Combinatorial Chemistry & High Throughput Screening - Volume 4, Issue 6, 2001

During the practice of combinatorial chemistry, it has been realized that molecular diversity is not the only essential feature in a synthetically feasible library. In addition, it is of utmost importance to enrich potential libraries with those molecules which could be converted to viable drug candidates. Given the enormous number of potentially synthesizable compounds, there is a need to design a subset of true drug-like compounds. In addition, a paradigm shift in drug discovery has resulted in the integration of pharmacokinetic and drug development activities into early stages of lead discovery. In particular, in silico filters are being developed and used to help identify and screen out compounds that are unlikely to become drugs. This paper highlights recent computational approaches towards the design of drug-like compound libraries, in particular, the prediction of drug-likeness in a more general sense as well as intestinal absorption through passive transport, the permeation of the blood-brain barrier and recent developments towards identification of potentially metabolically unstable molecules. Current computational tools for library design allow the incorporation of medicinal chemistry knowledge into library planning by a variety of methods, ranging from the use of privileged building blocks and simple counting of structural properties (e.g. number of hydrogen bonding partners) to relatively complex regression or neural network-based models to explain oral bioavailability and other pharmacokinetic properties by structural features. These tools are being incorporated more frequently into drug design according to the rule-of-five which refers to simple descriptors correlated to oral drug absorption. Combining experimental knowledge with effective computational filtering and prediction of various aspects of drug-likeness thus facilitates the rapid and cost-effective elimination of poor candidates prior to synthesis and helps focus attention on interesting molecules.

In recent years, there have been a growing number of examples of the successful isolation of peptide ligands for enzymes from phage-displayed combinatorial peptide libraries. These peptides typically bind at or near the active site of the enzymes and can inhibit their activity. We review the literature on peptide ligands that have been isolated for enzymes other than proteases as well as present data on peptide ligands we have identified for E. coli dihydrofolate reductase (DHFR) which bind at, or near, the same site as the known inhibitors methotrexate or trimethoprim. Thus, while the peptide ligand isolated from phage-displayed libraries may not resemble the chemical structure of the normal substrate of the enzyme, the peptide can be used as an inhibitor to evaluate the function of the enzyme or for drug discovery efforts (i.e., as a lead compound for peptidomimetic design or as displaceable probe in high-throughput screens of libraries of small molecules).

The technologies for screening peptide and protein libraries for studies in the fields of directed protein evolution and functional genomics have advanced with astonishing speed. For screening of functional proteins, three technologies are required: (i) the construction of a gene library (genotype), (ii) the establish-ment of a linkage between each protein (phenotype) and its encoding gene (genotype), and (iii) the selection of desired proteins (phenotype) from the library. This review highlights the genotype-phenotype linkage technologies, which can be classified into three types that is, cell-type linkage, virus-type linkage, and array-type linkage methods. These methods are summarized, and their advantages and disadvantages are discussed.

A method has been developed which allows direct measurement of partition coefficients (log D, log P) using liquid chromatography-mass spectrometry (LC-MS). The high throughput, microtiter plate based protocol uses small quantities of 10 mM analyte in DMSO solution and is therefore amenable to standard archive and screening formats. Single Ion Monitoring (SIM) mass spectrometry is used to achieve optimal sensitivity. Experimental log D values for 34 known drugs have been determined, with partition coefficients ranging from -2 to 5, giving data very similar to literature values. In these analyses, deviations from known values average less than 0.3 log units. The sample handling and data processing have been significantly automated, and the protocol has been applied to over 800 in-house lead molecules to date. In its format, sensitivity, throughput, and amenability to automation, it represents significant progress in the direct measurement of partitioning behavior [1].

As part of an ongoing lead discovery project we have developed a convenient method for the modification and substitution of indole moieties at the 3-position. Selective bromination of three different 2-carboxyindoles was followed by Suzuki cross-coupling with aryl and heteroaryl boronic acids on a Merrifield resin solid-phase. After column chromatography, yields of the 3- substituted indoles ranged from 42-98 percent,

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a kcat / Km of 5.9x104 M-1 s-1. Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.

The cloning of genes based on protein function has become a powerful tool for protein discovery and should play an important role in proteomics in general. We have recently reported a technique for the functional identification of protein targets by combining traditional affinity chromatography with cDNA phage display. This procedure, referred to as display cloning, directly couples biologically active natural products to the gene of their protein cellular target. We now report the cloning of a human gene, the a domain of F1 ATP synthase, using a synthetic scaffold molecule which serves as a prototype for a diverse chemical library. The ability to select genes from cDNA libraries using probes from combinatorial libraries would greatly increase the number of small molecule / protein interactions that can be identified. This method might prove valuable in furthering our understanding of biology and its application toward drug development.

Display methods development is currently extending the application of this strategy beyond the generation of ligand binding reagents for research, clinical, or biotechnological purposes to its use as a primary research tool. Peptide- and cDNA display methods have the potential to contribute to understanding the mechanisms of certain classes of drugs and to help map protein-protein interactions of physiological importance. Although the critical contribution of comprehensive amino acid sequence databases has been recognized, of equal importance might be structural genomics concepts in the application of display system technology to proteomics research. Lessons learned from the study of antibody-antigen interactions are reviewed here and applied to the field of display technology with the goal of delineating major factors involved in the successful mimicry of natural protein-ligand interactions.