Although targeting DNA had probably been the most successful anticancer concept
to date, the lack of selectivity of DNA reactive compounds remained a problem.
So commented Professor William Denny from the Auckland cancer society research
centre in New Zealand. Now, he said, researchers were beginning to understand
more about other pathways that presented better selective toxicity in cancer
therapy: one such pathway was cell signalling and signal transduction.
Normal cells were converted to cancer cells through the accumulation of damage
in several different ways, said Professor Denny. Many of these processes began
at the cell surface through the interaction of signal molecules with receptors.
Activation of many receptors, such as the protein tyrosine kinases, led to a
phosphorylation cascade of cellular proteins that culminated in gene activation
and cell division (see Panel). One of the most well understood groups among
the protein tyrosine kinases was the erb-B family. Several members of this transmembrane
receptor family and their ligands had been identified: erb-B1 (also known as
the HER-1 gene product or the epidermal growth factor receptor, EGFR), erb-B2
(HER-2), erb-B3 (HER-3) and erb-B4 (HER-4).
Many studies had correlated overexpression of the erb-B receptor family with
poor clinical prognosis in cancer, and provided a biochemical rationale for
shutting off signalling from the receptors with inhibitory agents, Professor
Denny continued. Structural information on the erb-B receptors had helped to
define possible options for drug targeting. The receptors were formed from external
and internal domains and a third regulatory domain. Ligand binding to the external
domain resulted in receptor dimerisation and autophosphorylation of the intracellular
regulatory domain. A subsequent conformational change enabled the intracellular
tyrosine kinase domain to phosphorylate tyrosine residues of the cellular substrate
using adenosine triphosphate (ATP) as a cofactor.
Already, monoclonal antibodies (MAbs) had been designed to target the external
domain of the receptors and were in advanced stages of clinical trials in breast
cancer, he said. Trastuzumab (Herceptin), an MAb raised against erb-B2, was
effective alone, conferred extra benefit on standard chemotherapy, and was well
tolerated by patients. “This work gave clinical proof of concept that blocking
the erb-B receptors was growth inhibitory,” Professor Denny said.
| Cell signalling and signal transduction explained | |
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With regard to targeting the intracellular domains of the erb-B receptors,
either the binding site of the intracellular substrate, or the ATP cofactor,
could be used. Targeting the substrate-binding site should provide a high degree
of selectivity as each substrate was different for any particular tyrosine kinase.
However, a series of tyrosine mimics based on sterically constrained analogues
of tyrphostin showed low potency (0.2-10µM) and poor in vivo stability.
Structure-activity relationships were flat and difficult to optimise, he said.
Conversely, six or seven years ago, some anilinopyrimidine-based compounds were
found to be highly potent and selective inhibitors of the erb-B ATP binding
site, said Professor Denny. The result was unexpected as such agents would have
to compete with high intracellular levels of ATP and could possibly block other
cellular ATP binding sites. Subsequently, pyridopyrimidine-based lead compounds
were developed and found to have tight structure-activity relationships, he
said. Using these relationships and structural information from a related protein,
a model of the ATP binding site was established and assisted rational improvement
of existing leads. For example, a region of “bulk tolerance” was apparent
in the active site and indicated that attachment of solubility enhancing substituents
would be possible at the C-6 position.
The optimised compounds were potent inhibitors of the tyrosine phosphorylating
activity of EGFR (erb-B1) and showed good cytostatic activity, said Professor
Denny. Other groups, he said, had also developed similar compounds. One inhibitor,
Iressa (Astra Zeneca), was now in phase II clinical trials in combination with
cytotoxics and appeared to further prove the principle of erb-B receptor modulation.
More recently, development had concentrated on irreversible inhibitors because
reversibly acting compounds required frequent dosing schedules as a result of
competition with circulating ATP, said Professor Denny. To this end, an acrylamide
substituent was incorporated into the lead compounds to bring about rapid and
specific alkylation of a cysteine residue (C773) that was unique to the active
site of the erb-B kinases: the acrylamide group had low chemical reactivity
preventing non-specific alkylation of other cellular components. These irreversible
compounds, of which one was now in clinical trial, constituted a new class of
agent, said Professor Denny. They were highly potent and displayed exceptional
activity in a range of xenograft models, good pharmacokinetics and tolerability.