A one-bead, one-stock solution approach to chemical genetics: part 2

BACKGROUND: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. RESULTS: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. CONCLUSIONS: Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.     

Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis

BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.     

Synthesis of a library of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives and identification of bioactive compounds

The design and synthesis of a library of novel families of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives is reported. The library was composed of 44 3-oxopiperazinium derivatives (11 of these compounds had a spiranic skeleton) and 22 perhydro-3-oxo-1,4-diazepinium compounds. The synthetic procedure involved a 6-step sequence carried out in solution, along with the use of solid-phase linked scavengers and microwave activation for the rapid removal of the excess of amine reagents. A final cyclization step performed under mild conditions led to the charged heterocyclic moiety. Screening of this library in two biological assays identified active compounds that inhibit the activity of the vanilloid receptor TRPV1 and modulators of the multidrug resistance phenomenon. Thus, this synthetic sequence represents a facile and convenient entry to unprecedented libraries of this sort of tetraalkylammonium derivatives that may be of use for identification of novel scaffolds of diverse biological activity.     

Evaluation of docking performance in a blinded virtual screening of fragment-like trypsin inhibitors

In this study, we have "blindly" assessed the ability of several combinations of docking software and scoring functions to predict the binding of a fragment-like library of bovine trypsine inhibitors. The most suitable protocols (involving Gold software and GoldScore scoring function, with or without rescoring) were selected for this purpose using a training set of compounds with known biological activities. The selected virtual screening protocols provided good results with the SAMPL3-VS dataset, showing enrichment factors of about 10 for Top 20 compounds. This methodology should be useful in difficult cases of docking, with a special emphasis on the fragment-based virtual screening campaigns.     

Schiff bases of indoline-2,3-dione (isatin) with potential antiproliferative activity

ABSTRACT: BACKGROUND: Cancer is one of the most dreaded diseases and it is a leading cause of mankind death worldwide. Recent reports documented a remarkable antiproliferative activity of isatin nucleus against various cancer cell lines. The current work describes the antiproliferative activity of Schiff bases of combinatorial mixtures of the isatin derivatives M1-M22 as well as the individual compounds 1-11(A-K) of these combinatorial mixtures. RESULTS: The designed combinatorial library composed from eleven hydrazides A-K and eleven isatin derivatives 1-11 has been synthesized to formally generate 22 mixtures, M1-M22 of 121 Schiff bases, and their antiproliferative activity against K562 chronic myelogenous leukemia cells was evaluated. The indexed method of analysis of the prepared library was applied to elucidate the active components in the tested mixtures M1-M22. The predictions from the crossing procedure was validated through evaluation of the antiproliferative activity of individual compounds 1-11(A-K) of the library. Individual compounds 1-11(A-K) were also evaluated against the non-tumorigenic MCF-12A cell line to investigate their selectivity. A pharmacophore model was developed to further optimize the antiproliferative activity among this series of compounds. CONCLUSIONS: Variable antiproliferative activity was revealed with the investigated mixtures M1-M22 and the individual compounds 1-11(A-K). Most of the tested mixtures and several individual Schiff bases displayed high potency with IC50 values in the low micromolar range. A considerable selectivity of some individual compounds to the tumorigenic K562 cell line compared with the non-tumorigenic MCF-12A cell line was observed as indicated by their selectivity index (SI).     

Identification of a minimal subset of receptor conformations for improved multiple conformation docking and two-step scoring

