Discovery of Potent, Selective and Reversible Caspase-3 Inhibitors

Recent studies towards understanding the molecular mechanisms of apoptosis have revealed the importance of a group of cysteinyl aspartate specific proteases, the caspases, in the programmed cell death process (Hengartner, M.O. Nature 2000, 407, 770). Caspase-3, in particular, has been characterized as the dominant effector caspase involved in the proteolytic cleavage of a variety of protein substrates including cytoskeletal proteins, kinases and DNA repair enzymes during apoptosis (Nicholson…     

Convergent synthesis of a complex oxime library using chemical domain shuffling

[reaction: see text] The synthesis of a complex hybrid oxime library is reported utilizing convergent ligation of alkoxyamine and carbonyl monomers via "chemical domain shuffling". Initial biological screening of the library against human small cell lung carcinoma (A549) cells led to the identification of a novel hybrid dimer in contrast to the corresponding monomeric compounds which were found to be inactive.     

Modeling collective behavior of molecules in nanoscale direct deposition processes

We present a theoretical model describing the collective behavior of molecules in nanoscale direct deposition processes such as dip-pen nanolithography. We show that strong intermolecular interactions combined with nonuniform substrate-molecule interactions can produce various shapes of molecular patterns including fractal-like structures. Computer simulations reveal circular and starlike patterns at low and intermediate densities of preferentially attractive surface sites, respectively. At large density of such surface sites, the molecules form a two-dimensional invasion percolation cluster. Previous experimental results showing anisotropic patterns of various chemical and biological molecules correspond to the starlike regime [P. Manandhar et al., Phys. Rev. Lett. 90, 115505 (2003); J.-H. Lim and C. A. Mirkin, Adv. Mater. (Weinheim, Ger.) 14, 1474 (2002); D. L. Wilson et al., Proc. Natl. Acad. Sci. U.S.A. 98, 13660 (2001); M. Su et al., Appl. Phys. Lett. 84, 4200 (2004); R. McKendry et al., Nano Lett. 2, 713 (2002); H. Zhou et al., Appl. Surf. Sci. 236, 18 (2004); G. Agarwal et al., J. Am. Chem. Soc. 125, 580 (2003)].     

Variable selection and specification of robust QSAR models from multicollinear data: arylpiperazinyl derivatives with affinity and selectivity for α<Subscript>2</Subscript>-adrenoceptors

Two QSAR models have been identified that predict the affinity and selectivity of arylpiperazinyl derivatives for alpha1 and alpha2 adrenoceptors (ARs). The models have been specified and validated using 108 compounds whose structures and inhibition constants (Ki) are available in the literature [Barbaro et al., J. Med. Chem., 44 (2001) 2118; Betti et al., J. Med. Chem., 45 (2002) 3603; Barbaro et al., Bioorg. Med. Chem., 10 (2002) 361; Betti et al., J. Med. Chem., 46 (2003) 3555]. One hundred and forty-seven predictors have been calculated using the Cerius 2 software available from Accelrys. This set of variables exhibited redundancy and severe multicollinearity, which had to be identified and removed as appropriate in order to obtain robust regression models free of inflated errors for the beta estimates - so-called bouncing betas. Those predictors that contained information relevant to the alpha2 response were identified on the basis of their pairwise linear correlations with affinity (-log Ki) for alpha2 adrenoceptors; the remaining variables were discarded. Subsequent variable selection made use of Factor Analysis (FA) and Unsupervised Variable Selection (UzFS). The data was divided into test and training sets using cluster analysis. These two sets were characterised by similar and consistent distributions of compounds in a high dimensional, but relevant predictor space. Multiple regression was then used to determine a subset of predictors from which to determine QSAR models for affinity to alpha2-ARs. Two multivariate procedures, Continuum Regression (the Portsmouth formulation) and Canonical Correlation Analysis (CCA), have been used to specify models for affinity and selectivity, respectively. Reasonable predictions were obtained using these in silico screening tools.     

