Lead Discovery Programs Research Articles

Discovery of Small Molecule Drugs Targeting the Biogenesis of microRNA-155 for the Treatment of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease that often leads to functional disorder in multiple organs, most often with symptoms related to skin lesions, cardiovascular disease and kidney damage. Although significant efforts have been made to find efficient therapies, it still remains uncured. Furthermore, the current therapy is often associated with adverse side effects and leads to a high economic burden for society. At Saverna Therapeutics, in collaboration with our partners, we initiated a lead discovery program that aims to modulate the biogenesis of miR-155. This non-coding RNA is upregulated in SLE patients and SLE mouse models. We used our drug discovery platform based on iterative fragment-based screening by nuclear magnetic resonance (NMR) and machine learning to identify ligands of pre-miR-155. After several iterations and chemical modifications, we have identified compounds that show structure-activity relationships in cellular assays. These inhibitors reduced the level of miR-155 as well as its associated inflammatory protein TNF α whereas the cells remained viable during exposure of the compounds.

Bioactivity-Based Molecular Networking for the Discovery of Drug Leads in Natural Product Bioassay-Guided Fractionation.

It is a common problem in natural product therapeutic lead discovery programs that despite good bioassay results in the initial extract, the active compound(s) may not be isolated during subsequent bioassay-guided purification. Herein, we present the concept of bioactive molecular networking to find candidate active molecules directly from fractionated bioactive extracts. By employing tandem mass spectrometry, it is possible to accelerate the dereplication of molecules using molecular networking prior to subsequent isolation of the compounds, and it is also possible to expose potentially bioactive molecules using bioactivity score prediction. Indeed, bioactivity score prediction can be calculated with the relative abundance of a molecule in fractions and the bioactivity level of each fraction. For that reason, we have developed a bioinformatic workflow able to map bioactivity score in molecular networks and applied it for discovery of antiviral compounds from a previously investigated extract of Euphorbia dendroides where the bioactive candidate molecules were not discovered following a classical bioassay-guided fractionation procedure. It can be expected that this approach will be implemented as a systematic strategy, not only in current and future bioactive lead discovery from natural extract collections but also for the reinvestigation of the untapped reservoir of bioactive analogues in previous bioassay-guided fractionation efforts.

The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery

ConspectusPrecise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible.The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify “successful” structures. The largest libraries made by conventional synthesis are currently of the order of 108 distinct molecules. To put this in context, there are 1013 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required.DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure—a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery—imagine being able to evolve molecules for light harvesting, or catalysts for CO2 fixation.The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous “DNA robots” have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution.

Boric acid and acetate anion binding to subclass B3 metallo-β-lactamase BJP-1 provides clues for mechanism of action and inhibitor design

Microbial infections represent a major risk to human health. In this respect, β-lactam antibiotics constitute a key therapeutic resource against such infections. However, we are facing increasing microbial resistance to antibiotic treatment and particularly worrisome is the emergence of resistant bacterial strains towards β-lactam antibiotics that can rapidly disseminate worldwide. β-lactamase enzymes are the main determinant of bacterial resistance and among them metallo-β-lactamases (MBLs) are most threatening, as exemplified by the recent resistance outbreaks due to New Delhi β-lactamase 1 (NDM-1) producing bacteria. MBLs are mono or di-zinc enzymes able to inactivate clinically important β-lactam antibiotics including carbapenems, which are used as a last resort therapy in severe infections. Under this scenery, the discovery of new potent inhibitors of MBLs becomes an urgent need and X-ray crystallography of MBLs in complex with small molecule inhibitors provides the possibility to accelerate the process of drug discovery. We present here the atomic-resolution crystal structures of BJP-1, a di-zinc MBL, in complex with two small molecules and their comparison with other MBL complexes with inhibitors. These structural data, besides providing hints about the mechanism of di-zinc MBLs, might be the starting point for a fragment-based lead-discovery program.

Erylusamides: Novel Atypical Glycolipids from Erylus cf. deficiens.

Among marine organisms, sponges are the richest sources of pharmacologically-active compounds. Stemming from a previous lead discovery program that gathered a comprehensive library of organic extracts of marine sponges from the off-shore region of Portugal, crude extracts of Erylus cf. deficiens collected in the Gorringe Bank (Atlantic Ocean) were tested in the innovative high throughput screening (HTS) assay for inhibitors of indoleamine 2,3-dioxygenase (IDO) and showed activity. Bioassay guided fractionation of the dichloromethane extract led to the isolation of four new glycolipids, named erylusamide A–D. The structures of the isolated compounds were established by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and chemical derivatization. The metabolites shared a pentasaccharide moiety constituted by unusual highly acetylated d-glucose moieties as well as d-xylose and d-galactose. The aglycones were unprecedented long chain dihydroxyketo amides. Erylusamides A, B and D differ in the length of the hydrocarbon chain, while erylusamide C is a structural isomer of erylusamide B.

On the prevalence of bridged macrocyclic pyrroloindolines formed in regiodivergent alkylations of tryptophan.

A Friedel-Crafts alkylation is described that efficiently transforms tryptophan-containing peptides into macrocycles of varying ring connectivity. Factors are surveyed that influence the distribution of regioisomers, with a focus on indole C3-alkylations leading to bridged endo-pyrroloindolines. We probe the stability and stereochemistry of these pyrroloindolines, study their rearrangement to C2-linked indolic macrocycles, and demonstrate a scalable, stereoselective synthesis of this compound class. Placing the macrocyclization in sequence with further template-initiated annulation leads to extraordinary polycyclic products and further demonstrates the potential for this chemistry to drive novel peptidomimetic lead discovery programs.

The impact of data integrity on decision making in early lead discovery.

Data driven decision making is a key element of today's pharmaceutical research, including early drug discovery. It comprises questions like which target to pursue, which chemical series to pursue, which compound to make next, or which compound to select for advanced profiling and promotion to pre-clinical development. In the following paper we will exemplify how data integrity, i.e. the context data is generated in and auxiliary information that is provided for individual result records, can influence decision making in early lead discovery programs. In addition we will describe some approaches which we pursue at Boehringer Ingelheim to reduce the risk for getting misguided.

New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: an open resource.

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host–pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.

Isolation and Structural Elucidation of Armeniaspirols A–C: Potent Antibiotics against Gram‐Positive Pathogens

In an antibiotic lead discovery program, the known strain Streptomyces armeniacus DSM19369 has been found to produce three new natural products when cultivated on a malt-containing medium. The challenging structural elucidation of the isolated compounds was achieved by using three independent methods, that is, chemical degradation followed by NMR spectroscopy, a computer-assisted structure prediction algorithm, and X-ray crystallography. The compounds, named armeniaspirol A-C (2-4), exhibit a compact, hitherto unprecedented chlorinated spiro[4.4]non-8-ene scaffold. Labeling experiments with [1-(13)C] acetate, [1,2-(13)C2] acetate, and [U-(13)C] proline suggest a biosynthesis through a rare two-chain mechanism. Armeniaspirols displayed moderate to high in vitro activities against gram-positive pathogens such as methicillin-resistant S. aureus (MRSA) or vancomycin resistant E. faecium (VRE). As analogue 2 was active in vivo in an MRSA sepsis model, and showed no development of resistance in a serial passaging experiment, it represents a new antibiotic lead structure.

Translational application of novel withanolides for cancer treatment from Physalis species and other Solanaceae

Natural products have been the most significant source of drugs and drug leads in history. Plant biodiversity provides a resource of unlimited structural diversity for drug lead and molecular probe discovery programs such as those currently underway at the University of Kansas. Technology is gradually overcoming the traditional difficulties encountered in natural products research by improving access to biodiverse resources and ensuring compatibility of samples with high throughput procedures. Our drug lead discovery effort is aimed at uncovering bioactive molecules from plants of the U.S. Great Plains. Approaches utilized in the selection of samples consist of biodiversity-based collections and ethnobotanical information on the medicinal uses in the areas of study. In order to identify antiproliferative compounds, a library of over 1,200 plant extracts was tested for antiproliferative activity against an array of cell lines [melanoma cell lines (B16F10, SKMEL28); human head and neck squamous cell carcinomas (HNSCC) cell lines (MDA1986, JMAR, UM-SCC-2, JHU011); medullary thyroid carcinoma (MTC) cell lines (TT and DRO81–1), and a non-malignant cell line (MRC5)]. In vitro experiments included evaluation of anticancer activity and effects on cell proliferation by MTS assay and trypan blue exclusion. Apoptosis and cell cycle arrest were characterized by annexin V-propidium iodide (PI) flow cytometry and confirmed by Western Blot analysis for activation of caspase 3 and cleavage of PARP as well as cell-cycle specific proteins. Analysis of specific pathways including modulation of Akt, MAP-kinase and RET phosphorylation were evaluated in vitro as well as ex vivo in treated mice using Western blot analysis. Physalis longifolia Nutt. and several related members in the Solanaceae were identified as the most active of the species tested in this study. This led to the discovery of over 30 withanolide-type steroidal lactones, 16 of which are new compounds. In addition to withaferin A (WA), 14 withanolides showed IC50 values in the range between 0.067 and 9.3µM in vitro by MTC with selectivity of 3 to 20 fold compared to normal cells. A mechanistic study showed that WA induced apoptosis and cell death in HNSCC and MTC cells as well as modulation of the cell cycle from G0/G1 arrest to a shift to G2/M and S-phase. Western blot analysis demonstrated that cancer cells treated with WA exhibited inhibition of Akt and caspase-3 expression, and cleavage of PARP. WA was further evaluated in vivo in a metastaic murine model of MTC. All treated animals were alive, showing tumor regression and growth delay without toxicity or weight loss at six weeks post-treatment. Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose dependent manner (almost complete inhibition with treatment of 5µM WA) as well as potent inhibition of phosphor-ERK and phosphor-Akt levels. WA was shown to be a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. This investigation constitutes the first report of the antiproliferative activity of WA and other withanolides against MTC and HNSCC. Procurement and screening protocols, isolation and structure determination, lead optimization, and antiproliferative activities of withanolides will be presented.

Cone snail milked venom dynamics – A quantitative study of Conus purpurascens

Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51–121.01 μM within single milked venom samples. This provides for a 6.37–20,965 fold-excess of toxin to induce apparent IC50 for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus.

Effectiveness of 2D Fingerprints for Scaffold Hopping

It has been suggested that similarity searching using 2D fingerprints may not be suitable for scaffold hopping. This article reports a detailed evaluation of the effectiveness of six common types of 2D fingerprints when they are used for scaffold-hopping similarity searches of the Molecular Design Limited Drug Data Report database, World of Molecular Bioactivity database and Maximum Unbiased Validation database. The results demonstrate that 2D fingerprints can be used for scaffold hopping, with novel scaffolds being identified in nearly every search that was carried out. The degree of enrichment depends on the structural diversity of the actives that are being sought, with the greatest enrichments often being obtained using the extended connectivity fingerprint encoding a circular substructure of diameter four bonds (ECFP4) fingerprint. 2D fingerprints provide a simple and computationally efficient way of identifying novel chemotypes in lead-discovery programs.

Novel Arylimidamides for Treatment of Visceral Leishmaniasis

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC(50)], <1 microM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC(50) < or = 0.12 microM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.

Synthesis, Inhibition Potency, Binding Mode, and Antiprotozoal Activities of Fluorescent Inhibitors of Trypanothione Reductase Based on Mepacrine‐Conjugated Diaryl Sulfide Scaffolds

Trypanothione reductase (TR) is a flavoenzyme unique to trypanosomatid parasites and a target for lead discovery programs. Various inhibitor scaffolds have emerged in the past, exhibiting moderate affinity for the parasite enzyme. Herein we show that the combination of two structural motifs of known TR inhibitors - diaryl sulfides and mepacrine - enables the simultaneous addressing of two hydrophobic patches in the active site. The binding efficacy of these conjugates is enhanced over that of the respective parent inhibitors. They show K(ic) values for the parasite enzyme down to 0.9+/-0.1 microm and exhibit high selectivity for TR over human glutathione reductase (GR). Despite their considerable molecular mass and in some cases permanent positive charges, in vitro studies revealed IC(50) values in the low micromolar to sub-micromolar range against Trypanosoma brucei rhodesiense and Trypanosoma cruzi, as well as the malaria parasite Plasmodium falciparum, which lack trypanothione metabolism. The inhibitors exhibit strong fluorescence due to their aminoacridine moiety. This feature allows visualization of the drugs in the parasite where high accumulation was observed by fluorescence microscopy even after short exposure times.

Receptor-Based Computational Screening of Compound Databases: The Main Docking-Scoring Engines

The processes used by academic and industrial scientists to discover new drugs have recently experienced a true renaissance with many new and exciting techniques. The number of protein structures and/or chemical ligands is constantly growing, through the use of parallel chemistry, X-ray crystallography, NMR or homology modeling methods and so is the theoretical understanding of protein-ligand interactions. As such, structure-based approaches to drug-design and in silico screening are becoming routine part of most modern lead discovery programs. Prioritization of compound libraries is an extremely important task that aims at the rapid identification of tight-binding ligands and ultimately new therapeutic compounds. These in silico approaches combined with other experimental methods facilitate the design of new medicines to treat cardiovascular, degenerative, infectious, and neoplastic diseases, among others. Here, we review key concepts and specific features of several selected ligand-receptor docking/scoring methods while several other topics pertaining to the field of in silico screening are reviewed in the following articles of this special issue of Current Protein and Peptide Science.

Fragmentary Solutions Astex Therapeutics Puts the Pieces Together

Sunesis Pharmaceuticals (www.sunesis.com), a 130 person company located in South San Francisco, focuses on cell-cycle inhibitors for cancer. Sunesis calls its fragment-based discovery approach “tethering” and is focusing on weak binding fragments by looking at the binding event itself. Sunesis also uses another approach called extended tethering, where fragments are linked by a covalent bond to the target protein. “In one instance you fish off the bank and the next you fish off the pier,” said Dr. James Wells, professor of pharmaceutical chemistry and cellular and molecular pharmacology at UCSF and Sunesis cofounder.Sunesis has three compounds in the pipeline; SNS-595, in phase II clinical trials; SNS-032, in phase I clinical trials; and SNS-314 (preclinical.) The company also has five strategic collaborations with Biogen Idec, Johnson & Johnson PRD, and Merck. Founded in 1998, Sunesis raised $200 million from various investors and went public in 2005. “I think it [fragment based discovery] is complementary to high-throughput screening,” said Robert McDowell, Ph.D, vice president, Discovery Chemistry.On the other side of the Bay, Plexxikon (www.plexxikon.com) is a 65 person company located in Berkeley. Plexxikon starts with fragments between 150 and 350 Da in molecular weight, then screens them using biochemical assays, looking for very weak activity. Subsequent cocrystallographic structural analysis sorts the wheat from the chaff. Sometimes, to get additional interaction information, Plexxikon employs diverse techniques like surface plasmon-enhanced Raman spectroscopy and NMR to weed out the false positives from each technique. “You use an assay just to identify a measure of biochemical activity at high concentration,” said Dean Artis, “We really let the crystals decide what is an interesting compound versus a confusing assay artifact. You get a very small, compact starting point.”Following this methodology, the company develops a “scaffold” that has binding affinity for multiple members of a protein family. Currently, Plexxikon uses several scaffolds to target each of three protein families (kinases, nuclear receptors, phosphodiesterases). Artis commented that Plexxikon has filed INDs for molecules generated using this approach. According to Artis, candidate compounds for preclinical testing can be generated in as little as 3–6 months. The company initiated collaborations with Genentech, Inc., in 2003 and Wyeth Pharmaceuticals in 2004.“I came to Plexxikon to make drugs,” Artis said. “This process gives you a very efficient path to do not just an exercise in technology, but to do drug design in a time frame that might actually help people sooner rather than later. Our first drug is in patients, and we've only been doing chemistry for four years.”What is the view of big pharma on this method? “We typically use it as the complementary method to HTS,” said Dr. Andreas Marzinzik, group leader, Integrated Lead Discovery Program, Novartis Institute of Biomedical Research, “we would really want to focus on targets where HTS delivers no promising hits.” Marzinzik cited, for example, non-ATP competitive inhibitors of kinases, which are hard to identify by high throughput screening. “The advantage is really that FBS technology is not prone to false positives.” According to Marzinzik, Novartis assembled a working group to advance fragment-based discovery technology in 2005. Novartis has an internal project and also collaborates with outside companies for specific projects. “It is definitely a tool for us here at Novartis,” Marzinzik said.It remains an open question, after the iterative optimizing of weak leads, if FBDD really shaves off that much time and effort from the discovery process. But it could pick up a few unprepossessing fragments that might grow up to be great drugs if they just got a bit of attention.

Unsupervised 3D Ring Template Searching as an Ideas Generator for Scaffold Hopping: Use of the LAMDA, RigFit, and Field-Based Similarity Search (FBSS) Methods

Crystal structures taken from the Cambridge Structural Database were used to build a ring scaffold database containing 19 050 3D structures, with each such scaffold then being used to generate a centroid connecting path (CCP) representation. The CCP is a novel object that connects ring centroids, ring linker atoms, and other important points on the connection path between ring centroids. Unsupervised searching in the scaffold and CCP data sets was carried out using the atom-based LAMDA and RigFit search methods and the field-based similarity search method. The performance of these methods was tested with three different ring scaffold queries. These searches demonstrated that unsupervised 3D scaffold searching methods can find not only the types of ring systems that might be retrieved in carefully defined pharmacophore searches (supervised approach) but also additional, structurally diverse ring systems that could form the starting point for lead discovery programs or other scaffold-hopping applications. Not only are the methods effective but some are sufficiently rapid to permit scaffold searching in large chemical databases on a routine basis.

Virtual Screening Using Binary Kernel Discrimination: Analysis of Pesticide Data

This paper discusses the use of binary kernel discrimination (BKD) for identifying potential active compounds in lead-discovery programs. BKD was compared with established virtual screening methods in a series of experiments using pesticide data from the Syngenta corporate database. It was found to be superior to methods based on similarity searching and substructural analysis but inferior to a support vector machine. Similar conclusions resulted from application of the methods to a pesticide data set for which categorical activity data were available.

Comparative Virtual and Experimental High-Throughput Screening for Glycogen Synthase Kinase-3β Inhibitors

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that has recently emerged as a key target for neurodegenerative diseases and diabetes. As an initial step of our lead discovery program, we developed a virtual screen to discriminate known GSK-3beta inhibitors and inactive compounds using FlexX, FlexX-Pharm, and FlexE. The maximal enrichment factor (EF = 28) suggests that our protocol identifies potential GSK-3beta inhibitors effectively from large compound collections. The effectiveness of our screening protocol was further investigated by comparative experimental and virtual high-throughput screens (HTSs) performed for the same subset of our corporate library. Enrichment factors, the significantly higher hit rate of virtual screening (12.9%) than that of the HTS (0.55%), and also the comparison of active clusters suggest that our virtual screening protocol is an effective tool in GSK-3beta-based library focusing. Head-to-head comparison of true/false positives and negatives revealed the two approaches to be complementary rather than competitive.

Sensitivity Improvement in 19F NMR-Based Screening Experiments: Theoretical Considerations and Experimental Applications

NMR-based binding and functional screening performed with FAXS (fluorine chemical shift anisotropy and exchange for screening) and 3-FABS (three fluorine atoms for biochemical screening) represents a potential alternative approach to high-throughput screening for the identification of novel potential drug candidates. The major limitation of this method in its current status is its intrinsic low sensitivity that limits the number of tested compounds. One approach for overcoming this problem is the use of a cryogenically cooled (19)F probe that reduces the thermal noise in the receiver circuitry. Sensitivity improvement in the two screening techniques achieved with the novel cryogenic (19)F probe technology permits an increased throughput, detection of weaker binders and inhibitors (relevant in a fragment-based lead discovery program), detection of slow binders, and reduction in protein and substrate consumption. These aspects are analyzed with theoretical simulations and experimental quantitative performance evaluation. Application of 3-FABS combined with the cryogenic (19)F probe technology to rapid screening at very low enzyme concentrations and the current detection limits reached with this approach are also presented.