It’s hard to imagine a biomedical field that’s not concerned with cell signaling. From immunologists studying GPCRs and neuroscientists investigating amyloid plaques, to cardiologists telling their patients to avoid caffeine, knowing what’s connected to what and which kinases are phosphorylating which others is all part of the daily grind.
At Cambridge Healthtech’s “Discovery on Target” conference, held earlier this month, pathways leading to and coming from PI3 kinase seemed to be in the forefront of many researchers’ minds. Yet, generating tools to assess and manipulate the status of all sorts of pathways—from those involved in cancer to those responsible for addictive behavior—was of interest no matter what your favorite signaling molecule.
It’s notoriously difficult to generate functional antibodies against complex cell-surface molecules. Antibodies are typically generated against “some kind of artificially expressed and folded GPCRs that don’t always have their native conformation,” remarked Sergej Kiprijanov, Ph.D., vp of research and preclinical development at Affitech.
Affitech wanted to antagonize GPCRs involved in cancer progression and inflammation. Using its cell-based antibody selection (CBAS) technology it screened its massive phage-display human antibody library against receptors (such as the CCR4 and CXCR4 chemokine receptors) expressed on the cell surface in their natural conformation, and identified several candidates able to impact the ability of these receptors to signal.
In the most advanced of six anti-GPCR programs, for example, an anti-CCR4 antibody was found that can deliver a strong response against CCR4+ tumors in xenografts. At the same time, the antibody is able to target CCR4+ regulatory T cells that can inhibit a robust imm?une response against the tumor.
Because of redundancy in the system, one chemokine receptor may have several natural ligands. So, unlike targeting the chemokines themselves, “by blocking the receptor with the same compound you can abolish the signal provided by three or four different chemokines,” explained Dr. Kiprijanov.
SHIP of PIP
Stephen Ward, Ph.D., works further inside immune cells, on signaling pathways involving PI3K. But rather than trying to directly inhibit the nearly ubiquitous kinase itself—risking substantial off-target effects—the University of Bath professor of pharmacy and pharmacology instead tries to focus efforts on a more targeted target.
Expression of the SHIP-1 is primarily restricted to cells of hematopoietic lineage. The phosphatase is responsible for degrading PIP3—the primary product of PI3K—to PIP2, essentially undoing what the kinase has done.
Dr. Ward’s group initially used short hairpin RNAs to silence expression of SHIP-1 in human T lymphocytes and was surprised to find no effect on directional cell migration and an impairment of basal cell mobility. They postulated that perhaps some of the molecule’s actions were the result of a scaffolding function distinct from domain-mediated effects. SHIP-1 encodes multiple structural domains that are known to interact with and recruit other molecules through protein-protein interaction and cellular relocalization.
A series of experiments using small molecules—activators and inhibitors of SHIP-1—revealed that the two approaches “don’t exactly phenocopy each other or the effect of SHIP-1 knock-down,” Dr. Ward noted. “One conclusion, therefore, is that the noncatalytic domain-mediated scaffolding functions may be avoided/missed by small molecule approaches.”
But it may not even be that simple. Some small molecules actually inhibit both basal and activated migration.
Like PIP3, PIP2 also interacts with pleckstrin homology (PH) domains within effector proteins, so altering SHIP-1 activity—whether by a shotgun molecular approach or a more targeted pharmaceutical one—may shift the balance between PIP3 and its degradation product. “And that is critical in determining overall motile response of T cells. The functional impact of disrupting this balance can be difficult to predict given the number of downstream effector molecules.”
Two for One
In tumors in which PI3K is implicated, another kinase, mTOR, is often found to be dysregulated as well. Interestingly, mTOR has never been found to be mutated, but it tends to be dysregulated by things other than mutations—such as a variety of upstream pathways that feed into it.
“These proteins don’t act in isolation,” explained David Matthews, Ph.D., vp for drug discovery and exploratory development at Pathway Therapeutics. “They form a complex signaling network with a lot of feedback loops and interactions between them and between various other cellular components.”
If mTOR is targeted on its own (for example, by a rapamycin analog), these feedback loops will actually serve to activate PI3K. Both proteins, Dr. Matthews said, really need to be targeted to have a robust effect on tumor cell signaling, and Pathway has developed just such a molecule. PWT33597 is an inhibitor of mTOR and PI3Kα, the isoform most frequently dysregulated in tumor cells. PWT33597 is very selective—“it doesn’t hit any other kinases that we can tell.”
The compound generates a balanced inhibition, leading to cell death, in cell-based assays, “and also nice tumor growth inhibition in xenograft models. And that’s the data that has led us to move this molecule forward into the clinic.”
PWT33597 is currently in a Phase I clinical study. In addition to demonstrating safety and tolerability, the company is also looking for pharmacodynamic markers of target inhibition. “To that end we put in place a panel of assays that lets us do this, monitoring PI3K and mTOR signaling in tumors, in blood, and in hair.”