 (PDLIM2) was validated through western blot in tissue and
cell lines from both cancers. Knock-down of PDLIM2 significantly reduced cell proliferation in
both meningioma and schwannoma cells, although no further in vivo experiments were
performed.
In a quite different setting, Zheng et al.[36] performed two-dimensional gel electrophoresis
followed by MS to investigate differences in the proteome of squamous cell carcinoma of
tongue cell lines (SCC-15) with acquired resistance to pingyangmycin in comparison to
parental cell lines. A total of 19 differential protein spots were identified (>2-fold change;
triplicate experiments). Western blot and ELISA confirmed significant increase of HSP27 in

11
cell extract and secretome of resistant cell lines in comparison to other SCCT cell lines,
respectively. Knock-down of HSP27 and treatment with anti-HSP27 antibody were able to
reverse the resistance. Further evaluation of the expression of HSP27 in tumor tissues in
two independent cohorts of patients with squamous cell carcinoma of tongue who received
pingyangmycin and/ or chemotherapy (cohort 1, n=84; cohort 2, n=81) revealed that high
level of HSP27 was associated with significantly shorter overall survival (p=0.0081 for cohort
1, and p=0.0024 for cohort 2).
From the above published reports, it is evident that proteomics has been extensively applied
in cell lines to investigate potential oncological targets. Even though, cell lines might not fully
reflect disease phenotype and also interactions of cancer cells with extracellular matrix, it is
frequently applied as it easily available, technically less challenging and better suited for
investigating secreted proteins.
iii. Cell line proteomics for discovery of downstream effectors
Apart from the application of proteomics for untargeted discovery of drug targets, global
analysis of the proteome was utilised to better understand and characterise protein partners/
effectors downstream of the targeted protein. The concept is of high interest, when
considering that downstream effectors can affect drug efficiency. Numerous studies take
advantage of MS (in many cases iTRAQ technology is applied) to profile downstream effects
of targets, targeting membrane proteins, known kinases and promising targets downstream
of the recently applied immunotherapy (PD1/ PDL1 axis).
In the study by Xie et al.[37] focusing on assessment of Putative HLA-DR-associated protein 1
(PHAP1) in glioma, immuno-based assays (n=30 human glioma tissues and n=12
specimens of non-tumour brain tissues) revealed significant increase of PHAP1 abundance
in tumoral vs non-tumoral tissue. In vitro investigation demonstrated that knock-down of
PHAP1 inhibited proliferation of glioma cells, while its overexpression had an opposite effect.
To investigate how PHAP1 regulates cell proliferation, U251 and U87 cells carrying PHAP1

12
knock-down and control cells were analysed using iTRAQ. 57 and 71 differentially abundant
proteins were identified through analysis of U251 and U87 cells, respectively; while 15
proteins were overlapping between two analyses. Western blot analysis confirmed that by
PHAP1 knock-down Stathmin (STMN1) was significant decreased. Further investigation of
p27- stathmin inhibitor, and its upstream regulator - phosphorylated Akt (S473) supported
the hypothesis that PHAP1 promotes proliferation of glioma cells through regulation of the
Akt/p27/stathmin pathway.
Along these lines, Programmed cell death protein 1 (PD-1) / ligand (PD-L1) inhibitors have
emerged as a promising strategy to treat various cancers. Considering the low response rate
to therapies targeting these proteins, there is an unmet need to better understand regulation
of its expression at the molecular level. Towards that end, Burr et al.[38] conducted a
genome-wide CRISPR-Cas9 screen in pancreatic cancer cell line to identify regulators of
PD-L1. Analysis resulted in identification of CKLF-like MARVEL transmembrane domain
containing protein 6 (CMTM6), of which depletion in resulted in reduction of PD-L1 level. Co-
immunoprecipitation experiments supported interaction between PD-L1 and CMTM6.
Tandem mass tag quantitative proteomics approach was applied to investigate membrane
proteome of wild type and CMTM6 depleted breast cancer cells. 1424 membrane proteins
were identified, and >2- fold decrease in PD-L1 abundance was observed. Further
experiments revealed that depletion of CMTM6 co-localises with PD-L1 at the plasma
membrane and prevents it from degradation in lysosomes. Moreover, in vitro and in vivo
experiments supported that CMTM6 acts as a regulator of T-lymphocyte-mediated anti-
tumour immunity.
In another study, Bai et al.[39] investigated the role of cadherin-18 (CADH18) in glioma.
Decrease in cadherin-18 from normal to stage IV gliomas was supported by publicly
available data and immunohistochemistry (n=453). Overexpression of CADH18 in glioma
cell lines (U87, U251) significantly decreased cell migration/ invasion (p<0.0001) in
comparison to controls. Overexpressing CADH18 in nude mice exhibited a slower rate of

