Notch signaling is an intercellular communication system coordinating developmental and homeostatic key processes in multiple tissue types. Perturbations in this pathway have been linked to pathogenesis and chemoresistance of multiple tumors, making its targeting one of the most promising anti-cancer therapeutic strategies. However, current Notch-interfering approaches under evaluation in clinical trials show drawbacks that limit their therapeutic use. Among the four Notch receptors (Notch1-4), inappropriate Notch3 signaling has been associated with the pathogenesis, poor prognosis and chemoresistance in specific subtypes of cancers, thus providing the rationale to develop novel Notch3 targeting-based therapies in such tumors. The aim of our proposal is to identify more effective novel Notch3-inhibitors with reduced associated toxicities with respect to the available therapeutic agents against Notch3-driven cancers. Our hypothesis is that small molecules docking within the Negative Regulatory Region (NRR) of Notch3 could stabilize the receptor in an auto-inhibited conformational status and provide signaling inhibition. Therefore, a library of natural products will be screened by molecular docking simulations against the computational structural model of the Notch3 NRR and the effectiveness of the most promising virtual hits will be investigated by assessing their strength in impairing Notch3 signaling and tumor growth in Notch3-dependent tumor contexts. Most promising compounds will be further optimized in terms of potency and specificity of action by recalling the computational modeling strategy, and structure-activity relationships will be afforded. The expected outcomes from this research proposal would provide the breakthrough in the design of novel single or combinatorial treatment of Notch3-addicted cancers.
Currently, a few classes of Notch inhibitors, characterized by different target specificity or mechanism of action, are under preclinical evaluation and some of them have been moved to the clinical trial investigation. At the state of art, the most promising class of agents blocking Notch activity includes small molecules of various chemical origins, which inhibit the final S3 proteolytic cleavage of Notch receptors (GSIs). Most of these chemicals have shown strong anti-cancer activity and favorable tissue penetration in numerous pre-clinical models, especially in combination with chemotherapy and some them are under clinical trial evaluation for the treatment of both pediatric and adult malignancies (1). However, the in vivo administration of GSIs is associated with serious adverse effects due to their own poor selectivity of action among different Notch receptors and to a plethora of additional gamma secretases substrates (2). The most common GSIs drawback is the goblet-cell metaplasia of the small intestinal epithelium due to perturbed intestinal stem cell function in the crypts caused by combined inhibition of Notch1 and Notch2 signaling (3). Notably, Notch3 has more restricted tissues distribution compared to Notch1 and Notch2. In fact, Notch3 is not expressed in the gut epithelium and has significantly less functional relevance in this context. (4). Therefore the selective therapeutic targeting of Notch3 may avoid the intestinal toxicity caused by PAN-Notch inhibition. Accordingly, expected results from our proposal of research, designed to discover novel specific Notch3 antagonist agents, could drive the development of novel therapeutic strategies, more effective and safe than GSIs, that could overcome the greatest challenge in the Notch therapeutic targeting field.
An alternative route to target Notch is the selective targeting of Notch receptors either with ligand-competitive antibodies (Abs) against the region of the receptor that encompasses the ligand-binding domain or with the most powerful allosteric Abs directed against the Notch NRR that stabilize the receptor in off state (1). Although these Abs inhibited the growth of cancer stem cell, promoted cell differentiation and disrupted tumor angiogenesis in preclinical experimental models, their effectiveness and safety have not yet been confirmed clinically. At present, the arsenal of Notch Abs under clinical evaluation for different tumors includes the Abs against the NRR of Notch1 (Trial ID: NCT01778439) and the cross-reactive Abs against Notch-2/3 (Trials ID: NCT01859741, NCT01647828, and NCT01277146). Despite the potential advantage in terms of target specificity, current therapeutic Abs are associated with several drawbacks limiting their widespread use. Antibodies limitations include inadequate pharmacokinetics and tissue accessibility, repeated treatment requirement, limited duration of action, undesired immune reactions and high costs of production (5).
Since Notch3 NRR activity perturbation by Abs appears to be promising for the treatment of solid cancers in several pre-clinical studies, the alternative or complementary targeting of Notch3-NRR by natural small-molecule agents could improve Notch3 targeting therapies in terms of delivery (as mABs do not penetrate the blood-brain barrier), cancer retention, pharmacokinetics and also in terms of cost. Overall, the aim of our project is to identify potential and context-dependent therapeutic agents against Notch3 to attenuate off-target and side effects of the conventional pan-Notch inhibitors and to avoid the limits of Abs. Although the selective Notch3 targeting in Notch3-dependent tumor, most likely, will not give us the aspired magic bullet to eradicate the tumor, the expected results from this research will have a significant impact in the field of the cancer-fighting drugs, providing, in the perspective of a personalized medicine, breakthrough in the design of novel combinatorial treatment of Notch-dependent cancers.
References:
1. Takebe, N, et al. (2014). Pharmacol Ther, 141(2), 140-149.
2. Haapasalo, A, and Kovacs, DM (2011). T J Alzheimers Dis, 25(1), 3-28.
3. Vooijs, M., et al. (2011). Gastroenterology, 141(2), 448-459.
4. Fre, S., et al (2011). PLoS One, 6(10), e25785.
5. Elloumi J et al. (2012) Recent patents on biotechnology;6(1):45K56.