extfieldsofstudy: Biology Medicine

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Characterization of two acetyltransferase genes in the pyripyropene biosynthetic gene cluster from Penicillium coprobium

Pyripyropenes potently and selectively inhibit acyl-CoA:cholesterol acyltransferase 2 (ACAT-2). Among multiple isomers of pyripyropene (A to R), pyripyropene A (PyA) has insecticidal properties in addition to its growth inhibition properties against human umbilical vein endothelial cells. Based on the predicted biosynthetic gene cluster of pyripyropene A, two genes (ppb8 and ppb9) encoding two acetyltransferases (ATs) were separately isolated and introduced into the model fungus Aspergillus oryzae, using the protoplast–polyethylene glycol method. The bioconversion of certain predicted intermediates in the transformants revealed the manner by which acetylation occurred in the biosynthetic pathway by the products expressed by these two genes (AT-1 and AT-2). The acetylated products detected by high-performance liquid chromatography (HPLC) in the extracts from AT-1 and AT-2 transformant clones were not present in the extract from the transformant clone with an empty vector. The HLPC charts of each bioconversion study exhibited high peaks at 12, 10.5 and 9 min, respectively. Further ultraviolet absorption and mass spectrometry analyses identified the products as PyE, PyO and PyA, respectively. AT-1 acetylated the C-1 of deacetyl-pyripyropene E (deAc-PyE), while AT-2 played an active role in acetylating the C-11 of 11-deAc-PyO and C-7 of deAc-PyA at two different steps of the biosynthetic pathway.

Introduction
Pyripyropenes are well-known as acyl-CoA:cholesterol acyltransferase inhibitors. Pyripyropene A (PyA) has been shown to be produced by Penicillium coprobium PF1169 [1] and to exhibit insecticidal properties. [2] Among known pyripyropene isomers, pyripyropene A, B and D from the marine-derived fungus Aspergillus sp. GF5 are reported to have selective growth-inhibiting properties against human umbilical vein endothelial cells (HUVECs). [3] Based on the findings with a newly developed cell-based assay using ACAT-1-or ACAT-2-expressing Chinese hamster ovary (CHO) cells, PyA was identified as a potent and selective inhibitor of ACAT-2. The in vivo efficacy of PyA in atherosclerosis has been also demonstrated in atherogenic mice. [4] PyA is also reported to exhibit insecticidal properties against Helicoverpa zea larvae in agricultural field conditions [2] as well as very strong ACAT-2 inhibitory activity [5] in health science experiments. Therefore, PyA would be a promising drug both in health science and agriculture.
Previous studies suggest that the biosynthesis of pyripyropenes involves polyketide synthase (PKS), which utilizes nicotinic acid-derived coenzyme A (CoA), nicotinyl-CoA, as a starter unit to form the pyrone moiety. The subsequent attachment of a farnesyl group by prenyltransferase (PT) is followed by epoxidation and cyclization to give the basic core structure. Both hydroxylation and acetylation-propionylation are then required to produce various pyripyropenes. [6] Previous biochemical studies partially delineated the pyripyropene A biosynthetic pathway. [7] The biosynthesis of deAc-PyE (the basic core structure of pyripyropenes) in Aspergillus fumigatus FO-1289 was precisely elucidated using a transgenic approach with a heterologous fungus. We previously described the formation of the 13-hydroxy group and 11-deAc-PyO by the actions of two P450 genes. [6] However, the mechanisms responsible for the formation of three acetyl groups currently remain unknown.
We deduced three steps to form the three acetyl groups in the biosynthesis pathway of PyA. Only two, out of the nine genes known to be involved in the biosynthesis of PyA, encoded proteins with predicted acetyltransferase (AT) function. Both potential acetyltransferases were Oacetyltransferase type. In the present study, we describe the two acetyltransferase genes involved in the biosynthesis of PyA. Each gene was expressed in the heterologous host fungus Aspergillus oryzae HL-1105, and functional analysis was performed by feeding several predicted intermediates of pyripyropenes into the culture medium of each transformant clone using high-performance liquid chromatography (HPLC) and liquid chromatogra-phyÀmass spectrometry (LCÀMS).

Bioinformatics analysis
The full PyA biosynthesis gene cluster for P. coprobium has been described previously. [6] Protein BLAST and nucleotide BLAST were performed at the National Center for Biotechnology Information (NCBI) website (http:// www.ncbi.nlm.nih.gov/), using the predicted amino acid sequences encoded by ppb8 and ppb9, respectively. The trees were constructed using MEGA version 5 [8] after a sequence alignment using ClustalW. The maximum likelihood phylogeny was constructed using MEGA version 5 software with a bootstrap of 500.
Isolation of genomic DNA from P. coprobium Genomic DNA was prepared using the QIAGEN Genomic DNA Kit (QIAGEN K. K, Tokyo, Japan) according to the protocol described previously. [6] Isolated DNA was evaluated for integrity on an 0.8% agarose gel, and concentrations were determined spectrophotometrically.

