TrkC Targeted Probes for Cancer Diagnosis and Therapeutics

This dissertation features a small molecule, non-peptidic, ligand designed to bind a cell surface receptor called tropomyosin receptor kinase C (TrkC). TrkC is overexpressed on various types of tumors including breast cancer and melanoma. It would be advantageous in clinical applications to conjugate the novel ligand with imaging or therapeutic agents then treat TrkC-positive cancers. For proof of concept, first, we used our ligand conjugated to a commercial cytotoxic drug, 6-mercaptopurine, then tested the agent in NIH-3T3 cells stably transfected with TrkC, and compared with wild type NIH-3T3 (TrkC-). A targeting effect was observed but we could not obtain an IC50 value for the conjugate due to lack of solubility at the concentrations that would have been required. Therefore, we chose a cytotoxic rosamine dye to conjugate with the targeting ligand. Selective cytotoxicity of the conjugate was observed, but high levels of non-specific binding also occurred. Conjugation of the TrkC-targeting ligand with a fluorescent dye may be useful for optical imaging in vivo, and they are also cytotoxic then these theranostics can be used to both image and treat the tumor. We hypothesized that BODIPY dye derivatives could be suitable candidates for this approach since they have excellent fluorescent characteristics and can be modified for photodynamic therapy (PDT). Results collected from cellular assays proved the selectivity of this probe and its fluorescence led us to where the conjugate functioned inside the living cells. Some cancer cell lines that naturally overexpress TrkC include metastatic breast cancer and metastatic melanoma. Consequently, this study was expanded from stable TrkC transfectants to include metastatic-breast and –melanoma. The results show the beneficial effects of TrkC targeting in histochemistry and cellular assays, including internalization and selective cell killing. In a mouse model, collaborators discovered our PDT probe at 10 mg/kg (one injection, intravenous tail vein) caused complete ablation of a tumor after 6 days post injection with no sign of metastasis to other organs. PDT can be effective if near-IR absorbing photosensitizers (λmax >700 nm) are used to excite organs located in the deeper tissue. For this reason, the cargo was modified to aza-BODIPY dyes, which can absorb light at a longer wavelength than BODIPYs. Simultaneously, the conjugate structure was changed because we hypothesized this might lead to decreased neurotoxicity. Histological studies showed the new probe selectively stained breast tissues leaving normal tissues unstained. In vivo optical imaging also proved that the new TrkC targeting probe was effective in a murine breast cancer model. Incidentally, the spinal cord of the mouse was nicely imaged by this same reagent, presumably because TrkC+ cells are highly concentrated in the peripheral nervous system. Finally, we hypothesized that the novel near-IR targeting probe could be applied to imaging and treatment of metastatic melanoma. A rare Sinclair Swine model, available at TAMU, was part of the plan for these studies. Histology results showed selective staining by the targeting probe on melanoma tissue, as compared with normal tissue obtained from the same pig. Similarly, our probe stained tissue from human patients with metastatic melanoma tissue, but it did not stain healthy skin

TrkC Targeted Probes for Cancer Diagnosis and Therapeutics

This dissertation features a small molecule, non-peptidic, ligand designed to bind a cell surface receptor called tropomyosin receptor kinase C (TrkC). TrkC is overexpressed on various types of tumors including breast cancer and melanoma. It would be advantageous in clinical applications to conjugate the novel ligand with imaging or therapeutic agents then treat TrkC-positive cancers. For proof of concept, first, we used our ligand conjugated to a commercial cytotoxic drug, 6-mercaptopurine, then tested the agent in NIH-3T3 cells stably transfected with TrkC, and compared with wild type NIH-3T3 (TrkC-). A targeting effect was observed but we could not obtain an IC50 value for the conjugate due to lack of solubility at the concentrations that would have been required. Therefore, we chose a cytotoxic rosamine dye to conjugate with the targeting ligand. Selective cytotoxicity of the conjugate was observed, but high levels of non-specific binding also occurred. Conjugation of the TrkC-targeting ligand with a fluorescent dye may be useful for optical imaging in vivo, and they are also cytotoxic then these theranostics can be used to both image and treat the tumor. We hypothesized that BODIPY dye derivatives could be suitable candidates for this approach since they have excellent fluorescent characteristics and can be modified for photodynamic therapy (PDT). Results collected from cellular assays proved the selectivity of this probe and its fluorescence led us to where the conjugate functioned inside the living cells. Some cancer cell lines that naturally overexpress TrkC include metastatic breast cancer and metastatic melanoma. Consequently, this study was expanded from stable TrkC transfectants to include metastatic-breast and –melanoma. The results show the beneficial effects of TrkC targeting in histochemistry and cellular assays, including internalization and selective cell killing. In a mouse model, collaborators discovered our PDT probe at 10 mg/kg (one injection, intravenous tail vein) caused complete ablation of a tumor after 6 days post injection with no sign of metastasis to other organs. PDT can be effective if near-IR absorbing photosensitizers (λmax >700 nm) are used to excite organs located in the deeper tissue. For this reason, the cargo was modified to aza-BODIPY dyes, which can absorb light at a longer wavelength than BODIPYs. Simultaneously, the conjugate structure was changed because we hypothesized this might lead to decreased neurotoxicity. Histological studies showed the new probe selectively stained breast tissues leaving normal tissues unstained. In vivo optical imaging also proved that the new TrkC targeting probe was effective in a murine breast cancer model. Incidentally, the spinal cord of the mouse was nicely imaged by this same reagent, presumably because TrkC+ cells are highly concentrated in the peripheral nervous system. Finally, we hypothesized that the novel near-IR targeting probe could be applied to imaging and treatment of metastatic melanoma. A rare Sinclair Swine model, available at TAMU, was part of the plan for these studies. Histology results showed selective staining by the targeting probe on melanoma tissue, as compared with normal tissue obtained from the same pig. Similarly, our probe stained tissue from human patients with metastatic melanoma tissue, but it did not stain healthy skin

