The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1 H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo. The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.

Anti-apoptotic proteins, like the Bcl-2 family proteins, present an important therapeutic cancer drug target. Their activity is orchestrated through neutralization upon interaction of pro-apoptotic protein counterparts that leads to immortality of cancer cells. Therefore, generating compounds targeting these proteins is of immense therapeutic importance. Herein, Induced Fit Docking (IFD) and Molecular Dynamics (MD) simulations were performed to rationally design quercetin analogues that bind in the BH3 site of the Bcl-xL protein. IFD calculations determined their binding cavity while Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) calculations provided an insight into the binding enthalpies of the analogues. The quercetin analogues were synthesized and their binding to Bcl-xL was verified with fluorescence spectroscopy. The binding affinity and the thermodynamic parameters between Bcl-xL and quercetin-glutamic acid were estimated through Isothermal Titration Calorimetry. 2D H-N HSQC NMR chemical shift perturbation mapping was used to chart the binding site of the quercetin analogues in the Bcl-xL that overlapped with the predicted poses generated by both IFD and MD calculations. Furthermore, evaluation of the four conjugates against the prostate DU-145 and PC-3 cancer cell lines, revealed quercetin-glutamic acid and quercetin-alanine as the most potent conjugates bearing the higher cytostatic activity. This pinpoints that the chemical space of natural products can be tailored to exploit new hits for difficult tractable targets such as protein-protein interactions.

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Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT 2 R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT 2 R. We repurposed EMA401, an AT 2 R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT 2 R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT 2 R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or β-arrestin recruitment. The architecture and interactions of EMA401 in AT 2 R differ drastically from complexes of AT 2 R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2–tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

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