Discovery of enzyme inhibitors through combinatorial chemistry

Summary This review serves to highlight the recent examples of combinatoric methodology as applied to the discovery and optimization of enzyme inhibitors. Early research efforts focused on the identification of polypeptides from libraries as inhibitors of proteases. As solution- and solid-phase chemistries gain in sophistication, libraries containing less peptidic structural motifs have been created. A recurring design stratagem relies on the synthesis of libraries incorporating pharmacophores with known affinity for the target enzyme. Screening of these structure-based libraries has led to the discovery of small-molecule inhibitors of both proteolytic and non-proteolytic enzymes alike. Two tables are provided listing the enzyme targeted libraries through 1996. A name, generic structure and size is given for each library citation, accompanied by the enzyme screen and the structure and potency of the most active library member.     

Application of computer assisted combinatorial chemistry in antivirial,antimalarial and anticancer agents design

Combinatorial chemistry and technologies have been developed to a stage where synthetic schemes are available for generation of a large variety of organic molecules. The innovative concept of combinatorial design assumes that screening of a large and diverse library of compounds will increase the probability of finding an active analogue among the compounds tested. Since the rate at which libraries are screened for activity currently constitutes a limitation to the use of combinatorial technologies, it is important to be selective about the number of compounds to be synthesized. Early experience with combinatorial chemistry indicated that chemical diversity alone did not result in a significant increase in the number of generated lead compounds. Emphasis has therefore been increasingly put on the use of computer assisted combinatorial chemical techniques. Computational methods are valuable in the design of virtual libraries of molecular models. Selection strategies based on computed physicochemical properties of the models or of a target compound are introduced to reduce the time and costs of library synthesis and screening. In addition, computational structure-based library focusing methods can be used to perform in silico screening of the activity of Compounds against a target receptor by docking the ligands into the receptor model. Three case studies are discussed dealing with the design of targeted combinatorial libraries of inhibitors of HIV-1 protease, P. falciparum plasmepsin and human urokinase as potential antivirial, antimalarial and anti-cancer drugs. These illustrate library focusing strategies.     

Libraries of 2β-(N-substituted piperazino)-5α-androstane-3α, 17β-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes

Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R¹ (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R² (library E: 168 members). The screening of these aminosteroids revealed interesting structure–activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 μM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A–D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC₅₀ determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC₅₀ (lymphocytes)/IC₅₀ (HL-60 cells) = 5 - 55) were obtained.     

On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.     

Towards a full reference library of MS(n) spectra. II: A perspective from the library of pesticide spectra extracted from the literature/Internet

To gain perspective on building full transferable libraries of MS(n) spectra from their diverse/numerous collections, a new library was built from 1723 MS(>1) spectra (mainly MS2 spectra) of 490 pesticides and related compounds. Spectra acquired on different types of tandem instruments in various experimental conditions were extracted from 168 literature articles and Internet sites. Testing of the library was based on searches where 'unknown' and reference spectra originated from different sources (mainly from different laboratories) were cross-compared. The NIST 05 MS2 library was added to the reference spectra. The library searches were performed with all the test spectra or were divided into different subsamples containing (a) various numbers of replicate spectra of test compounds or (b) spectra acquired from different instrument types. Thus, the dependence of true/false search (identification) result rates on different factors was explored. The percentage of 1st rank correct identifications (true positives) for the only 'unknown' mass spectrum and two and more reference spectra and matching precursor ion m/z values was 89%. For qualified matches, above the cut-off match factor, that rate decreased to 80%. The corresponding rates based on the best match for two and more 'unknown' and reference spectral replicates were 89-94%. For quadrupole instruments, the rates were even higher: 91-95% (one 'unknown' spectrum) and 90-100% (two and more such spectra). This study shows that MS2 spectral libraries generated from the numerous literature/Internet sources are not less efficient for the goal of identification of unknown compounds including pesticides than very common EI-MS1 libraries and are almost as efficient as the most productive from current MS2 spectral databases. Such libraries may be used as individual reference databases or supplements to large experimental spectral collections covering many groups of abundant compounds and different types of tandem mass spectrometers.     

