Hydration and Structural Adaptations of the Human CYP1A1, CYP1A2, and CYP1B1 Active Sites by Molecular Dynamics Simulations.

Cytochromes CYP1A1, CYP1A2, and CYP1B1, the members of the cytochrome P450 family 1, catalyze the metabolism of endogenous compounds, drugs, and non-drug xenobiotics which include substances involved in the process of carcinogenesis, cancer chemoprevention, and therapy. In the present study, the interactions of three selected polymethoxy-trans-stilbenes, analogs of a bioactive polyphenol trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) with the binding sites of CYP1 isozymes were investigated with molecular dynamics (MD) simulations. The most pronounced structural changes in the CYP1 binding sites were observed in two substrate recognition sites (SRS): SRS2 (helix F) and SRS3 (helix G). MD simulations show that the number and position of water molecules occurring in CYP1 APO and in the structures complexed with ligands are diverse. The presence of water in binding sites results in the formation of water-protein, water-ligand, and bridging ligand-water-protein hydrogen bonds. Analysis of the solvent and substrate channels opening during the MD simulation showed significant differences between cytochromes in relation to the solvent channel and the substrate channels 2c, 2ac, and 2f. The results of this investigation lead to a deeper understanding of the molecular processes that occur in the CYP1 binding sites and may be useful for further molecular studies of CYP1 functions.

Polymorphism of Butyl Ester of Oleanolic Acid-The Dominance of Dispersive Interactions over Electrostatic.

Oleanolic (OA) and glycyrrhetinic acids (GE), as well as their derivatives, show a variety of pharmacological properties. Their crystal structures provide valuable information related to the assembly modes of these biologically active compounds. In the known-to-date crystals of OA esters, their 11-oxo derivatives, and GE ester crystals, triterpenes associate, forming different types of ribbons and layers whose construction is based mainly on van der Waals forces and weak C-H···O interactions. New crystal structures of 11-oxo OA methyl ester and the polymorph of OA butyl ester reveal an alternative aggregation mode. Supramolecular architectures consist of helical chains which are stabilized by hydrogen bonds of O-H···O type. It was found that two polymorphic forms of butyl OA ester (layered and helical) are related monotropically. In a structure of metastable form, O-H···O hydrogen bonds occur, while the thermodynamically preferred phase is governed mainly by van der Waals interactions. The intermolecular interaction energies calculated using CrystalExplorer, PIXEL, and Psi4 programs showed that even in motifs formed through O-H···O hydrogen bonds, the dispersive forces have a significant impact.

Weak intermolecular interactions in a series of biologically active 4'-methylthio-trans-stilbenes.

The crystal structures of nine methoxy-substituted 4'-methylthiostilbenes, which are potential inhibitors of human recombinant cytochrome P450 enzymes, were determined. These compounds included two mono-methoxy-substituted derivatives: 2-methoxy-4'-methylthio-trans-stilbene {systematic name: 1-[(E)-2-(2-methoxyphenyl)ethenyl]-4-(methylsulfanyl)benzene} (1) and 3-methoxy-4'-methylthio-trans-stilbene (2), both C16H16OS; four dimethoxy derivatives: 2,3-dimethoxy-4'-methylthio-trans-stilbene (3), 2,5-dimethoxy-4'-methylthio-trans-stilbene (4), 3,5-dimethoxy-4'-methylthio-trans-stilbene (5) and 2,4-dimethoxy-4'-methylthio-trans-stilbene (6), all C17H18O2S; and three trimethoxy compounds: 2,4,5-trimethoxy-4'-methylthio-trans-stilbene (7), 3,4,5-trimethoxy-4'-methylthio-trans-stilbene (8) and 2,4,6-trimethoxy-4'-methylthio-trans-stilbene (9), all C18H20O3S. The geometries of the compounds in the crystal structures were compared with those found during docking studies at the active site of the receptor, and some relevant differences were identified. Intermolecular interactions were analyzed using three different methods. First, the (3,-1) critical points of the gradient field of the electron density were identified, and then the appropriate contacts were analyzed using their geometrical characteristics and interaction energy calculations. The results confirmed the importance of weak delocalized interactions in the construction of the crystal structures, and the results of different methods (PIXEL and DFT) were comparable in the absence of strong well-defined intermolecular interactions.

