Abstract-Angiotensin-converting enzyme 2 (ACE2) is a key renin-angiotensin system enzyme involved in balancing the adverse effects of angiotensin II on the cardiovascular system, and its overexpression by gene transfer is beneficial in cardiovascular disease. Therefore, our objectives were 2-fold: to identify compounds that enhance ACE2 activity using a novel conformation-based rational drug discovery strategy and to evaluate whether such compounds reverse hypertension-induced pathophysiologies. We used a unique virtual screening approach. In vitro assays revealed 2 compounds (a xanthenone and resorcinolnaphthalein) that enhanced ACE2 activity in a dose-dependent manner. Acute in vivo administration of the xanthenone resulted in a dose-dependent transient and robust decrease in blood pressure (at 10 mg/kg, spontaneously hypertensive rats decreased 71Ϯ9 mm Hg and Wistar-Kyoto rats decreased 21Ϯ8 mm Hg; PϽ0.05). Chronic infusion of the xanthenone (120 g/day) resulted in a modest decrease in the spontaneously hypertensive rat blood pressure (17 mm Hg; 2-way ANOVA; PϽ0.05), whereas it had no effect in Wistar-Kyoto rats. Strikingly, the decrease in blood pressure was also associated with improvements in cardiac function and reversal of myocardial, perivascular, and renal fibrosis in the spontaneously hypertensive rats. We conclude that structure-based screening can help identify compounds that activate ACE2, decrease blood pressure, and reverse tissue remodeling. Administration of ACE2 activators may be a valid strategy for antihypertensive therapy. T he recent discovery of angiotensin-converting enzyme (ACE) 2 1,2 and its role in the control of renin-angiotensin system activity is relevant as a potentially valuable target for antihypertensive therapies. ACE2 is a zinc-dependent monocarboxypeptidase that plays a central role in balancing vasoconstrictor and proliferative actions of angiotensin (Ang) II with the vasodilatory and antiproliferative effects of Ang-(1-7). 3 Altered expression of this enzyme is associated with cardiac, vascular, and renal dysfunctions. 4,5 In addition, blocking the synthesis of Ang II by Ang-converting enzyme inhibitors or its actions by Ang II receptor blockers has been shown to increase cardiac ACE2 expression. 6,7 Furthermore, overexpression of ACE2 by gene transfer 8 protects the heart from hypertension-induced cardiac remodeling. It was demonstrated that ACE2 is an effective enzyme in attenuating fibrosis and structural remodeling. 8 Based on these observations, we hypothesize that pharmacological enhancement of ACE2 activity would have beneficial effects on the cardiovascular system and would protect against hypertensioninduced pathophysiology. Therefore, our objectives in this study were 2-fold: to identify compounds that enhance ACE2 activity and to determine the effects of ACE2 enhancers on hypertension and associated pathophysiology. Materials and MethodsSynthesis of xanthenone, measurement of ACE2 activity, histological analysis, and statistical analysis are described in the sup...

In vivo optical imaging shows that a fluorescent imaging probe, comprised of a near-infrared fluorophore attached to an affinity group containing two zinc(II)-dipicolylamine (Zn-DPA) units, targets prostate and mammary tumors in two different xenograft animal models. The tumor selectivity is absent with control fluorophores whose structures do not have appended Zn-DPA targeting ligands. Ex vivo biodistribution and histological analyses indicate that the probe is targeting the necrotic regions of the tumors, which is consistent with in vitro microscopy showing selective targeting of the anionic membrane surfaces of dead and dying cells.There is a major ongoing research effort to identify oligonucleotide and protein biomarkers of malignant disease. 1 Phospholipid biomarkers are less common, however, there is increasing evidence that the membrane surfaces of certain cells and particles of biomedical significance, smith.115@nd.edu. Supporting Information Available: Experimental details and additional imaging data. The information is available free of charge via the Internet at http://pubs.acs.org. Synthetic zinc(II)-dipicolylamine (Zn-DPA) coordination complexes are known to associate with multianionic phosphorylated biomolecules, 14 and we have discovered that they can be converted into optical imaging probes that target the outer surfaces of anionic vesicle and cell membranes.15 Fluorescent Zn-DPA probes can distinguish dead and dying mammalian cells from healthy cells in a cell culture,16 and also selectively target bacteria in heterogeneous biological media.17 Furthermore, we have recently demonstrated that the near-IR fluorescent probe 1 can be used to image bacterial infections in living mice, 18 indicating that probe 1 has a notable ability to selectively target anionic cells over other anionic sites in the bloodstream and extracellular matrix. Here, we greatly expand the animal imaging capability of probe 1 by showing that it can also target the anionic dead and dying cells within xenograft tumors in rat and mouse models. The structure of probe 1 includes a near-IR carbocyanine fluorophore whose absorption and emission wavelengths of 794 and 810 nm, respectively, are within the optimal window for maximum penetration through skin and tissue. 19 The high tumor selectivity of 1 is demonstrated by comparison to the less-selective imaging that is achieved by using control near-IR fluorophores 2 and 3 whose structures do not have Zn-DPA targeting ligands. The expected ability of probe 1 to selectively target dead and dying cells with exposed anionic phosphatidylserine was confirmed with in vitro fluorescence microscopy studies of mammalian cells treated with a cytotoxic agent. 16 Specifically, treatment of Jurkat cells (T lymphocytes) with camptothecin induced significant amounts of cell death, and as shown in Figure 1, the near-IR probe 1 stained the same cells as fluorescently labeled Annexin V. Using procedures that were approved by the appropriate institutional animal care and use committee, two tumor...

Optical imaging of bacterial infection in living animals is usually conducted with genetic reporters such as light emitting enzymes or fluorescent proteins. However, there are many circumstances where genetic reporters are not applicable, and there is a need for exogenous synthetic probes that can selectively target bacteria. The focus of this study is a fluorescent imaging probe that is composed of a bacterial affinity group conjugated to a near infrared dye. The affinity group is a synthetic zinc (II) coordination complex that targets the anionic surfaces of bacterial cells. The probe allows detection of Staphylococcus aureus infection (5 × 10 7 cells) in a mouse leg infection model using whole animal near infrared fluorescence imaging. Region of interest analysis showed that the signal ratio for infected leg to uninfected leg reaches 3.9 ± 0.5 at 21 h post-injection of the probe. Ex vivo imaging of the organs produced a signal ratio of 8 for infected to uninfected leg. Immunohistochemical analysis confirmed that the probe targeted the bacterial cells in the infected tissue. Optimization of the imaging filter set lowered the background signal due to autofluorescence and substantially improved imaging contrast. The study shows that near infrared molecular probes are amenable to non-invasive optical imaging of localized S. aureus infection.