Docking and scoring are critical issues in virtual drug screening methods. Fast and reliable methods are required for the prediction of binding affinity especially when applied to a large library of compounds. The implementation of receptor flexibility and refinement of scoring functions for this purpose are extremely challenging in terms of computational speed. Here we propose a knowledge-based multiple-conformation docking method that efficiently accommodates receptor flexibility thus permitting reliable virtual screening of large compound libraries. Starting with a small number of active compounds, a preliminary docking operation is conducted on a large ensemble of receptor conformations to select the minimal subset of receptor conformations that provides a strong correlation between the experimental binding affinity (e.g., Ki, IC50) and the docking score. Only this subset is used for subsequent multiple-conformation docking of the entire data set of library (test) compounds. In conjunction with the multiple-conformation docking procedure, a two-step scoring scheme is employed by which the optimal scoring geometries obtained from the multiple-conformation docking are re-scored by a molecular mechanics energy function including desolvation terms. To demonstrate the feasibility of this approach, we applied this integrated approach to the estrogen receptor alpha (ERalpha) system for which published binding affinity data were available for a series of structurally diverse chemicals. The statistical correlation between docking scores and experimental values was significantly improved from those of single-conformation dockings. This approach led to substantial enrichment of the virtual screening conducted on mixtures of active and inactive ERalpha compounds.     

Telomerase inhibitors identified by a forward chemical genetics approach using a yeast strain with shortened telomere length

Telomerase has been proposed as a selective target for cancer chemotherapy. We established a forward chemical genetics approach using a yeast strain with shortened telomere length. Since this strain rapidly enters cell senescence in the absence of active telomerase, compounds that induce selective growth defects against telomere-shortened yeast could be candidates for drugs acting on telomeres and telomerase. We screened our microbial products library and identified three structurally unrelated antibiotics, chrolactomycin, UCS1025A, and radicicol, as active compounds. Detailed analysis showed that chrolactomycin inhibited human telomerase in a cell-free assay as well as in a cellular assay. Long-term culture of cancer cells with chrolactomycin revealed population-doubling-dependent antiproliferative activity accompanied by telomere shortening. These results suggest that chrolactomycin is a telomerase inhibitor, and that the yeast-based assay is useful for discovering the small molecules acting on human telomerase.     

Analysis of image-based phenotypic parameters for high throughput gene perturbation assays

Although image-based phenotypic assays are considered a powerful tool for siRNA library screening, the reproducibility and biological implications of various image-based assays are not well-characterized in a systematic manner. Here, we compared the resolution of high throughput assays of image-based cell count and typical cell viability measures for cancer samples. It was found that the optimal plating density of cells was important to obtain maximal resolution in both types of assays. In general, cell counting provided better resolution than the cell viability measure in diverse batches of siRNAs. In addition to cell count, diverse image-based measures were simultaneously collected from a single screening and showed good reproducibility in repetitions. They were classified into a few functional categories according to biological process, based on the differential patterns of hit (i.e., siRNAs) prioritization from the same screening data. The presented systematic analyses of image-based parameters provide new insight to a multitude of applications and better biological interpretation of high content cell-based assays.     

Cytotoxicity of guanine-based degradation products contributes to the antiproliferative activity of guanine-rich oligonucleotides

Guanine-rich oligonucleotides (GROs) have attracted considerable attention as anticancer agents, because they exhibit cancer-selective antiproliferative activity and can form G-quadruplex structures with higher nuclease resistance and cellular uptake. Recently, a GRO, AS1411 has reached phase II clinical trials for acute myeloid leukemia and renal cell carcinoma. The antiproliferative activity of GROs has been associated with various protein targets; however the real mechanisms of action remain unclear. In this study, we showed evidence that antiproliferative activity of GROs (including AS1411) is mainly contributed by the cytotoxicity of their guanine-based degradation products, such as monophosphate deoxyguanosine (dGMP), deoxyguanosine (dG) and guanine. The GROs with lower nuclease resistance exhibited higher antiproliferative activity. Among nucleotides, nucleosides and nucleobases, only guanine-based compounds showed highly concentration-dependent cytotoxicity. Our results suggest that it is necessary to reconsider the cancer-selective antiproliferative activity of GROs. Since guanine-based compounds are endogenous substances in living organisms, systematic studies of the cytotoxicity of these compounds will provide new information for the understanding of certain diseases and offer useful information for drug design.     

