An optimization of the guanidylation process by verifying the efficacy of common guanylation reagents in order to obtain the guanidine derivatives of indolo[2,3-b]quinoline has been performed. As a result, a high-yield procedure using N,N′-di-Boc-N′′-triflylguanidine was applied to synthesize the guanidine derivative of indolo[2,3-b]quinoline 1 in a gram scale for specific in vitro and in vivo biological research. Extensive studies on the antiproliferative activity against eight human tumor cell lines were completed. Compound 1 revealed the highest activity against A549 lung adenocarcinoma and MCF7 breast cancer cell lines. Thus, 1 was evaluated for the in vivo anticancer activity against 4T1 mammary gland carcinoma and KLN205 murine lung carcinoma in mouse models. The anticancer effect was observed in the KLN205 model with a 37% tumor growth inhibition at the 20 mg/kg dose. This anticancer activity of 1 was comparable to that of cyclophosphamide which inhibited murine lung tumor growth in the range of 27–43% at the dose of 100 mg/kg. The biochemistry research after 1 admission, including measurements of blood parameters like alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and urea and creatinine, were also performed.

Targeting of the TRAIL‐DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD‐O51.4, which is a TRAIL equipped with positively charged VEGFA‐derived effector peptides. The study was performed in multiple cancer cell line‐ and patient‐derived xenografts. A pharmacokinetic profile was established in monkeys. AD‐O51.4 strongly inhibits tumor growth, even leading to complete long‐term tumor remission. Neither mice nor monkeys treated with AD‐O51.4 demonstrate symptoms of drug toxicity. AD‐O51.4 exhibits a satisfactory half‐life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD‐O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD‐O51.4‐driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL‐sensitive and TRAIL‐resistant cancer cells, respectively. The FADD‐dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD‐O51.4 is capable of bypassing the refractoriness of TRAIL. AD‐O51.4‐driven cell death, which exceeds TRAIL activity, is achieved due to the N‐terminally fused polypeptide, containing VEGFA‐derived effector peptides. The high anticancer efficiency of AD‐O51.4 combined with its safety has led to the entry of AD‐O51.4 into toxicological studies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors became promising molecules for selective targeting of tumor cells without affecting normal tissue. Unfortunately, cancer cells have developed a number of mechanisms that confer resistance to TRAIL\Apo2L-induced apoptosis, which substantiates the need for development of alternative therapeutic strategies. Here we present a recombinant variant of TRAIL\Apo2L peptide, named AD-O53.2, fused to the peptide-derived from Smac/Diablo protein-the natural inhibitor of the apoptotic X-linked IAP (XIAP) protein considered as a pro-apoptotic agent. The proposed mechanism of action for this construct involves specific targeting of the tumor by TRAIL\Apo2L followed by activation and internalization of pro-apoptotic peptide into the cancer cells. While in the cytoplasm , the Smac\Diablo peptide inhibits activity of X-linked IAP (XIAP) proteins and promotes caspase-mediated apoptosis. AD-O53.2 construct was expressed in E.coli and purified by Ion Exchange Chromatography (IEC). Derived protein was initially characterized by circular dichroism spectroscopy (CD), HPLC-SEC chromatography, surface plasmon resonance, protease activation and cell proliferation assays. Our Smac/Diablo-TRAIL fusion variant was tested against a panel of cancer cells (including lung, colorectal, pancreatic, liver, kidney and uterine) and showed a potent cytotoxic effect with the IC50 values in femtomolar range for the most sensitive cell lines, while it remained ineffective against non-transformed HUVEC cells as well as isolated normal human and rat hepatocytes. Importantly, the construct was well tolerated by animals and significantly reduced the rate of the tumor growth in colon and lung adenocarcinoma animal models.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising antitumor agent, but its clinical utility in human malignancies is limited by the resistance mechanism evolved in many cancers types, including overexpresion of antiapoptotic Bcl-2 like proteins and lack or disfunction of proapoptotic agents. Bid is well known proapoptotic factor able to inhibit Bcl-2 proteins after activation by caspases 8 or 10. Bid may also promote apoptosis by forming selective voltage dependent anion-channels in mitochondrial membrane through interaction with Bax protein. AD-O57.4 is a fusion protein consisting of a short peptide derived from Bid linked to an amino terminus of soluble TRAIL domain. The separating sequence contains additional motif recognized by MMPs and uPa proteases that enables cutting-off and releasing the proapoptotic peptide in tumor environment. Activity of AD-O57.4 was examined using a MTT assay on NCI panel and primal, patient derived cancer cell lines. Apoptosis was analyzed by caspase 3 activation and mitochondrial membrane potential change. The expression of Bid was determined by Western Blot analysis. Various of tested cell lines were very sensitive to AD-O57.4 protein with IC50 value between 0.06-173 ng/ml, whereas 5 cell lines showed weak sensitivity or remained resistant. Cytotoxic activity of TRAIL was significantly lower in comparison with AD-O57.4 protein. AD-O57.4 induced relatively strong mitochondrial depolarization both in moderate sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549). Analysis of caspase 3 activation and expression of Bid protein showed very fast induction of apoptosis by AD-O57.4 in comparison to TRAIL protein. Strong antitumor activity of AD-O57.4 fusion protein was analyzed on the xenograft models of series of human cancer cell lines regarding TRAIL sensitive human colorectal carcinoma Colo205, TRAIL resistant, hepatocellular carcinoma HepG2 and human uterine sarcoma (MES-SA/Dx5) - a multidrug resistant cell line where it caused almost complete tumor regression and showed much higher efficacy than TRAIL alone or standard chemotherapeutic agent. We demonstrated that AD-O57.4 protein has broad in vitro cytotoxic activity against a panel of cancer cell lines and in vivo antitumor activity on xenograft model. Sensitization by AD-O57.4 was dependent on short peptide derived from Bid protein indicating its role for mitochondrial signal amplification in the proapoptotic TRAIL activity. The use of a peptide derived from Bid protein causes a significant increase in potency of TRAIL activity and therefore TRAIL resistant cancer cells can be sensitized by the fusion peptide which can connect extrinsic and intrinsic pathways of apoptosis. Citation Format: Jerzy S. Pieczykolan, Sebastian D. Pawlak, Bartlomiej M. Zerek, Piotr K. Rozga, Anna Pieczykolan, Michal Szymanik, Albert Jaworski, Marlena Galazka, Wojciech Strozek, Katarzyna Wiciejowska, Malgorzata Teska-Kaminska, Lukasz Kutner. Fusion protein capable to initiate two apoptotic pathways in cancer cells resistant to classical PARA's. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5488. doi:10.1158/1538-7445.AM2013-5488

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