Biased and unbiased strategies to identify biologically active small molecules

Small molecules are central players in chemical biology studies. They promote the perturbation of cellular processes underlying diseases and enable the identification of biological targets that can be validated for therapeutic intervention. Small molecules have been shown to accurately tune a single function of pluripotent proteins in a reversible manner with exceptional temporal resolution. The identification of molecular probes and drugs remains a worthy challenge that can be addressed by the use of biased and unbiased strategies. Hypothesis-driven methodologies employs a known biological target to synthesize complementary hits while discovery-driven strategies offer the additional means of identifying previously unanticipated biological targets. This review article provides a general overview of recent synthetic frameworks that gave rise to an impressive arsenal of biologically active small molecules with unprecedented cellular mechanisms.     

Target identification of biologically active small molecules via in situ methods

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The screening of some microorganisms for their ability to N-dealkylate drug molecules

Summary Selected microorganisms were screened for their ability to N-dealkylate drug molecules. The compounds studied enabled the investigation of N-alkyl groups in different chemical environments, including alkylaminoalkyl chains, saturated cyclic structures and in amide functions. Transformation products were extracted from the transformation mixtures, derivatised and analysed by gas liquid chromatography (GLC). For the purposes of screening, important transformation products were identified by comparison of their GLC retention data with similar derivatives of authentic standards. N-demethylation was effected by all test strains of the fungus Cunninghamella, except C. elegans, and also by three of the Streptomyces species tested. The Cunninghamella demonstrated the widest spectrum of transformation activity, N-demethylating the alkylaminoalkyl side chains of amitriptyline and chlorpromazine, the N-methylpiperidine function of codeine, and the cyclic amide function of diazepam. The N-demethylation of the latter substrate, which is only slightly water soluble, occurred in surprisingly high yield. There was no evidence that bulky groups such as N-dimethylallyl were celaved and selectivity for N-demethylation rather than O-demethylation was demonstrated when both N-and O-methyl functions were present in the same molecule. Comparisons are made with the mammalian metabolic pathways of the drug compounds studied.     

Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

Culture-independent studies on environmental samples indicate that most bacteria are not readily cultured in the laboratory. The small fraction of bacteria that have been successfully cultured from environmental samples have been a very rewarding source of novel biologically active natural products. The introduction of DNA extracted directly from environmental samples into easily cultured bacteria and the screening of these large libraries for clones that produce biologically active small molecules is one means to access natural products encoded by the genomes of previously uncultured bacteria. This protocol provides detailed procedures for cloning DNA directly from environmental samples and screening these clones for the production of antibacterially active natural products. The entire protocol, from soil sample to the identification of antibacterially active environmental DNA clones, will take approximately 2 weeks.     

Mucoralean fungi for sustainable production of bioethanol and biologically active molecules

Applied Microbiology and Biotechnology - Mucoralean fungi are suitable microorganisms for the sustainable production of food, fodder, and fuels from inexpensive natural resources. Ethanol-producing...     

未培养环境微生物培养方法的研究进展

自然界中微生物种类繁多、功能多样、分布广泛,对人类健康安全、生态稳定和物种进化等发挥不可替代的作用.尽管微生物培养技术至今已有一百多年,然而由于各种限制因素的制约,目前成功分离培养的微生物仅占0.1％-1.0％,自然界中仍有十分丰富的微生物资源有待挖掘和开发利用.如何理解难培养微生物的制约因素并探索作用机制,同时借此开...     

Use of recombinant Salmonella strains for supplying the macro-organism with biologically active molecules

The characterization of known Salmonella vaccine strains and different attenuated mutants used for developing new vaccines is presented. The use of attenuated Salmonella strains as vaccine vector for the supply of heterologous antigens opens prospects for the creation of effective and commonly available vaccines which approximate the "ideal" vaccine in their qualitative characteristics. The possibility of the genetic modification of attenuated strains permits their targeted reconstruction, considering the specific features of the formation of immune response to the definite heterologous antigen supplied to the body by the bacterial vector.     

Joint cultivation of the producers of biologically active substances with other microorganisms (a review)

The review surveys data on the combined cultivation of producers of biologically active substances with various microorganisms. It is emphasized that the combined cultivation of strains-producers with microorganisms-partners may increase the synthesis and improve the quality of many useful products.     

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.     

Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria: IDENTIFICATION OF THE INDUCER OF KstR REPRESSOR*

Cholesterol degradation plays a prominent role in Mycobacterium tuberculosis infection; therefore, to develop new tools to combat this disease, we need to decipher the components comprising and regulating the corresponding pathway. A TetR-like repressor (KstR) regulates the upper part of this complex catabolic pathway, but the induction mechanism remains unknown. Using a biophysical approach, we have discovered that the inducer molecule of KstR in M. smegmatis mc²155 is not cholesterol but 3-oxo-4-cholestenoic acid, one of the first metabolic intermediates. Binding this compound induces dramatic conformational changes in KstR that promote the KstR-DNA interaction to be released from the operator, retaining its dimeric state. Our findings suggest a regulatory model common to all cholesterol degrading bacteria in which the first steps of the pathway are critical to its mineralization and explain the high redundancy of the enzymes involved in these initial steps.     