13
growth and smaller volume. iTRAQ analysis resulted in identification of 201 and 331
differentially expressed proteins (p≤0.05, ≥1.5-fold change). Cytochrome b-c1 complex
subunit 2, mitochondrial (UQCRC2) was selected as a potential downstream target of
CADH18. Down-regulation of UQCRC2 in CADH18 overexpressed glioma cell lines, partly
reversed the inhibition of invasion/migration ability and chemoresistance. [39] Xu et al.[40]
investigated the impact of human antigen R on the progression of esophageal squamous cell
carcinoma (ESCC). Human antigen R showed to be overexpressed in tumor tissues (n=112)
in comparison to the matched adjacent tissues (n=52) using IHC. Knock-down of human
antigen R decreased proliferation and clonogenicity of ESCC cell lines (TE-1 and Eca-109),
along with a significant reduction of cell invasion and migration. Tandem mass tags- based
proteomics allowed for identification of 100 up-regulated and 122 down-regulated proteins
after human antigen R knock-down. Interleukin-18 (IL18) was identified among the up-
regulated proteins. Treatment of ESCC cells with IL18 led to inhibition of cell proliferation
and metastasis; while in vitro luciferase assay revealed that 3'-UTR of IL18 have a binding
site for human antigen R. IHC supported significant decrease of IL18 in ESCC vs tumor-
adjacent tissues.
Cheung et al.[41] investigated functional network of minichromosome maintenance protein 2
(MCM2) in lung cancer cell lines. Phosphoproteomics analysis resulted in identification of
215 and 107 phosphosites that were significantly regulated (>1.5-fold change) in response to
MCM2 overexpression and silencing, respectively; while proteomics analysis of MCM2
silenced cells led to identification of 46 differentially abundant proteins in comparison to
controls. Moreover, phosphoproteomics analysis revealed that MCM2 promotes cell
proliferation via regulation of high mobility group protein HMG-I/HMG-Y phosphorylation and
the impact of phosphorylation of HMGA1 was supported by Western blot and in vitro
[42]
experiments. Weinberg et al. focused on the characterisation of RIO kinases (RIOK) in
cancer. In vitro studies of isogenic colon-, breast- and lung cancer cell lines revealed that
silencing of RIOK1 inhibited cancer cells proliferation and invasiveness in 2D and 3D culture

14
system. To further investigate the protein network controlled by RIOK1, proteome of NCI-
H1299 cells expressing and depleted in RIOK1 was analysed using SILAC. Depletion of
RIOK1 affected expression and phosphorylation of proteins involved in multiple biological
processes including ribosomal biogenesis, cell cycle progression, metabolic activity, NF-κB
signaling and metastases. Moreover, in vivo studies demonstrated that mice inoculated with
NCI-H1299 cells expressing RIOK1 exhibited colony formation in lungs, which was not
observed after inoculation of cells depleted in RIOK1. IHC analysis of lung (n=30) and breast
cancer (n=22) tissue samples supported differential expression of RIOK1.
[43]
Li et al. employed immunoprecipitation combined with MS to investigate mechanisms of
anaplastic lymphoma kinase (ALK) signalling in neuroblastoma. Analysis of Ba/F3 cells
expressing FLAG-tagged wildtype ALK (ALKWT-FLAG) and neuroblastoma-associated
F1174L ALK (ALKF1174L-FLAG) resulted in identification of tyrosine kinase Bruton’s tyrosine
kinase (BTK) as a novel ALK interaction partner, further confirmed by co-
immunoprecipitation. BTK expression was detected in both neuroblastoma cell lines and
tumour tissue samples (n=4). Importantly, treatment of neuroblastoma cell lines with BTK
inhibitor (ibrutinib) inhibited cell proliferation, and growth of neuroblastoma xenograft in nude
mice; whereas combination of ibrutinib with ALK inhibitor (crizotinib) further enhance the
inhibitory effect.
In another study, the interaction partners of 14-3-3ζ, a known protein for its overexpression
in non–small cell lung cancer (NSCLC) were investigated by proteomics analysis,[44] after
immunoprecipitation in three different cells lines. 16 proteins that overlapped between all cell
lines, were considered, among those, heat shock protein 27 (Hsp27) was investigated with
interference experiments in vitro. Importantly, validation in human tissue specimens revealed
that combination of 14‐3‐3ζ and Hsp27 expression was an independent prognostic indicator
of OS (p=0.036).[44]

15
[45]
Additional efforts have been made to analyse the cancer stromal cells. Principe et al.
performed proteomics profiling of fibroblasts isolated from tumor and adjacent tissue from
patients with oral tongue squamous cell carcinoma, focusing on identification of stromal-
derived secreted factors. LC-MS/MS analysis was performed on cell lysates, conditioned
media and exosomes in four matched pairs of cancer associated fibroblast and matched
adjacent fibroblast. Further analysis of proteins identified in conditioned media and
exosomes, revealed 43 and 47 proteins consistently up-regulated (≥1.5-fold, p<0.05) in
fibroblasts derived from tumors. Microfibrillar-associated protein 5 (MFAP5) and Protein
canopy homolog 3 (CNPY3) overlapped between exosomes and conditioned media
analyses, with MFAP5 found more abundant in secretome (conditioned medium, exosomes)
in comparison to total cell lysate. Overexpression of MFAP5 in cancer-associated stroma
was confirmed by IHC (n=69 tongue cancer). Treatment of cancer cells with human
recombinant MFAP5 resulted in increased cell proliferation and migration, likely via induction
of MAPK and AKT pathways.