Cavity resonators of metal-coated dielectric nanorods

Metallic nanoparticles bridge the length scale between atoms and crystals, exhibiting mesoscopic properties unique to their size. Thus, they have generated much interest for their potential applications as chemical or biological sensors and particularly as waveguides for light in nanoscale structures. [Y. W. C. Cao, R. C. Jin, and C. A. Mirkin, Science 297, 1536 (2002); H. J. Lezec et al., Science 297, 820 (2002); S. A. Maier, P. G. Kik, and H. A. Atwater, Appl. Phys. Lett. 81, 1714 (2002); J. M. Oliva and S. K. Gray, Chem. Phys. Lett. 379, 325 (2003)]. One important direction of research into the properties of individual metal nanoparticles involves the controlled variation of their geometry, which can yield new and tunable optical properties that simple spherical configurations do not possess. [T. S. Ahmadi, Z. L. Wang, T. C. Green, A. Henglein, and M. A. Ei-Sayed, Science 272, 1924 (1996)]. A prime example of this is the core-shell nanostructure that has a central material surrounded by differing cladding layer.     

The Role of Small Molecule–small Molecule Interactions in Overcoming Biological Barriers for Antibacterial Drug Action

The ineffectiveness of antibiotics against bacteria can be caused by multidrug resistance (MDR) or by an outer membrane, which restricts the penetration of amphipathic compounds into Gram-negative bacteria. Remarkable activities of plant antimicrobials in the presence of MDR modulators have been observed against a series of MDR and Gram-negative bacteria (Tegos et al., Antimicrob Agents Chemother 46:3133, 2002). Assuming that modulators of MDR might form complexes with substrates of efflux pumps Zloh et al., Biogr Med Chem Lett 14:881, 2004), we have evaluated interaction energies between antimicrobials and MDR modulators reported in Tegos et al. (Antimicrob Agents Chemother 46:3133, 2002). In this paper, we can confirm that modulation activity against the efflux pump NorA in Staphylococcus aureus correlates with the interaction energies between MDR modulator INF271 and antibacterials. Additionally, the change of log P of complexes might be responsible for overcoming the membrane impermeability in Gram-negative bacteria and increasing the antibacterial activity in the presence of the modulator MC207110. This suggests that interactions between small molecules may play an important role in overcoming biological barriers in bacteria.     

Neural network studies : Part 3. Prediction of partition coefficients

In a previous paper (N. Bodor, A. Harget and M.-J. Huang, J. Am. Chem. Soc., 113 (1991) 9480) we demonstrated the utility of a neural network approach in the estimation of the aqueous solubility of organic compounds. This approach has now been extended to the prediction of partition coefficients. A training set of AM1 calculated properties and experimental values for 302 compounds was used and, after training, the neural network was tested for its ability to predict the partition coefficients of 21 compounds not included in the training set. We also tested six more compounds with molecular properties out of the training set property range. A comparison was made with the results obtained from a previous study which had used a regression analysis approach (N. Bodor and M.-J. Huang, J. Pharm. Sci., 81 (1992) 272). The neural network results compared favorably with those given by the regression analysis approach, both for the training set and for the new compounds.     

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP® LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a “scheduled” survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP^® hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization–MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography–mass spectrometry, and liquid chromatography–diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).     

A peptoid "antibody surrogate" that antagonizes VEGF receptor 2 activity

We report a two-color, cell-based screen to identify specific receptor-binding compounds in a combinatorial library of peptoids displayed on beads. We apply this strategy to the isolation of vascular endothelial growth factor receptor 2 (VEGFR2)-binding peptoids. A dimeric derivative of one of these lead compounds is shown to be an antagonist of VEGFR2 activity both in vitro and in vivo. This methodology provides a potentially general route to synthetic molecules that bind integral membrane receptors with affinities and specificities similar to those of antibodies, but which are far smaller and easier to make and manipulate.     

Synthesis and screening of peptoid arrays on cellulose membranes

Rapid synthesis and screening of compound libraries enables the accelerated identification of novel protein ligands in order to support processes like analysis of protein interactions, drug target discovery or lead structure discovery. SPOT synthesis—a well established method for the rapid preparation of peptide arrays—has recently been extended to the field of nonpeptides. In this contribution we report on the systematic evaluation of the SPOT technique for the assembly of N-alkylglycine (peptoid) library arrays. In the course of this investigation bromoacetic acid 2,4-dinitrophenylester (1a) was identified to be the most suited agent for bromoacetylation in terms of yield and N-selectivity enabling straightforward submonomer synthesis on hydroxy-group rich cellulose membranes. The potential of this method for the rapid identification of novel nonpeptidic protein ligands was demonstrated by synthesis and screening of a library consisting of 8000 peptoids and peptomers (i.e. their hybrids with α-substituted amino acids) allowing the identification of micromolar ligands for the monoclonal antibody Tab-2.     

