The Application of the Combination of Monte Carlo Optimization Method based QSAR Modeling and Molecular Docking in Drug Design and Development.

In recent years, one of the promising approaches in the QSAR modeling Monte Carlo optimization approach as conformation independent method, has emerged. Monte Carlo optimization has proven to be a valuable tool in chemoinformatics, and this review presents its application in drug discovery and design. In this review, the basic principles and important features of these methods are discussed as well as the advantages of conformation independent optimal descriptors developed from the molecular graph and the Simplified Molecular Input Line Entry System (SMILES) notation compared to commonly used descriptors in QSAR modeling. This review presents the summary of obtained results from Monte Carlo optimization-based QSAR modeling with the further addition of molecular docking studies applied for various pharmacologically important endpoints. SMILES notation based optimal descriptors, defined as molecular fragments, identified as main contributors to the increase/ decrease of biological activity, which are used further to design compounds with targeted activity based on computer calculation, are presented. In this mini-review, research papers in which molecular docking was applied as an additional method to design molecules to validate their activity further, are summarized. These papers present a very good correlation among results obtained from Monte Carlo optimization modeling and molecular docking studies.

Biological Degradation of Aflatoxin B1 by Cell-Free Extracts of Bacillus velezensis DY3108 with Broad PH Stability and Excellent Thermostability.

(1) Background: Aflatoxin contamination in food and grain poses serious problems both for economic development and public health protection, thus leading to a focus on an effective approach to control it; (2) Methods: Aflatoxin B1 (AFB1) degrading bacteria were isolated using a medium containing coumarin as the sole carbon source, and the biodegradation of AFB1 by the isolate was examined by high performance liquid chromatography, and liquid chromatography mass spectrometry; (3) Results: a bacterial strain exhibiting strong AFB1 degradation activity (91.5%) was isolated and identified as Bacillusvelezensis DY3108. The AFB1 degrading activity was predominantly attributed to the cell-free supernatant of strain DY3108. Besides, it was heat-stable and resistant to proteinase K treatment but sensitive to sodium dodecyl sulfate treatment. The optimal temperature for the maximal degradation of AFB1 was 80 °C. Even more notable, the supernatant showed a high level of activity over a broad pH (4.0 to 11.0) and exhibited the highest degradation (94.70%) at pH 8.0. Cytotoxicity assays indicated that the degradation products displayed significantly (p < 0.05) lower cytotoxic effects than the parent AFB1; (4) Conclusions: B.velezensis DY3108 might be a promising candidate for exploitation in AFB1 detoxification and bioremediation in food and feed matrices.

Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation.

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.

Simultaneous assessment of absorption characteristics of coumarins from Angelicae Pubescentis Radix: In vitro transport across Caco-2 cell and in vivo pharmacokinetics in rats after oral administration.

Angelicae Pubescentis Radix (APR), a well-known traditional Chinese medicine, is widely used for the treatments of rheumatism and headache for centuries. To assess the absorption characteristics of coumarins from APR, a sensitive and reliable UPLC-MS/MS method was established for the simultaneous determination of sixteen coumarins from APR, including psoralen, xanthotoxin, bergapten, bergaptol, isoimperatorin, imperatorin, columbianetin, columbianetin acetate, columbianadin, oxypeucedanin hydrate, angelol B, umbelliferone, scopoletin, osthole, meranzin hydrate and nodakenetin. The specificity, linearity, sensitivity, precision, accuracy, recovery, matrix effect and stability of the method were all validated to be satisfactory. The method was then applied to the in vitro transport of APR extract (APRE) across human intestinal epithelial Caco-2 cell and in vivo pharmacokinetics in rats after oral administration of APRE. All of the tested coumarins were well or moderately absorbed across Caco-2 monolayers, and could be quickly absorbed into rat blood circulation after oral administration. Columbianetin was the most easily absorbed compound across Caco-2 cell, and also had extremely highest plasma concentration in vivo. Excellent correlation between in vitro absorption across Caco-2 cell monolayers and in vivo pharmacokinetics of coumarins from APRE was well verified. The results provided valuable information for the overall absorption characteristics of the coumarins from APR, as well as for its further studies of in vivo active substances in the further.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Assessment of substrate inhibition of bacterial oligopeptidase B.

Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria, Stenotrophomonas maltophilia (Stm) and Serratia marcescens (Sem), and the Gram-positive bacterium Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100-500 times more active than SemOPB and 3-30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35 µM did not inhibit SemOPB. On the other hand, the same substrates at only 5 µM inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine.

Assessment of caspase-4 released free AFC by RP-HPLC and fluorescence detection.

A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.

A simple strategy for mitigating the effect of data variability on the identification of active chemotypes from high-throughput screening data.

Among the several goals of a high-throughput screening campaign is the identification of as many active chemotypes as possible for further evaluation. Often, however, the number of concentration response curves (e.g., IC(50)s or K(i)s) that can be collected following a primary screen is limited by practical constraints such as protein supply, screening workload, and so forth. One possible approach to this dilemma is to cluster the hits from the primary screen and sample only a few compounds from each cluster. This introduces the question as to how many compounds must be selected from a cluster to ensure that an active compound is identified, if it exists at all. This article seeks to address this question using a Monte Carlo simulation in which the dependence of the success of sampling is directly linked to screening data variability. Furthermore, the authors demonstrate that the use of replicated compounds in the screening collection can easily assess this variability and provide a priori guidance to the screener and chemist as to the extent of sampling required to maximize chemotype identification during the triage process. The individual steps of the Monte Carlo simulation provide insight into the correspondence between the percentage inhibition and eventual IC(50) curves.