Two new polymorphic forms of combretastatin A-4, an antitumour agent.

Two new polymorphic forms of combretastatin A-4 {systematic name: 2-methoxy-5-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenol, C18H20O5, CA-4}, an inhibitor of tubulin polymerization at the colchicine binding site, were identified. A number of crystallization attempts led to the orthorhombic form, with two molecules in the asymmetric part of the unit cell; obtaining a different form required the experiment to be moved to another laboratory. None of the attempts resulted in the monoclinic form described earlier. The three different forms contain molecules of significantly different geometries, which can be related to conformational freedom, postulated as the result of biological studies. In addition, the packing modes in all three forms are basically different. The structural differences at both the molecular and the supramolecular level have also been studied via calculations of energies and a topological analysis of the electron density. The results confirm the role of weak interactions in the determination of crystal architecture and additionally hint at an explanation for the results of crystallization attempts: the new monoclinic form has significantly lower energy than the form reported earlier.

Structure-Based Drug Design for Cytochrome P450 Family 1 Inhibitors.

Cytochromes P450 are a class of metalloproteins which are responsible for electron transfer in a wide spectrum of reactions including metabolic biotransformation of endogenous and exogenous substrates. The superfamily of cytochromes P450 consists of families and subfamilies which are characterized by a specific structure and substrate specificity. Cytochromes P450 family 1 (CYP1s) play a distinctive role in the metabolism of drugs and chemical procarcinogens. In recent decades, these hemoproteins have been intensively studied with the use of computational methods which have been recently developed remarkably to be used in the process of drug design by the virtual screening of compounds in order to find agents with desired properties. Moreover, the molecular modeling of proteins and ligand docking to their active sites provide an insight into the mechanism of enzyme action and enable us to predict the sites of drug metabolism. The review presents the current status of knowledge about the use of the computational approach in studies of ligand-enzyme interactions for CYP1s. Research on the metabolism of substrates and inhibitors of CYP1s and on the selectivity of their action is particularly valuable from the viewpoint of cancer chemoprevention, chemotherapy, and drug-drug interactions.

Cytotoxic, tubulin-interfering and proapoptotic activities of 4'-methylthio-trans-stilbene derivatives, analogues of trans-resveratrol.

The aim of this study was to evaluate the cytotoxicity of a series of seven 4'-methylthio-trans-stilbene derivatives against cancer cells: MCF7 and A431 in comparison with non-tumorigenic MCF12A and HaCaT cells. The mechanism of anti-proliferative activity of the most cytotoxic trans-resveratrol analogs: 3,4,5-trimethoxy-4'-methylthio-trans-stilbene (3,4,5-MTS) and 2,4,5-trimethoxy-4'-methylthio-trans-stilbene (2,4,5-MTS) was analyzed and compared with the effect of trans-resveratrol. All the compounds that were studied exerted a stronger cytotoxic effect than trans-resveratrol did. MCF7 cells were the most sensitive to the cytotoxic effect of trans-resveratrol analogs with IC50 in the range of 2.1-6.0 µM. Comparing the cytotoxicity of 3,4,5-MTS and 2,4,5-MTS, a significantly higher cytotoxic activity of these compounds against MCF7 versus MCF12A was observed, whereas no significant difference was observed in cytotoxicity against A431 and HaCaT. In the series of 4'-methylthio-trans-stilbenes, 3,4,5-MTS and 2,4,5-MTS were the most promising compounds for further mechanistic studies. The proapoptotic activity of 3,4,5-MTS and 2,4,5-MTS, estimated with the use of annexin-V/propidium iodide assay, was comparable to that of trans-resveratrol. An analysis of cell cycle distribution showed a significant increase in the percentage of apoptotic cells and G2/M phase arrest in MCF7 and A431 as a result of treatment with 3,4,5-MTS, whereas trans-resveratrol tended to increase the percentage of cells in S phase, particularly in epithelial breast cells MCF12A and MCF7. Both trans-stilbene derivatives enhanced potently tubulin polymerization in a dose-dependent manner with sulfur atom participating in the interactions with critical residues of the paclitaxel binding site of ß-tubulin.

