Combinatorial Chemistry & High Throughput Screening - Volume 14, Issue 1, 2011

The drug discovery landscape is at cross roads with profound changes looming in the horizon. Open innovation has become the new ‘mantra’ for reinvigorating the pharmaceutical R&D's lackluster drug candidate pipeline. To fill this void, academia has now ventured from its traditional role of exploring the fundamental aspects of disease biology in to the high throughput screening arena in a big way. Thanks to the NIH Roadmap Initiative and the EuOpen Screen program for facilitating this transition. A look at the manuscripts published in Combinatorial Chemistry & High Throughput Screening is reflective of this transition, with 75% of articles coming from Academia across the globe. We have published a total of 90 articles in volume 13, 2010, averaging 9 articles per issue, with 33 articles from Europe, 22 from North America, 16 from Asia and the remainder coming from the Middle East and other countries. These facts attest to the truly global reach of Combinatorial Chemistry & High Throughput Screening. Articles in the theme-based ‘Hot Topic’ issues are gaining in prominence, and are becoming increasingly the most cited ones. We have lined up quite an exciting array of hot topic issues for 2011, which range from bioluminescence to LIMS and screen data management. From looking at the kind and range of articles that have been submitted for publication in Combinatorial Chemistry & High Throughput Screening, it is clear that a number of articles are related to pharmacognosy. It is indeed a welcome sign as many drugs have historically been derived from natural products and that there exists a vast amount of natural product chemistry to be explored for their relevance in serving as drug leads. To fully support the review process of pharmacognosy-related manuscripts, we will soon be introducing a new section in Combinatorial Chemistry & High Throughput Screening on pharmacognosy, managed by an expert in natural products. The new changes we introduced at the beginning of the year, restructuring the Editorial Board and the introduction of Section Editors, have turned out to be good moves and benefitted the manuscript review process enormously. It has also become clear that there was a certain amount of duplicity in the roles of the Regional Editor vs Section Editor. In streamlining the entire editorial review process, I have decided to empower the Section Editors to wholly manage their sections, irrespective of the regional origin of the articles; and hence discontinue the Regional Editorial process. I have not reached this decision lightly. I would like to thank the Regional Editors for the years of service, expertise, time and effort that they have tirelessly extended in helping me and Richard in bringing Combinatorial Chemistry & High Throughput Screening issues month after month, year after year. We are continuing to strive for publishing high quality articles and that could only be done with outstanding manuscript submissions, and expert peer review process. Just completed Volume 13 was our best, and my first as the Editor-in-Chief. My gratitude to all those who have helped, the authors, reviewers, section and regional editors and publishing staff, for their time, advice, expertise, and tireless effort.

An efficient synthesis of stable phosphonate ylides and phosphonate esters is described via a one-pot reaction between activated acetylenes and triphenylphosphite in the presence of sulfonamides and heterocyclic NH-acids. Single X-ray diffraction analysis and NMR studies were used in characterizing the ylides and phosphonate ester products. Dynamic NMR studies performed on a phosphonate ylide allowed the calculation of the free energy barrier for the interconversion between the geometrical isomers (E) and (Z).

Bacteriophage (phage) display has been exploited for the purpose of discovering new cancer specific targeting peptides. However, this approach has resulted in only a small number of tumor targeting peptides useful as in vivo imaging agents. We hypothesize that in vivo screening for tumor uptake of fluorescently tagged phage particles displaying multiple copies of an in vivo selected tumor targeting peptide will expedite the development of peptide based imaging agents. In this study, both in vivo selection and in vivo screening of phage displaying foreign peptides were utilized to best predict peptides with the pharmacokinetic properties necessary for translation into efficacious in vivo imaging agents. An in vivo selection of phage display libraries was performed in SCID mice bearing human PC-3 prostate carcinoma tumors. Eight randomly selected phage clones and four control phage clones were fluorescently labeled with AlexaFluor 680 for subsequent in vivo screening and analyses. The corresponding peptides of six of these phage clones were tested as 111Inlabeled peptide conjugates for single photon emission computed tomography (SPECT) imaging of PC-3 prostate carcinomas. Two peptide sequences, G1 and H5, were successful as in vivo imaging agents. The affinities of G1 and H5 peptides for cultured PC-3 cells were then analyzed via cell flow cytometry resulting in Kd values of 1.8 μM and 2.2 μM, respectively. The peptides bound preferentially to prostate tumor cell lines compared to that of other carcinoma and normal cell lines, and H5 appeared to possess cytotoxic properties. This study demonstrates the value of in vivo screening of fluorescently labeled phage for the prediction of the efficacy of the corresponding 111In-labeled synthetic peptide as an in vivo SPECT tumor imaging agent.

