Development of novel receptor tyrosine kinase inhibitors by a chemocentric approach
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Date
08/07/2017Author
Myers, Samuel Harry
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Abstract
In recent years, there has been a major movement in the pharmaceutical industry towards the
development of molecules that selectivity inhibit a previously-validated specific target. This
is referred to as target-based drug discovery. It was hoped that adopting this approach would
usher in a new golden age of drug discovery. However, this has not been the case, with
issues arising such as the target’s mechanism of action being poorly understood, with it not
playing the expected role in the disease progression, or feedback resistance mechanisms
causing the target to lose its role in the disease. In contrast to this, in the past 20 years it has
been argued that developing drugs in a target-agnostic way and screening them against an
expressed phenotype i.e. phenotypic drug discovery, has been more successful, despite fewer
programs being run in the manner.
The AXL kinase is a receptor tyrosine kinase (RTK) and a member of the TAM family,
along with MER and TYRO3. AXL has long been associated with numerous types of
cancer. Having been first discovered in 1991 in acute myeloid leukaemia (AML), it has gone
on to be more associated with advanced solid tumours such as brain, breast, and lung, with
the trend being that increased AXL correlates with a poorer prognosis for the patient. Upon
the activation of AXL by the vitamin K ligand GAS6, a series of downstream pathways are
activated that go on to encourage cell survival, proliferation, and migration. In addition to
this, AXL has been shown to be involved in crosstalk with other kinase pathways, resulting
in AXL expression being associated with chemoresistance and survival mechanisms. Despite
the promising outlook for AXL inhibitors, to date only one selective AXL inhibitor, BGB324
(formally R428) has entered clinical trials, with selective AXL inhibitors being difficult to
develop due to a lack of a crystal structure or a reliable homology model.
To address the aforementioned issues that target-based approaches can suffer from, and due
to AXL lacking a crystal structure, the work in this thesis utilised a pragmatic drug design
method that started from ligands/existing scaffolds known to inhibit the target from the
literature (publications, clinical trials and patents). A series of small libraries were prepared
and then tested against a selected phenotype e.g. cell viability, in at least two cell types: one
that expressed the target (e.g. AXL) and one that did not. Hits were optimised for potency
against the desired phenotype. The compounds then went through target deconvolution
(kinase screening) to confirm the target of the inhibitors.
Employing this approach, we initially synthesised two small libraries of potential AXL
inhibitors. The potency of these compounds was tested using cell-based phenotypic assays,
by evaluating cell viability in both native and chemo-resistant breast cancer cells. These
libraries were optimised through focused combinatorial synthesis and phenotypic screening,
to yield a small collection of antiproliferative hits. These hits were then profiled against a
panel of twelve select kinases. The first library, while giving some important structural
information, did not inhibit the kinases screened in a meaningful manner. However, the
second library gave several potent compounds, inhibiting AXL, FLT3, and RET, with one
compound being selective for AXL. The leads from this series were optimised further,
through SAR studies, gaining important structural information in order to improve potency
and selectivity of the compounds. The flexibility of the phenotypic cell-based approach
allowed the pursuit of FLT3 inhibitors, resulting in the synthesis of one of the most potent
FLT3 inhibitors synthesised to date.