Design, synthesis, and biological evaluation of novel combretastatin A-4 thio derivatives as microtubule targeting agents.

A series of novel combretastatin A-4 (CA-4) thio derivatives containing different molecular cores, namely α-phenylcinnamic acids (core 1), (Z)-stilbenes (core 2), 4,5-disubstituted oxazoles (core 3), and 4,5-disubstituted N-methylimidazoles (core 4), as cis-restricted analogues were designed and synthesized. They were selected with the use of a parallel virtual screening protocol including the generation of a virtual combinatorial library based on an elaborated synthesis protocol of CA-4 analogues. The selected compounds were evaluated for antiproliferative activity against a panel of six human cancer cell lines (A431, HeLa, MCF7, MDA-MB-231, A549 and SKOV) and two human non-cancer cell lines (HaCaT and CCD39Lu). Moreover, the effect of the test compounds on the inhibition of tubulin polymerization in vitro was estimated. In the series studied here, oxazole-bridged analogues exhibited the most potent antiproliferative activity. Compounds 23a, 23e, and 23i efficiently inhibited tubulin polymerization with IC50 values of 0.86, 1.05, and 0.85 µM, respectively. Thio derivative 23i, when compared to its oxygen analogue 23j, showed a 5-fold higher inhibitory impact on tubulin polymerization. Compounds 23e and 23i, which showed both best cytotoxic and antitubulin activity, were further studied in terms of their effect on cell cycle distribution and proapoptotic activity. Compound 23e induced a statistically significant block of the cell cycle at the G2/M phase in A431, HaCaT, HeLa, MCF-7, MDA-MB-231, and SKOV-3 cells to an extent comparable to that observed in CA-4. In HeLa and SKOV-3 cells incubated with 23i, a concentration-dependent block of the G2/M phase was observed. The proapoptotic effect of 23e and 23i in A431, HaCaT, MCF-7, MDA-MB-231, and SKOV-3 was demonstrated with ELISA assay and double staining with Annexin V-FITC/PI. The results indicated that compound 23e and 23i may serve as novel lead compounds in research on more effective anticancer agents.

Synthesis, in vitro and in silico evaluation of novel trans-stilbene analogues as potential COX-2 inhibitors.

25 new trans-stilbene and trans-stilbazole derivatives were investigated using in vitro and in silico techniques. The selectivity and potency of the compounds were assessed using commercial ELISA test. The obtained results were incorporated into 2D QSAR assay. The most promising compound 4-nitro-3',4',5'-trihydroxy-trans-stilbene (N1) was synthetized and its potency and selectivity were confirmed. N1 was classified as preferential COX-2 inhibitor. Its ability to inhibit COX-2 in MCF-7 cell line was established and its cytotoxicity by MTT test was assessed. The compound was more cytotoxic than celecoxib within studied concentration range. Finally, the investigated trans-stilbene was docked into COX-1 and COX-2 active sites using "CDOCKER" protocol.

Synthesis, biological evaluation and docking studies of trans-stilbene methylthio derivatives as cytochromes P450 family 1 inhibitors.

Cytochromes P450 family 1 (CYP1) are responsible for the metabolism of procarcinogens, for example polycyclic aromatic hydrocarbons and aromatic and heterocyclic amines. The inhibition of CYP1 activity is examined in terms of chemoprevention and cancer chemotherapy. We designed and synthesized a series of trans-stilbene derivatives possessing a combination of methoxy and methylthio functional groups attached in different positions to the trans-stilbene skeleton. We determined the effects of synthesized compounds on the activities of human recombinant CYP1A1, CYP1A2 and CYP1B1 and, to explain the variation of inhibitory potency of methoxystilbene derivatives and their methylthio analogues, we employed computational analysis. The compounds were docked to CYP1A1, CYP1A2 and CYP1B1 binding sites with the use of Accelrys Discovery Studio 4.0 by the CDOCKER procedure. For CYP1A2 and CYP1B1, values of scoring functions correlated well with inhibitory potency of stilbene derivatives. All compounds were relatively poor inhibitors of CYP1A2 that possess the most narrow and flat enzyme cavity among CYP1s. For the most active CYP1A1 inhibitor, 2-methoxy-4'-methylthio-trans-stilbene, a high number of molecular interactions was observed, although the interaction energies were not distinctive.

