Combinatorial Chemistry & High Throughput Screening - Volume 9, Issue 7, 2006

African trypanosomes are extracellular blood parasites that cause sleeping sickness in humans and Nagana in cattle. The therapeutics used to control and treat these diseases are very ineffective and thus, the development of new drugs is urgently needed. We have previously suggested to use trypanosome-specific RNA aptamers as tools for the development of novel trypanocidal compounds. Here, we report the selection of a 2'-NH2-modified RNA aptamer that binds to live trypanosomes with an affinity of 70 ± 15 nM. The aptamer adopts a stable G-quartet structure and has a half-life in human serum of ≥30 h. RNA binding is restricted to the flagellar attachment zone, located between the cell body and the flagellum of the parasite. We demonstrate that antigen-tagged preparations of the aptamer can bind to live trypanosomes and that they can be used to re-direct immunoglobulins to the parasite surface.

The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for endpoint measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.

In the past few years, NMR has been extensively utilized as a screening tool for drug discovery using various types of compound libraries. The designs of NMR specific chemical libraries that utilize a fragment-based approach based on drug-like characteristics have been previously reported. In this article, a new type of compound library will be described that focuses on aiding in the functional annotation of novel proteins that have been identified from various ongoing genomics efforts. The NMR functional chemical library is comprised of small molecules with known biological activity such as: co-factors, inhibitors, metabolites and substrates. This functional library was developed through an extensive manual effort of mining several databases based on known ligand interactions with protein systems. In order to increase the efficiency of screening the NMR functional library, the compounds are screened as mixtures of 3-4 compounds that avoids the need to deconvolute positive hits by maintaining a unique NMR resonance and function for each compound in the mixture. The functional library has been used in the identification of general biological function of hypothetical proteins identified from the Protein Structure Initiative.

The automated parallel solid-phase synthesis of a 17-member library of metallothionein-mimic decapeptides carrying a Lariat ether group is described. The peptides were synthesized in good yield and the identity and quality of each product were performed by mass spectrometry and IR. Subsequently, in the presence of europium(III) ions as fluorescent reporter, each compound was screened, both attached to the resin and cleaved off, for their sensing behavior towards metal ions (Cd2+, Hg2+, Cu2+, Mg2+ and Ca2+) using fluorimetric techniques. Several of these Cys-enriched synthetic peptides showed surprisingly low detection limits for Cd2+ and Hg2+. The analytical potential of these metallothioneinmimic decapeptides as metal ion recognition materials for sensor development is outlined. Finally, the sensing response mechanism, based on an energy transfer process and a metal ion allosteric interaction, is proposed.

The development of structure-activity relationships (SARs) relating to the function of a biological protein is often a long and protracted undertaking when using an iterative medicinal chemistry approach. High throughput screening of ECLiPS®(Encoded Combinatorial Libraries on Polymeric Support) libraries can be used to simplify this process. In this paper we illustrate how a large ECLiPS library of 26,908 compounds, based on a tricyclic core structure, was used to define a multitude of SARs for the oncogenic target, farnesyltransferase (FTase). This library, FT-2, was prepared using a split-and-pool approach in which small molecules are constructed on resin that contains tag/linker constructs to track the synthetic process [1-5]. Highly defined SARs were produced from this screen that enhanced our understanding of FTase binding site interactions. The pivotal compounds culled from this library were potent in both cell-free and cell-based FTase assays, selective over the closely related enzyme, geranylgeranyltransferase I (GGTase I), and inhibited the adherent- independent growth of a transformed cell line.

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.

Phosphoinositide 3-kinases (PI3Ks) comprise a family of kinases that transfer the terminal phosphate of adenosine triphosphate to phosphoinositides at the 3-hydroxyl of the inositol ring to form phosphoinositide (3,4,5) triphosphate (PIP3). The PI3Ks have been shown to play key roles in cell growth, motility, morphology, and survival and thus are of interest as targets in anti-inflammatory and anti-oncogenic drug development. To facilitate identification of novel and selective inhibitors of PI3Ks, we have developed a TR-FRET assay that uses directly labeled reagents. The assay makes use of the high affinity binding of phosphoinositides to a Pleckstrin homology (PH) domain in the general receptor for phosphoinositides 1 (Grp1) protein. It monitors PIP3 produced from the enzymatic reaction by measuring its competition with Bodipy®-FL-labeled PIP3 for binding to Terbium chelate-labeled Grp1. By using directly labeled reagents, this assay configuration offers higher sensitivity and faster binding/dissociation kinetics than existing non-radioactive assays, which are critical for competitive assay formats. The assay is homogenous, robust (Z' = 0.88), and simple and, thus, compatible with high throughput screening (HTS) processes.