Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant Gram-positive pathogens identified from a mixture-based combinatorial library

The rapid rise in antibiotic-resistant Gram-positive bacterial infections prompted us to explore the development of novel strategies for synthesis of large chemical libraries amenable to high-throughput screening for antimicrobial activities. Here we report the solid-phase synthesis of a 738,192 member pyrrolidine bis-cyclic guanidine chemical library with 26 different amino acids at three positions of diversity and 42 carboxylic acids at the fourth position. This synthetic combinatorial library was developed for positional scanning and screened for bacteriostatic and bactericidal activities against the important human pathogen methicillin-resistant Staphylococcus aureus (MRSA). The eight compound mixtures exhibiting bactericidal activity (10 microg/mL) against MRSA were used to direct the synthesis of 36 individual compounds that were then screened for activity against MRSA, vancomycin-resistant Enterococcus faecalis (VRE), and two Gram-negative bacterial species. At least 20 individual compounds were bactericidal for MRSA at 2.5 microg/mL, with a subset of these compounds showing bactericidal activities (10 microg/mL) against the other species tested. This approach demonstrates the capability to synthesize and screen a complex library to yield promising antimicrobials that address a critical need for novel infectious disease therapeutics.     

Identification of potent and highly selective chiral tri-amine and tetra-amine mu opioid receptors ligands: an example of lead optimization using mixture-based libraries

The generation of chiral polyamine libraries has been successfully accomplished in our laboratory following exhaustive reduction of resin-bound peptides. Herein, we report the synthesis and screening results of a positional scanning mixture-based library of chiral hepta-amines in a radioreceptor assay for the opioid receptor. The positional scanning hepta-amine library was generated by the exhaustive reduction of a library of 34,012,070 hexapeptides. Following screening of the entire library, combinations of the most active functionalities found at each position were used to synthesize and screen 40 individual hepta-amines and served as starting 'hits' for further SAR studies. The individual compounds showed IC(50) values ranging from 14 to 345 nM. As might be anticipated by the known studies of mu opiate antagonists, the identified active hepta-amines possessed aromatic rings derived from phenylalanine and tyrosine amino acid side chains. Following SAR studies, a truncation analog, reduced and permethylated YYF-NH(2), was found to be highly active (0.5 nM) as a selective mu antagonist in the guinea pig ileum bioassay.     

Label-free primary screening and affinity ranking of fragment libraries using parallel analysis of protein panels

The authors present fragment screening data obtained using a label-free parallel analysis approach where the binding of fragment library compounds to 4 different target proteins can be screened simultaneously using surface plasmon resonance detection. They suggest this method as a first step in fragment screening to identify and select binders, reducing the demanding requirements on subsequent X-ray or nuclear magnetic resonance studies, and as a valuable "clean-up" tool to eliminate unwanted promiscuous binders from libraries. A small directed fragment library of known thrombin binders and a general 500-compound fragment library were used in this study. Thrombin, blocked thrombin, carbonic anhydrase, and glutathione-S-transferase were immobilized on the sensor chip surface, and the direct binding of the fragments was studied in real time. Only 12 microg of each protein is needed for screening of a 3000-compound fragment library. For screening, a binding site-blocked target as reference facilitates the identification of binding site-selective hits and the signals from other reference proteins for the elimination of false positives. The scope and limitations of this screening approach are discussed for both target-directed and general fragment libraries.     

Potent antibacterial lysine-peptoid hybrids identified from a positional scanning combinatorial library

In this paper, we describe the synthesis and screening of a biased positional scanning library made up of peptoids (N-alkylglycines) and lysines. The library consisted of 100 mixtures divided into four sub-libraries; OXXXKKK, XOXXKKK, XXOXKKK, and XXXOKKK, O being a defined peptoid building block and X a mixture of 25 peptoid building blocks. A theoretical number of 390,625 compounds were synthesized. The compound mixtures were screened against the American Type Culture Collection (ATCC) Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 bacterial strains, and the cytotoxic activities were assessed using a human blood hemolytic assay. The results from each sub-library were examined to identify the most potent amine at each position. On the basis of this knowledge eight new lysine-peptoid hybrids were synthesized and tested in the biological assays. One compound in particular, [N-(cyclohexylmethyl)glycyl]-[N-(1-methylhexyl)glycyl]-[N-(4-methylbenzyl)glycyl]-[N-(2-(3-chlorophenyl)ethyl)glycyl]-lysyl-lysyl-lysine amide, showed high antibacterial activity and low toxicity toward red blood cells.     

