1. Solid phase synthesis of Smac/DIABLO-derived peptides using a 'Safety-Catch' resin: identification of potent XIAP BIR3 antagonists
Mohamed A Elsawy, Lorraine Martin, Irina G Tikhonova, Brian Walker Bioorg Med Chem. 2013 Sep 1;21(17):5004-11. doi: 10.1016/j.bmc.2013.06.055. Epub 2013 Jul 2.
The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide 'Safety-Catch' resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1μM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.
2. Apoptosis-inducing peptide loaded in PLGA nanoparticles induces anti-tumor effects in vivo
Dwi L Priwitaningrum, Julian Jentsch, Ruchi Bansal, Sima Rahimian, Gert Storm, Wim E Hennink, Jai Prakash Int J Pharm. 2020 Jul 30;585:119535. doi: 10.1016/j.ijpharm.2020.119535. Epub 2020 Jun 10.
Induction of apoptosis in tumor cells specifically within the complex tumor microenvironment is highly desirable to kill them efficiently and to enhance the effects of chemotherapy. Second mitochondria-derived activator of caspase (Smac) is a key pro-apoptotic pathway which can be activated with a Smac mimetic peptide. However, in vivo application of peptides is hampered by several limitations such as poor pharmacokinetics, rapid elimination, enzymatic degradation, and insufficient intracellular delivery. In this study, we developed a nanosystem to deliver a Smac peptide to tumor by passive targeting. We first synthesized a chimeric peptide that consists of the 8-mer Smac peptide and a 14-mer cell penetrating peptide (CPP) and then encapsulated the Smac-CPP into polymeric nanoparticles (Smac-CPP-NPs). In vitro, Smac-CPP-NPs were rapidly internalized by 4T1 mammary tumor cells and subsequently released Smac-CPP into the cells, as shown with fluorescence microscopy. Furthermore, Smac-CPP-NPs induced apoptosis in tumor cells, as confirmed with cell viability and caspase 3/7 assays. Interestingly, combination of Smac-CPP-NPs with doxorubicin (dox), a clinically used cytostatic drug, showed combined effects in vitro in 4T1 cells. The effect was significantly better than that of SMAC-CPP-NPs alone as well as empty nanoparticles and dox. In vivo, co-treatment with Smac-CPP-NPs and free dox reduced the tumor growth to 85%. Furthermore, the combination of Smac-CPP-NPs and free dox showed reduced proliferating tumor cells (Ki-67 staining) and increased apoptotic cells (cleaved caspase-3 staining) in tumors. In conclusion, the present study demonstrates that the intracellular delivery of Smac-mimetic peptide using nanoparticle system can be an interesting strategy to attenuate the tumor growth and to potentiate the therapeutic efficacy of chemotherapy in vivo.
3. Combination therapy using Smac peptide and doxorubicin-encapsulated MUC 1-targeted polymeric nanoparticles to sensitize cancer cells to chemotherapy: An in vitro and in vivo study
Mojgan Nejabat, Farhad Eisvand, Fatemeh Soltani, Mona Alibolandi, Seyed Mohammad Taghdisi, Khalil Abnous, Farzin Hadizadeh, Mohammad Ramezani Int J Pharm. 2020 Sep 25;587:119650. doi: 10.1016/j.ijpharm.2020.119650. Epub 2020 Jul 15.
Targeting inhibitors of apoptosis proteins (IAPs) family comprising high level expression in many cancer cells, could sensitize tumor cells to conventional chemotherapies. In the present study, we designed both doxorubicin and SmacN6 (an antagonist of the IAPs) encapsulated polymeric nanoparticles (NPs) and investigated their synergistic effect of combination therapy in vitro and in vivo. According to the results, NPs-SmacN6 significantly enhanced the cytotoxicity effect of NPs-DOX and reduced its IC50 in MCF-7, 4T1 and C26 cancer cells. Western blot analysis confirmed mechanism of cell apoptosis via caspase activation through intrinsic and also extrinsic pathways. Moreover, 5TR1 aptamer-modified NPs could effectively deliver DOXor SmacN6 to C26 cancer cells (MUC1 positive) in comparison with the non-targeted one (p < 0.001). However, they could not be efficiently internalized into CHO cells (MUC1 negative), showing less cytotoxicity in this cell line. In vivo experiments in BALB/c mice bearing C26 tumor indicated that Apt-NPs-DOX in combination with Apt-NPs-SmacN6 had significant tumor growth inhibition in comparison with mice receiving either free DOX or Apt-NPs-DOX with p < 0.0001 and p < 0.05, respectively. Our results revealed that combination therapy of DOX and SmacN6 via Apt-modified nanoparticles can lead to improvement of therapeutic index of DOX in MUC1 positive cancer cells.