Comparative performance of several flexible docking programs and scoring functions: enrichment studies for a diverse set of pharmaceutically relevant targets

Virtual screening by molecular docking has become a widely used approach to lead discovery in the pharmaceutical industry when a high-resolution structure of the biological target of interest is available. The performance of three widely used docking programs (Glide, GOLD, and DOCK) for virtual database screening is studied when they are applied to the same protein target and ligand set. Comparisons of the docking programs and scoring functions using a large and diverse data set of pharmaceutically interesting targets and active compounds are carried out. We focus on the problem of docking and scoring flexible compounds which are sterically capable of docking into a rigid conformation of the receptor. The Glide XP methodology is shown to consistently yield enrichments superior to the two alternative methods, while GOLD outperforms DOCK on average. The study also shows that docking into multiple receptor structures can decrease the docking error in screening a diverse set of active compounds.     

A 3D mammalian cell separator biochip

The dissimilar cytoskeletal architecture in diverse cell types induces a difference in their deformability that presents a viable approach to separate cells in a non-invasive manner. We report on the design and fabrication of a robust and scalable device capable of separating a heterogeneous population of cells with variable degree of deformability into enriched populations with deformability above a certain threshold. The three dimensional device was fabricated in fused silica by femtosecond laser direct writing combined with selective chemical etching. The separator device was evaluated using promyelocytic HL60 cells. Using flow rates as large as 167 μL min(-1), throughputs of up to 2800 cells min(-1) were achieved at the device output. A fluorescence-activated cell sorting (FACS) viability analysis on the cells revealed 81% of the population maintain cellular integrity after passage through the device.     

Antiproliferative Effect of Colonic Fermented Phenolic Compounds from Jaboticaba (Myrciaria trunciflora) Fruit Peel in a 3D Cell Model of Colorectal Cancer

Jaboticaba is a Brazilian native berry described as a rich source of phenolic compounds (PC) with health promoting effects. PC from jaboticaba peel powder (JPP) have low intestinal bio-accessibility and are catabolized by gut microbiota. However, the biological implication of PC-derived metabolites produced during JPP digestion remains unclear. This study aimed to evaluate the antiproliferative effects of colonic fermented JPP (FJPP) in a 3D model of colorectal cancer (CRC) composed by HT29 spheroids. JPP samples fermented with human feces during 0, 2, 8, 24 or 48 h were incubated (10,000 µg mL⁻¹) with spheroids, and cell viability was assessed after 72 h. Chemometric analyses (cluster and principal component analyses) were used to identify the main compounds responsible for the bioactive effect. The antiproliferative effect of FJPP in the CRC 3D model was increased between 8 h and 24 h of incubation, and this effect was associated with HHDP-digalloylglucose isomer and dihydroxyphenyl-γ-valerolactone. At 48 h of fermentation, the antiproliferative effect of FJPP was negligible, indicating that the presence of urolithins did not improve the bioactivity of JPP. These findings provide relevant knowledge on the role of colonic microbiota fermentation to generate active phenolic metabolites from JPP with positive impact on CRC.     

Improved Antiproliferative Activities of a New Series of 1,3,4-Thiadiazole Derivatives Against Human Leukemia and Breast Cancer Cell Lines

Twenty-two novel 1,3,4-thiadiazole derivatives were synthesized using different aromatic acids as starting materials, followed by cyclization, coupling and deprotection reaction. The structures of all the target compounds were identified by means of ¹H nuclear magnetic resonance(NMR), ¹³C NMR and high resolution mass spectrometer(HRMS). Further biological evaluations were performed for chronic myelogenous leukemia cell and breast cancer cell. The results suggest that most of the target compounds exhibit potent anti-proliferative activities. Especially, compound 5b shows better antiproliferative activities against MDA-MB-231 and K562 cell lines compared with gossypol.     