An iterative knowledge-based scoring function to predict protein-ligand interactions: II. Validation of the scoring function

We have developed an iterative knowledge-based scoring function (ITScore) to describe protein-ligand interactions. Here, we assess ITScore through extensive tests on native structure identification, binding affinity prediction, and virtual database screening. Specifically, ITScore was first applied to a test set of 100 protein-ligand complexes constructed by Wang et al. (J Med Chem 2003, 46, 2287), and compared with 14 other scoring functions. The results show that ITScore yielded a high success rate of 82% on identifying native-like binding modes under the criterion of rmsd < or = 2 A for each top-ranked ligand conformation. The success rate increased to 98% if the top five conformations were considered for each ligand. In the case of binding affinity prediction, ITScore also obtained a good correlation for this test set (R = 0.65). Next, ITScore was used to predict binding affinities of a second diverse test set of 77 protein-ligand complexes prepared by Muegge and Martin (J Med Chem 1999, 42, 791), and compared with four other widely used knowledge-based scoring functions. ITScore yielded a high correlation of R2 = 0.65 (or R = 0.81) in the affinity prediction. Finally, enrichment tests were performed with ITScore against four target proteins using the compound databases constructed by Jacobsson et al. (J Med Chem 2003, 46, 5781). The results were compared with those of eight other scoring functions. ITScore yielded high enrichments in all four database screening tests. ITScore can be easily combined with the existing docking programs for the use of structure-based drug design.     

Natural products in parallel chemistry--novel 5-lipoxygenase inhibitors from BIOS-based libraries starting from alpha-santonin

Recently, we developed a concept known as biology-oriented synthesis (BIOS), which targets the design and synthesis of small- to medium-sized compound libraries on the basis of genuine natural product templates to provide screening compounds with high biological relevance. We herein describe the parallel solution phase synthesis of two BIOS-based libraries starting from alpha-santonin (1). Modification of the sesquiterpene lactone 1 by introduction of a thiazole moiety followed by a Lewis-acid-mediated lactone opening yielded a first library of natural product analogues. An acid-mediated dienone-phenol rearrangement of 1 and a subsequent etherification/amidation sequence led to a second natural product-based library. After application of a fingerprint-based virtual screening on these compounds, the biological screening of 23 selected library members against 5-lipoxygenase resulted in the discovery of four potent novel inhibitors of this enzyme.     

Optimization and testing of mass spectral library search algorithms for compound identification

Five algorithms proposed in the literature for library search identification of unknown compounds from their low resolution mass spectra were optimized and tested by matching test spectra against reference spectra in the NIST-EPA-NIH Mass Spectral Database. The algorithms were probability-based matching (PBM), dot-product, Hertz et al. similarity index, Euclidean distance, and absolute value distance. The test set consisted of 12,592 alternate spectra of about 8000 compounds represented in the database. Most algorithms were optimized by varying their mass weighting and intensity scaling factors. Rank in the list of candidatc compounds was used as the criterion for accuracy. The best performing algorithm (75% accuracy for rank 1) was the dot-product function that measures the cosine of the angle between spectra represented as vectors. Other methods in order of performance were the Euclidean distance (72%), absolute value distance (68%) PBM (65%), and Hertz et al. (64%). Intensity scaling and mass weighting were important in the optimized algorithms with the square root of the intensity scale nearly optimal and the square or cube the best mass weighting power. Several more complex schemes also were tested, but had little effect on the results. A modest improvement in the performance of the dot-product algorithm was made by adding a term that gave additional weight to relative peak intensities for spectra with many peaks in common.     