Identification of novel antitumor agents from mixture-based synthetic combinatorial libraries using cell-based assays

A new strategy is presented here which integrates combinatorial library technology with the antitumor in vitro screening system at the National Cancer Institute in the search for novel antitumor agents. Mixture-based synthetic combinatorial libraries (SCLs) representing hundreds of thousands to millions of individual compounds were screened against the cell-based assay, which evaluates compounds for their ability to inhibit the growth of 60 different human tumor cell lines. Five different SCLs, composed of peptides, peptidomimetics, polyamines or small molecules were first tested against three cell lines to identify the most active SCLs. Two SCLs, namely the N-perbenzylated pentamine and the N-acylated permethylated triamine, were deconvoluted to yield individual compounds having significant activities against the 60 tumor cell lines. Active compounds were tested in mice to determine the maximum tolerated dose, followed by in vivo testing in a hollow fiber assay. Using this strategy, three different compounds identified directly from SCLs are currently being evaluated in human tumor xenografts. This study demonstrates for the first time the use of in vitro cell-based assays to identify antitumor lead compounds from mixture-based combinatorial libraries.     

Sequence-selective nonmacrocyclic two-armed receptors for peptides

Summary Tweezer-like receptor molecules have proven their potential for molecular recognition on several occasions. We decided to make twofold use of this receptor design: firstly to learn whether simple molecular forceps consisting of two peptide chains linked by a spacer are able to selectively bind to small peptides, and secondly to investigate the importance of structural preorganization for the characteristics of the receptors. We prepared two encoded combinatorial libraries based on this design, featuring two combinatorial tripeptide chains held by different scaffolds: the use of chenodeoxycholic acid as spacer provided a rigid scaffold for the forceps, whereas linking the peptide chains by a pentamethylene chain yielded a very flexible forceps structure. Molecules from the cholic acid library recognize and discriminate various enkephalins with micromolar affinities. Molecules from the flexible library show distinct interactions with the enkephalins as well, but the specificity and affinity are clearly diminished. Thus, although the interactions of molecular forceps with peptides are not crucially dependent on structural preorganization, receptors with a rigid design are clearly superior to flexible molecular forceps. Supplementary Material comprising procedures for the preparation and spectroscopical data of compounds5 through8, as well as a listing of the 104 decoded sequences from the assay of the flexible library with⁵Leu-enkephalin/Disperse Red, can be obtained from the author on request.     

第一性原理软件北京原子技术模拟工具包的重构进展

基于第一性原理的理论方法的研究,代表了材料计算、分子模拟等领域的科学高地,相应的第一性原理计算软件直接关系到该领域相关理论、算法的积累.本文汇报了我们在重构第一性原理计算模拟软件—北京原子技术模拟工具包(BSTATE)的一些最新进展.重构的核心思想是降低用户使用门槛、扩展软件适用范围、增加软件对于流行计算框架的支持.基于此思路,在BSTATE原有Makefile编译系统的基础上添加了CMake编译环境,并支持各种数学函数库的自动和交互式配置;通过在原有内置泛函基础上增添Libxc泛函库的支持,使BSTATE支持的泛函数量有了数量级上的增长;分析测试BSTATE在集群的并行特点,并以更新数学库接口(FFTW3、Cufftw)的形式提供对于流行异构框架的初步支持.     

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: Deconvolution strategy

Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X₁–X₂–hinge–X₃–X₄–NH₂ (capped) and X₁–hinge–X₂–X₃ (uncapped), where X₁ through X₄ are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.     