Synergistic Interactions between Tocol and Phenolic Extracts from Different Tree Nut Species against Human Cancer Cell Lines

Tree nuts are rich in polar (phenolic compounds) and non-polar (tocols) antioxidants, with recognized effects in the prevention of diseases such as cancer. These biomolecules possess antiproliferative activity on cancer cells; however, the combined effect of both types of compounds has been scarcely studied, and this approach could give valuable information on the real anticancer potential of tree nuts. In the present study, the antiproliferative activity of pure tocols and phenolic compounds, tocol- and phenolic-rich extracts (TRE and PRE, respectively) from tree nuts and the extracts combinations, was evaluated in four cancer (HeLa, MCF7, PC3, A549) and one control (ARPE) cell lines. The most sensible cell lines were HeLa and MCF7. TRE and PRE from nuts were chemically characterized; γ and δ tocopherols, total tocols, total tocopherols and total phenolic compounds were negatively correlated with cell viability in MCF7 cells. In HeLa cells, only δ and total tocopherols were negatively correlated with cell viability. TRE and PRE had a low effect in reducing cell viability of the cancer cell lines, the most effective extracts were those of emory oak acorn (EOA), pecan nut (PEC) and walnut (WAL), and these were further studied for their pharmacological interactions, using the combination index and the isobologram methods. Combinations of both extracts showed a synergistic and strongly synergistic behavior in the three nuts (EOA, PEC and WAL), with combination indexes between 0.12 and 0.55. These results highlight the need to understand the interactions among components found in complex natural extracts or food products in order to fully understand their bioactivities.     

In Vitro Assessment of the Cytotoxic and Antiproliferative Profile of Natural Preparations Containing Bergamot,Orange and Clove Essential Oils

Medicinal plants and essential oils (EOs), in particular, were intensively studied in recent years as viable alternatives for antiproliferative chemical synthetic agents. In the same lines, the present study focuses on investigating the effects of natural preparations (emulsions) based on EOs obtained from Citrus bergamia Risso (bergamot-BEO), Citrus sinensis Osbeck (orange-OEO), and Syzygium aromaticum Merill et L. M. Perry (clove-CEO) on different healthy (human immortalized keratinocytes—HaCaT and primary human gingival fibroblasts—HGF) and human tumor cell lines (human melanoma—A375 and oral squamous carcinoma—SCC-4) in terms of the cells’ viability and cellular morphology. The obtained results indicate that the CEO emulsion (ECEO) induced a dose-dependent cytotoxic in both healthy (HaCaT and HGF) and tumor (A375 and SCC-4) cells. OEO emulsion (EOEO) increased cell viability percentage both for HaCaT and A375 cells and had an antiproliferative effect at the highest concentration in HGF and SCC-4 cells. BEO emulsion (EBEO) decreased the viability percentage of SCC-4 tumor cells. By associating OEO with CEO as a binary mixture in an emulsified formulation, the inhibition of tumor cell viability increases. The E(BEO/OEO) binary emulsion induced an antiproliferative effect on oral health and tumor cells, with a minimal effect on skin cells. The non-invasive tests performed to verify the safety of the test compound’s emulsions at skin level indicated that these compounds do not significantly modify the physiological skin parameters and can be considered safe for human skin.     

Untargeted LC-MS/MS-Based Multi-Informative Molecular Networking for Targeting the Antiproliferative Ingredients in Tetradium ruticarpum Fruit

The fruit of Tetradium ruticarpum (TR) is commonly used in Chinese herbal medicine and it has known antiproliferative and antitumor activities, which can serve as a good source of functional ingredients. Although some antiproliferative compounds are reported to be present in TR fruit, most studies only focused on a limited range of metabolites. Therefore, in this study, the antiproliferative activity of different extracts of TR fruit was examined, and the potentially antiproliferative compounds were highlighted by applying an untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based multi-informative molecular networking strategy. The results showed that among different extracts of TR fruit, the EtOAc fraction F2-3 possessed the most potent antiproliferative activity against HL-60, T24, and LX-2 human cell lines. Through computational tool-aided structure prediction and integrating various data (sample taxonomy, antiproliferative activity, and compound identity) into a molecular network, a total of 11 indole alkaloids and 47 types of quinolone alkaloids were successfully annotated and visualized into three targeted bioactive molecular families. Within these families, up to 25 types of quinolone alkaloids were found that were previously unreported in TR fruit. Four indole alkaloids and five types of quinolone alkaloids were targeted as potentially antiproliferative compounds in the EtOAc fraction F2-3, and three (evodiamine, dehydroevodiamine, and schinifoline) of these targeted alkaloids can serve as marker compounds of F2-3. Evodiamine was verified to be one of the major antiproliferative compounds, and its structural analogues discovered in the molecular network were found to be promising antitumor agents. These results exemplify the application of an LC-MS/MS-based multi-informative molecular networking strategy in the discovery and annotation of bioactive compounds from complex mixtures of potential functional food ingredients.     

