Development of Near-Infrared Fluorophore (NIRF)-Labeled Activity-Based Probes for <i>in Vivo</i> Imaging of Legumain

Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease

Porphyrin–Peptoid Conjugates: Face-to-Face Display of Porphyrins on Peptoid Helices

Distance, orientation, and number controlled porphyrin–peptoid conjugates (PPCs) were efficiently synthesized. Cofacial (<b>1</b>, <b>2</b>, and <b>4</b>), slipped-cofacial (<b>3</b>), and unstructured (<b>5</b>) arrangements of porphyrins provided distinct optical and electronic properties characterized by UV–vis and circular dichroism spectroscopy. In addition, ECCD spectra confirmed the handedness of peptoid helices

A Steroid-Conjugated Contrast Agent for Magnetic Resonance Imaging of Cell Signaling

We have synthesized the first steroid hormone−MR contrast agent conjugate designed to track the cell signaling process upon binding to a gene switch system. The derivative has a high relaxivity and when tested in vitro is active as a progesterone antagonist (RU-486). By combining a transcriptional system and a noninvasive imaging technology, such as MRI, it would be a powerful tool to research the cell signaling pathway in vivo

Mitochondria-Targeting Peptoids

Mitochondria-specific delivery methods offer a valuable tool for studying mitochondria-related diseases and provide breakthroughs in therapeutic development. Although several small-molecule and peptide-based transporters have been developed, peptoids, proteolysis-resistant peptidomimetics, are a promising alternative to current approaches. We designed a series of amphipathic peptoids and evaluated their cellular uptake and mitochondrial localization. Two peptoids with cyclohexyl residues demonstrated highly efficient cell penetration and mitochondrial localization without significant adverse effects on the cells and mitochondria. These mitochondria-targeting peptoids could facilitate the selective and robust targeted delivery of bioactive compounds, such as drugs, antioxidants, and photosensitizers, with minimal off-target effects

Table_1_Effects of Virtual Reality Education on Procedural Pain and Anxiety During Venipuncture in Children: A Randomized Clinical Trial.DOCX

BackgroundVenipuncture is one of the most frequent and frightening medical procedures for children. This randomized clinical trial aimed to evaluate whether pre-procedural immersive virtual reality (VR) education could decrease pain and anxiety during venipuncture procedure of children.MethodsSixty children scheduled for venipuncture at the phlebotomy unit were randomized into either the control or VR group. Before the procedure, children of the control group received conventional simple verbal instructions, whereas those of the VR group experienced a 4-min VR education regarding venipuncture. The primary outcome was the pain and anxiety of pediatric patients assessed with the children’s hospital of eastern ontario pain scale. Secondary outcomes were parental satisfaction, venipuncture time, repeated procedure and procedural difficulty rated by phlebotomists.ResultsThe pain and anxiety score during the procedure was significantly lower in the VR group than in the control group (median [IQR], 6.0 [5.0–7.0] vs. 8.0 [6.0–9.8], P = 0.001). Parental satisfaction about the procedural process were higher in the VR group than in the control group (P = 0.029), and the degree of procedural difficulty was lower in the VR group, compared to the control group (P = 0.026).ConclusionThe preprocedural VR education significantly reduced pain and anxiety of children and decreased the procedural difficulty of phlebotomists during venipuncture procedure.Clinical Trial RegistrationUniversity hospital Medical Information Network Clinical Trials Registry (registration number: UMIN000042968, date of registration: January 9, 2021, URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000049043).</p

Discovery of an Orally Bioavailable Gonadotropin-Releasing Hormone Receptor Antagonist

We developed a compound library for orally available gonadotropin-releasing hormone (GnRH) receptor antagonists that were based on a uracil scaffold. On the basis of in vitro activity and CYP inhibition profile, we selected <b>18a</b> (SKI2496) for further in vivo studies. Compound <b>18a</b> exhibited more selective antagonistic activity toward the human GnRH receptors over the GnRHRs in monkeys and rats, and this compound also showed inhibitory effects on GnRH-mediated signaling pathways. Pharmacokinetic and pharmacodynamic evaluations of <b>18a</b> revealed improved bioavailability and superior gonadotropic suppression activity compared with Elagolix, the most clinically advanced compound. Considering that <b>18a</b> exhibited highly potent and selective antagonistic activity toward the <i>h</i>GnRHRs along with favorable pharmacokinetic profiles, we believe that <b>18a</b> may represent a promising candidate for an orally available hormonal therapy

Discovery of an Orally Bioavailable Gonadotropin-Releasing Hormone Receptor Antagonist

We developed a compound library for orally available gonadotropin-releasing hormone (GnRH) receptor antagonists that were based on a uracil scaffold. On the basis of in vitro activity and CYP inhibition profile, we selected <b>18a</b> (SKI2496) for further in vivo studies. Compound <b>18a</b> exhibited more selective antagonistic activity toward the human GnRH receptors over the GnRHRs in monkeys and rats, and this compound also showed inhibitory effects on GnRH-mediated signaling pathways. Pharmacokinetic and pharmacodynamic evaluations of <b>18a</b> revealed improved bioavailability and superior gonadotropic suppression activity compared with Elagolix, the most clinically advanced compound. Considering that <b>18a</b> exhibited highly potent and selective antagonistic activity toward the <i>h</i>GnRHRs along with favorable pharmacokinetic profiles, we believe that <b>18a</b> may represent a promising candidate for an orally available hormonal therapy

Functional Imaging of Legumain in Cancer Using a New Quenched Activity-Based Probe

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions

Synthesis and Biological Evaluation of Aryloxazole Derivatives as Antimitotic and Vascular-Disrupting Agents for Cancer Therapy

A series of aryloxazole, thiazole, and isoxazole derivatives was synthesized as vascular-targeting anticancer agents. Antiproliferative activity and tumor vascular-disrupting activity of all of the synthesized compounds were tested in vitro using various human cancer cell lines and HUVECs (human umbilical vein endothelial cells). Several compounds with an arylpiperazinyl oxazole core showed excellent cytotoxicity and metabolic stability in vitro. Among this series, two representative compounds (<b>6</b>-<b>48</b> and <b>6</b>-<b>51</b>) were selected and tested for the evaluation of anticancer effects in vivo using tumor-bearing mice. Compound <b>6</b>-<b>48</b> effectively reduced tumor growth (42.3% reduction in size) at the dose of 100 mg/kg. We believe that compound <b>6</b>-<b>48</b> will serve as a good lead compound for antimitotic and vascular-disrupting agents; further investigation to improve the in vivo efficacy of this series is underway

Coupling Protein Engineering with Probe Design To Inhibit and Image Matrix Metalloproteinases with Controlled Specificity

Matrix metalloproteinases (MMPs) are zinc endopeptidases that play roles in numerous pathophysiological processes and therefore are promising drug targets. However, the large size of this family and a lack of highly selective compounds that can be used for imaging or inhibition of specific MMPs members has limited efforts to better define their biological function. Here we describe a protein engineering strategy coupled with small-molecule probe design to selectively target individual members of the MMP family. Specifically, we introduce a cysteine residue near the active-site of a selected protease that does not alter its overall activity or function but allows direct covalent modification by a small-molecule probe containing a reactive electrophile. This specific engineered interaction between the probe and the target protease provides a means to both image and inhibit the modified protease with absolute specificity. Here we demonstrate the feasibility of the approach for two distinct MMP proteases, MMP-12 and MT1-MMP (or MMP-14)