A rapid synthesis of lavendustin-mimetic small molecules by click fragment assembly

Lavendustin-mimetic small molecules modifying the linker –CH₂–NH– with an 1,2,3-triazole ring have been synthesized via a click chemistry. Two pharmacophoric fragments of lavendustin were varied to investigate chemical space and the auxophoric –CH₂–NH– was altered to an 1,2,3-triazole for rapid click conjugation. The small molecules were evaluated against HCT116 colon cancer and CCRF-CEM leukemia cell lines. Among 28 analogues, 3-phenylpropyl ester 26b inhibited CCRF-CEM leukemia cell growth with GI₅₀ value of 0.9 μM.     

Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions

DNA-encoded chemical libraries are increasingly being employed for the identification of binding molecules to protein targets of pharmaceutical relevance. Here, we describe the synthesis and characterization of a DNA-encoded chemical library, consisting of 4000 compounds generated by Diels-Alder cycloaddition reactions. The compounds were encoded with unique DNA fragments which were generated through a stepwise assembly process and serve as amplifiable bar codes for the identification and relative quantification of library members.     

Design, synthesis and preliminary biological evaluation of a focused combinatorial library of stereodiverse carbohydrate-scaffold-based peptidomimetics

A focused combinatorial library of 126 mimetics of the RGD sequence based on sugar scaffolds have been rationally constructed using molecular modeling, with a particular emphasis on the stereodiversity of the library. A liquid phase, mix and divide synthesis was used, active compounds being identified by using orthogonal libraries and recursive deconvolution strategies.     

Design of inhibitors using a combinatorial library for HIV-Nef and human SH3 domain interaction

The HIV-1 Nef protein has the ability to down regulate important molecules at the immune synapse. These include class I and classII (Human Leukocyte Antigen) HLA on the Antigen Presenting Cells (APC). The receptors in these molecules consist of SH-3domain and their interaction with the HIV-1 Nef is critical. Therefore, it is important to inhibit this HIV-Nef and human SH3domain interaction. Thus, we used a combinatorial library to screen for molecules to inhibit this interaction. The exercise identifieda group of top ranking compounds for further consideration.     

Rational combinatorial chemistry-based selection,synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.     

Solution-phase synthesis of a combinatorial monocyclic β-lactam library: Potential protease inhibitors

A 126-member library of monocyclic β-lactams was generated in parallel fashion by solution-phase Ugi four-component condensation reaction between β-amino acids, aldehydes, and isocyanides. The library was designed to identify potential human leukocyte elastase inhibitors. The approach is also capable of optimizing the lead compounds generated in the original library.     

PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays

Permeable resins cross-linked with long PEG chains were synthesized for use in solid-phase enzyme library assays. High molecular weight bis-amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido–PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6–9 ). Furthermore, acryloyl–sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13–19 ). N,N-Dimethylacrylamide was used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene; no swelling was observed in diethyl ether. The PEGA resins (Type I ) with an amino acid group capacity between 0.07 and 1.0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries were synthesized on the new polymer using a multiple column library synthesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II ) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Development of a herbicide biosensor using a peptide receptor screened from a combinatorial library

The purpose of this study was to screen for peptides that bind herbicides with a chlorinated aniline chemical structure. A tetrapeptide library was constructed using a solid phase split synthesis approach. Peptide beads were suspended in a buffer containing fluorescent-labeled dichloroaniline (DCA) as the bait. Eighteen fluorescent peptide beads were selected which bound to the bait after two rounds of staining screenings. The beads were then stained and suspended in a solution containing an excess of DCA and five quenched peptide beads were subsequently selected that recognized the DCA moiety. The screened peptides had many sequence similarities. The binding affinity of the screened peptides to herbicides was analyzed using surface plasmon resonance (SPR). N′-(3,4-dichlorophenyl)-N,N-dimethylurea [3-(3,4-dichlorophenyl)-1,1-dimethylurea] solution was injected over the peptide immobilized SPR chip. The SPR signal was found to increase in proportion to the DCMU concentration, whereas no signal was obtained from the negative control, 2-(2-methyl-4-chlorophenoxy) propionic acid (MCPP). From these results it is suggested that the screened peptide selectively recognizes the chemical structure of DCA.     

Solid-phase synthesis and anti-infective activity of a combinatorial library based on the natural product anisomycin

The solid-phase synthesis of a library based on the natural product anisomycin is described. The resulting library was tested against a panel of bacterial and fungal targets, and active compounds were identified in a Staphylococcus aureus whole-cell assay and an efflux-deficient fungal whole-cell assay.     

Design and synthesis of a library of chemokine antagonists

A library of chemokine antagonists has been synthesized using a combination of solid and solution-phase chemistry. Structures of known chemokine antagonists were used to produce a pharmacophore which served to guide monomer selection. Several combinations of monomers have resulted in providing novel chemokine antagonists which in some cases display dual chemokine receptor antagonism.     