Is resection of the tendon edge necessary to enhance the healing process? An evaluation of the expression of collagen type I, IL-1ß, IFN-γ, IL-4, and IL-13 in the distal 1 cm of a torn supraspinatus tendon: part II.

BACKGROUND: Type I collagen proin pro-in expression in a damaged supraspinatus tendon is thought to be dependent on the distance from the edge of the tear and the local expression of pro-inflammatory, anti-proliferative, and pro-proliferative cytokines. The study evaluates the expression of type I collagen, pro-inflammatory interleukin (IL) 1ß, anti-proliferative interferon-γ (IFN-γ), and pro-proliferative IL-4 and IL-13 cytokines along a 1-cm section taken from the edge of a torn supraspinatus tendon. Three sections were taken: 3 mm distal to the tear, 3 mm proximal to the tear, and the 4-mm middle section between them. METHODS: Nine patients (average age, 58 years) were included in the study. All fulfilled strict inclusion criteria regarding tear morphology and reconstruction technique. Samples were taken from the ruptured supraspinatus tendon at the time of arthroscopic repair. Quantitative real-time polymerase chain reaction assay was used for analysis. RESULTS: The expression of type I collagen, IL-4, and IL-13 significantly increased and that of IL-1ß and IFN-γ decreased from the distal to the proximal parts of the tendon edge (P < .05). CONCLUSIONS: The expression of type I collagen is dependent on the distance from the edge of the torn supraspinatus tendon, the balance between anti-proliferative IFN-γ and pro-proliferative IL-4 and IL-13, and the expression of pro-inflammatory IL-1ß. Hence, whereas resection of the distal 3 mm of the torn supraspinatus tendon edge eliminates its least valuable part, resection between 4 and 7 mm may enhance the healing process by reaching a reasonable compromise between the mechanical features of the tendon characterized by collagen type I expression and the technical abilities of reconstruction.

Is resection of the tendon edge necessary to enhance the healing process? An evaluation of the homeostasis of apoptotic and inflammatory processes in the distal 1 cm of a torn supraspinatus tendon: part I.

BACKGROUND: We hypothesize that the expression of proapoptotic and antiapoptotic molecules and cytokines is dependent on the distance from the torn supraspinatus tendon edge and this expression may influence its potential for healing. The aim of this work is to evaluate the expression of proapoptotic Bax molecule and caspases 3, 8, and 9; antiapoptotic Bcl-2 molecule; and proinflammatory tumor necrosis factor (TNF) α and anti-inflammatory interleukin 10 (IL-10) in 3 sections taken from a 1-cm section of the edge of a torn supraspinatus tendon: 3 mm distal and 3 mm proximal, as well as the remaining 4-mm middle section between them. METHODS: Nine patients, with a mean age of 58 years, were included in the study. All fulfilled strict inclusion criteria regarding the morphology of the tear and reconstruction technique. Samples were taken from the ruptured supraspinatus tendon at the time of arthroscopic repair. Quantitative real-time polymerase chain reaction assay was used for analysis. RESULTS: The expression of caspases 9, 8 and 3; Bax; and TNF-α significantly decreased from the distal to the proximal parts of the tendon edge (P < .05). However, a significant increase in Bcl-2 and IL-10 expression was also found in the same direction (P < .05). CONCLUSIONS: Tenocytes can reduce the expression of proapoptotic caspases 3, 8, and 9 and Bax, as well as proinflammatory TNF-α, by increasing the expression of Bcl-2 and IL-10 within 1 cm of the supraspinatus edge in a distal to proximal direction. Resection 4 to 7 mm from the edge of the torn supraspinatus tendon may enhance the healing process by reaching a reasonable compromise between molecular homeostasis of apoptotic and inflammatory processes and mechanical aspects of rotator cuff reconstruction.

Design, synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1, CYP1A2 and CYP1B1.