[46]
In the study by Ale kovi et al., SILAC was applied to investigate secretome from lung
metastatic breast and melanoma cancer cell lines. Up- and down-regulated cancer-specific
lung metastasis secretome signatures were established through the inclusion of proteins
found to be 2-fold up/ down regulated in at least one of the two sublines in comparison to
parental cell line. A signature of 149 and 210 up-regulated as well as 243 and 92 down-
regulated proteins in breast cancer and melanoma were established, respectively. 20 up-
regulated and 16 down-regulated proteins were common in both analyses. Overexpression
of Nidogen 1 (NID-1) enhanced cell migration, invasion, adhesion to the endothelium,
vascular permeability, and tube formation. NID-1 was also found to be upregulated in
patients with metastatic breast cancer and melanoma in comparison to patients without
metastases and its expression was correlated with poor outcome.
Altogether the above studies (as evident from the number of the identified proteins
downstream of a protein target) demonstrate the complexity of targeting one protein and the

16
potential of proteomics in identifying several downstream effectors/ pathways, moving from a
targeted one- pathway view to a more holistic approach of overlapping molecular pathways.
iv. Proteomics-driven drug discovery in other diseases
Oncology evidently covers the majority of the discovery studies, as tumorous tissue can be
excised from patients. Apart from cancer research though, several groups have employed
high resolution proteomics to investigate the pathophysiology of other chronic implications,
with very promising results in the pre-clinical setting.
Likely, the most successful example is the study of Qi et al.[47] attempting to introduce novel
drug targets for diabetic nephropathy. For this purpose, the authors analysed proteome of
glomeruli from two groups of individuals: 1) no or mild Diabetic nephropathy, Diabetic
nephropathy class 0–I and 2) severe Diabetic nephropathy, Diabetic nephropathy class IIb–
III. Comparative analysis indicated that 88 proteins were found to be up-regulated in group 1
(≥1.5-fold; p ≤ 0.05). Pathway enrichment analysis of these proteins revealed that most of
the top 12 top-ranked pathways were related to glucose metabolism and glycolysis. Pyruvate
kinase M2 (PKM2), up-regulated in group 1, was considered for further investigation
considering the prominent fold change, and possibility to modulate its action either via
pharmacological and genetic approaches.[47] Further in vitro and in vivo experiments
supported that activation of PKM2 likely protect against Diabetic nephropathy through
increase of glucose metabolic flux, inhibition of the production of toxic glucose metabolites
and induction of mitochondrial biogenesis, suggesting the potential of the protein as a drug
target.[47, 48]
The above studies have formed the basis for molecular treatment of diabetic
nephropathy through targeting of PKM2 activators.
Mulligan et al.[49] investigated novel therapeutic targets for Chronic rhinosinusitis with nasal
polyps (CRSwNP), focusing on complement component C3. Proteomics analysis of nasal
mucus samples (n=17) resulted in identification of 466 proteins, including 146 differentially

17
expressed proteins. 24 and 122 proteins exhibited an increase and decrease in the
abundance between CRSwNP and control group, respectively. Pathway analysis of the
proteins with increased abundance in CRSwNP identified initial triggering of complement
and complement cascade as top hit. Moreover, sinonasal mucus levels of complement C3
were correlated with disease severity. Investigation of the sinonasal tissue showed also an
increase levels of C3 and C3a in CRSwNP in comparison to controls. Further in vitro and in
vivo studies demonstrated that inhibition of the complement C3a receptor reduces
inflammation and CRS development in vivo, supporting its potential as novel therapeutic
target.
In the cardiovascular area, subendothelial retention of LDL is the early stage of
atherosclerosis and calcific aortic valve disease. In an effort to characterise LDL interaction
[50]
with proteoglycans, quantitative proteomics via iTRAQ coupled to MS was performed in
animal derived valve and diverter tissues (n=6 aortic valve leaflets and n=6 renal ostia) after
enrichment with LDL affinity columns. Matrix Proteoglycans and collagens were enriched
[50]
(decorin: 3.3 fold-change; biglycan: 4.4 fold-change . Specificity and affinity of decorin to
LDL was further verified, demonstrating a potential as therapeutic target [50].

[51]
In an investigation involving X-linked lymphoproliferative disease, Peled et al. analysed
molecular alteration downstream of PD-1. Using affinity purification and MS, signalling
lymphocytic activation molecule associated protein (SAP) was identified among proteins
interacting with PD-1. Knock-down of SAP in Jurkat T cells enhanced PD-1 function, while
its overexpression had an opposite effect on PD-1. Furthermore, evaluation of T cells from
patients lacking functional SAP – harbouring X-linked lymphoproliferative disease, indicated
a high hypersensitivity to PD-1 signalling, supporting role of SAP as an inhibitor of PD-1.
E. Profiling drug effects on proteome
i. Drug effects in cancer studies

18
Closer to drug development process, there are proteomics studies that investigate
mechanisms of drug action through characterisation of drug- affected proteins. This
information contributes to gain a better understanding of therapeutic potential of drugs, and
off-target effects. As concluded by the selected studies below, the majority of the proteomics
applications up to date, involve proteomic profiling upon treatment with kinase inhibitors to
therapeutically target malignancies.
In an effort to profile the alterations upon treatment with cyclin-dependent kinases (CDK)
[52]
inhibitors in neuroblastoma , Delehouse and collaborators applied roscovitine and CR8 in
neuroblastoma cell lines, both with amplified (IMR32) and not amplified MYCN (SH-SY5Y) .
iTRAQ labeling in combination LC-MS analysis, in the treated cells, resulted in the
quantification of 1320 proteins, out of those, expression of 25 proteins was altered by both
compounds. Of those, c-MYC and p27Kip1 were verified by western blot, suggesting a
downregulation of MYC signalling pathway downstream of CDK inhibition. To further confirm
this finding in vivo, IMR32 bearing mice (where MYC is amplified), were treated with CR8
and the tumor size and growth was decreased massively.[52] In another study, the proteomics
alterations downstream of BI-TK/GCV (Bifidobacterium infantis thymidine kinase/nucleoside
analogue ganciclovir) treatment system were investigated by iTRAQ- followed by MS
analysis for bladder cancer.[53] Sixty tumor- bearing animals (rats) were treated and further
proteomics analysis resulted in 402 differentially expressed proteins (1.3-fold change; 192
down-regulated and 210 up-regulated) after BI-TK/GCV treatment. Of the most
downregulated proteins, Peroxiredoxin-I (Prx-I) was verified by Western blotting and IHC,
while the effect of knocking down of the protein was also investigated in a human cell line
(T24), resulting in decrease of phospho-NF-κB p50 and p65. The results showed a
mechanistic insight of treatment with BI-TK/GCV, likely through Prx-I and NF-kB apoptotic
pathway.[53]

