The Anticancer Activity of a First-in-class Small-molecule Targeting PCNA.

PURPOSE: Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. EXPERIMENTAL DESIGN: By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. RESULTS: AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. CONCLUSIONS: These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.

Facile Preparation of a Thiol-Reactive (18)F-Labeling Agent and Synthesis of (18)F-DEG-VS-NT for PET Imaging of a Neurotensin Receptor-Positive Tumor.

UNLABELLED: Accumulating evidence suggests that neurotensin receptors (NTRs) play key roles in cancer growth and survival. In this study, we developed a simple and efficient method to radiolabel neurotensin peptide with (18)F for NTR-targeted imaging. METHODS: The thiol-reactive reagent (18)F-(2-(2-(2-fluoroethoxy)ethoxy)ethylsulfonyl)ethane ((18)F-DEG-VS) was facilely prepared through 1-step radiofluorination. After high-pressure liquid chromatography purification, (18)F-DEG-VS was incubated with the c(RGDyC) and c(RGDyK) peptide mixture to evaluate its specificity toward the reactive thiol. Thiolated neurotensin peptide was then labeled with (18)F using this novel synthon, and the resulting imaging probe was subjected to receptor-binding assay and small-animal PET studies in a murine xenograft model. The imaging results and metabolic stability of (18)F-DEG-VS-NT were compared with the thiol-specific maleimide derivative N-[2-(4-(18)F-fluorobenzamido)ethyl]maleimide-neurotensin ((18)F-FBEM-NT). RESULTS: (18)F-DEG-VS was obtained in high labeling yield. The reaction of (19)F-DEG-VS was highly specific for thiols at neutral pH, whereas the lysine of c(RGDyK) reacted at a pH greater than 8.5. (18)F-DEG-VS-c(RGDyC) was the preferred product when both c(RGDyK) and c(RGDyC) were incubated together with (18)F-DEG-VS. Thiolated neurotensin peptide (Cys-NT) efficiently reacted with (18)F-DEG-VS, with a 95% labeling yield (decay-corrected). The radiochemical purity of the (18)F-DEG-VS-NT was greater than 98%, and the specific activity was about 19.2 ± 4.3 TBq/mmol. Noninvasive small-animal PET demonstrated that (18)F-DEG-VS-NT had an NTR-specific tumor uptake in subcutaneous HT-29 xenografts. The tumor-to-muscle, tumor-to-liver, and tumor-to-kidney ratios reached 30.65 ± 22.31, 11.86 ± 1.98, and 1.91 ± 0.43 at 2 h after injection, respectively, based on the biodistribution study. Receptor specificity was demonstrated by blocking experiment. Compared with (18)F-FBEM-NT, (18)F-DEG-VS-NT was synthesized with fewer steps and provided significantly improved imaging quality in vivo. CONCLUSION: We have established a facile (18)F-labeling method for site-specific labeling of the Cys-NT. Using this method, we synthesized an NTR-targeted PET agent, which demonstrated high tumor-to-background contrast.

Spirooxindole derivative SOID-8 induces apoptosis associated with inhibition of JAK2/STAT3 signaling in melanoma cells.

Melanoma is generally refractory to current chemotherapy, thus new treatment strategies are needed. In this study, we synthesized a series of spirooxindole derivatives (SOID-1 to SOID-12) and evaluated their antitumor effects on melanoma. Among the 12 spirooxindole derivatives, SOID-8 showed the strongest antitumor activity by viability screening. SOID-8 inhibited viability of A2058, A375, SK-MEL-5 and SK-MEL-28 human melanoma cells in a dose- and time-dependent manner. SOID-8 also induced apoptosis of these tumor cells, which was confirmed by positive Annexin V staining and an increase of poly(ADP-ribose) polymerase cleavage. The antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was downregulated and correlated with SOID-8 induced apoptosis. In addition, SOID-8 reduced tyrosine phosphorylation of Signal Tansducer and Activator of Transcription 3 (STAT3) in both dose- and time-dependent manners. This inhibition was associated with decreased levels of phosphorylation of Janus-activated kinase-2 (JAK2), an upstream kinase that mediates STAT3 phosphorylation at Tyr705. Accordingly, SOID-8 inhibited IL-6-induced activation of STAT3 and JAK2 in melanoma cells. Finally, SOID-8 suppressed melanoma tumor growth in a mouse xenograft model, accompanied with a decrease of phosphorylation of JAK2 and STAT3. Our results indicate that the antitumor activity of SOID-8 is at least partially due to inhibition of JAK2/STAT3 signaling in melanoma cells. These findings suggest that the spirooxindole derivative SOID-8 is a promising lead compound for further development of new preventive and therapeutic agents for melanoma.

Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development.

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that overexpress active Stat3, leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.