Biological Properties of New Chiral 2-Methyl-5,6,7,8-tetrahydroquinolin-8-amine-based Compounds

The synthesis of a small library of 8-substituted 2-methyl-5,6,7,8-tetrahydroquinoline derivatives is presented. All the compounds were tested for their antiproliferative activity in non-cancer human dermal microvascular endothelial cells (HMEC-1) and cancer cells: human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa), human dermal microvascular endothelial cells (HMEC-1), colorectal adenocarcinoma (HT-29), ovarian carcinoma (A2780), and biphasic mesothelioma (MSTO-211H). Compounds 3a, 5a, and 2b, showing significant IC₅₀ values against the whole panel of the selected cells, were further synthesized and tested as pure enantiomers in order to shed light on how their stereochemistry might impact on the related biological effect. The most active compound (R)-5a was able to affect cell cycle phases and to induce mitochondrial membrane depolarization and cellular ROS production in A2780 cells.     

Novel 4-azaandrostenes as prostate cancer cell growth inhibitors: Synthesis,antiproliferative effects,and molecular docking studies

In this study, synthesis, structural characterization, molecular docking studies, and antiproliferative effects in four different cell lines of several novel 16-arylidene-4-azaandrost-5-ene compounds are reported. These compounds were prepared by oxidative cleavage of the enone system of androstenedione followed by an azacyclization reaction and an aldol condensation with various aldehydes at C16. In the androgen-dependent LNCaP cells, the most relevant antiproliferative effects were observed with the 16-phenyl, 16-p-tolyl, and 16-p-nitrophenyl derivatives. Compound 16E-[(4-methylphenyl)methylidene]-4-azaandrost-5-ene-3,17-dione was the most potent in these cells (IC₅₀ = 28.28 μM), having lower antiproliferative effects in the androgen-independent PC-3 cells (IC₅₀ = 45.31 μM). In addition, an interesting selectivity toward cancer cell lines was found for all compounds because a generally low cytotoxicity was detected in healthy human fibroblasts. Furthermore, the 16-p-tolylazaandrostene steroid induced a reduction of viability in LNCaP cells similar to that observed with finasteride, a clinically used 5α-reductase inhibitor. Moreover, molecular docking studies predicted that these 4-azaandrostene derivatives can interact with 5β-reductase, which has a high level of similarity to 5α-reductase enzyme, and with other common targets of steroidal drugs, particularly the enzyme 17α-hydroxylase/17,20-lyase.     

Modular synthesis and preliminary biological evaluation of stereochemically diverse 1,3-dioxanes

Modular synthesis and substrate stereocontrol were combined to furnish 18,000 diverse 1,3-dioxanes whose distribution in chemical space rivals that of a reference set of over 2,000 bioactive small molecules. Library quality was assessed at key synthetic stages, culminating in a detailed postsynthesis analysis of purity, yield, and structural characterizability, and the resynthesis of library subsets that did not meet quality standards. The importance of this analysis-resynthesis process is highlighted by the discovery of new biological probes through organismal and protein binding assays, and by determination of the building block and stereochemical basis for their bioactivity. This evaluation of a portion of the 1,3-dioxane library suggests that many additional probes for chemical genetics will be identified as the entire library becomes biologically annotated.