Construction of expression plasmids
The expression vector pUSA [9] with an a-amylase promoter was used to express the acetyltransferase 1 (AT-1) gene and AT-2 gene in A. oryzae. Each gene was amplified by polymerase chain reaction (PCR) with a template of the fosmid clone G7-9 from a genomic library, and then inserted into pUSA at KpnI/SmaI (see Figure S1 in the Online Supplemental data). The sC gene on the plasmid was used as the selectable marker for fungal transformation as described previously. [6] Transformation of Aspergillus oryzae A. oryzae protoplasts were obtained and transformed according to standard methods. [10] All compressed genes were placed under the a-amylase promoter and plasmids were introduced into the adenosine triphosphate (ATP) sulphurylase mutant A. oryzae HL-1105 strain, using the protoplastÀpolyethylene glycol method to give transformants, and were then selected for ATP sulphurylase. An average 10 mg expression plasmid was used for transformation. Each independent transformant was grown, harvested and analyzed. The DNA for each transformant clone was extracted in accordance with the modified protocol. [11] Transformants carrying the respective AT genes were verified by PCR analysis using MightyAmp TM DNA polymerase (Takara Bio. Inc., Japan) with the primers listed in Table 1. The primer pairs 40F and AT R with Kpn were used for the transformant harbouring ppb8. The primer pairs 70F and infusion R of toxin were used to verify the transformant harbouring ppb9. Reactions were cycled at 98 C for 2 min and 35 cycles of 98 C for 10 s, 60 C for 15 s and 68 C for 1 min.

Metabolite analysis
To determine the function of each open reading frame, wild type A. oryzae HL-1034 and the positive transformants of A. oryzae HL-1105 with plasmids containing AT genes were grown at 30 C on CzapekÀDox medium for 7 days. One hundred microliters of the fungal spores (10 4 spores mL ¡1 ) of each transformant were transferred into a 50 mL flask with 10 mL YDP, which contained 1% maltose, and were fed with the predicted intermediates: 11-deAc-PyO, deAc-PyA and deAc-PyE, separately. The culture was agitated at 25 C. The bioconversion products of the transformants were processed after 24À96 h using culture samples from the mycelia and broth taken at each time point. All these processes were performed as described previously. [6] The mycelia and broth mixture was treated with acetone and thoroughly mixed. Acetone was removed by a centrifugal concentrator and ethyl acetate was added to extract the total products. Then the ethyl acetate extract was concentrated in vacuum to dryness. Individual extracts were dissolved in 1 mL of methanol and analyzed with LCÀMS on a Micromass ZQ (Waters, MA, USA), 2996 Photodiode array (Waters), 2695 Separation module (Waters) and Xterra C18 column (w 4.5 mm £ 50 mm, 5 mm; Waters). A linear gradient (eluent: acetonitrile-water (v/v) 20% to 100% in 26 min, flow rate 0.8 mL min ¡1 at 40 C) was used for analysis of bioconversion products. All were detected at 320 nm.