19
Similarly, the effect of Polo-like kinase 1 (Plk1) inhibition was investigated in melanoma.
Cholewa et al.[54] treated BRAFV600E mutant melanoma cell lines with a Plk1
(serine/threonine kinase) inhibitor, namely BI 6727. Label-free nano-LC–MS/MS analysis
was performed on a Q-Exactive instrument, revealing alterations on cellular metabolism,
proteasome degradation, and p53 translation, directing towards apoptosis and cell survival.
Particularly, proteasome subunits, a3, β2, β5, and β1 (PSMA3, PSMB2, PSMB5, and
PSMB1) were identified as downregulated upon MS analysis. For the catalytic subunits, β2,
β5, and β1, the result was verified by western blot following Plk1 inhibition.[54] Ferruci et al[55]
investigated angiogenesis in myeloma progression,[55] focusing on HGF/cMET pathway.
SU11274, a cMET inhibitor was applied together with other anti-myeloma drugs, Bortezomib
and lenalidomide. Upon SU11274 inhibition, 14 proteins were significantly altered (2-fold
change) among those four angiogenic proteins, like annexin A4 (ANXA4), prohibitin (PHB),
peroxiredoxin-6 (PRDX6), and annexin A2 (ANXA2) were downregulated after gel-based
proteomics analysis (2D coupled to MS), while calpain small subunit 1 (CPNS1) was
upregulated. Verification of these 5 proteins was further performed with real-time RT-PCR.[55]
A novel small-molecule inhibitor SKLB-163 (benzothiazole-2-thiol derivative) was developed
based on in silico design for cancer treatment.[56] Apart from the anti-tumor benefit of SKLB-
163 in vivo, proteomics analysis was performed to profile drug’s action in treated human
melanoma cells (A375). 21 proteins were identified with significantly altered expression, with
Rho GDP-dissociation inhibitor 1 (RhoGDI) to be shown with very significant decrease (3.6-
fold), was further validated by western blot, while overexpression rescued tumor
phenotype.[56]
Armstrong et al.[57] combined proteomics and transcriptomics analysis to investigate the
mechanism of action for AUY922 (next generation Hsp90 inhibitor) in prostate cancer in
explants from prostate cancer patients (n=6 for RNA Seq and n=12 for 2D-DIGE). Pathway
analysis of differentially expressed genes and proteins pointed towards enrichment in
regulation of actin cytoskeleton in AUY922 treated explants. AUY922 treatment notably

20
affected cell morphology and inhibited cell motility and cell invasion, in comparison to vehicle
or 17-AAG (first-generation inhibitor). Assessment of cytoskeletal and ECM-related proteins
revealed a most notable induction of fibronectin by AUY922. Interference with fibronectin
decreased the invasive potential of prostate cancer cell lines, similarly to AUY922. Jain et
al.[58] investigated changes in the proteome of U87 glioma cells after treatment with S3I201
(STAT3 inhibitor) using two dimensional gel electrophoresis and iTRAQ, resulting in
identification of six differentially expressed protein spots between treated and untreated
cells. Cytochrome C oxidase subunit 5A, peroxiredoxin-6, Prot DJ-1, chloride intra-cellular
channel protein 1 were identified via MS. iTRAQ approach enabled identification of 134
significantly altered proteins expressed (1% FDR and fold≥1.5). Differential expression of
two proteins involved in cellular metabolism i.e. PGAM1 and TPI1 was investigated by
Western blot. An increased expression of PGAM1 was observed in glioma cells treated with
S3I201, in line with proteomics analysis.

[59]
Roolf et al. investigated effect of sorafenib, inhibitor targeting receptor tyrosine kinase
FLT3, on a panel of thirteen Acute Myeloid Leukemia cell lines that differs in FLT3 status [i.e.
FLT3 wild-type and internal tandem duplication (ITD) mutated cell lines]. Treatment of cells
with increased concentration of Sorafenib revealed that FLT3-ITD+ cells are generally more
sensitive to sorafenib than FLT3 wild-type cells. Phosphoproteomics was applied to
investigate mechanisms underlying sensitivity to sorafenib in both FTL3 wild-type and
mutated cell lines. For that purpose, SILAC labelled MV4-11 (Sorafenib sensitive and FLT3-
ITD+), MONO-MAC-1 (Sorafenib sensitive and FLT3 wild-type), and SKM-1 (Sorafenib
insensitive and FLT3 wild-type) cell lines were treated with 0.01 M sorafenib, and 0.2 M
sorafenib. Phosphoproteomics analysis and in silico pathway analysis indicated different
pathways being affected in Sorafenib sensitive FLT3 wild-type and FLT3-ITD+ cells. In a
FLT3 wildtype cells Sorafenib inhibited MEK/ERK signaling pathway, while in the FLT3-ITD+
cell line, Sorafenib treatment supressed mTOR signalling.