A series of trans-stilbene derivatives containing 4'-methylthio substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. Additionally, for CYP1A2 and CYP1B1 molecular docking analysis was carried out to provide information on enzyme-ligand interactions and putative site of metabolism. 3,4,5-Trimethoxy-4'-methylthio-trans-stilbene, an analogue of DMU-212 (3,4,5,4'-tetramethoxy-trans-stilbene) was an effective inhibitor of all CYP1 enzymes. On the other hand, 2,3,4-trimethoxy-4'-methylthio-trans-stilbene, appeared to be the most selective inhibitor of the isozymes CYP1A1 and CYP1B1, displaying extremely low affinity towards CYP1A2. Molecular modeling suggested that the most probable binding poses of the methylthiostilbene derivatives in CYP1A2 active sites are those with the methylthio substituent directed towards the heme iron. Products of CYP1A2-catalyzed oxidation of 2,4,5-trimethoxy-4'-methylthiostilbene and 3,4,5-trimethoxy-4'-methylthiostilbene were identified as monohydroxylated compounds. Other studied derivatives appeared to be poor substrates of CYP1A2. Structure-activity relationship analysis rendered better understanding of the mechanism of action of xenobiotic-metabolizing enzymes crucial at the early stage of carcinogenesis.

Potential aluminium(III)- and gallium(III)-selective optical sensors based on porphyrazines.

Porphyrazines possessing non-coordinating alkyl (propyl) and aralkyl (4-tert-butylphenyl) groups in the periphery were studied as optical sensors for a set of mono-, di- and trivalent cations. Investigated porphyrazines in the UV-Vis monitored titrations revealed significant responses towards aluminium and gallium cations, unlike other metal ions studied. Additionally, porphyrazine possessing 4-tert-butylphenyl peripheral substituents showed sensor property towards ruthenium cation and was chosen for further investigation. The presence of isosbestic points in absorption spectra for its titration with aluminium, gallium and ruthenium cations, accompanied by a linear Benesi-Hildebrand plot, proved complex formation. The continuous variation method was used to determine binding stoichiometry in 1:1 porphyrazine-metal ratio. X-Ray studies and density functional theory calculations were employed to investigate octa(4-tert-butylphenyl)porphyrazine structure. The results helped to explain the observed selectivity towards certain ions. Interaction between ion and porphyrazine meso nitrogen in a Lewis acid-Lewis base manner is proposed.

Experimental and computational study on the reactivity of 2,3-bis[(3-pyridylmethyl)amino]-2(Z)-butene-1,4-dinitrile, a key intermediate for the synthesis of tribenzoporphyrazine bearing peripheral methyl(3-pyridylmethyl)amino substituents.

ABSTRACT: An earlier developed alkylating path leading to tetraalkylated diaminomaleonitrile derivatives was explored. Attempts to explain the reactivity of the representative dialkylated diaminomaleonitrile 2,3-bis[(3-pyridylmethyl)amino]-2(Z)-butene-1,4-dinitrile during the alkylation reaction were performed using X-ray and density functional theory (DFT) studies. The condensed Fukui functions accompanied by softness indices were found to be useful in explaining its reactivity observed during the reaction. The values of the Fukui functions and condensed softness for electrophilic attack calculated from Mulliken, Löwdin, and natural population analyses closely corresponded to the experimental observations. When 2,3-bis[(3-pyridylmethyl)amino]-2(Z)-butene-1,4-dinitrile disodium salt was treated with dimethyl sulfate at lower temperatures the alkylation reaction prevailed, whereas at higher temperatures the alkylating agent acted as a hydride anion acceptor, which favored the elimination reaction. The tetraalkylated dinitrile 2,3-bis[methyl(3-pyridylmethyl)amino]-2(Z)-butene-1,4-dinitrile was used in the synthesis of tribenzoporphyrazine bearing methyl(3-pyridylmethyl)amino groups, which was subsequently subjected to solvatochromic and metallation studies. The changes observed during metallation seem to result from the coordination of the 3-pyridyl group by a palladium ion. This could influence the configuration of the methyl(3-pyridylmethyl)amino moiety, causing more effective donation of a lone pair of electrons from peripheral nitrogen to the macrocyclic ring. GRAPHICAL ABSTRACT: .