Results and discussion
To identify all the biosynthetic genes needed to produce PyA, the putative biosynthetic gene cluster that contained both PKS and PT genes was obtained based on the genomic DNA database of P. coprobium through 454 FLX sequencing. [6] We previously identified a gene cluster that spanned 24 kb with nine genes, including CoA ligase, PKS, cytochrome P450 (P450-1), P450-2, integral membrane protein (IMP), flavin adenine dinucleotide (FAD)dependent monooxygenase (FMO), prenyltransferase (PT) and acetyltransferase (AT-1 and AT-2). Four R groups were identified for pyripyropenes: C-1, C-11, C-7 and C-13. The inhibition of ACAT-2 from a structureactivity relationship (SAR) study of PyA derivatives showed that: (1) the 13-hydroxy group was necessary; (2) 1,11-dihydroxy position could be modified and the acyl group at the 7-O-hydroxyl position was necessary. [12,13] Bioinformatics analysis and phylogenetic analysis We here focused on two genes encoding ATs. Based on the deduced amino acid sequence, the molecular weight of AT-1 (522 amino acids) was calculated to be approximately 57.7 kD and that of AT-2 (434 amino acids) was approximately 49 kD (Table 2). When analyzed by blastx with genomic DNA sequences, AT-1 showed 98% coverage to the acetyltransferase of Neosartorya fischeri NRRL 181 and 72% identity, while AT-2 showed 96% coverage to A. fumigatus A1163 and 75% identity. Regarding the genomic structure of the two AT genes, the AT-2 gene had two introns, whereas the AT-1 gene had none. The phylogenetic analysis of the acetyltransferases from Aspergillus, Neosartorya and Legionella revealed that the ppb8 gene encoding AT-1 of P. coprobium shared strong homology to A. fumigatus Af293 O-acetyltransferase (Figure 1(A)), and the ppb9 gene encoding AT-2 shared very strong homology to toxin biosynthesis protein   Tri7 (Figure 1(B)). That is, the sequence of P. coprobium acetyltransferase AT-1 was 23205À24773 from GenBank accession no. FW308713. The sequence of P. coprobium toxin biosynthesis protein Tri7 (AT-2) was 25824À27178 from GenBank accession no. FW308713. The results of the bioinformatics analyses indicated that the protein products of these two genes may be involved in acyltransferase reactions.
Generation of AT transformants in the A. oryzae host To characterize the function of both gene products, each protein was expressed in the heterologous fungal host A. oryzae [14,15] HL-1105 rather than disrupting each gene in the cluster of the producer strain P. coproium PF1169. This fungal expression system was used successfully to characterize fungal genes functionally, such as the P450 genes, brlA [16] and abaA. [17] Thus, the function of each enzyme was characterized directly, and novel meroterpenoids were produced by the incorporation of different starter units or intermediates in the pathway.
The earliest candidate single clone was found on the third day. The candidates were selected until day 7, and the average number of selected clones in transformation experiments was approximately 10À15. The candidate single clonal transformants were then transferred to separate dishes and cultured for functional analysis. Before the bioconversion analysis, the transformants harbouring AT genes were confirmed through genomic PCR (see Figure  S2 in the Online Supplemental data).

Bioconversion analysis
The expression of the predicted AT-1 gene (ppb8) was carried out in the A. oryzae host. Three transformant clones were cultured in YDP media supplemented with maltose to induce expression, and deAc-PyE, 11-deAc-PyO and deAc-PyA (final concentration of 20 mg mL ¡1 ) Figure 2. (Continued) were fed into the induction culture separately. The transformants were cultured for 48À96 h. A product was detected by HPLC analysis at a retention time (RT) of 12 min, but was not found in the control transformant with an empty vector (Figure 2(A)). Further characterization was performed by LCÀMS and the structure of the product was identified. It gave a quasimolecular ion peak at m/z 452 [MCH] C as PyE, which has already been identified, [18] and its molecular formula was determined to be C 27 H 33 NO 5 . It also showed the same ultraviolet (UV) absorption at 210 and 320 nm as reported previously. [18] The expected PyE was detected in the culture fed with deAc-PyE. Neither PyO, nor PyA was detected in the extract of the culture when fed with 11-deAc-PyO or deAc-PyA. There was no difference among the three transformant clones. These results suggest that the ppb8 gene encodes a protein with acetyltransferase activity that selectively acted on the 1-hydroxy position of deAc-PyE.
The two other expected acetylation steps at C-7 and C-11 should be a result of the activity of one or more other acetyltransferase genes. First, the AT-2 gene (ppb9) encoding a predicted toxin biosynthesis protein Tri7 was introduced into the host fungi as described above. Three transformant clones were also fed with deAc-PyE, 11-deAc-PyO and deAc-PyA, respectively. The expected product PyE was not detected in feeding with deAc-PyE ( Figure 3). In the extract of the transformants supplied with 11-deAc-PyO, the peak at RT D 10.5 min became clear after 48 h of cultivation (Figure 2(C)). LCÀMS analysis gave a quasimolecular ion peak at m/z 510 [MCH] C as PyO, which has already been identified, [19] and the molecular formula was determined to be C 27 H 35 NO 7 . It also showed the same UV absorption at 231 and 320 nm as reported previously. [19] When the transformants expressing the ppb9 gene were fed with deAc-PyA, the HLPC chart exhibited a high peak at RT D 9 min from the  (Figure 2(B)). The compound gave a quasimolecular ion peak at m/z 584 [MCH] C as PyA, which has already been identified, [20] and the molecular formula was determined to be C 31 H 37 NO 10 . It also showed the same UV absorption at 231 and 320 nm as PyA. [20] These three transformant clones also showed no difference in bioconversion analysis. The presented results of the biotransformation analysis indicate that the  Steps (1)À(8) were reported previously by Itoh et al. [21] Based on the results of bioconversion, the next steps were deduced to be acetylation by AT-1 and AT-2 and hydroxylation by two P450s.
ppb9 gene encodes a protein with acetyltransferase activity that is involved in C-7 and C-11 acetylation in two separate steps of the PyA biosynthesis pathway.
Including our previous findings, the basic core structure, deAc-PyE, was first acetylated at 1-hydroxy by the expression of AT-1 and PyE was formed (Figure 4). It was subsequently hydroxylated by P450-1 to form 11-deAc-PyO, [6] which was subsequently converted to PyO by the activity of AT-2 at the 11-hydroxy group. By expressing P450-2, the C-13 and C-7 of PyO were hydroxylated to form deAc-PyA. [6] The 7-hydroxy group was then acetylated by the activity of AT-2. Thus, we elucidated the later steps of the fungal meroterpenoid biosynthetic gene cluster for PyA from P. coprobium, which has important potential activities. Combined with the biosynthetic pathway of the basic core structure reported by Itoh's group, [21] the biosynthesis of PyA was clarified. The isolation and cloning of these enzymes will greatly enhance our understanding of these processes and will allow engineering of novel hybrid molecules for industrial applications.