Prediction of protein tertiary structure using PROFESY, a novel method based on fragment assembly and conformational space annealing

A novel method for ab initio prediction of protein tertiary structures, PROFESY (PROFile Enumerating SYstem), is proposed. This method utilizes the secondary structure prediction information of a query sequence and the fragment assembly procedure based on global optimization. Fifteen-residue-long fragment libraries are constructed using the secondary structure prediction method PREDICT, and fragments in these libraries are assembled to generate full-length chains of a query protein. Tertiary structures of 50 to 100 conformations are obtained by minimizing an energy function for proteins, using the conformational space annealing method that enables one to sample diverse low-lying local minima of the energy. We apply PROFESY for benchmark tests to proteins with known structures to demonstrate its feasibility. In addition, we participated in CASP5 and applied PROFESY to four new-fold targets for blind prediction. The results are quite promising, despite the fact that PROFESY was in its early stages of development. In particular, PROFESY successfully provided us the best model-one structure for the target T0161.     

Design,synthesis, and biological evaluation of novel histone deacetylase 1 inhibitors through click chemistry

We report the design, synthesis, and biological evaluation of a new series of HDAC1 inhibitors using click chemistry. Compound 17 bearing a phenyl ring at meta-position was identified to show much better selectivity for HDAC1 over HDAC7 than SAHA. The compond 17 also showed better in vitro anticancer activities against several cancer cell lines than that of SAHA. This work could serve as a foundation for further exploration of selective HDAC inhibitors using the compound 17 molecular scaffold.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Asymmetric proteome equalization of the skeletal muscle proteome using a combinatorial hexapeptide library

Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method.     

Acquisition of a specific and potent PTP1B inhibitor from a novel combinatorial library and screening procedure

Protein-tyrosine phosphatases (PTPases) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTPase activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases including cancers and diabetes. For example, recent gene knockout studies in mice identify PTP1B as a promising target for anti-diabetes/obesity drug discovery. Thus, there is intense interest in obtaining specific and potent PTPase inhibitors for biological studies and pharmacological development. However, given the highly conserved nature of the PTPase active site, it is unclear whether selectivity in PTPase inhibition can be achieved. We describe a combinatorial approach that is designed to target both the active site and a unique peripheral site in PTP1B. Compounds that can simultaneously associate with both sites are expected to exhibit enhanced affinity and specificity. We also describe a novel affinity-based high-throughput assay procedure that can be used for PTPase inhibitor screening. The combinatorial library/high-throughput screen protocols furnished a small molecule PTP1B inhibitor that is both potent (K(i) = 2.4 nm) and selective (little or no activity against a panel of phosphatases including Yersinia PTPase, SHP1, SHP2, LAR, HePTP, PTPalpha, CD45, VHR, MKP3, Cdc25A, Stp1, and PP2C). These results demonstrate that it is possible to acquire potent, yet highly selective inhibitors for individual members of the large PTPase family of enzymes.     

Chemoenzymatic construction of a four-component Ugi combinatorial library

The chemoenzymatic preparation of a nine-member Ugi condensation library is described. The carboxylic acid and amine precursors are based on 3-hydroxybutyrate and 4-amino-1-butanol, respectively, and have been acylated selectively using a variety of acyl donors catalyzed by porcine pancreatic lipase. The enzyme is selective for the hydroxyl functionalities on both precursors, thereby yielding 3-acyl-butyric acid and 4-amino-1-acyl compounds. These enzymatically generated derivatives were then subjected to a four-component Ugi condensation reaction in the presence of acetaldehyde and methyl isocyanoacetate. Isolated yields of the alpha-(acylamino)amide Ugi products ranged from 72-95%. The inherent chemoselectivity of enzymatic catalysis may play an increasingly important role in expanding the structural diversity that can be achieved by chemical multicomponent condensation reactions.     

Identification of novel SIRT2-selective inhibitors using a click chemistry approach

A series of 114 SIRT inhibitor candidates was assembled using ‘click chemistry’, by reacting two alkynes bearing 2-anilinobenzamide pharmacophore with 57 azide building blocks in the presence of Cu(I) catalyst. Screening identified two SIRT2-selective inhibitors, which were more SIRT2-selective than AGK2, a known SIRT2 inhibitor. These findings will be useful for further development of SIRT2-selective inhibitors.     

Design, combinatorial chemical synthesis and in vitro characterization of novel urea based gelatinase inhibitors.

A novel series of hydroxamate/urea-based inhibitors of gelatinases has been discovered via solid-phase combinatorial chemistry. SAR of P1', P2', and P3' has been exploited and structures different from traditional succinate-based MMP inhibitors have been found.