Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

Metagenome-Guided Analogue Synthesis Yields Improved Gram-Negative-Active Albicidin- and Cystobactamid-Type Antibiotics.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics. Our search for PABA-specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio-informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition.

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Analysis of HIF-1 inhibition by manassantin A and analogues with modified tetrahydrofuran configurations.

We have shown that manassantin A downregulated the HIF-1alpha expression and inhibited the secretion of VEGF. We have also demonstrated that the 2,3-cis-3,4-trans-4,5-cis-configuration of the tetrahydrofuran is critical to the HIF-1 inhibition of manassantin A.

Design of Peptoid-peptide Macrocycles to Inhibit the ß-catenin TCF Interaction in Prostate Cancer.

New chemical inhibitors of protein-protein interactions are needed to propel advances in molecular pharmacology. Peptoids are peptidomimetic oligomers with the capability to inhibit protein-protein interactions by mimicking protein secondary structure motifs. Here we report the in silico design of a macrocycle primarily composed of peptoid subunits that targets the ß-catenin:TCF interaction. The ß-catenin:TCF interaction plays a critical role in the Wnt signaling pathway which is over-activated in multiple cancers, including prostate cancer. Using the Rosetta suite of protein design algorithms, we evaluate how different macrocycle structures can bind a pocket on ß-catenin that associates with TCF. The in silico designed macrocycles are screened in vitro using luciferase reporters to identify promising compounds. The most active macrocycle inhibits both Wnt and AR-signaling in prostate cancer cell lines, and markedly diminishes their proliferation. In vivo potential is demonstrated through a zebrafish model, in which Wnt signaling is potently inhibited.

CTSA Consortium Consensus Scientific Review Committee (SRC) Working Group Report on the SRC Processes.

Human research projects must have a scientifically valid study design, analytic plan, and be operationally feasible in order to be successfully completed and thus to have translational impact. To ensure this, institutions that conduct clinical research should have a scientific review process prior to submission to the Institutional Review Committee (IRB). This paper reports the Clinical and Translational Science Award (CTSA) Consortium Scientific Review Committee (SRC) Consensus Working Group's proposed framework for a SRC process. Recommendations are provided for institutional support and roles of CTSAs, multisite research, criteria for selection of protocols that should be reviewed, roles of committee members, application process, and committee process. Additionally, to support the SCR process effectively, and to ensure efficiency, the Working Group recommends information technology infrastructures and evaluation metrics to determine outcomes are provided.

Synthesis of α,α'-trans-oxepanes through an organocatalytic oxa-conjugate addition reaction.

Oxepanes are found in a wide range of natural products; however, they are challenging synthetic targets due to enthalpic and entropic barriers. Organocatalytic oxa-conjugate addition reactions promoted by the gem-disubstituent (Thorpe-Ingold) effect stereoselectively provided α,α'-trans-oxepanes. In addition, the potential of an organocatalytic tandem oxa-conjugate addition/α-oxidation was demonstrated in a rapid generation of molecular complexity. These organocatalytic oxa-conjugate addition reactions would provide powerful tools for the synthesis of natural products that contain highly functionalized oxepanes.

Contraceptive method selection by women with inflammatory bowel diseases: a cross-sectional survey.

OBJECTIVE: Women with inflammatory bowel diseases (IBDs) utilize contraception at a lower rate than the general population. We sought to identify factors associated with contraceptive use and selection of more effective methods in IBD patients at risk for unintended pregnancy. STUDY DESIGN: An online survey was distributed to women with IBD in January 2013. Contraceptive methods were categorized by effectiveness and associations with use explored by demographics, disease characteristics and reproductive goals. RESULTS: A total of 162 respondents were analyzed: 62% had Crohn's disease and 38% ulcerative colitis. Mean age was 31 (range 20-45), 97% identified as White, and 53% were nulliparas. Seventy-four percent were currently using IBD medications. A quarter of participants (23%) used no contraception, 17% used highly effective methods, 41% used short-term hormonal methods, and 19% chose barrier/behavioral methods. Prior IBD-related surgery, biologic therapy use and low education attainment were associated with no contraception use. Of contraceptive users, age, parity, insurance status, IBD surgery and prior immunomodulator use were associated with highly effective method selection. CONCLUSIONS: A quarter of women with IBD at risk for pregnancy in this study population reported no contraceptive method use. Higher levels of IBD activity influence contraceptive use and method selection, which could guide future patient and provider educational interventions. IMPLICATIONS: Pregnancy planning is important for women with inflammatory bowel diseases to avoid adverse outcomes in a disease-poor state. Use of contraception assists in avoidance of unintended pregnancy. IBD characteristics are targets for educational interventions to improve uptake of highly effective contraceptive methods.