Conclusions
Together with previous findings, the results from this study helped to identify the later steps of the fungal meroterpenoid biosynthetic gene cluster for PyA from P. coprobium. The ppb8 gene was shown to encode a protein with acetyltransferase activity that selectively acts on the 1-hydroxy position of deAc-PyE, and the ppb9 gene to encode a protein with acetyltransferase activity that is involved in C-7 and C-11 acetylation in two separate steps of the PyA biosynthesis pathway. The elucidation of the steps involved in the biosynthesis of PyA and the isolation and cloning of these enzymes will allow engineering of novel promising hybrid molecules for industrial applications both in health science and agriculture.


Experimental and theoretical studies on the molecular properties of ciprofloxacin, norfloxacin, pefloxacin, sparfloxacin, and gatifloxacin in determining bioavailability

The aim of this investigation is to identify, by in silico and in vitro methods, the molecular determinants, e.g., solubility in an aqueous medium and lipophilic properties, which have an effect on the bioavailability of five selected fluoroquinolones. These properties were estimated by analysis of the electrostatic potential pattern and values of free energy of solvation as well as the partition coefficients of the studied compounds. The study is based on theoretical quantum-chemical methods and a simple experimental shake-flask technique with two immiscible phases, n-octanol and phosphate buffer. The solvation free energy values of compounds in both environments appeared to be negative. The wide range of electrostatic potential from negative to positive demonstrates the presence of dipole–dipole intermolecular interactions, while the high electron density at various sites indicates the possibility of hydrogen bond formation with solvent molecules. High partition coefficient values, obtained by summing the atomic contributions, did not take various correction factors into account and therefore were not accurate. Theoretical partition coefficient values based on more accurate algorithms, which included these correction factors (fragmental methods), yielded more accurate values. Theoretical methods are useful tools for predicting the bioavailability of fluoroquinolones.