21
[60]
On a study investigating resistance to chemotherapy, Tripathi et al. studied mechanisms
of chemoresistance of small-cell lung cancer (SCLC) cell lines via LC-MS/MS analysis. Five
cell surface receptors exhibiting the most robust difference in abundance between
chemoresistant and chemosensitive cells were further validated by WB and its expression
was assessed in patients’ derived xenografts. Among them, upregulation of Cell surface
glycoprotein MUC18 (MCAM) in chemoresistant tumors was supported by IHC. Knock-down
of MCAM in chemoresistant cells significantly decreased cell proliferation (p<0.001) and
colony-formation (p<0.01). Additional experiments suggested that the MCAM-mediated
sensitization to chemotcherapeuic agents occurred through SOX2-dependent up-regulation
of mitochondrial 37S ribosomal protein 1/ATP-binding cassette subfamily C member 1
(MRP1/ABCC1) and the PI3/AKT pathway.
ii. Proteomics drug effects’ investigation in other pathological conditions
Filipovic´ et al.[61] investigated mechanisms of Fluoxetine action, an anti-depressive drug,
using GeLC-MS/MS analysis of hippocampal tissue excised from treated and untreated rats.
83 proteins were identified as differentially expressed in cytosolic hippocampal proteome
after Fluoxetine treatment, with majority of proteins being down-regulated in comparison to
vehicle-treated controls; 63 proteins were identified as differentially expressed in
mitochondrial proteome, with most of them being up-regulated after treatment. Differential
expression of Calretinin was confirmed by Western Blot, while the change in Parvalbumin
expression was supported by immunofluorescence.
Bowen et al.[62] focused on identification of compounds able to inhibit Muscle ring finger 1 –
titin interaction, previously shown to be activated in numerous conditions associated with
skeletal muscle wasting. Using AlphaScreen assay, 9 compounds have shown to have a
high Muscle ring finger 1- titin specificity. From these, three compounds inhibited also
Muscle ring finger 1 activity, with ID#704946 compound characterised by low cytotoxicity on

22
myoblasts or myotubes. Animal experiments with ID#704946, showed that the compound
prevented atrophy in myotubes and attenuated fibre atrophy and contractile dysfunction in
mice during cardiac cachexia. Proteomics analysis of muscle tissue was applied to
investigate proteins/ pathways affected by this compound, and 51 proteins were found to be
differentially expressed between MCT group (cachexia model) vs sham (p<0.01), and 70
proteins were found as differentially expressed between MCT group treated with ID#704946
vs MCT group (p<0.01). Analysis revealed that compound attenuated proteins involved in
stress pathways (e.g. Muscle ring finger 1), apoptosis (BAX), protein synthesis (elF2B-delta),
along with inhibition actin ubiquitinylation and proteasome activity.[62]
F. Chemical proteomics for drug selectivity and inhibitor binding
Chemical proteomics platforms have been widely applied to support drug discovery process,
with activity-based protein profiling (ABPP) in combination with MS to be the most common
technique to discover selective and in vivo-active inhibitors for enzymes.[63] After evaluating
the proteomics studies (that are described in detail below), a popular field of research
involves quantitative MS protocols to profile drug targets of covalent kinase inhibitors, based
on conserved active cysteines.[63] A common approach is based on Kinobead technology,[64]
through which, a broad-range of kinase inhibitors is immobilized to beads for purification of
kinases from tissue samples or cells. In combination with MS analysis, this approach allows
for simulations measurement of multiple protein targets.[65] Along these lines, combination of
chemical proteomics with fragment-based ligand discovery was established as a platform to
map interactions of small molecules and proteins in human cells.[66] Last but not least,
chemical proteomics approach has been used to assess lipid-protein interactions.[67]
i. Chemical proteomics coupled to mass spectrometry in oncology

23
Kinase inhibitors represent a major class of drug targets. Recently, efforts have been
undertaken to characterize target space of 243 clinically evaluated kinase inhibitors. For that
purpose, Klaeger et al.[65] applied kinobead technology in combination with MS on cancer
lysate cells.[65] As a result, the largest resource for clinical drug-target interactions has been
established[65]. Along these lines, tyrosine kinase inhibitors (TKIs) have become an important
therapeutic option for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal
growth factor receptor (EGFR), is in clinical use for treating NSCLC harboring activating
EGFR mutations. A chemical proteomic approach[68] comprising from kinobeads and
quantitative MS was applied for the identification of kinase inhibitor resistance mechanisms
in cancer cells. Previously described amplification of MET and found Ephrin type-A receptor
2 (EPHA2) was confirmed to be more than 10-fold overexpressed (p <0.001) in gefitinib-
resistant HCC827 cells suggesting a potential role in developing resistance. siRNA-mediated
EPHA2 knock-down or treating cells with the multikinase inhibitor dasatinib restored
sensitivity to gefitinib. Of all dasatinib targets, EPHA2 exhibited the most drastic effect (p
<0.001). In addition, EPHA2 knock-down or ephrin-A1 treatment of resistant cells decreased
FAK phosphorylation and cell migration.[68] In another study, Heinzlmeir et al. [69]
aimed at
development of EPHA2 inhibitors with improved target profile, with dasatinib used as a basis
of a lead structure. For that purpose, chemoproteomics-aided medicinal chemistry approach
was employed. Kinobeads pulldown assays in combination with MS analysis was used to
assess the affinity of identified inhibitors as well as investigate their selectivity in comparison
to parent lead compound. Furthermore, the effect of the inhibitor candidates on proliferation
of SF-268 glioblastoma cell line was assessed, and one compound showed a strong anti-
proliferative potential and warrants further investigation.

