December 1, 2006 (Vol. 26, No. 21)

Using the Focused Diversity Set to Discover Small Molecule Antagonists

It has been noted that there are several features shared between stem and tumor cells, including self-renewal and replication potential. It is believed that the early stage of oncogenesis involves acquisition of mutations in a single cell accumulated during its lifespan. This hypothesis puts stem cells in the focus of attention as possible sources of various cancers.

There are several developmental pathways that are involved in the deregulated signaling in stem cells resulting in tumorigenesis. Namely, aberrant activation of the Hedgehog (Hh) pathway has been associated with malignancies, including basal cell carcinoma, medulloblastoma, and prostate, pancreatic, and breast cancer. In vivo evidence suggests the antagonism of excessive Hh signaling may provide a route to unique mechanism-based anticancer therapies.

Hh signaling is triggered by lipid-modified Hh proteins that exert their activity via a series of transmembrane receptors (Patched, Ptc and Smoothened, Smo). Smo is a 7-TM protein reported to be the most druggable target in the Hh-signaling cascade. Control of Smo activity in cells is realized via the 12-TM protein Ptc. Ptc is negatively modulated by direct binding of Hh.

The downstream effectors of Hh signaling have been studied to a lesser extent. Drosophila data suggests that a 155-residue protein Ci (Ci155) forms the Hh-signaling complex (HSC) with Fu (Fused, S/T kinase), suppressor-of-fused (Su(fu)), and a kinesin-like protein Coastal-2 (Cos-2). In the absence of the Hh signal, the components are believed to be associated with microtubules.

Veratrum alkaloids jervine and cyclopamine were among the first identified Hh pathway antagonists, found by following cases of lamb cyclopia. ChemDiv ( completed a successful screening campaign for the Hh pathway modulators. Specifically, pathway activation was measured in both mouse embryonic fibroblasts C3H10T1/2 and Shh-LIGHT2 cells using the control medium described.

Further profiling of the hits included whole-cell binding (293T cells stably over-expressing Smo; Figure 1). As a screening library, the ChemDiv Focused Diversity collection of 5,000 individual molecules was used, enticing a manageable set of compounds with high IP potential and annotated against ca. 60 orthogonal biological targets and pathways (Figure 2). These targets included distinct, structurally unrelated branches of the same target class and/or different classes of targets. Examples of the former proteins include CAMK and TK branches of kinome. The latter biomolecules are represented by GPCR’s and ligand- and voltage-gated ion channels.

A special effort has been made to select templates and respective compounds with good IP potential, as evidenced by Beilstein, SciFinder, and Markush sub-structure searches. Components of this screening set included both traditional (kinases, nuclear hormone receptors) as well as eclectic biological targets (apoptosis, cell cycle), signaling pathways (RTK/Ras), or protein fragments (AVPI, RGD). Chemical epistasis studies in cells stably transfected with Smo were used to determine the molecular level of action for the molecules signaling downstream of Smo.

Specifically, an Hh-blocking antibody, a Smo antagonist cyclopamine and its fluorescent derivatives, and a synthetic Smo antagonist CUR-61414 was used. Further, structure-activity relationship (SAR) studies were constructed for the series of compounds, confirming feasibility of hit optimization in the cell-based reporter assay.

Seven distinct series of potent, drug-like, trackable chemical series that antagonize Hh signaling were identified. These classes have been further expanded via SAR effort to yield compounds with ca. 5–100-nM functional activity in both C3H10T1/2 and Shh-LIGHT2 assays. These series did not have nonspecific cytotoxicity. Competition analysis performed with sHh along with reported synthetic agonists and antagonists of Hh pathway linked Chemdiv’s compounds to three distinct signaling points in the cascade, namely disruption of Hh signaling at the Hh/Ptch, Smo, and downstream of Smo levels (Figure 3).

With the availability of diverse potent small molecule antagonists, the long-term effects of Hh-signaling blockade on the developed organism could be investigated in detail. Small molecule modulators of the Hh pathway may prove to be resistant to the activating mutations in Smo, affect intracellular targets on one or multiple levels of the pathway, and possess tunable efficacy in vivo.

Figure 1

Alex S. Kiselyov is executive vp, R&D, for ChemDiv. Web: Phone: (858) 794-4860. E-mail: [email protected].

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