the lower and upper respiratory tract. Their primary mechanism of action is inactivation of bacterial DNA topoisomerase II. They also interact weakly with DNA topoisomerase IV, thus inhibiting replication of bacterial cells and leading to their death. Fluoroquinolones have a broad antibacterial spectrum. First-generation fluoroquinolones are, with a few exceptions (e.g., pipemidic acid), becoming less commonly used because of their poor bioavailability, narrow spectrum of activity, development of microbial resistance, and more side effects than those induced by fluoroquinolones of newer generations. The range of antimicrobial activity exhibited by newer fluoroquinolones includes most Gram-negative bacteria (Salmonella, Shigella), Gram-positive bacteria (Streptococci and Staphylococci) and atypical strains such as Chlamydia, Mycoplasma, Legionella, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Drugs of the second, third, and fourth generations contain an additional fluorine atom at position 6 of the quinolone core, which affects the pharmacokinetic profile of fluoroquinolones in polar environments and improves their bioavailability and antibacterial activity [1][2][3].
Diprotic molecules like fluoroquinolones contain acidic and basic groups and, depending on the pH of the aqueous environment, they can occur in four protonated forms: cationic, zwitterionic, neutral, and anionic. The equilibrium between the forms can be shifted by pH changes. At the pH corresponding to the isoelectric point, zwitterions and neutral molecules are present at their highest concentrations. Neutral molecules exhibit the lowest solubility in water and in other polar solvents. However, in non-polar media, such as lipids in the cell membrane, their solubility is higher, so they can easily pass through cell membranes and show good bioavailability. By contrast, zwitterions have a greater affinity for polar environments and penetrate easily through pores in bacterial cell walls, after which they readily bind to bacterial gyrase [4][5][6][7].
The bioavailability of a drug substance is determined by the first and second stage of the drug's fate in the body (liberation and absorption). Here, the aim of the study is to evaluate the usefulness of simple and inexpensive experimental and theoretical methods to estimate the bioavailability of the studied compounds. We calculated the thermodynamic and electrostatic properties of ciprofloxacin, norfloxacin, and its methylene derivatives pefloxacin, sparfloxacin, and gatifloxacin (Fig. 1). These properties are: the free energy of solvation in water and diethyl ether, the electrostatic potential surface of the molecule, the logarithm of the n-octanol/water partition coefficient (P o/w ) and the logarithm of the apparent n-octanol/buffer partition coefficient.
The theoretical method comprised quantum-chemical calculations through which the lipophilicity parameters of fluoroquinolones were determined. For theoretical evaluations of lipophilicity, the following determinants were calculated: the electrostatic potential on the surface of a drug molecule, which, inter alia, indicates the number of electron donor sites as a measure of the electrostatic interactions of the molecules with the solvent and possible formation of hydrogen bonds, the free energy of solvation of the molecule in water or diethyl ether and its partition coefficient between the two media. In drug design, it is the n-octanolwater partition coefficient, log P o/w , which is commonly used as the standard lipophilicity parameter [8]. P o/w is directly related to the free energy of binding [9,10]. P o/w is an equilibrium constant for solute transfer between octanol and water, as evidenced by the following equation: where T is the absolute temperature, R the gas constant, and ΔG°the binding free energy variation measured under standard conditions (298 K, 1 atm, and 1 M concentration for both reagents and products).
To experimentally determine the apparent partition coefficient of the drug substance, we used the shake-flask technique with two immiscible phases, n-octanol and phosphate buffer at pH 6.8. Aqueous buffer was used instead of water as it provided an environment similar to that in the gastrointestinal tract, the site of drug absorption.
The equipment used included a JASCO 530 UV-Vis spectrophotometer, a mechanical shaker from Janke & Kunkel, IKA Labortechnik, HS 250, a Mettler-Toledo MP 225 model pH meter, R program v. 2.14.2 with R Studio graphic overlay v. 0.95.262, an Intel Core i5 CPU computer with an AMD Radeon HD 6800 series graphic card, Spartan 08 software package, ACD/ChemSketch freeware v.12 and the Virtual Computational Chemistry Laboratory (VCCLAB) on-line software package (ALOGPS 2.1).

Calculation of thermodynamic parameters
In the first stage of the study, the conformational space was searched for each compound in order to determine the most stable, lowest energy conformers. These calculations were performed by the Monte Carlo method with an implemented MMFF94 force field [11]. The lowest energy conformer was the initial structure in the later geometry optimization stage of the calculations. Next, the free energy of solvation in water and diethyl ether was determined for the most stable conformers of all the compounds by employing the SM8 continuum solvent model and the density functional theory (DFT) method with the gradient corrected using the B3LYP (Beck-Lee-Yang-Parr) functional and the 6-31*G basis set as implemented in the Spartan 08 software package [12]. The free solvation energy was calculated as the difference in binding energy between the molecule in the gas phase and the molecule in solution; more precisely, this difference involves a change in all possible points of the molecular surface interaction when the molecule is in the gas phase, then it is placed in the selected liquid environment. Diethyl ether was selected because its dielectric constant is close to that of n-octanol, which is not parameterized within Spartan 08. The electrostatic potential on a surface of equal electron density was calculated using the same functional and basis set as for the geometry optimization, in order to determine the contribution of electron donor and electron acceptor sites into the electrostatic pattern of the molecule. In addition, the value of the electrostatic potential was checked using the 6-31 G** basis set.