Repositioning of Quinazolinedione-Based Compounds on Soluble Epoxide Hydrolase (sEH) through 3D Structure-Based Pharmacophore Model-Driven Investigation

The development of new bioactive compounds represents one of the main purposes of the drug discovery process. Various tools can be employed to identify new drug candidates against pharmacologically relevant biological targets, and the search for new approaches and methodologies often represents a critical issue. In this context, in silico drug repositioning procedures are required even more in order to re-evaluate compounds that already showed poor biological results against a specific biological target. 3D structure-based pharmacophoric models, usually built for specific targets to accelerate the identification of new promising compounds, can be employed for drug repositioning campaigns as well. In this work, an in-house library of 190 synthesized compounds was re-evaluated using a 3D structure-based pharmacophoric model developed on soluble epoxide hydrolase (sEH). Among the analyzed compounds, a small set of quinazolinedione-based molecules, originally selected from a virtual combinatorial library and showing poor results when preliminarily investigated against heat shock protein 90 (Hsp90), was successfully repositioned against sEH, accounting the related built 3D structure-based pharmacophoric model. The promising results here obtained highlight the reliability of this computational workflow for accelerating the drug discovery/repositioning processes.     

Agonists of the follicle stimulating hormone receptor from an encoded thiazolidinone library

The design, synthesis, characterization, and screening of a large, encoded thiazolidinone library are described. Three sets of 35 building blocks were combined by encoded split-pool synthesis to give a library containing more than 42 000 members. Building block selection was based in part on a novel small molecule follicle stimulating hormone receptor agonist hit and in part for diversity. HPLC/MS techniques were applied at the single-bead level to build confidence in the reliability of library construction. Application of two distinct screening strategies resulted in the identification of compounds with significantly improved potency over the initial hit. This work demonstrates the versatility of encoded libraries for preparing a large number of analogues of a given hit while simultaneously generating a large collection of compounds for screening against other targets.     

In silico studies toward the discovery of new anti-HIV nucleoside compounds through the use of TOPS-MODE and 2D/3D connectivity indices. 2. Purine derivatives

The TOPological Substructural MOlecular DEsign (TOPS-MODE) approach has been used to predict the anti-HIV activity in MT-4 assays (Estrada et al., 2002) of a diverse range of purine-based nucleosides. A database of 206 nucleosides has been selected from the literature and a theoretical virtual screening model has been developed. The model is able of discriminating between compounds that have anti-HIV activity and those that do not, with a good classification level of 85% in the training and 82.8% in the cross-validation series. On the basis of the information generated by the model, the correct classification of practically 80% of compounds from an external prediction set has been achieved using the theoretical model. Furthermore, the contribution of a range of molecular fragments to the pharmacological action has been calculated and this could provide a powerful tool in the design of nucleoside analogues that show activity against the HIV. Finally, a QSAR model has been developed that allows quantitative data to be obtained regarding the pharmacological potency shown by this type of compound.     

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.     

A selective intramolecular 5-exo-dig or 6-endo-dig cyclization en route to 2-furanone or 2-pyrone containing tricyclic scaffolds

Ringfused bicyclic 2-pyridones exhibit interesting biological properties against pili assembly in uropathogenic Escherichia coli (Pinkner, J. S. et al. Proc. Natl. Acad. Sci. U. S. A.2006, 103, 17897-17902; ?berg, V. et al. Org. Biomol. Chem.2007, 5, 1827-1834) as well as curli formation (Cegelski, L. et al. Nat. Chem. Biol.2009, 5, 913-919). In the search for new ring-fused central fragments, highly selective synthetic routes to the 2-furanone or 2-pyrone containing tricyclic scaffolds 1 and 2 have been developed.     

Screening of cardioprotective diseases bioactive components from Danshen extracts and LC–MS analysis

A rapid and efficient analysis and screening method is adopted for cell affinity capture coupled with HPLC–MS (CAC–HPLC–MS) analysis of bioactive components that have possible efficiency against cardiovascular diseases. This method involves affinity capture, concentration, and separation of bioactive components from Danshen library using oxidatively damaged endothelial cells induced by H₂O₂, as well as analysis and identification of targeted compounds with HPLC and MS. It combines the specific interaction between cell membrane receptors and bioactive components with the powerful analysis and identification function of HPLC–MS. The CAC–HPLC–MS method was also used for analysis and screening of bioactive components from crude extracts of Danshen. A total of 19 components were found to be bound to oxidatively damaged endothelial cells with seven of these identified. Existing literature confirms that these seven components have many activities related to cardioprotective diseases. Therefore, the combination of biological affinity capture with HPLC–MS should be regarded as an attractive method with great potential for rapid and efficient screening of bioactive components related to anti-cardiovascular diseases from natural product libraries.