微机械生化传感器

自从Clark和Lyons在1962年研制出第一个生物传感器以来，探测各种生物和化学分子的生化传感器相继问世。这类传感器的基本原理是通过生化敏感层，被分析分子在敏感层上的物理或化学吸附被换能器转化为电信号。在众多的设计中，将活泼的生化敏感材料涂镀在硅器件表面是一个最有新意的设想。以往的硅生化传感器多设计为膜片式，器件的灵敏度受到限制。硅微机械悬臂梁是一种灵敏度极高的器件，近年来在传感器领域受到关注。文章总结了目前世界上硅基微悬梁生化传感器的最新发展动态。对几种硅悬臂梁的设计方法和工作原理进行了讨论，并给出了几种新型微生化气体和液体传感器检测不同有机分子和生物分子的结果。     

Uniqueness: skews bit occurrence frequencies in randomly generated fingerprint libraries

Requiring that randomly generated chemical fingerprint libraries have unique fingerprints such that no two fingerprints are identical causes a systematic skew in bit occurrence frequencies, the proportion at which specified bits are set. Observed frequencies (O) at which each bit is set within the resulting libraries systematically differ from frequencies at which bits are set at fingerprint generation (E). Observed frequencies systematically skew toward 0.5, with the effect being more pronounced as library size approaches the compound space, which is the total number of unique possible fingerprints given the number of bit positions each fingerprint contains. The effect is quantified for varying library sizes as a fraction of the overall compound space, and for changes in the specified frequency E. The cause and implications for this systematic skew are subsequently discussed. When generating random libraries of chemical fingerprints, the imposition of a uniqueness requirement should either be avoided or taken into account.     

Peptomers: A Versatile Approach for the Preparation of Diverse Combinatorial Peptidomimetic Bead Libraries

This report describes a versatile approach in thegeneration of peptidomimetic bead libraries. Themethod is based on the preparation of peptide–peptoidhybrids using the portioning–mixing procedure, whichgives diverse peptidomimetic bead libraries composedof peptides, peptoids and peptide–peptoid hybrids. Weterm these peptomers, from peptide–peptoidhybrid polymers. The synthesis of the peptomersis easily accomplished by adapting the peptoidsynthesis strategy, in which a primary amine reactswith bromoacetic acid, and we combine this methodologywith conventional peptide synthesis. The sequence ofthe active compound is deduced by conventionalmicrosequencing using Edman degradation chemistry,thus avoiding the synthesis of a coding structure orthe addition of molecular tags. We demonstrate theutility of the peptomer approach by the synthesis ofa bead library together with the identification ofnovel peptidomimetic ligands binding to themacromolecular targets streptavidin and the insulin receptor.     

Construction and screening of M13 phage libraries displaying long random peptides

Summary We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.     

Rational selection of structurally diverse natural product scaffolds with favorable ADME properties for drug discovery

Natural product analogs are significant sources for therapeutic agents. To capitalize efficiently on the effective features of naturally occurring substances, a natural product-based library production platform has been devised at Aurigene for drug lead discovery. This approach combines the attractive biological and physicochemical properties of natural product scaffolds, provided by eons of natural selection, with the chemical diversity available from parallel synthetic methods. Virtual property analysis, using computational methods described here, guides the selection of a set of natural product scaffolds that are both structurally diverse and likely to have favorable pharmacokinetic properties. The experimental characterization of several in vitro ADME properties of twenty of these scaffolds, and of a small set of designed congeners based upon one scaffold, is also described. These data confirm that most of the scaffolds and the designed library members have properties favorable to their utilization for creating libraries of lead-like molecules.These authors have contributed equally to this work.     

