In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway have been under-explored. To date, there are no small molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 (Maml1) to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization.

In this report, we demonstrate that the internal disulfide bridge in human neuroglobin modulates structural changes associated with ligand photo-dissociation from the heme active site. This is evident from time-resolved photothermal studies of CO photo-dissociation, which reveal a 13.4+/-0.9 mL mol(-1) volume expansion upon ligand photo-release from human neuroglobin, whereas the CO dissociation from rat neuroglobin leads to a significantly smaller volume change (DeltaV=4.6+/-0.3 mL mol(-1)). Reduction of the internal disulfide bond in human neuroglobin leads to conformational changes (reflected by DeltaV) nearly identical to those observed for rat Ngb. Our data favor the hypothesis that the disulfide bond between Cys46 and Cys55 modulates the functioning of human neuroglobin.

In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.