The mTOR Inhibitor RAD001 Sensitizes Tumor Cells to DNA-Damaged Induced Apoptosis through Inhibition of p21 Translation

SummaryAlthough DNA damaging agents have revolutionized chemotherapy against solid tumors, a narrow therapeutic window combined with severe side effects has limited their broader use. Here we show that RAD001 (everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53, but not mutant p53 tumor cells. The use of isogenic tumor cell lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. We further show that RAD001 sensitizes cells to cisplatin by inhibiting p53-induced p21 expression. Unexpectedly, this effect is attributed to a small but significant inhibition of p21 translation combined with its short half-life. These findings provide the molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors

A critical assessment of the synthesis and biological activity of p53/Hdm2 stapled peptide inhibitors

The covalent linkage of amino acid side chains to conformationally constrain -helices has been employed as a powerful mean to enhance the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cell entry including cytoplasmic targeting. In particular, stapled peptides inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein were of interest for this approach. Here, we scrutinized to which degree the pharmacokinetic characteristics are a function of the staple and differ from those of a standard cationic cell-penetrating peptide (CPP). Stapled peptides and their linear counterparts were synthesized to verify activity in biochemical and cellular assays. All peptides showed potent sub-nanomolar potency to Hdm2. Assessing uptake for carboxyfluorescein-labeled variants, for short incubation times, there was only little difference in uptake efficiency for the stapled peptides and their linear counterpart and both were taken up less efficiently than the prototypic CPP nonaarginine (R9). Fluorescence was restricted to vesicular structures. Only following long-term incubation, and for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides and also the linear counterparts, albeit to a lesser degree, showed an enhanced cytoplasmic and nuclear accumulation. For HeLa cells, lacking target expression no such accumulation was observed. These findings demonstrate that the cytosolic and nuclear accumulation are not an intrinsic property of the stapled peptide but result from capture by the target Hdm2 once leaking out of the endo-lysosomal compartment

Discovery of dihydroisoquinolinone derivatives as novel inhibitors of the p53-MDM2 interaction with a distinct binding mode

Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogues. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors

The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels.

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCFSkp2 ubiquitin ligase has been reported to mediate p27Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27Kip1, and prevent cellular proliferation. Elevation of p27Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCFSkp2) ubiquitin ligase substrate p27Kip1, but has no concomitant effect on the level of IkBalpha and beta-catenin, which are known substrates of a closely related SCF ligase

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects

Discovery of a novel class of highly potent inhibitors of the p53–MDM2 interaction by structure-based design starting from a conformational argument

The p53–MDM2 interaction is an anticancer drug target under investigation in the clinic. Our compound NVP-CGM097 is one of the small molecule inhibitors of this protein–protein interaction currently evaluated in cancer patients. As part of our effort to identify new classes of p53–MDM2 inhibitors that could lead to additional clinical candidates, we report here the design of highly potent inhibitors having a pyrazolopyrrolidinone core structure. The conception of these new inhibitors originated in a consideration on the MDM2 bound conformation of the dihydroisoquinolinone class of inhibitors to which NVP-CGM097 belongs. This work forms the foundation of the discovery of HDM201, a second generation p53–MDM2 inhibitor that recently entered phase I clinical trial

A double blinded, placebo-controlled pilot study to examine reduction of CD34+/CD117+/CD133+ lymphoma progenitor cells and duration of remission induced by neoadjuvant valspodar in dogs with large B-cell lymphoma [version 3; referees: 2 approved]

We previously described a population of lymphoid progenitor cells (LPCs) in canine B-cell lymphoma defined by retention of the early progenitor markers CD34 and CD117 and “slow proliferation” molecular signatures that persist in the xenotransplantation setting. We examined whether valspodar, a selective inhibitor of the ATP binding cassette B1 transporter (ABCB1, a.k.a., p-glycoprotein/multidrug resistance protein-1) used in the neoadjuvant setting would sensitize LPCs to doxorubicin and extend the length of remission in dogs with therapy naïve large B-cell lymphoma. Twenty dogs were enrolled into a double-blinded, placebo controlled study where experimental and control groups received oral valspodar (7.5 mg/kg) or placebo, respectively, twice daily for five days followed by five treatments with doxorubicin 21 days apart with a reduction in the first dose to mitigate the potential side effects of ABCB1 inhibition. Lymph node and blood LPCs were quantified at diagnosis, on the fourth day of neoadjuvant period, and 1-week after the first chemotherapy dose. Valspodar therapy was well tolerated. There were no differences between groups in total LPCs in lymph nodes or peripheral blood, nor in event-free survival or overall survival. Overall, we conclude that valspodar can be administered safely in the neoadjuvant setting for canine B-cell lymphoma; however, its use to attenuate ABCB1+ cells does not alter the composition of lymph node or blood LPCs, and it does not appear to be sufficient to prolong doxorubicin-dependent remissions in this setting