The biosynthesis of biologically active proteins in mRNA-microinjected Xenopus oocytes

The basic properties of mRNA-injected Xenopus oocytes as a heterologous system for the production of biologically active proteins will be reviewed. The advantages and limitations involved in the use of this in ovo system will be discussed, as compared with in vitro cell-free translation systems and with in vivo microinjected mammalian cells in culture. The different assay systems that have been utilized for the identification of the biological properties of oocyte-produced proteins will be described. This section will review the determination of properties such as binding of natural ligands, like heme or alpha-bungarotoxin; immunological recognition by antibodies; subcellular compartmentalization and/or secretion; various enzymatic catalytic activities; and induction in ovo of biological activities that affect other living cells in culture, such as those of interferon and of the T-cell receptor. The limitations involved in interpretation of results obtained using mRNA-injected oocytes will be critically reviewed. Special attention will be given to the effect of oocyte proteases and of changes in the endogenous translation rate on quantitative measurements of oocyte-produced proteins. In addition, the validity of the various measurement techniques will be evaluated. The various uses of bioassays of proteins produced in mRNA-injected Xenopus oocytes throughout the last decade will be reviewed. Nuclear and cytoplasmic injections, mRNA and protein turnover measurements and abundance calculations, and the use of in ovo bioassays for molecular cloning experiments will be discussed in this section. Finally, potential future uses of the oocyte system in various fields of research, such as immunology, neurobiology, and cell biology will be suggested.     

Prediction of location of active sites in biologically active peptides

In a previous paper we demonstrated that the short-range compact regions in atrial natriuretic factor (-hANF) predicted by the average distance map (ADM) correspond to its active sites [Kikuchi,J. Protein Chem.11, 579–581 (1992)]. In the present paper we apply the same method to other bioactive peptides and peptidic enzyme inhibitors. We again observe that active sites in each peptide are contained in short-range compact regions predicted by the ADM for the peptide. This demonstrates that the ADM method predicts the possible location of active sites in biologically active peptides in general. The possibility of practical application of the present method to rational drug design is also discussed.     

The chemistry of vitamin B12. The coordination of biologically important molecules

The following equilibrium constants (given as logK in units of m⁻¹) were determined for the substitution of co-ordinated H₂O in aquocobalamin by glycine (bound through N) 5.8, cysteine (bound through S) 6.0 or 8.3, depending on the value chosen for the pK of the thiol group, and phenolate 2.9. The spectrum of the phenolate cobalamin shows an additional intense absorption band at 468nm with a molar extinction coefficient of 1.1×10⁴, which is assigned to a charge transfer from the phenolate to the cobalt ion. Equilibrium constants have also been determined for the equilibria between adenylcobamide cyanide and CN⁻, HO⁻ and H⁺, which show that the adenine is more easily displaced by CN⁻ and HO⁻ than is 5,6-dimethylbenziminazole in vitamin B₁₂, but can be protonated by acid while still remaining co-ordinated to the cobalt. It is shown that in the binding of corrinoids to proteins and polypeptides the formation of hydrogen bonds is far more important than co-ordination by the metal.     

Glycosyltransferases: managers of small molecules

Studies of the glycosyltransferases (GTs) of small molecules have greatly increased in recent years as new approaches have been used to identify their genes and characterize their catalytic activities. These enzymes recognize diverse acceptors, including plant metabolites, phytotoxins and xenobiotics. Glycosylation alters the hydrophilicity of the acceptors, their stability and chemical properties, their subcellular localisation and often their bioactivity. Considerable progress has been made in understanding the role of GTs in the plant and the utility of GTs as biocatalysts, the latter arising from their regio- and enantioselectivity and their ability to recognize substrates that are not limited to plant metabolites.     

ٍSome biologically active microorganisms have the potential to suppress mosquito larvae (Culex pipiens,Diptera: Culicidae)

Malaria is a disease caused by protozoan species of the genus Plasmodium. It is widespread and becoming a challenge in several African countries in the tropical and subtropical regions. In 2010, a report was published showing that over 1.2 million death cases were occurred globally due to malaria in just one year. The transmission of the disease from one person to another occurs via the bite of the Anopheles female. It is known that Plasmodium ovale, P. vivax, P. malariae, P. falciparum, and P. knowlesi are the highly infective malaria species. The problem of this disease is the absence of any effective medical treatment or vaccine, making the mosquito control is the only feasible way for disease prevention. Pesticides are currently the most widely used method for mosquito control, despite its well-known negative effects, including health hazards on human, the increasing insecticidal resistance, and the negative impact on the environment and beneficial organisms. Biological control (also called: biocontrol) of insects has been a promising method to overcome the negative effects of using chemical insecticides, as it depends on just using the natural enemies of pests to either minimize their populations or eradicate them. This article provides an overview of the recent and effective biological means to control malaria, such as bacteria, fungi, viruses, larvivorous fish, toxorhynchites larva and nematodes. In addition, the importance, advantages, and disadvantages of the biocontrol methods will be discussed in comparison with the traditionally used chemical methods of malaria control with special reference to nanotechnology as a novel method for insects’ control.     

The formation of biologically active beta-galactosidase inclusion bodies in Escherichia coli

Culture conditions affecting the formation of beta-galactosidase inclusion bodies in E. coli were examined. High temperature, early induction, high salt concentration and low aeration were all found to favour an increase of insoluble beta-galactosidase and the formation of visible inclusion bodies. The ratio of soluble to insoluble beta-galactosidase decreased during the course of cell growth. When assayed for beta-galactosidase activity, the inclusion bodies were enzymatically active with a specific activity of one third that of soluble beta-galactosidase. The activity remained associated with the inclusion bodies on washing with detergent and high ionic strength buffers. These results suggest that inclusion bodies can contain correctly folded protein.