Identification of novel polyphenol oxidase inhibitors by enzymatic one-pot synthesis and deconvolution of combinatorial libraries

The feasibility of enzymatic synthesis of combinatorial libraries using multifunctional starting materials [i.e., 2,4-dihydroxy-N-(2-hydroxyethyl)benzamide, 1; 4-hydroxyphenethyl alcohol, 2; 3,5-dihydroxybenzyl alcohol, 3; and 4-hydroxybenzyl alcohol, 4] with six vinyl esters, in a one-pot reaction, was investigated. Candida antarctica lipase was employed as a biocatalyst. The resulting 24-compound library contained all the expected species with no significant bias toward particular combinations of substrates. As expected, the library contained a substance(s) that showed significant inhibition of polyphenol oxidase, which was used as a model target. The deconvolution was accomplished via resynthesis of ten partial libraries, which were prepared with either an equimolar mixture of the four alcohols and a single vinyl ester, or a single alcohol and equimolar mixture of the activated esters. Analysis of the inhibition pattern observed with these partial libraries suggested that 4-hydroxybenzyl benzoate (4e) should be the most potent inhibitor. This conclusion was confirmed by the preparation and comparison of all 24 components of the initial library. Finally, it was shown that 4e was a competitive inhibitor of polyphenol oxidase, with a K(i) of 40 microM. This value compared favorably with a K(i) of 400 microM, which was determined for parent phenol 4.     

Antimalarial and cytotoxic activities of chiral triamines

Chiral triamine antimalarial compounds have been identified following the screening of mixture-based positional scanning libraries made up of 31,320 compounds against P. falciparum. The library, namely N-methyl triamine (TPI 762) was generated following exhaustive reduction of resin-bound acylated dipeptides. Using the PSCL approach, individual compounds were rapidly identified which were only 10 times less active than the standard drugs chloroquine (CQ) and Artemisinin (Artes).     

Identification of novel peptide ligands for the cancer‐specific receptor mutation EFGRvIII using a mixture‐based synthetic combinatorial library

We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer‐specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer, in particular, ovarian, glioblastomas, and breast cancer, but not in normal tissue. The library consisted of six individual positional sublibraries in the format, H‐O_1–6XXXXX‐NH₂, O being one of the 19 proteinogenic amino acids (cysteine omitted) and X an equimolar mixture of these. The library consisted of 114 mixtures in total. Using a biotin‐streptavidin assay, the binding of each sublibrary to NR6M, NR6W‐A, and NR6 cells was tested. These cells express EGFRvIII, EGFR, and neither of the receptors, respectively. The result from each sublibrary was examined to identify the most active amino acid residue at each position. On the basis of this knowledge, eight peptides were synthesized and tested for binding to EGFRvIII. We identified one peptide, H‐FALGEA‐NH₂, that showed more selective binding to the mutated receptor than the EGFRvIII specific peptide PEPHC1. This study demonstrates the value of using mixture‐based combinatorial positional scanning libraries for the identification of novel peptide ligands targeted against the cancer‐specific EGFRvIII. Our best candidate H‐FALGEA‐NH₂ will be radioactively labeled and evaluated as an imaging agent for positron emission tomography investigation for diagnosis, staging, and monitoring of therapy of various types of cancer. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 201–206, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.     

A strategy to profile prime and non-prime proteolytic substrate specificity

A strategy was developed to determine the prime and non-prime substrate specificity of serine, threonine and cysteine proteases. ACC positional scanning technology was employed to determine the P4-P1 non-prime site substrate specificity. The data was used to synthesize biased donor-quencher positional scanning libraries to profile the P1'-P4' prime site substrate specificity. Directed sorting using the Irori Nanokan system allowed for the archiving of multiple P1'-P4' positional scanning libraries. From these libraries focused donor-quencher libraries incorporating P4-P1 data for each protease under study could be rapidly prepared. The profiling of thrombin and caspase-3 P4-P4' substrate specificity, comparison of the library specificity data to single substrates, and the analysis of physiological cleavage sites are described.     