[70]
Along the same lines , Wang et al. profiled the protein targets of andrographolide (Andro)
a natural product with anti-inflammation and anti-cancer effects. The authors introduced a
combination of state-of-art chemical and proteomics techniques, combining ABPP with bio-
orthogonal click chemistry and iTRAQ to identify drug targets in vitro and in vivo. 75 potential

24
Andro targets were identified with high confidence and two targets, NF-κB and β-actin, were
validated by pull-down assays and WB, while NF-κB p50 protein, was investigated for the
PTM site after Andro treatment, by MS/MS.[70] Roberts et al.[71] combined chemical genetic
screening with chemoproteomics platform to identify novel therapeutic compounds for
pancreatic cancer and identify its targets. Considering the ability of compounds to impair
pancreatic cancer cell survival or proliferation as well as nonspecific toxicity, chemical
genetic screening pointed out three compounds (DKM 2−67, DKM2−83, and DKM 2−93),
and DKM 2−93 was selected for further investigations in vivo and for targets identification
using chemoproteomics. It has been shown that DKM 2−93 impairs tumor growth in vivo,
while thorough chemoproteomics approach cysteine 250 on ubiquitin-like modifier activating
enzyme 5 (UBA5) was identified as a druggable hotspot targeted by DKM 2−93. Moreover,
knock-down of UBA5 in pancreatic cancer cell lines mimicked the effect of DKM 2−93
treatment. In a similar way, Bateman et al.[72] identified compound that impairs colorectal
cancer cell pathogenicity via targeting cystein 1101 on Reticulon 4. Knock-down of Reticulon
4 or treatment of cells with DKM 3-30 affected also morphology of endoplasmic reticulum
and nuclear envelope. Along these lines, Grossman et al.[73] applied the same
chemoproteomic platform to discover drugabble hotspots targeted by withaferin A, an anti-
cancer natural product, followed by covalent ligand screening to support the translation of
natural products into therapeutics, through development of more synthetically tractable
compounds. Chemoproteomics analysis showed that withaferin A specifically targets
cysteine 377 of a regulatory subunit within the tumor-suppressor protein phosphatase 2A,
leading to its activation, and inactivation of AKT signalling. This impairs breast cancer
pathogenicity, as supported by in vitro and in vivo investigation. JNS 1-40 was identified as a
ligand interacting with C377 of PPP2R1A through covalent ligand screening, having similar
effects to withaferin A.
ii. Proteomics drug profiling in other conditions

25
Excepting the cancer studies, Sulaiman et al. employed chemical proteomics to identify
protein targets of SH-11037, an homoisoflavonoid derivative, with antiangiogenic activity in
mouse model of wet age-related macular degeneration. Pull-down proteins were separated
by SDS-PAGE, followed by MS analysis used for protein identification and Soluble epoxide
hydrolase was identified as a target of SH-1103. In silico docking analysis showed that SH-
1103 occupies the entire active site of Soluble epoxide hydrolase. Soluble epoxide hydrolase
was inhibited by SH-11037 in vitro and in vivo, although the effect was not that prominent as
for known inhibitors of Soluble epoxide hydrolase. Activity and level of Soluble epoxide
hydrolase in the eyes of a choroidal neovascularization mouse model significantly was up-
regulated as well as the enzyme was also overexpressed in human wet age-related macular
degeneration eyes.
[74]
Tian et al. investigated targets of calenduloside E, being triterpenoid saponin compound,
and signaling pathways underlying its protective activity on endothelial cells using chemical
proteomics. 587 proteins were identified (at least 1 unique peptide, score >0) through biotin–
neutravidin pull-down assay followed by geLC-MS/MS. Numerous proteins were involved in
cell survival signaling, with some of these pathways being associated with anti-apoptotic
activity such as PI3K-AKT, VEGF, MAPK and ER stress. In vitro assay supported that biotin
conjugated calenduloside E analogue protects HUVEC cells against ox-LDL-induced injury.
G. Structural insights by using mass spectrometry
Mass spectrometry analysis has been also employed to confirm the chemical structure of
synthesized compounds/ ligands targeting proteins of interest. Kadasi et al. [75] performed a
structure based design of benzofuran-3(2H)-one analogues targeting HSP90. In silico
docking study was conducted followed by chemical synthesis of the ligands. Chemical
structure of the compounds was confirmed by IR, NMR and MS. Subsequent in vitro
investigation in breast cancer and melanoma cells highlighted 2-(4-(4-fluorobenzyloxy)-