This paper describes a stripping method for the determination of nevirapine at the submicromolar concentration levels The method is based on controlled adsorptive accumulation of nevirapine at thin-film mercury electrode, followed by a linear cyclic scan voltammetry measurement of the surface species. Optimal experimental conditions include a 2.0 × 10-3 mol L-1 NaOH solution (supporting electrolyte), an accumulation potential of -0.20 V, and a scan rate of 100 mV s-1. The response of nevirapine is linear over the concentration range 0.01 - 0.14 ppm. For an accumulation time of 6 minutes, the detection limit was found to be 0.87 ppb (3.0 × 10- 9 mol L-1). More convenient methods to measure the nevirapine in presence of the efavirenz, acyclovir, didanosine, indinavir, nelfinavir, saquinavir, lamivudine, zidovudine and metals ions were also investigated. The utility of this method is demonstrated by the presence of nevirapine together with ATP or DNA.

This paper reports on a systematic method - called PLUXter - developed for screening and data mining of large numbers of potential metal-organic framework compounds that have been synthesized then subsequently analyzed with high throughput synchrotron radiation X-ray powder diffraction. The first part of the method utilizes principal components analysis (PCA), which allows materials to be ranked in order of crystallinity so that undesirable amorphous materials may be identified and eliminated. The second part allows structural grouping within and between samples to be observed using hierarchical cluster analysis (HCA). Classification using a single linkage distance produced unsatisfactory clusters however the dendrogram's structural relationships were used to establish and guide the boundaries of groups. The resultant grouping identities allowed further structure-property studies to be undertaken on representative structures from the clusters, significantly reducing time and the use of resources.

OPLS discriminant analysis (OPLS-DA) was successfully applied for the selection of a limited number of gene transcripts necessary to discriminate PTPN11 and RAS mutated cells in acute lymphoblastic leukaemia (ALL) patients. The original set of 273 variables with VIP (1) values higher than 2.0 in the OPLS-DA model could be further reduced to 200 by elimination of less informative variables in the PCA class models adopted for SIMCA classification. The above 200 transcripts not only achieve a satisfactory discrimination accuracy between PTPN11 and RAS mutated cells but also indicate clearly that wild type samples belong to none of the mutated class models. In this list it was possible to identify candidate genes that could be involved in the molecular mechanisms discriminating PTPN11 and RAS mutations in ALL. Among them CBFA2T2, a member of the “ETO” family, is known because of its homology and association with the product of RUNX1-CBFA2T1 gene fusion generated by t(8;21) translocation, one frequent cause of acute myeloid leukemia.

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.

A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 107 cells and ∼105 of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate <1% of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased Km (22.0 vs 18.1 mM) and a 2.7 fold increased kcat (150 s-1 vs 54.8 s-1) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s-1) it has a slightly increased Km and 1.8 times increased kcat.

In this paper we propose a matrix depiction and two new invariants of DNA sequences. This approach is illustrated on the primate mitochondrial DNA sequences for 11 different species and 80 different H5N1 avian influenza virus DNA sequences. We also construct the dendrogram tree for them. These phylogenies obtained are generally consistent with evolutionary trees constructed in previous studies.