The CTSA Consortium's Catalog of Assets for Translational and Clinical Health Research (CATCHR).

The 61 CTSA Consortium sites are home to valuable programs and infrastructure supporting translational science and all are charged with ensuring that such investments translate quickly to improved clinical care. Catalog of Assets for Translational and Clinical Health Research (CATCHR) is the Consortium's effort to collect and make available information on programs and resources to maximize efficiency and facilitate collaborations. By capturing information on a broad range of assets supporting the entire clinical and translational research spectrum, CATCHR aims to provide the necessary infrastructure and processes to establish and maintain an open-access, searchable database of consortium resources to support multisite clinical and translational research studies. Data are collected using rigorous, defined methods, with the resulting information made visible through an integrated, searchable Web-based tool. Additional easy-to-use Web tools assist resource owners in validating and updating resource information over time. In this paper, we discuss the design and scope of the project, data collection methods, current results, and future plans for development and sustainability. With increasing pressure on research programs to avoid redundancy, CATCHR aims to make available information on programs and core facilities to maximize efficient use of resources.

The effects of total dissolved solids on egg fertilization and water hardening in two salmonids--Arctic Grayling (Thymallus arcticus) and Dolly Varden (Salvelinus malma).

Previous studies have indicated that salmonid fertilization success may be very sensitive to elevated concentrations of total dissolved solids (TDS) with effects at concentrations as low as 250mgl(-1) being reported. However, interpretation of these studies is complicated by poor control performance and variable concentration response relationships. To address this, a series of experiments were performed to evaluate TDS effects on Arctic Grayling (Thymallus arcticus) and Dolly Varden (Salvelinus malma) fertilization success and identify possible mechanisms for previously observed test variability and any observed effects of TDS. Results indicate that some of the experiments reported here were likely confounded by extended milt holding times prior to experiment initiation. Milt holding times >6h were shown to significantly reduce control fertilization and corresponding concentration response relationships were variable. When milt holding time was minimized during fertilization experiments, consistent control performance with >90% control fertilization was achieved and consistent concentration response relationships were observed for both species examined. Experiments performed under these conditions indicate that Arctic Grayling and Dolly Varden fertilization success is not sensitive to elevated TDS with EC20s (concentration causing 20% effect) of >2782 and >1817mgl(-1) (the highest concentrations tested), respectively. However, TDS was shown to significantly affect embryo water absorption during the water hardening phase immediately following fertilization. The lowest observable effect concentrations (LOECs) for this endpoint were 1402 and 964mgl(-1) for Arctic Grayling and Dolly Varden, respectively. The effect of reduced embryo turgidity, due to impaired water absorption, on resistance to mechanical damage under real world conditions needs further investigation in order to understand the implications of this observed effect.

The end game of chemical genetics: target identification.

The use of classical genetic and molecular biology methods along with the sequencing of many genomes has proven crucial for elucidating complex biological processes. Despite being invaluable tools, their limitations have led to a search for more versatile alternatives and, thus, to the use of small molecules. Chemical genetics is a rapidly emerging field that uses small-molecule techniques to probe biological systems and is composed of three parts: natural product or small-molecule libraries, phenotypic screening and target identification. Currently, the biggest hurdle in the overall process of chemical genetics is target identification. Efforts to overcome this obstacle have led to advances in the areas of affinity chromatography, yeast haploinsufficiency, complementary DNA (cDNA) overexpression, DNA microarray, small-molecule microarray and RNA interference (RNAi) technologies. While these technologies continue to undergo further optimization, they have been integral in the identification and/or confirmation of many cellular targets and have seen an increase in applications to the drug-development process.

Nucleophilic addition of organozinc reagents to 2-sulfonyl cyclic ethers: stereoselective synthesis of manassantins A and B.

A convergent route to the synthesis of manassantins A and B, potent inhibitors of HIF-1, is described. Central to the synthesis is a stereoselective addition of an organozinc reagent to a 2-benzenesulfonyl cyclic ether to achieve the 2,3-cis-3,4-trans-4,5-cis-tetrahydrofuran of the natural products. Preliminary structure-activity relationships suggested that the (R)-configuration at C-7 and C-7''' is not critical for HIF-1 inhibition. In addition, the hydroxyl group at C-7 and C-7''' can be replaced with a carbonyl group without loss of activity.