In silico models
Values of the partition coefficient were calculated using associated neural networks and other combined models to predict hydrophilic and lipophilic properties [13,14]. These calculations rely on two basic rules: that (1) molecules are cut into fragments or atoms (fragmental methods and atomic-contribution methods or surface areas) and (2) each fragment is then summed up. The fragmental method developed by Rekker and Mannhold shows that this lipophilicity descriptor can be calculated as the sum of the fragment values taking into account certain correction factors. These correction factors can be related to tautomerization effects, dipolar effects, ion effects, proximity effects, electronic interactions within one fragment, conjugated multi-hetero atomic effects, steric effects, H-bonding interactions, and hydrophobic constants [15,16].
According to this method, the log P value was calculated according to the equation: where k is the number of repeats of fragment f of type 1, and l is the number of repeats of correction factor F of type 2 [17].
The fragment values include averaged contributions of simple fragments derived from a large database of experimentally measured log P values. The ACD/log P, AB/log P algorithms included in ACD/ChemSketch freeware v.12 I-Lab 2.0 (v 5.0.0.184) were used according to the fragmentation rules. MiLog P from http://www.molinspiration. com/services/logp.html takes into account charge interactions as well. A similar method, AC/log P, from the VCCLAB on-line calculator, is based on Hansch and Leo's approach and takes structure-dependent correction values from their database into account [9,18].
In the Ghose/Crippen approach, values of log P can be verified with the use of atomiccontribution methods [19].
The method to obtain log P is represented by the following equation: where m 1 is the number of atoms of type 1 and n 1 is the contribution of an atom of type 1. These algorithms, among others, are accessible in the VCCLAB online software package (ALOGPS 2.1) and implemented in Spartan 08 software and were used in our study. It is worth mentioning that atomic-contribution methods do not take correction factors into account [16,[20][21][22][23].

Experimental determination of log P o/w
Determinations were performed using the shake-flask technique [24]. The temperature of the separation media was controlled and kept at 24ºC. The amount of the drug substance used to determine the partition coefficient in n-octanol/buffer was fixed in such a way as not to exceed a concentration of 0.01 mol/l. Each substance was dissolved in 0.1 M phosphate buffer at pH 7.4 and then the pH of the solution was adjusted to pH 6.8 with 1 M HCl. After dilution of the samples, concentration vs. UV absorbance calibration curves were drawn for five different concentrations of each compound. The absorbance was measured at a specific UV wavelength λ max for each compound. For each test substance, the Pearson linear correlation coefficient was not less than 0.9993, whereas the p values were in the range of 1.35 × 10 −6 to 7.55 × 10 −6 [25]. Before the shake-flask experiments, the buffered samples were pre-saturated with 0.1 M noctanol. The samples were shaken for a few hours in a flask and then separated in a separator and, after reaching the steady state, the aqueous phase was centrifuged at 650 × g. The concentration of the compound in the aqueous phase was determined based on the calibration curve. Then, the concentration of the compound in n-octanol and the apparent partition coefficient were calculated. Each of the values calculated is an average of three parallel measurements.

Thermodynamic potential of solvation and electrostatic potential on the surfaces of the molecules
In the first stage, using the DFT method with the gradient corrected using the B3LYP functional and the 6-31*G basis set as implemented in the Spartan software package, it was decided to analyze the values of the calculated free energy of solvation. As shown in Table 1, the solvation free energy values of the compounds in water and the organic solvent were largely negative. Thus, values for cationic forms of ciprofloxacin, norfloxacin, and pefloxacin were determined, as well as zwitterionic/neutral forms for ciprofloxacin, pefloxacin, norfloxacin, sparfloxacin, and gatifloxacin. The negative free energy of solvation in diethyl ether, representing the lipophilic solvent, was higher than in water. Also, the negative free energy of solvation of the cationic forms in diethyl ether was higher than the energy of solvation of the zwitterionic/neutral forms. For ciprofloxacin, the difference between the energy of solvation in water and diethyl ether was higher compared to the other compounds.

Theoretical and experimental partition coefficient
In the next stage of the study, the logarithm of the n-octanol/water partition coefficient was calculated using various in silico models, such as ACD/log P, ALOGP, MLOGP, AB/log P, AC/log P, or miLog P ( Table 2) and the experimental shake-flask method ( Table 3). The experimentally obtained pefloxacin partition coefficient value was greater than zero and showed the good properties of this lipophilic compound in a physiological medium. The negative values of norfloxacin, ciprofloxacin, sparfloxacin, and gatifloxacin log P indicate a high affinity of the compounds into a polar environment at a given pH (6.8). The theoretical partition coefficient calculated values were fairly different. The results obtained using the fragmentary and atomic contribution methods showed considerable discrepancy.

