Advances in Combinatorial Chemistry & High Throughput Screening

This review summarizes the use of selected partial order ranking (POR) techniques for the assessment of chemicals. Simple partial order ranking may advantageously be applied to give the single chemicals investigated an identity in relation to other substances. Thus, it constitutes an effective tool for the prioritization of chemicals, e.g., based on their PBT (Persistence, Bioaccumulating, Toxicity) characteristics. In more elaborate cases where a larger number of descriptors are taken into account, e.g., comprising physico-chemical characteristics, atmospheric parameters, geospecific factors and possibly socio-economic factors hierarchical partial order ranking (HPOR) may be applied. Thus, in a first ordering step a series of meta-descriptors are generated that later subsequently being used as descriptors in a subsequent ordering. HPOR allows a sensible ranking model even if a relative high number of descriptors are included. Assessing chemicals can, taken the actual situation into account, be based on a variety of parameters/descriptors. Thus, it might be valuable information to know the mutual importance of the applied descriptor. This information may appropriately be retrieved by a sensitivity study applying a designed module in the PyHasse software package. Finally accumulation partial order ranking (APOR) is illustrated. Accumulating partial APOR is a technique where data from a series of individual tests of various characteristics are aggregated, however, maintaining the basics of the partial order ranking methodology. APOR offers prioritization based on mutual probabilities derived from the aggregated data. Alternatively prioritization may be achieved based on average ranks derived from the APOR. The application APOR is demonstrated by an assessment of a series of potential PBT substances. In contrast to simple ranking techniques, partial order ranking does not automatically lead to an absolute rank of the single substances being studied. However, a weak linear order may in all cases be achieved based on the average ranks of the single substances. Additionally ranking probabilities can be derived based on Monte Carlo simulations.

This article reviews the application of fragment descriptors at different stages of virtual screening: filtering, similarity search, and direct activity assessment using QSAR/QSPR models. Several case studies are considered. It is demonstrated that the power of fragment descriptors stems from their universality, very high computational efficiency, simplicity of interpretation and versatility.

A fluorescent probe is a fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus. Fluorescent probes have been used for nearly a century to study cellular processes due to their exquisite sensitivity and selectivity. Fluorescent probes have also gained in popularity as safety and environmental concerns over the use of radioactive probes have grown. At the same time, cellular assays are being more widely used now than ever before. This review will give a broad overview of types of fluorescent probes, types of fluorescent assays, and their application in cellular assays for a number of pharmaceutically relevant target classes.

G protein-coupled receptors (GPCRs) have been proven to be the largest family of druggable targets in the human genome. Given the importance of GPCRs as drug targets and the de-orphanization of novel targets, GPCRs are likely to remain the frequent targets of many drug discovery programs. Traditionally, molecular assays dominate in the process of GPCR drug discovery and development. With recent advances in instrumentation and pathway deconvolution of GPCR ligand-induced biosensor signatures, label-free biosensor-enabled cell-based assays have become a very active area for GPCR screening. This article reviews the principles, current status and future directions of leading label-free technology platforms for GPCR drug discovery.

In the demanding field of proteomics, there is a great need for affinitycatcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterization of specific antibodies represent a key issue to meet success. Alternatively, it is expected that novel affinity reagents generated in fast, cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligandreceptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the “intelligent” design and synthesis of chemical libraries of lowmolecular- weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterization of leads is a most relevant task. Immunoassays are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.

Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating an urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element (ERE) luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs using bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested.

The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high-throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gα_i1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z´-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z´-factor of 0.80. To determine the screening factor window (Zfactor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC¹²⁸⁰ collection was screened three times with every library compound being tested over a range of concentrations following the quantitative highthroughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.

Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive state in which GDP-bound Gα is complexed with the Gβγ dimer, and an active state in which GTP-bound Gα is freed of its Gβγ binding partner. Structural studies have illustrated the basis for the distinct conformations of these states which are regulated by alterations in three precise ‘switch regions’ of the Gα subunit. Discrete differences in conformation between GDP- and GTP-bound Gα underlie its nucleotide-dependent protein-protein interactions (e.g., with Gβγ/receptor and effectors, respectively) that are critical for maintaining their proper nucleotide cycling and signaling properties. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Gα (and free Gβγ) in nucleotidedependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Gα and Gβγ signaling, and serving as biosensor tools in assays for Gα activation including high-throughput drug screening. In this review, we highlight some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides.