A centralized approach to tandem mass spectrometry method development for high-throughput ADME screening

A centralized approach to acquisition and dissemination of tandem mass spectrometry (MS/MS) conditions within an ADME-screening bioanalytical mass spectrometry group has been developed. The method development process uses two automated software products (Autoscan and Automaton) specifically designed for mass spectrometers manufactured by MDS Sciex. Both provide the ability to quickly determine selected reaction monitoring (SRM) transitions for hundreds of compounds per day. In addition, Autoscan determines optimal polarity and collision energy (CE). Automaton also determines the optimal declustering potential (DP) as well as the CE. The resulting optimized conditions are loaded into a central database for access by LC/MS/MS bioanalysis workstations in the group. The effect of DP and CE on the sensitivity was investigated. Optimization of DP improved signal response about 27% on average. For approximately 10% of compounds, signal enhancement was greater than 50% compared to the generic setting. A generic setting of DP = 25 V can be used for the majority of ADME-screening applications. Optimization of CE can have a much larger impact on signal intensity and a minimum of three CE settings should be tested. We have determined that CE values of 1, 30 and 45 V provide adequate coverage for most small molecule drug discovery analytes.     

Polymorphism - integrated approach from high-throughput screening to crystallization optimization

Crystal structure (polymorphism) as well as crystal shape (morphology) and size have a huge practical and commercial impact on active substances all the way from research to manufacture of the final product. For an optimal development process, it is important to have an integrated approach to these issues ranging from a systematic polymorphism screening to a controlled scale-up of the crystallization process. The polymorphism program has to be tailored according to the development stage. Particularly suitable for an early development stage is a high-throughput polymorphism screening, which is the basis for a more thorough investigation if the product proceeds further in development. Such a comprehensive polymorphism investigation involves further crystallization experiments and extensive physicochemical characterization of the various forms. In this article the high-throughput polymorphism screening method that we have developed is described. Using carbamazepine as an example, the power of this high-throughput polymorphism screening system is demonstrated. Not only were all published forms found, but also new forms were identified. In the second part of the article, important considerations for crystallization optimization are discussed, again using the example of carbamazepine. This revised version was published online in July 2006 with corrections to the Cover Date.     

Novel statistical approach for primary high-throughput screening hit selection

The standard activity threshold-based method (the "top X" approach), currently widely used in the high-throughput screening (HTS) data analysis, is ineffective at identifying good-quality hits. We have proposed a novel knowledge-based statistical approach, driven by the hidden structure-activity relationship (SAR) within a screening library, for primary hit selection. Application to an in-house ultrahigh-throughput screening (uHTS) campaign has demonstrated it can directly identify active scaffolds containing valuable SAR information with a greatly improved confirmation rate compared to the standard "top X" method (from 55% to 85%). This approach may help produce high-quality leads and expedite the hit-to-lead process in drug discovery.     

Electrochemistry-enabled fabrication of orthogonal nanotopography and surface chemistry gradients for high-throughput screening

Gradient surfaces are emerging tools for investigating mammalian cell-surface interactions in high throughput. We demonstrate the electrochemical fabrication of an orthogonal gradient platform combining a porous silicon (pSi) pore size gradient with an orthogonal gradient of peptide ligand density. pSi gradients were fabricated via the anodic etching of a silicon wafer with pore sizes ranging from hundreds to tens of nanometers. A chemical gradient of ethyl-6-bromohexanoate was generated orthogonally to the pSi gradient via electrochemical attachment. Subsequent hydrolysis and activation of the chemical gradient allowed for the generation of a cyclic RGD gradient. Whilst mesenchymal stem cells (MSC) were shown to respond to both the topographical and chemical cues arising from the orthogonal gradient, the MSC's responded more strongly to changes in RGD density than to changes in pore size during short-term culture.     

A high-throughput approach to lanthanide complexes and their rapid screening in the ring opening polymerisation of caprolactone

Libraries of lanthanide complexes supported by nitrogen and oxygen containing ligands have been synthesised using a high-throughput approach. The complexes were employed in the ring-opening polymerisation of epsilon-caprolactone, in some cases giving polycaprolactone of controlled molecular weight and narrow polydispersity. The libraries, based on twenty-one ligands and eight lanthanide reagents, were developed in order to determine the best combination of lanthanide metal and ligand. They were prepared via transamination reactions of [Ln[N(SiMe(3))(2)](3)] complexes with tetradentate dianionic ligands containing oxygen and nitrogen donors. 1H NMR spectroscopy was used to screen polymerisation activity. The steric demand of the ligand has a significant effect on the polymerisation process, as do the type of nitrogen donor and the size of the central Ln(3+) ion. Ligands containing aryl rings with bulky substituents such as tert-pentyl groups afforded species capable of performing controlled polymerisation of caprolactone, whereas less bulky groups such as methyl were not effective. Yttrium and mid-sized lanthanides such as samarium showed increased activity compared with the larger lanthanides, lanthanum and praseodymium, and the smaller lanthanides like ytterbium. X-ray crystal structures of a sterically demanding chelating amine-bis((2-hydroxyaryl)methyl) ligand and a chloride bridged dinuclear gadolinium complex are reported. The centrosymmetric molecule contains gadolinium in distorted capped trigonal prismatic environments bonded to two amine, two phenolate, one THF and two chloride donors.     

A pseudo-ligand approach to virtual screening

A virtual screening method is presented that is grounded on a receptor-derived pharmacophore model termed "virtual ligand" or "pseudo-ligand". The model represents an idealized constellation of potential ligand sites that interact with residues of the binding pocket. For rapid virtual screening of compound libraries the potential pharmacophore points of the virtual ligand are encoded as an alignment-free correlation vector, avoiding spatial alignment of pharmacophore features between the pharmacophore query (i.e., the virtual ligand) and the candidate molecule. The method was successfully applied to retrieving factor Xa inhibitors from a Ugi three-component combinatorial library, and yielded high enrichment of actives in a retrospective search for cyclooxygenase-2 (COX-2) inhibitors. The approach provides a concept for "de-orphanizing" potential drug targets and identifying ligands for hitherto unexplored or allosteric binding pockets.     

A high-throughput screening assay for hydroxynitrile lyase activity

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.     

A scalable approach to diaminopyrazoles using flow chemistry

This Letter reports on how the combination of microwave and continuous flow chemistry facilitated the convenient preparation of aminopyrazoles from commercial aryl halides. The method was applied to a variety of substrates with good to excellent yields and further extended toward the complete flow synthesis of 5,7-dimethyl-3-phenylpyrazolo[1,5-a]pyrimidin-2-amine.     

A multiple replica approach to simulate reactive trajectories

A method to generate reactive trajectories, namely equilibrium trajectories leaving a metastable state and ending in another one is proposed. The algorithm is based on simulating in parallel many copies of the system, and selecting the replicas which have reached the highest values along a chosen one-dimensional reaction coordinate. This reaction coordinate does not need to precisely describe all the metastabilities of the system for the method to give reliable results. An extension of the algorithm to compute transition times from one metastable state to another one is also presented. We demonstrate the interest of the method on two simple cases: A one-dimensional two-well potential and a two-dimensional potential exhibiting two channels to pass from one metastable state to another one.     

A novel approach to high-throughput quality control of parallel synthesis libraries

Combinatorial chemistry is a powerful tool to enhance drug discovery efforts in the pharmaceutical industry. One type of combinatorial chemistry, parallel synthesis, is now widely used to prepare numerous compounds of structural diversity. A novel high-throughput method for quality control of parallel synthesis libraries has been developed. The method uses flow injection MS, for proof of structure and estimation of purity, and a novel direct injection CLND technique for quantitation of amount. Following the synthesis of a small molecule library, compounds analyzed using this technique were characterized by mass spectrometry, and an accurate concentration of the compound was assessed by CLND. Characterization of one compound is completed in 60 s, allowing for up to 1000 compounds to be analyzed in a single day. The data is summarized using pass/fail criteria using internally developed software.     

Combinatorial chemistry approach to chiral catalyst engineering and screening: rational design and serendipity

An efficient asymmetric catalyst relies on the successful combination of a large number of interrelated variables, including rational design, intuition, persistence, and good fortune-not all of which are necessarily well-understood; this renders such practice largely empirical. As a result, the possibility of using combinatorial chemistry methods in asymmetric catalysis research has been widely recognized to be highly desirable. In this account, we attempt to show the principle and application of combinatorial approach in the discovery of chiral catalysts for enantioselective reactions. The concept focuses on the strategy for the creation of a modular chiral catalyst library by two-component ligand modification of metal ions on the basis of molecular recognition and assembly. The self-assembled chiral catalyst with two different ligands indeed exhibited synergistic effects in terms of both enantioselectivity and activity in comparison with its corresponding homocombinations in many reactions. The examples described in this paper demonstrated the powerfulness of combinatorial approach for the discovery of novel chiral catalyst systems, particularly for the development of highly efficient, enantioselective, and practical catalysts for enantioselective reactions. We hope this concept will stimulate further work on the discovery of more highly efficient and enantioselective catalysts, as well as unexpected classes of catalysts or catalytic enantioselective reactions in the future with the help of a combinatorial chemistry approach.     

A fragment based click chemistry approach towards hybrid G-quadruplex ligands: design,synthesis and biophysical evaluation

A library of hybrid oxazole–triazole based compounds containing contiguously linked aromatic units were synthesised as G-quadruplex binding ligands. The design of these ligands was based upon combining features of our first generation of G-quadruplex bis-triazole ligands and the natural product telomestatin. The syntheses and biophysical studies of these ligands are described.     

The semiempirical approach to chemistry

Two kinds of quantum mechanical treatment are needed in chemistry: First, a treatment accurate enough to provide definitive solutions of chemical problems, in particular, the prediction of reactions and reaction mechanisms, without reference to experiment. As yet, treatments of this degree of accuracy are restricted to a few small atoms and molecules. Second, a treatment that chemists can use as a practical aid to their own research, on the same basis as mass spectrometry or NMR . Here, the need has been largely met by the series of progressively better semiempirical methods, in particular, those developed by our group. Since only the best and most expensive ab initio procedures are superior to ours and since the computing time they need is far greater, their use as a practical aid in chemistry is restricted. Our procedures, therefore, serve as a useful supplement to ab initio ones in areas where the latter cannot be applied effectively, as the leading ab initioists now fully recognize. Since the semiempirical approach is capable of almost unlimited further development, this situation is likely to continue in the foreseeable future. © 1992 John Wiley & Sons, Inc.     

A practical approach to metrology in chemistry and biology

 The inherent difficulties of measurement in chemistry and biology have made it necessary to open discussion forums solely devoted to bench level work. This contribution summarises the conclusions of the Standards, Measurements and Testing Project (SMT4-CT96-6505) of the European Commission, devoted to the application of metrology in chemistry and biology. The objective of this project was to provide orientations and some recommendations which could partially satisfy end-users needs in the near future. Received: 29 September 1998 · Accepted: 21 December 1998     

A massively parallel approach to the quasiclassical reactive scattering

The suitability of massively parallel architectures for carrying out efficient calculations of quasiclassical rate constants for atom-diatom reactive processes has been investigated. Problems related to the parallel structuring of the computational procedure, fixed and scaled speed-ups, efficiency factors and their dependence upon the size of the problem, and the number of processors are discussed.     

A 2DE approach for high-throughput antigen separation applicable to mAb production

A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein-protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development of mAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 μg of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available for monoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the high-throughput monoclonal antibody process providing a vast array of high quality antigens at very low cost.