Discussion
The free energy of solvation, as mentioned earlier, is an important parameter that takes into account both the enthalpy and entropy of non-covalent bond formation between a dissolution molecule and its environment, in fact, the interaction that occurs between molecules of the solute and the solvent. The free energy of binding affects both the hydrophilic and lipophilic properties of molecules. Negative values of the free energy of solvation show that the dissolution reaction occurs spontaneously and there is no obstacle to the substance dissolving in the aqueous environment and passing through biological membranes. As in the gastrointestinal tract the pH environment at the site of penetration is below the isoelectric point of the compound [5], it was decided to test the free energy for a possible ionization state at a physiologically relevant pH. The data (Table 1) show that the studied molecules in the case of both cationic and zwitterionic/neutral forms interact more strongly with an organic solvent than with an aqueous solvent. The compounds in which the nitrogen of the piperazine group is protonated provided significantly higher negative free energy values than those with the charge distributed uniformly for both end groups, or those that were neutral. The electrostatic potential pattern is adequate to explain the interactions between the solvent and the solute [28]. A map of electrostatic potentials provides information on possible solvent molecule distributions around the functional groups of the dissolved molecule (Fig. 2a, b). The pattern of its electrostatic potential field determines the polarity of a compound and predetermines the interactions of the molecule with an aqueous medium. The wide range of electrostatic potential around the studied molecules indicates an interaction with the polar solvent based on the formation of dipole-dipole or dipole-induced dipole type electrostatic interactions (Fig. 2a, b, red and blue areas of the molecule). A number of electron donor sites in the molecule allow the creation of hydrogen bonds with the polar solvent. The range of the positive electrostatic potential around the cationic form indicates the inability to form hydrogen bonds necessary for the interaction of the test substance and water molecules (Table 1). It is true that a more advanced basis set choice may slightly or insignificantly impact the electrostatic potential values. However, the differences in the calculated values of electrostatic potential in the gas phase and in the presence of the solvent are not more important here because the selected model did not exactly describe the dispersion interactions. In the polarizable continuum model, the second shell is more diffused and there is less orientation of the dipoles of the solvent molecules on the dissolved molecule. This model does not take into account specific interactions, such as hydrogen bonding between solute and solvent molecules, but only considers the average effect of solvation, which the Coulomb forces simulate and is affected by dipole-induced dipole interactions.
High log P values, obtained by summing the atomic contributions, showed the imperfections of algorithms that do not take various correction factors into account. It is evident that the correction factors, such as steric (Hansch constant) and electronic (Hammett constant) parameters and atomic hydrophobic contributions should play a key role in theoretical calculations [23,29]. The theoretical partition coefficient values based on more accurate algorithms, which included the correction factors (fragmental methods) [16,17] such as AB/log P, AC/log P, or miLog P (Table 2), yielded values only slightly different than the experimentally obtained values in this study or reference values obtained by the authors [30][31][32][33]. These values seem to be more comparable, although there are still some notable differences. These differences result from the use of various correction factors, specific to each method. Some approaches develop a purely atom-based procedure, thereby avoiding correction factors [16]. Pefloxacin seems to be strongly lipophilic, while norfloxacin and ciprofloxacin seem to be strongly hydrophilic, despite the large value of negative thermodynamic potential of solvation in the apolar solvent.
In contrast, sparfloxacin appears to be moderately lipophilic, which has been confirmed by other authors [29,[31][32][33]. The lipophilicity of gatifloxacin (the fourth-generation group) is controversial. The difference value of the partition coefficient between norfloxacin and pefloxacin shows that even a small change in the molecule (addition of a methyl group to the nitrogen of the piperazine ring) can greatly change the bioavailability properties of a compound. The discrepancies between log P values can be explained by the changing states of ionization [26]. Even in an environment close to the isoelectric point, the zwitterionic form predominates [34]. Interestingly, the zwitterionic form, despite the high electric field produced by each dipole, passes through the membrane mainly via passive transport [27]. The answers to these considerations require insights into intramolecular and intermolecular interactions. Any change in the charge distribution, formed in the presence of electronegative atoms such as oxygen or nitrogen, raises the dipole moment between the carbon atoms in the molecule; intramolecular hydrogen bonds additionally stabilize the molecule [35]. Interesting conclusions have been put forward by other authors [36]. They claim to have observed that neutral and zwitterionic ciprofloxacin form agglomerates. Neutral molecules were present inside liposomes, where there is a polar environment. However, zwitterions formed stacks in the presence of the hydrophobic part of the membrane through which they pass. The creation of such clusters is due to hydrophobic interactions and hydrogen bonds, and may play an important role in their activity [36,37]. As noted by Cramariuc et al. [36], if particles are dipoles and have an electric field, then they are able to arrange in an antiparallel fashion, thus reducing the electric field and increasing their activity in an apolar environment. This feature should improve their lipophilicity and thus enable them to pass through the membrane by passive transport. Further studies by these authors have demonstrated that such agglomerates move between the polar and apolar environment by intermolecular transfer of protons, during which the particles do not lose their stability and retain their solvation ability. Other authors [38] have noted that the difference between the theoretical partition coefficients obtained based on calculations as opposed to experimental studies may be due to interactions occurring between the cationic form of solute molecules and the anionic buffer; the formation of such an ion complex may result in these differences.

