RNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures.

We optimized our previously reported proline-based STAT3 inhibitors into an exciting new series of ( R )-azetidine-2-carboxamide analogues that have sub-micromolar potencies. 5a , 5o , and 8i have STAT3-inhibitory potencies (IC 50 ) of 0.55, 0.38, and 0.34 μM, respectively, compared to potencies greater than 18 μM against STAT1 or STAT5 activity. Further modifications derived analogues, including 7e , 7f , 7g , and 9k , that addressed cell membrane permeability and other physicochemical issues. Isothermal titration calorimetry analysis confirmed high-affinity binding to STAT3, with K D of 880 nM ( 7g ) and 960 nM ( 9k ). 7g and 9k inhibited constitutive STAT3 phosphorylation and DNA-binding activity in human breast cancer, MDA-MB-231 or MDA-MB-468 cells. Furthermore, treatment of breast cancer cells with 7e , 7f , 7g , or 9k inhibited viable cells, with an EC 50 of 0.9–1.9 μM, cell growth, and colony survival, and induced apoptosis while having relatively weaker effects on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively active STAT3.

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Rational structure‐based drug design relies on a detailed, atomic‐level understanding of protein–ligand interactions. The chiral nature of drug binding sites in proteins has led to the discovery of predominantly chiral drugs. A mechanistic understanding of stereoselectivity (which governs how one stereoisomer of a drug might bind stronger than the others to a protein) depends on the topology of stereocenters in the chiral molecule. Chiral graphs and reduced chiral graphs, introduced here, are new topological representations of chiral ligands using graph theory, to facilitate a detailed understanding of chiral recognition of ligands/drugs by proteins. These representations are demonstrated by application to all ≈14 000+ chiral ligands in the Protein Data Bank (PDB), which will facilitate an understanding of protein–ligand stereoselectivity mechanisms. Ligand modifications during drug development can be easily incorporated into these chiral graphs. In addition, these chiral graphs present an efficient tool for a deep dive into the enormous chemical structure space to enable sampling of unexplored structural scaffolds.

Signal Transducer and Activator of Transcription (STAT)3 is a member of the STAT family of transcription factors that is frequently aberrantly-activated in breast cancer and many other cancers. STAT3 represents a valid target for the development of novel anticancer therapeutics. However, it has posed significant challenges to the discovery efforts to identify potent and suitable small molecule inhibitors. As a result, there is currently no anti-STAT3 drugs in clinical application to realize the potential clinical benefits of inhibiting abnormal STAT3 function. Herein is a new series of (R)-azetidine-2-carboxamide-based small molecules, exemplified by H098, H127, H152, H169, H174, H182, and H203, which selectively inhibit STAT3 DNA-binding activity, with in vitro potency (IC50) in the range of 0.28-0.66 μM, compared to potencies greater than 18 µM against the activities of the family members, STAT1 and STAT5. Equally notable is the high affinity binding to STAT3, as shown by KD of 1 pM and 3.7 nM for H169 and H174, respectively. Treatment of human triple-negative breast cancer (TNBC) cells with H182 or H169 inhibited constitutive STAT3 phosphorylation (pY705STAT3) and DNA-binding activity in both time- and dose-dependent manner. Furthermore, both inhibitors potently inhibited anchorage-dependent and independent growth, colony survival, and migration of TNBC cells in vitro, with potencies, EC50 of 1.0-1.9 μM, and the compounds also induced apoptosis of same cells. Additionally, the expression of STAT3 target genes, including c-Myc, VEGF, and survivin, were suppressed in the compound-treated TNBC cells subsequent to the inhibition of constitutive STAT3 activation, thereby validating the inhibition of constitutively-active STAT3 and its downstream targets as being responsible for the antitumor cell responses. Moreover, H169 and docetaxel combinatorial treatment led to enhanced suppression of TNBC cell viability. By contrast, treatments with the new compounds had minimal effects on the induction of STAT1, Janus kinase 2, epidermal growth factor receptor, extracellular signal-regulated kinases (Erk1/2), Src, Shc phosphatase, or Akt in TNBC cells, and they were relatively weak on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively-active STAT3. Significantly, H182 delivered at 5-20 mg/kg via oral gavage or intra-peritoneal route inhibited growth of TNBC tumor xenograft in mice, with no evidence of toxicity. These studies identify novel, sub-micromolar potent azetidine-based small molecule STAT3 inhibitors that have shown antitumor efficacy against human TNBC in xenograft models. H182 and H169 represent suitable novel chemical entities for developing new therapeutics against TNBC and other cancers that harbor constitutively-active STAT3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Christine Brotherton-Pleiss, Wenzhen Fu, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1230.