26
benzylidene)-5-chloro-6-hydroxybenzofuran-3(2H)-one (SY3) as the promising anticancer
agent that enabled significant decrease of cell proliferation, when comparing to first-class
HSP90 inhibitor, 17-AAG.
H. Outlook
Through a systematic literature search, in this article, we attempted to provide an overview
on the most promising proteomics studies categorized according to their application in
different fields of drug discovery and development, covering the following areas: target
identification for drug discovery, proteome profiling for drug effects, chemical proteomics for
drug selectivity and inhibitor binding and structural proteomics studies for assisting in silico
docking. The aim of the evaluation was to provide a critical view on how close the
proteomics results are to being implemented in drug development pipeline. Collective
elements based on the literature search showed that high-resolution MS-based proteomics
has been employed in a plethora of studies, involving patient-derived patients, cell lines and
already developed drugs. As evident from the number of the retrieved studies, most of the
proteomics investigations are in the context of oncology. This observation is, to certain
extent, expected considering that access to tissue biospecimens is generally more
permissive in tumor affected areas and after the surgical removal of the malignant tissue.
Currently, most of the cancer drugs that are developed show low response rates and are
associated with side-effects and/or mechanisms of resistance, which are frequently adopted.
Considering this aspect, global proteomic profiling, particularly for identifying downstream
effectors and drug’s mechanism of action are of vital importance, as they enable a more
comprehensive description of molecular alterations, not only at the level of individual
proteins, but also at the level of pathways and their relationship. Moreover, combination of
knowledge at the molecular level with pathology can improve on the drug discovery and
development process, as also schematically depicted in Figure 2.

27
Nevertheless, the proteomic studies, at least as derived by this systematic search are not
free of limitations. Frequently the number of samples (particularly the patient-derived tissue
specimens) is low, as the availability of the tissue may be also limited. This has an impact on
the analysis in terms of statistical power, and application of correction for multiple testing is
challenging. Moreover, cell line models cannot always accurately reflect the human
physiological conditions, as diseases like cancer can be highly heterogeneous among
different patients. More importantly, apart from the technical limitations, the field is yet
noticeably not that advanced in terms of proteomics-derived drugs that have reached the
phase of clinical trials, and ultimately clinical implementation. On a positive note, certain very
promising studies, as in the case of diabetic nephropathy through the targeting of PKM2 and
bladder cancer, through EIF3D pave the way towards molecular therapy based on the
proteomics profiling. Certainly, recent breakthroughs in thermal proteome profiling assays
are expected to change drug development landscape, particularly regarding drug screening.
In an effort to move from a more static observation (likely this is mostly the challenge in
genomics analysis) to a more dynamic profiling and in order to improve real time monitoring,
novel innovative proteomics platforms have been recently introduced. Along these lines, the
advantages of chemical proteomics (i.e. ABPP) and cellular thermal shift assay (CETSA) are
now combined with high resolution MS, allowing for characterization of drug’s action in an in
vitro environment that is closer to the physiological conditions in the cell.
Ideally, in a future clinical scenario (as depicted in Figure 3) where personalised medicine is
followed, proteomics is envisioned to have an important role in all parts from drug targeting
till section of appropriate intervention. Proteomics profiling can at first indicate altered
pathways, providing insights at the molecular level, beyond the clinical and/ histological
phenotype. Then, drugs can be directly predicted based on molecular profiling data and can
be suggested considering the probability to reverse the clinical phenotype. As through
proteomics and drug mechanism networks, drug targets can be linked with biomarkers,
especially markers to predict and monitor treatment response can be subsequently applied.

28
Collectively, by this article we provide an overview on the status of proteomics in the field of
drug discovery, exemplified by the most prominent studies in the field of chronic diseases.
Acknowledgements
The work was supported by the BioMedBC Project (Project ID: 752755) and PCaProTreat
Project (Project ID: 800048), funded by the EU Commission, under the MSCA-IF-2016
Individual Fellowships.
Conflicts of interest
Harald Mischak is the founder and co-owner of Mosaiques Diagnostics GmbH. Maria Frantzi
and Agnieszka Latosinska are employed by Mosaiques Diagnostics GmbH.
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32
Figure Legends
Figure 1. Areas of application for proteomics generally cover the discovery of potential drug
targets, investigations into mechanisms of drug action and protein-protein interactions, and
the discovery and application of stratification biomarkers.

33
Figure 2. Knowledge at the molecular level and pathology can improve on the drug
discovery and development process.

34
Figure 3. Envisioned clinical setting where proteomics can guide drug selection and
intervention.

35
Supplementary Table 1. List of the 1625 studies, as retrieved by the literature search.
Harald Mischak
Harald Mischak is the director of Mosaiques diagnostics, which he founded in 2002. In

addition, he is a Professor at the University of Glasgow. He received his PhD from the
Technical University of Vienna and has been involved in proteomics during the last 30 years,
with focus on deciphering signaling processes and identifying biomarkers and therapeutic
targets. He initiated the use of urinary proteomics and capillary electrophoresis coupled
mass spectrometry for clinical application, and is a leading authority in clinical proteomics
and biomarker identification, with over 300 scientific publications on signaling and
proteomics that have been cited over 20000 times. Among his achievements in this field are
the development of guidelines for clinical proteome analysis, the demonstration of successful
application of clinical proteomics in the diagnosis and prognosis of several diseases, and the
initiation of proteomics-guided randomized controlled clinical trials.

36
Maria Frantzi
Maria Frantzi received a MSc (2012) and a PhD (2016) in Molecular Medicine from Athens
Medical School. During her PhD studies, she received a Marie Skłodowska-Curie fellowship,
during which she shared her time between University of Glasgow (UK), Biomedical
Research Foundation of Academy of Athens (BRFAA, Greece) and Mosaiques diagnostics
GmbH (Germany). She currently works in Mosaiques diagnostics GmbH with main research
focus on proteomics, cancer diagnostic and stratification biomarkers. Through a second
post-doctoral Marie Skłodowska-Curie fellowship, she has expanded her research towards
personalized medicine and -omics data integration, through the application of proteomics
technologies and system biology approaches for investigating disease pathophysiology and
molecularly driven drug targets.