混合评价核数据库HENDL1.0/MG/MC研制

根据世界几个主要核评价数据库,如ENDF/B 6(美国)、JEF 2.2(欧盟)、JENDL 3.2(日本)、BROND 2.2(俄罗斯)、CENDL 2.1(中国)和FENDL 2(IAEA/NDS),兼顾聚变、裂变以及聚变 裂变次临界混合堆设计研究的多种需要,经过甄别、筛选,最后集成为包含213个核素的基本评价文件,名为HENDL1.0/E的核评价数据库.在此基础上,利用目前流行的群常数加工程序系统NJOY和输运截面制备程序TRANSX制作两套用于中子或/和光子输运计算的输运截面工作库:①参考Vitamin J能群结构制作了175群中子和42群光子、中子 光子耦合多群工作数据库HENDL1.0/MG,可用于离散纵标Sn法程序计算;②连续能群结构、紧凑ENDF(ACE)格式中子截面库HENDL1.0/MC,可用于蒙特卡罗方法输运计算,如MCNP.另外还制作了可用于燃耗(嬗变)计算的燃耗库BURNUP.LIB和响应函数库RESPONSE.LIB两个专用数据库.同时,也对HENDL1.0综合评价核数据库的有效性进行了抽样测试、基准检验和初步确认. A Hybrid Evaluated Nuclear Data Library(HENDL) named as HENDL1.0 has been developed by Fusion Design Study (FDS) team of Institute of Plasma Physics, Academia Sinica (ASIPP) to take into account the requirements in design and research relevant to fusion, fission and fusion-fission sub-critical hybrid reactor. HENDL1.0 contains one basic evaluated sub-library naming HENDL1.0/E and two processed working sub-libraries naming HENDL1.0/MG and HENDL1.0/MC, respectively. Through carefully comparing...     

Comparison of conventional and ultrasonically assisted extractions of pharmaceutically active compounds from Salvia officinalis

Conventional as well as ultrasonically assisted extractions of biologically active compounds from Salvia officinalis using 65% ethanol have been studied. Cineole, Thujone and Borneol were used as standards for the GLC-MS evaluation of the extracts. The effect of temperature, stirring and mode of sonication (ultrasonic bath or horn system) have been studied. The results indicate that ultrasonically assisted extraction with mechanical stirring at room temperature in a period of 12 h produces a substantial improvement over conventional methodology.     

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified (\(\sim \)5 % hit rate, best inhibitory activity: 16 \(\upmu \hbox {M}\)). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.     

Adaptive higher-order finite element methods for transient PDE problems based on embedded higher-order implicit Runge–Kutta methods

We present a new class of adaptivity algorithms for time-dependent partial differential equations (PDE) that combine adaptive higher-order finite elements (hp-FEM) in space with arbitrary (embedded, higher-order, implicit) Runge–Kutta methods in time. Weak formulation is only created for the stationary residual, and the Runge–Kutta methods are specified via their Butcher’s tables. Around 30 Butcher’s tables for various Runge–Kutta methods with numerically verified orders of local and global truncation errors are provided. A time-dependent benchmark problem with known exact solution that contains a sharp moving front is introduced, and it is used to compare the quality of seven embedded implicit higher-order Runge–Kutta methods. Numerical experiments also include a comparison of adaptive low-order FEM and hp-FEM with dynamically changing meshes. All numerical results presented in this paper were obtained using the open source library Hermes (http://www.hpfem.org/hermes) and they are reproducible in the Networked Computing Laboratory (NCLab) at http://www.nclab.com.     

Implementation of REDIM reduced chemistry to model an axisymmetric laminar diffusion methane–air flame

The goal of this work is to analyze the use of automatically reduced chemistry by the Reaction–Diffusion Manifold (REDIM) method in simulating axisymmetric laminar coflow diffusion flames. Detailed chemical kinetic models are usually computationally prohibitive for simulating complex reacting flows, and therefore reduced models are required. Automatic reduction model approaches usually exploit the natural multi-scale structure of combustion systems. The novel REDIM approach applies the concept of invariant manifolds to treat also the influence of the transport processes on the reduced model, which overcomes a fundamental problem of model reduction in neglecting the coupling of molecular transport with thermochemical processes. We have considered a previously well studied atmospheric pressure nitrogen-diluted methane–air flame as a test case to validate the methodology presented here. First, one-dimensional and two-dimensional REDIMs were computed and tabulated in lookup tables. Then, the full set of governing equations are projected on the REDIM and implemented in the object-oriented C++ Gascoigne code with a new add-on library to deal with the REDIM tables. The projected set of governing equations have been discretized by the Finite Element Method (FEM) and solved by a GMRES iteration preconditioned by a geometric multigrid method. Local grid refinement, adaptive mesh and parallelization are applied to ensure efficiency and precision. The numerical results obtained using the REDIM approach have shown very good agreement with detailed numerical simulations and experimental data.     

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage

Summary We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 × 10⁹ members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of -endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v -ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.