The Design, Synthesis and Biological Testing of
Potential CCL2 and EBNA1 Therapeutics

Abstract

Chemokine ligand 1 (CCL2) is an inflammatory protein that induces the chemotaxis of
leukocytes to sites of inflammation or infection through binding with its receptor CCR2.
Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for the DNA replication and
episome maintenance of the Epstein-Barr virus (EBV) which is estimated to have infected
over 90% of the worldwide population. Both CCL2 and EBNA1 are associated with
autoimmune diseases and cancer and thus they represent potential therapeutic targets.
The emerging field of proteolysis targeting chimeras (PROTACs) presents an alternative
therapeutic approach to small molecule drug discovery, with the key benefits of targeting
‘undruggable’ proteins and transient binding modes with the target of interest. The primary
aims of this thesis were to design, synthesise and evaluate the first examples of CCL2
and EBNA1-targeting PROTACs.
2,5-Diketopiperazines (DKPS) have been shown in previous work to inhibit CCL2-
mediated chemotaxis, with CCL2 binding as their proposed mode of action. In Chapter 2
a library of 13 DKPs, that incorporate fluorinated or unnatural amino acids, were
synthesised using solid phase or solution phase peptide synthesis. From subsequent
Boyden chamber (chemotaxis) assays, an improved chemotaxis inhibitor (43) was
discovered in which THP-1 migration was reduced to approximately 51% (at 50 μM). Next,
the mode of action in which the DKPs reduced THP-1 chemotaxis was evaluated using
CCR2 internalisation assays. In the work presented in Chapter 2, it was shown that DKP
43 (at 100 μM and 50 μM) and previous lead 27 (100 μM) were neither agonists for CCR2
internalisation nor were they outcompeting CCL2 for the orthosteric site. The mode of
action was further assessed through the analysis of DKP-CCL2 binding in Chapter 3. The
first step was hsCCL2 overexpression in E. coli which produced moderate amounts of high
purity protein. Subsequently, DKP ligand-binding assays with hsCCL2, as well as
btnCCL2, were undertaken using surface plasmon resonance (SPR) with no binding
events observed (up to 200 μM). The work in both Chapter 2 and Chapter 3 indicated
v
that DKPs were not CCR2 orthosteric antagonists or CCL2 binders thus the new proposed
mode of action is that DKPs likely act as allosteric CCR2 antagonists. In addition, the
CCL2 binding of a group of fluorinated, small molecule fragments was assessed using
SPR assays (with hsCCL2 and btnCCL2) with fragment 80 as the most promising binder
(KD = 89 μM for btnCCL2). This was an important discovery as few CCL2 small molecule
binders are currently known. Chapter 4 reported the synthesis and biological testing of
three potential CCL2-PROTACs 105 - 107 (work undertaken prior to biophysical studies
detailed in Chapter 3) incorporating hydroxyl-containing DKPs. The reduction of CCL2
levels in response to each PROTAC (100 nM) was determined using a novel CCL2
degradation assay, however a consistent reduction in CCL2 was not observed. Finally, in
Chapter 5, the synthesis of YFMF-NH2 incorporating PROTACs, with the potential to
degrade EBNA1 via the CRBN, were synthesised. Initial in vivo EBNA1-degradation
assays indicated 147, 149 and 153 as potential hits, however this work is still in its infancy
and is an ongoing project in the Cobb group.
In summary, the design, synthesis, and biological testing of potential CCL2 and EBNA1
PROTACs was undertaken. Although the PROTACs prepared were unable to degrade
CCL2, the newly discovered CCL2 binders and the emerging alternative approaches to
targeted protein degradation (e.g. LYTACs) still make CCL2 a viable target. The synthesis
of 13 EBNA1-PROTACs was achieved, and their initial biological evaluation has identified
some promising leads for further development.