Lead compounds discovered from libraries: part 2

Many lead compounds with the potential to progress to viable drug candidates have been identified from libraries during the past two years. There are two key strategies most often employed to find leads from libraries: first, high-throughput biological screening of corporate compound collections; and second, synthesis and screening of project-directed libraries (i.e. target-based libraries). Numerous success stories, including the discovery of several clinical candidates, testify to the utility of chemical library collections as proven sources of new leads for drug development.     

Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ～10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.     

Construction of the novel conformationally-restricted peptide library for screening of peptides that control the interaction between nucleobases

A unique conformationally-restricted peptide library was constructed using a loop structure as a structural scaffold. This library was used for the screening of the amino acid sequences that control the interaction between nucleobase triplets. The peptides have PNAs at the C-terminus as the recognition site and the random amino acid sequence at the N-terminus as the effector for the interaction between PNA and its complementary DNA triplets. From the peptide libraries constructed by the positional scanning method, the sequences that affect the interaction between PNA and complementary DNA were selected. The difference in the characteristic results by using A-T and G-C pairs was presented. This study would also give us some useful information about interaction between peptides and nucleic acids, such as relevances between these biomolecules in a prebiotic era.     

Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library

The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV-induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The "hit" rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro-compounds which will serve as excellent lead candidates for further evaluation. At a 10-microM concentration, 3 compounds with selective indexes (SI50) of > 53 were discovered.     

Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries.

We describe here a conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL), that can be used for the rapid (i.e., a single day) identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor molecules. The PS-SPCL described here is made up of six individual positional peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixtures. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.     

A multidimensional overpressured layer chromatographic method for the characterization of tetrazine libraries

The recent combinatorial approach in synthetic organic chemistry started a new age in drug discovery. The generation of compound libraries in combination with high-throughput screening has become the method of choice for the production of new pharmacological leads for chemical optimization. Characterization and separation of such pool of compounds have been lagging behind the synthetic and screening methodologies. Overpressured layer chromatography (OPLC) is an instrumentalized planar liquid chromatographic technique associated with the use of optimized layers prepared from particles of narrow particle size distribution and small diameter. On one hand, uni-directional OPLC allows the simultaneous separation of large number of samples in minutes. On the other hand, two-dimensional OPLC offers multidimensional separation on a single layer. This paper shows the complete multidimensional separation of a tetrazine library prepared by parallel combinatorial synthesis. In general, this approach may become the method of choice for the characterization of compound libraries.     

High-throughput screening of novel peptide inhibitors of an integrin receptor from the hexapeptide library by using a protein microarray chip

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, ProteoChip, in new drug discovery. Integrin alpha(v)beta(3) microarray immobilized on the ProteoChip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin alpha(v)beta(3)-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner and was inhibited not only by the synthetic RGD peptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and high-throughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the ProteoChip is a promising tool for high-throughput screening of lead molecules in new drug development.     

One-bead–one-structure combinatorial libraries

Combinatorial libraries employing the one-bead–one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect “coding” methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and “scaffold” libraries, together with a new approach for the discovery of a pentide binding motif using a “library of libraries,” are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries. © 1994 John Wiley & Sons, Inc.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

Differential assay for high-throughput screening of antibacterial compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.     

De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library

Highly active fluorescent compounds having kappa opioid activity were identified following the screening in a kappa-specific radioligand binding assay of a positional scanning tetrapeptide combinatorial library in which every tetrapeptide was fluorescently labeled. Lissamine rhodamine B sulfonyl chloride was coupled to the N terminal of a mixture-based tetrapeptide positional scanning library made up of over 7.3 million tetrapeptides. Upon determination of the most active mixtures for each position of the library in the kappa binding assay, individual rhodamine labeled tetrapeptides were then synthesized and tested to determine their activities. Eight individual rhodamine labeled peptides were identified that were specific for the kappa opioid receptor, having binding affinities ranging from 5-20 nM. These peptides were poor inhibitors at the mu and delta receptors (K(i)>5,000 nM). Furthermore, neither rhodamine itself nor these same tetrapeptides lacking the N-terminal rhodamine had any significant activity at the kappa receptor, indicating that both the tetrapeptide sequence and the rhodamine moiety are required for receptor binding. This study has demonstrated that novel fluorescent compounds with intrinsic activity can be identified through the use of combinatorial chemistry.