Assessment of cryopreserved hepatocytes as an alternative to fresh hepatocytes for comparative interspecies metabolism studies with suitable acceptance criteria.

Fresh hepatocytes have been the choice for interspecies comparative drug metabolism studies. Cryopreserved hepatocytes represent a readily available alternative when combined with acceptance limits based on the metabolic turnover of 7-ethoxycoumarin. Results for the ten NCEs examined show that the metabolites formed were strongly correlated in fresh and cryopreserved hepatocytes.

Coumarin metabolism, toxicity and carcinogenicity: relevance for human risk assessment.

The metabolism, toxicity and results of tests for carcinogenicity have been reviewed with respect to the safety for humans of coumarin present in foodstuffs and from fragrance use in cosmetic products. Coumarin is a natural product which exhibits marked species differences in both metabolism and toxicity. The majority of tests for mutagenic and genotoxic potential suggest that coumarin is not a genotoxic agent. The target organs for toxicity and carcinogenicity in the rat and mouse are primarily the liver and lung. Moreover, the dose-response relationships for coumarin-induced toxicity and carcinogenicity are non-linear, with tumour formation only being observed at high doses which are associated with hepatic and pulmonary toxicity. Other species, including the Syrian hamster, are seemingly resistant to coumarin-induced toxicity. There are marked differences in coumarin metabolism between susceptible rodent species and other species including humans. It appears that the 7-hydroxylation pathway of coumarin metabolism, the major pathway in most human subjects but only a minor pathway in the rat and mouse, is a detoxification pathway. In contrast, the major route of coumarin metabolism in the rat and mouse is by a 3,4-epoxidation pathway resulting in the formation of toxic metabolites. The maximum daily human exposure to coumarin from dietary sources for a 60-kg consumer has been estimated to be 0.02 mg/kg/day. From fragrance use in cosmetic products, coumarin exposure has been estimated to be 0.04 mg/kg/day. The total daily human exposure from dietary sources together with fragrance use in cosmetic products is thus 0.06 mg/kg/day. No adverse effects of coumarin have been reported in susceptible species in response to doses which are more than 100 times the maximum human daily intake. The mechanism of coumarin-induced tumour formation in rodents is associated with metabolism-mediated, toxicity and it is concluded that exposure to coumarin from food and/or cosmetic products poses no health risk to humans.

Imidocarb residues in edible bovine tissues and in vitro assessment of imidocarb metabolism and cytotoxicity.

Imidocarb residues in liver and muscle were measured by HPLC after a single therapeutic dose to cattle (3 mg imidocarb dipropionate kg-1). Imidocarb and 7-ethoxycoumarin metabolism were compared in three different in vitro systems prepared from bovine liver: cultures of hepatocyte monolayers, precision-cut liver slices, and microsomes. The potential hepatotoxicity of imidocarb residues was tested on hepatocyte monolayers and assessed using the neutral red and lactate dehydrogenase leakage assays. The concentration of imidocarb (mean +/- SD) decreased between days 14 and 224 after treatment from 5.40 +/- 0.61 to 0.12 +/- 0.01 and from 1.05 +/- 0.31 to 0.06 +/- 0.02 microgram g-1 in liver and muscle, respectively. The depletion kinetics of imidocarb fitted a two-compartment model with alpha- and beta-phase half-lives of 31.7 and 48.5 days in liver and 34.9 and 120.7 days in muscle, respectively. Imidocarb metabolites were not detected in any in vitro system. 7-Ethoxycoumarin metabolism was found in all in vitro systems; the predominant metabolite produced by hepatocyte and liver slice cultures was umbelliferone glucuronide. Cytotoxicity of imidocarb (100 microM) to hepatocyte monolayers was maximal after 72 hr treatment and dose-dependent above 10 microM imidocarb. It is most likely that the hepatotoxicity of imidocarb is caused by the parent compound, because no evidence for imidocarb metabolism was found.

A novel nonpeptide HIV-1 protease inhibitor: elucidation of the binding mode and its application in the design of related analogs.

HIV-1 protease has been identified as a significant target enzyme in AIDS research. While numerous peptide-derived inhibitors have been described, the identification of a nonpeptide inhibitor remains an important goal. Using an HIV-1 protease mass screening technique, 4-hydroxy-3-(3-phenoxypropyl)-2H-1-benzopyran-2-one (1) was identified as a nonpeptide competitive inhibitor of the enzyme. Employing a Monte Carlo-based docking procedure, the coumarin was docked in the active site of the enzyme, revealing a binding mode that was later confirmed by the X-ray crystal analysis. Several analogs were prepared to test the binding interactions and improve the overall binding affinity. The most active compound in the study was 4,7-dihydroxy-3-[4-(2-methoxyphenyl)butyl]-2H-1-benzopyran-2-one (31).

Interaction of 7-n-alkoxycoumarins with cytochrome P-450(2) and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients.

A study was made of the binding of 7-ethoxy-, 7-n-propoxy- and 7-n-pentoxy-coumarin to cytochrome P-450(2) reconstituted into large unilamellar liposomes composed of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants Ksapp. increased linearly with increasing proteoliposomal concentration. When both cytochrome P-450(2) and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants Kmapp. for O-dealkylation of 7-methoxy-, 7-ethoxy- and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-n-butoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.