37
Agnieszka Latosinska
Agnieszka Latosinska graduated from Jagiellonian University, Poland in 2012, and received
a PhD from the Charité-Universitätsmedizin Berlin, Germany in 2016. In parallel, she worked
as an Early Stage Researcher in the Marie Curie BCMolMed (Molecular Medicine for
Bladder Cancer) EID Program. During the programme she was performing research in the
proteomic laboratory in the Biomedical Research Foundation of the Academy of Athens
(Greece) and in Mosaiques Diagnostics in Hannover (Germany). She is currently holding a
Marie Skłodowska-Curie Individual Fellowship at Mosaiques Diagnostics, with the main line
of research focused on the identification of therapeutic agents and drug targets through the
knowledge gained based on molecular characterization of Prostate Cancer.

38

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com Page 1 Proteomics - Clinical Applications

Proteomics in drug development: the dawn of a new era?

Maria Frantzi1*, Agnieszka Latosinska1* and Harald Mischak1,2

*equally contributing first authors

Prof. Harald Mischak

Mosaiques Diagnostics GmbH

D-30659 Hannover, Germany

Phone: +49 (0)511 55 47 44 13

Fax: +49 (0)511 55 47 44 31

Keywords: Drug discovery, Mass Spectrometry, Proteomics

List of Abbreviations (excl. standard abbreviations):

ABPP- activity-based protein profiling; ALK- anaplastic lymphoma kinase; ANGPTL2-

Angiopoietin-related protein 2; ANXA- annexin A BI-TK/GCV- Bifidobacterium infantis

thymidine kinase/nucleoside analogue ganciclovir; BTK- Bruton’s tyrosine kinase; CALU-

dependent kinases; CMTM6- CKLF-like MARVEL transmembrane domain containing protein

Received: 10 18, 2018; Revised: 01 13, 2019; Accepted: 02 04, 2019

This article is protected by copyright. All rights reserved.

IL1RAP- Interleukin 1 Receptor Accessory Protein; IL13RA2- Interleukin-13 receptor subunit

8; KRT17- Keratin 17; LGALS3BP- Galectin-3-binding protein; MCAM- Cell surface

glycoprotein MUC18; MCM2- minichromosome maintenance protein 2; MFAP5-

Microfibrillar-associated protein 5; MIBC- muscle invasive bladder cancer; NAP1L1-

Nucleosome assembly protein 1-like 1; NID-1- Nidogen-1; NKCC1- Sodium-potassium-

chloride cotransporter 1; NMIBC- non-muscle invasive bladder cancer; NSCLC- Non–small

component 1; PHAP1- Putative HLA-DR-associated protein 1; PHB- prohibitin; PKM2 -

PSMA3- proteasome subunit a3; PTPRZ1- Receptor-type tyrosine-protein phosphatase

Scaffold attachment factor B; SAP- Signalling lymphocytic activation molecule associated

protein; STMN1-Stathmin; TAGLN2- Transgelin-2; TGF‐β1- Transforming growth factor beta

1; TGF‐β3- Transforming growth factor beta 1; UBA5- Ubiquitin-like modifier activating

enzyme 5; UQCRC2- Cytochrome b-c1 complex subunit 2, mitochondrial

functional status of a group of healthy or disease individuals, according to the different

This article is protected by copyright. All rights reserved.

to be improved in terms of efficacy, a mechanistic insight for downstream effectors can be

to implementation, therefore particular emphasis is given in studies conducted in human

diseased population and further verified in vitro or in vivo.

A. Concept of improving drug discovery and development by proteomics

proteomics is a key technology to support development of specific drugs to stop or reverse

effects of the disease.[2]

In principle, proteomic analysis by mass spectrometry is based on the identification of

ionised compounds in a sample mixture by recording their mass-to-charge ratio.[3] The

translational modifications (PTMs) and protein interactions. Compared to genome, which is

considering the complexity of the proteome,[4] interpretation of proteomics data is certainly

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proteins can be measured simultaneously in complex samples, in an unbiased, untargeted

proteomics in the pharmaceutical industry, which can be categorised to: a) discovery of

and to reverse them to a normal condition. Therefore, gaining a holistic understanding of

interactions. Discovery investigations involve the identification of proteins whose expression

identification of systemic biomarkers.[9] As a result of our increased understanding at the

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covered in recent reviews.[10, 11]

B. New technological advancements

Latest technological breakthroughs have revolutionized drug development, particularly

recently introduced,[12-15] as an evolution to the traditional fluorometric/ calorimetric thermal

drug’s mechanism of action through comprehensive screening in living cells.[16] The

denaturation temperature of a protein under different conditions is representative of protein-

assay (CETSA), activity-based protein profiling (ABPP),[17] photoaffinity labeling (PAL),[18]

be covalently linked through UV irradiation[18] and in ABPP methodology through biotinylated

greatest breakthrough technology, however, is cellular thermal shift assay (CETSA),[7] as

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C. From proteomics to personalized medicine: Literature review

Science database (22/08/18), as follows: TOPIC: "drug discover*" OR "drug target" OR

"therapeutic target" AND TOPIC: proteom*. Only manuscripts defined as “ARTICLE” OR

article we focused on chronic diseases. Therefore, biomarker studies, any investigations in

Maria Frantzi1, Agnieszka Latosinska1 and Harald Mischak1,2