Successful Proof-of-Concept for Topical Delivery of Novel Peptide ALM201 with Potential Usefulness for Treating Neovascular Eye Disorders.

Purpose: To evaluate the therapeutic benefit of a novel peptide, ALM201, in ocular pathologic vascularization. Design: Experimental study in mouse, rat, and rabbit animal models. Participants: Ten-week-old Lister Hooded male rats, 8-week-old Brown Norway male rats, 9-day-old C57BL/6J mice, and 12-month-old New Zealand male rabbits. Methods: Corneal vascularization was scored for vessel density and vessel distance to suture in a rat corneal suture model. Ocular penetration and biodistribution were evaluated by matrix-assisted laser desorption/ionization mass spectrometry imaging after topical ALM201 application to rabbit eyes. A mouse choroidal sprouting assay, with aflibercept as positive control, was used to evaluate choroidal neovascularization (CNV) in the posterior segment tissue. Efficacy of topical ALM201 was assessed using a rat laser CNV model of neovascular age-related macular degeneration. Main Outcome Measures: Clinical scoring and histologic analysis of vascularized corneas, sprouting area, lesion size, and vessel leakiness in posterior segments. Results: Assessment of ALM201 treatment in the rat corneal suture model showed a significant decrease in vessel density (P = 0.0065) and vessel distance to suture (P = 0.021) compared with vehicle control (phosphate-buffered saline [PBS]). Infiltration of inflammatory cells into the corneal stroma also was reduced significantly compared with PBS (724.5 ± 122 cells/mm2 vs. 1837 ± 195.9 cells/mm2, respectively; P = 0.0029). Biodistribution in rabbit eyes confirmed ALM201 bioavailability in anterior and posterior ocular segments 1 hour after topical instillation. ALM201 treatment significantly suppressed choroid vessel sprouting when compared with PBS treatment (44.5 ± 14.31 pixels vs. 120.9 ± 33.37 pixels, respectively; P = 0.04) and was not inferior to aflibercept (65.63 ± 11.86 pixels; P = 0.7459). Furthermore, topical ALM201 significantly improved vessel leakiness (leakage scores: 2.1 ± 0.7 vs. 2.9 ± 0.1; P = 0.0274) and lesion size (144,729 ± 33,239 µm3 vs. 187,923 ± 28,575 µm3; P = 0.03) in the rat laser CNV model when compared with topical PBS vehicle. Conclusions: ALM201 is a promising novel molecule with anti-inflammatory and antivascularization activity and is a strong candidate to meet the clinical need of a new, topically delivered therapeutic agent for treating inflammation and pathologic vascularization in the anterior and posterior segments of the eye.

Effects of vitamin D as a regulator of androgen intracrinology in LNCAP prostate cancer cells.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumor regression, but commonly restricted by the eventual emergence of a more lethal 'castrate resistant' form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids, are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). In this study, we explored the capacity of the active vitamin D hormone to interact and elicit changes upon this prostatic intracrine pathway at a metabolic level. We used androgen dependent LNCaP cells cultured under steroid-depleted conditions and assessed the impact of vitamin D-based compounds upon intracrine pathways that convert exogenously added DHEA to relevant metabolites, through Mass Spectrometry (MS). Changes in relevant metabolism-related gene targets were also assessed. Our findings confirm that exposure to vitamin D based compounds, within LNCaP cells, elicits measurable and significant reduction in the intracrine conversion of DHEA to T, DHT and other intermediate metabolites within the androgenic pathway. The aassessment and validation of the biological model and analytical platforms were performed by pharmacological manipulations of the SRD5α and HSD-17ß enzymes. The data provides further confirmation for how a vitamin D-based regime may be used to counter intracrine mechanisms contributing to the emergence of castrate-resistant tumors.

Copper Catalysis in Living Systems and In†Situ Drug Synthesis.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has proven to be a pivotal advance in chemical ligation strategies with applications ranging from polymer fabrication to bioconjugation. However, application in vivo has been limited by the inherent toxicity of the copper catalyst. Herein, we report the application of heterogeneous copper catalysts in azide-alkyne cycloaddition processes in biological systems ranging from cells to zebrafish, with reactions spanning from fluorophore activation to the first reported in situ generation of a triazole-containing anticancer agent from two benign components, opening up many new avenues of exploration for CuAAC chemistry.

Ferric ion (hydr)oxo clusters in the "Venus flytrap" cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies.

Isothermal calorimetric studies of the binding of iron(III) citrate to ferric ion binding protein from Neisseria gonorrhoeae suggested the complexation of a tetranuclear iron(III) cluster as a single step binding event (apparent binding constant K(app) (ITC) = 6.0(5) × 10(5) M(-1)). High-resolution Fourier transform ion cyclotron resonance mass spectrometric data supported the binding of a tetranuclear oxo(hydroxo) iron(III) cluster of formula [Fe(4)O(2)(OH)(4)(H(2)O)(cit)](+) in the interdomain binding cleft of FbpA. The mutant H9Y-nFbpA showed a twofold increase in the apparent binding constant [K(app) (ITC) = 1.1(7) × 10(6) M(-1)] for the tetranuclear iron(III) cluster compared to the wild-type protein. Mössbauer spectra of Escherichia coli cells overexpressing FbpA and cultured in the presence of added (57)Fe citrate were indicative of the presence of dinuclear and polynuclear clusters. FbpA therefore appears to have a strong affinity for iron clusters in iron-rich environments, a property which might endow the protein with new biological functions.

Lactate, a product of glycolytic metabolism, inhibits histone deacetylase activity and promotes changes in gene expression.

Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell's aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription.

Selective chemical intervention in the proteome of Caenorhabditis elegans.

We present the first study of protein regulation by ligands in Caenorhabditis elegans. The ligands were peptidyl-prolyl isomerase inhibitors of cyclophilins. Up-regulation is observed for several heat shock proteins and one ligand in particular caused a greater than 2-fold enhancement of cyclophilin CYN-5. Additionally, several metabolic enzymes display elevated levels. This approach, using label-free relative quantification, provides an extremely attractive way of measuring the effect of ligands on an entire proteome, with minimal sample pretreatment, which could be applicable to large-scale studies. In this initial study, which compares the effect of three ligands, 54 unique proteins have been identified that are up- (51) or down- (3) regulated in the presence of a given ligand. A total of 431 C. elegans proteins were identified. Our methodology provides an intriguing new direction for in vivo screening of the effects of novel and untested ligands at the whole organism level.

Gel free analysis of the proteome of intracellular Leishmania mexicana.

Investigating the proteome of intracellular Leishmania amastigotes has recently become possible due to the exploitation of fluorescence activated intracellular parasite sorting. Here, we employed this technology in combination with gel free analysis to greatly improve proteome coverage and suggest proteins putatively secreted by the parasites. In total, 1764 proteins were identified of which 741 had not been reported before. Protein abundance indices were calculated to rank individual proteins according to their abundance in vivo. Using the LeishCyc resource, an overview of metabolically relevant proteins was produced that integrated protein abundance data. Bioinformatic analysis identified 143 proteins possibly secreted by L. mexicana amastigotes, half of which have no known function. The data provide a useful resource, e.g. for modelling metabolic flux or selecting novel vaccine antigens.