Unique structural solution from a VH3-30 antibody targeting the hemagglutinin stem of influenza A viruses

Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV

A Modular Class of Fluorescent Difluoroboranes: Synthesis, Structure, Optical Properties, Theoretical Calculations and Applications for Biological Imaging.

Ten borylated bipyridines (BOBIPYs) have been synthesized and selected structural modifications have been made that allow useful structure-optical property relationships to be gathered. These systems have been further investigated using DFT calculations and spectroscopic measurements, showing blue to green fluorescence with quantum yields up to 41 %. They allow full mapping of the structure to determine where selected functionalities can be implemented, to tune the optical properties or to incorporate linking groups. The best derivative was thus functionalised with an alkyne linker, which would enable further applications through click chemistry and in this optic, the stability of the fluorophores has been evaluated

Tropomyosin Receptor Kinase C Targeted Delivery of a Peptidomimetic Ligand-Photosensitizer Conjugate Induces Antitumor Immune Responses Following Photodynamic Therapy

Tropomyosin receptor kinase C (TrkC) targeted ligand-photosensitizer construct, IYIY-diiodo-boron-dipyrromethene (IYIY-I(2)-BODIPY) and its scrambled counterpart YIYI-I(2)-BODIPY have been prepared. IYIY-I(2)-BODIPY binds TrkC similar to neurotrophin-3 (NT-3), and NT-3 has been reported to modulate immune responses. Moreover, it could be shown that photodynamic therapy (PDT) elevates antitumor immune responses. This prompted us to investigate the immunological impacts mediated by IYIY-I(2)-BODIPY in pre- and post-PDT conditions. We demonstrated that IYIY-I(2)-BODIPY (strong response) and YIYI-I(2)-BODIPY (weak response) at 10 mg/kg, but not I(2)-BODIPY control, increased the levels of IL-2, IL-4, IL-6 and IL-17, but decreased the levels of systemic immunoregulatory mediators TGF-β, myeloid-derived suppressor cells and regulatory T-cells. Only IYIY-I(2)-BODIPY enhanced the IFN-γ(+) and IL-17(+) T-lymphocytes, and delayed tumor growth (~20% smaller size) in mice when administrated daily for 5 days. All those effects were observed without irradiation; when irradiated (520 nm, 100 J/cm(2), 160 mW/cm(2)) to produce PDT effects (drug-light interval 1 h), IYIY-I(2)-BODIPY induced stronger responses. Moreover, photoirradiated IYIY-I(2)-BODIPY treated mice had high levels of effector T-cells compared to controls. Adoptive transfer of immune cells from IYIY-I(2)-BODIPY-treated survivor mice that were photoirradiated gave significantly delayed tumor growth (~40–50% smaller size) in recipient mice. IYIY-I(2)-BODIPY alone and in combination with PDT modulates the immune response in such a way that tumor growth is suppressed. Unlike immunosuppressive conventional chemotherapy, IYIY-I(2)-BODIPY can act as an immune-stimulatory chemotherapeutic agent with potential applications in clinical cancer treatment

Near-IR Absorbing BODIPY Derivates as Glutathione-Activated Photosensitizers for Selective Photodynamic Action

Cataloged from PDF version of article.Enhanced spatiotemporal selectivity in photonic sensitization of dissolved molecular oxygen is an important target for improving the potential and the practical applications of photodynamic therapy. Considering the high intracellular glutathione concentrations within cancer cells, a series of BODIPY-based sensitizers that can generate cytotoxic singlet oxygen only after glutathione-mediated cleavage of the electron-sink module were designed and synthesized. Cell culture studies not only validate our design, but also suggest an additional role for the relatively hydrophobic quencher module in the internalization of the photosensitizer

Bacopa monnieri extract increases rat coronary flow and protects against myocardial ischemia/reperfusion injury

Background: This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. Methods: In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 μg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca2+-currents (ICa, L) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. Results: In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 μg/ml) and 216 ± 21% (100 μg/ml), compared to control (5 ± 3%) (n = 8–10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 μg/ml), 82 ± 10% (100 μg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 μg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 μg/ml) reduced ICa, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50–1000 μg/ml) B. monnieri. Conclusions: B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy