Solution-phase Screening Research Articles

Designer Gelators for the Crystallization of a Salt Active Pharmaceutical Ingredient-Mexiletine Hydrochloride.

We report an approach to obtain drug-mimetic supramolecular gelators, which are capable of stabilizing metastable polymorphs of the pharmaceutical salt mexiletine hydrochloride, a highly polymorphic antiarrhythmic drug. Solution-phase screening led to the discovery of two new solvated solid forms of mexiletine, a type C 1,2,4-trichlorobenzene tetarto-solvate and a type D nitrobenzene solvate. Various metastable forms were crystallized within the gels under conditions which would not have been possible in solution. Despite typically crystallizing concomitantly with form 1, a pure sample of form 3 was crystallized within a gel of ethyl methyl ketone. Various type A channel solvates were crystallized from gels of toluene and ethyl acetate, in which the contents of the channels varied from those of solution-phase forms. Most strikingly, the high-temperature-stable form 2 was crystallized from a gel in 1,2-dibromoethane: the only known route to access this form at room temperature. These results exemplify the powerful stabilizing effect of drug-mimetic supramolecular gels, which can be exploited in pharmaceutical polymorph screens to access highly metastable or difficult-to-nucleate solid forms.

Affinity selection-mass spectrometry for the discovery of pharmacologically active compounds from combinatorial libraries and natural products.

Invented to address the high-throughput screening (HTS) demands of combinatorial chemistry, affinity selection-mass spectrometry (AS-MS) utilizes binding interactions between ligands and receptors to isolate pharmacologically active compounds from mixtures of small molecules and then relies on the selectivity, sensitivity, and speed of mass spectrometry to identify them. No radiolabels, fluorophores, or chromophores are required. Although many variations of AS-MS have been devised, three approaches have emerged as the most flexible, productive, and popular, and they differ primarily in how ligand-receptor complexes are separated from nonbinding compounds in the mixture. These are pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS). PUF and SEC AS-MS are solution-phase screening approaches, and MagMASS uses receptors immobilized on magnetic microbeads. Because pools of compounds are screened using AS-MS, each containing hundreds to thousands of potential ligands, hundreds of thousands of compounds can be screened per day. AS-MS is also compatible with complex mixtures of chemically diverse natural products in extracts of botanicals and fungi and microbial cultures, which often contain fluorophores and chromophores that can interfere with convention HTS. Unlike conventional HTS, AS-MS may be used to discover ligands binding to allosteric as well as orthosteric receptor sites, and AS-MS has been useful for discovering ligands to targets that are not easily incorporated into conventional HTS such as membrane-bound receptors.

A Synergistic Combinatorial and Chiroptical Study of Peptide Catalysts for Asymmetric Baeyer-Villiger Oxidation.

We report an approach to the asymmetric Baeyer-Villiger oxidation utilizing bioinformatics-inspired combinatorial screening for catalyst discovery. Scaled-up validation of our on-bead efforts with a circular dichroism-based assay of alcohols derived from the products of solution-phase reactions established the absolute configuration of lactone products; this assay proved equivalent to HPLC in its ability to evaluate catalyst performance, but was far superior in its speed of analysis. Further solution-phase screening of a focused library suggested a mode of asymmetric induction that draws distinct parallels with the mechanism of Baeyer-Villiger monooxygenases.

Jeffamine Derivatized TentaGel Beads and Poly(dimethylsiloxane) Microbead Cassettes for Ultrahigh-Throughput in Situ Releasable Solution-Phase Cell-Based Screening of One-Bead-One-Compound Combinatorial Small Molecule Libraries

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Development of Hydrogel TentaGel Shell−Core Beads for Ultrahigh Throughput Solution-Phase Screening of Encoded OBOC Combinatorial Small Molecule Libraries

The one-bead one-compound (OBOC) combinatorial library method enables the rapid generation and screening of millions of discrete chemical compounds on beads. Most of the OBOC screening methods require the library compounds to remain tethered to the bead during screening process. Methods have also been developed to release library compounds from immobilized beads for in situ solution phase or "lawn" assays. However, this latter approach, while extremely powerful, is severely limited by the lack of suitable solid supports for such assays. Here, we report on the development of a novel hydrogel TentaGel shell-core (HTSC) bead in which hydrogel is grafted onto the polystyrene-based TentaGel (TG) bead as an outer shell (5-80 mum thick) via free radical surface-initiated polymerization. This novel shell-core bilayer resin enables the preparation of encoded OBOC combinatorial small molecule libraries, such that the library compounds reside on the highly hydrophilic outer layer and the coding tags reside in the polystyrene-based TG core. Using fluorescein as a model small molecule compound, we have demonstrated that fluorescein molecules that have been linked covalently to the hydrogel shell via a disulfide bond could readily diffuse out of the hydrogel layer into the bead surrounding after reduction with dithiothreitol. In contrast, under identical condition, the released fluorescein molecules remained bound to unmodified TG bead. We have prepared an encoded OBOC small molecule library on the novel shell-core beads and demonstrated that the beads can be readily decoded.

High-Throughput Sequence Determination of Cyclic Peptide Library Members by Partial Edman Degradation/Mass Spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.

New dsDNA binding unnatural oligopeptides with pyrimidine selectivity

Solid phase peptide library screening followed by extension of a lead recognition element for binding to a dsDNA sequence (NF binding site of IL6) using solution phase screening, delivered a new DNA binding peptide, Ac-Arg-Ual-Sar-Chi-Chi-Tal-Arg-CONH 2. In the present research, the contribution of the different amino acid side chains to the binding strength of the peptide to dsDNA was investigated using an ethidium bromide displacement test. Based on these results, the lead structure was optimized by deconvolution. Eight new unnatural amino acids were evaluated at two positions of the heptapeptide replacing the Ual-Sar fragment. The strongest dsDNA binding was observed using {[(3-chlorophenyl)methyl]amino}acetic acid (Cbg) and β-cyclohexyl-l-alanine (Cha) respectively, at those two positions. A 10-fold increase in affinity compared to the Ual-Sar sequence was obtained. Further enhancement of dsDNA binding was obtained with hybrid molecules linking the newly developed peptide fragment to an acridine derivative with a flexible spacer. This resulted in ligands with affinities in the μM range for the dsDNA target ( K d of 2.1×10 −6 M). DNase I footprinting with the newly developed oligopeptide motifs showed the presence of a pronounced pyrimidine specificity, while conjugation to an intercalator seems to redirect the interaction to mixed sequences. This way, new unnatural oligopeptide motifs and hybrid molecules have been developed endowed with different sequence selectivities. The results demonstrate that the unnatural peptide library approach combined with subsequent modification of selected amino acid positions, is very suited for the discovery of novel sequence-specific dsDNA binding ligands.

Selection of New Sequence-Selective Unnatural Peptides Binding to Double-Stranded Deoxyribonucleic Acids (dsDNA) by Means of a Gel-Retardation Experiment for Library Analysis

Previously, we developed a methodology for the solid-phase screening of peptide libraries for interaction with double-stranded deoxyribonucleic acids (dsDNA). In the search for new and more-potent DNA ligands, we investigated the strategy of solution-phase screening of chemical libraries consisting of unnatural oligopeptides. After synthesis of the selected amino acid building blocks, libraries were constructed with the general structure Ac-Arg-Ual-Sar-X1-X2-X3-Arg-NH2, where X represents each of twelve unnatural or natural amino acids. Optimization of the sequence of binding peptides was performed with an iterative deconvolution procedure. Selection of interacting peptides was carried out in solution by means of gel-retardation experiments, starting with libraries of 144 compounds. A 14-base-pair double-stranded DNA fragment was chosen as the target. After several cycles of synthesis and screening of libraries and individual peptides, an oligopeptide was selected with an apparent dissociation constant of 9⋅10−5 M, as determined by gel-retardation experiments. This peptide was studied by NMR spectroscopy. A certain degree of conformational pre-organization of the peptides was shown by temperature-dependent circular-dichroism experiments. Finally, DNase-I-footprinting studies indicated a preferential interaction with a 6-base-pair mixed sequence 5′-CTGCAT-3′. This study demonstrates that gel-shift experiments can be used for the solution-phase screening of library mixtures of peptides against dsDNA. In general, this technique allows the selection of new sequence-selective dsDNA-interacting molecules. Furthermore, novel dsDNA-binding unnatural oligopeptides were developed with affinities in the 0.1 mM range.

Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides.

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

Immunoaffinity Ultrafiltration with Ion Spray HPLC/MS for Screening Small-Molecule Libraries

A solution-phase screening method for libraries of pharmaceutically relevant molecules is presented. The technique is applicable to screening combinatorial libraries of 20-30 closely related molecules. In this report, individual benzodiazepines are selected from a multicomponent library mixture by formation in solution of noncovalent immunoaffinity complexes with antibodies raised to therapeutically proven drugs such as nitrazepam, temazepam, or oxazepam. Captured compounds are separated from nonspecifically bound library components by centrifugal ultrafiltration. The specifically selected molecules retained on the filter are subsequently liberated from the antibodies by acidification and analyzed by HPLC coupled with pneumatically assisted electrospray (ion spray) ionization mass spectrometric detection. Competition by the benzodiazepines for limited antibody binding sites is controlled by varying the stoichiometry of the complexation mixture. This procedure selects library components with the greatest affinity for a particular antibody. Specific capture of benzodiazepines is demonstrated by screening both a pool of structurally similar benzodiazepines and a more complex mixture of benzodiazepines with an additional set of unrelated compounds. Affinity ultrafiltration and electrospray mass spectrometry complement each other to enhance screening and identification of pooled drug candidates and potentially can be extended to other small-molecule combinatorial libraries and macromolecular receptor preparations.

One Bead, One Chemical Compound: Use of the Selectide Process for Anticancer Drug Discovery

A technology for chemical synthesis and testing of libraries of millions of chemical entities has been developed for rapid molecular and cellular screening for drug leads. Each individual compound in the library is on a separate resin bead. Screening for binding activity can be conducted directly on the beads. Biological activity is assessed in solution phase assay by cleaving a portion of the compound from each bead. The molecular structure of the compound of interest is obtained by automated peptide sequencing from the bead of origin. We have applied this technology to anticancer drug discovery as well as to other pharmaceutical targets. For anticancer drug development, current molecular targets include B-cell lymphoma, the EGF receptor, and the HER2-neu receptor. Solution phase screening with dual cleavable libraries is being used for growth inhibition of human tumor cell lines. Initial in vitro leads have been identified in each of these areas of anticancer drug discovery.

A rapid solution-phase screening technique for hybridoma culture supernatants using radiolabeled antigen and a solid-phase immunoadsorbent

The solid-phase immunoassay commonly used to screen hybridoma supernatants at times gave falsely positive results. A solution-phase screening technique, which uses radiolabeled antigen and a solid-phase immunoadsorbent, is described. This technique overcomes the problem of false positives and can be easily adopted for other soluble antigens.