Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals.

Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.

Mitochondrial copper depletion suppresses triple-negative breast cancer in mice.

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo.

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

A Universal Assay for Aminopeptidase Activity and Its Application for Dipeptidyl Peptidase-4 Drug Discovery.

Aminopeptidases, such as dipeptidyl peptidase-4 (DPP-4, CD26), are potent therapeutic targets for pharmacological interventions because they play key roles in many important pathological pathways. To analyze aminopeptidase activity in vitro (including high-throughput screening [HTS]), in vivo, and ex vivo, we developed a highly sensitive and quantitative bioluminescence-based readout method. We successfully applied this method to screening drugs with potential DPP-4 inhibitory activity. Using this method, we found that cancer drug mitoxantrone possesses significant DPP-4 inhibitory activity both in vitro and in vivo. The pharmacophore of mitoxantrone was further investigated by testing a variety of its structural analogues.

Intracellular cargo delivery by an octaarginine transporter adapted to target prostate cancer cells through cell surface protease activation.

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.

Releasable luciferin-transporter conjugates: tools for the real-time analysis of cellular uptake and release.

The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.

Anticancer and antithrombin activity of Russian plants.

A chromogenic bioassay was utilized to determine the antithrombin activity of the methylene chloride and methanol extracts prepared from forty-five plants of Russia. Mouse leukemia cells (L1210) were utilized to screen these extracts for activity against cancer. The results indicated that eight plant extracts demonstrated 90% or higher activity in the inhibition of thrombin. Also, nine methanol extracts demonstrated activity of 90% or higher in the inhibition of mouse leukemia L1210 cells. The methanol extracts of Quercus robur and Sanguisorba officinalis demonstrated high activity against both thrombin and cancer.