Conclusions
Ampholytes, such as fluoroquinolones, make it very difficult to accurately assess their lipophilicity on the basis of theoretical methods that do not address all the thermodynamic phenomena occurring between the molecules and the environment in which the compound is located, as well as intramolecular interactions between the functional groups. The elimination of the high total charge of the electric field that allows the passage of fluoroquinolone molecules through biological membranes explains the phenomenon of agglomerate formation by zwitterions. The higher negative values of the thermodynamic solvation potential in diethyl ether compared to that in water confirms the good lipophilic properties of these compounds and demonstrates their high intrinsic lipophilicity.
It is worth noting that the methods used in this study can also be used as a convenient tool for initial and rapid substance classification into the four classes of the Biopharmaceutical Classification System and serve to enlarge this system [39,40]. To date, the WHO has classed only three active substances in this group of drugs, i.e., ciprofloxacin hydrochloride, ofloxacin, and levofloxacin, with ciprofloxacin in the third class and levofloxacin and ofloxacin in the first [41]. The correctness of the classification of ciprofloxacin to the third BCS class seems to confirm the experimentally determined log P o/w and theoretical results obtained only with the algorithms AB/log P and miLog P, as well as the electrostatic potential and free enthalpy of solvation in water, while the value of free solvation enthalpy in diethyl ether provides evidence of its high intrinsic lipophilicity.
Theoretical methods are good tools in the prognosis of the thermodynamic properties of molecules and thus in screening tests to evaluate the liberation and absorption of new therapeutic substances.
Disclosures The corresponding author certifies on behalf of all authors that there are no relevant interests to disclose.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.


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Cytotoxic Illudalane Sesquiterpenes from the Wood-Decay Fungus Granulobasidium vellereum (Ellis & Cragin) Jülich

Seven illudalane sesquiterpenes were obtained from the wood decomposing fungus Granulobasidium vellereum: granuloinden A, granuloinden B and dihydrogranuloinden, along with the previously known compounds radulactone, pterosin M, echinolactone A and D. Granuloinden B showed potent cytotoxic activity against the Huh7 and MT4 tumor cell lines (CC50 values of 6.7 and 0.15 µM, respectively), whereas granuloinden A and dihydrogranuloinden had no or moderate activity.

Introduction
Illudalane sesquiterpenes have primarily been isolated from ferns of the family Pteridaceae [1] and fungi of the phylum Basidiomycota [2][3][4]. Biological activities that have been ascribed to the illudalane type sesquiterpenes include among others, cytotoxicity [5][6][7]. The illudalanes produced by ferns are sometimes glycosylated, which has been reported to increase the cytotoxicity of the compounds, though there are examples of some potent cytotoxins among the non-glycosylated illudalanes [6,7].

OPEN ACCESS
The wood decomposing fungus Granulobasidium vellereum (Ellis & Cragin) Jülich (syn. Hypochnicium vellereum (Ellis & Cragin) Parmasto) has previously been found to produce protoilludane sesquiterpenes with moderate biological activity [8,9]. Here we describe the isolation and characterization of seven illudalane sesquiterpenes produced by G. vellereum, of which three have not previously been described, and among which one showed potent cytotoxic activity.

Results and Discussion
Seven illudalane sesquiterpenes were isolated from the saprotrophic fungus G. vellereum using solid-phase extraction (SPE) and preparative HPLC: granuloinden A (1), dihydrogranuloinden (2), granuloinden B (3), radulactone, pterosin M, echinolactone A and D ( Figure 1). The structures were determined with spectroscopic techniques and the cytotoxic effects of 1-3 were evaluated against the Huh7 and MT4 cancer cell lines. The determinations of the structures of radulactone, pterosin M and echinolactone A and D were based on the comparison of their MS, NMR and polarimetric data to those reported in the literature [2][3][4]10,11]. Pterosin M has been isolated as the aglycon of pteroside M, but has not previously been reported as a natural product [10], whereas radulactone had been isolated from the fungus Radulomyces confluens [2] and echinolactone A and D from Echinodontium japonicum [3,4].
Compound 1 was obtained as a light yellow crystalline compound and was the most abundant of the seven identified illudalanes, with a maximum yield of 77.3 mg/L filtrate. The molecular composition was C 14 H 18 O 3 according to HRMS, indicating a degree of unsaturation of six and since the 13 C-NMR spectrum displayed the presence of eight sp 2 carbons, the structure must be bicyclic (Table 1). Through COSY and HSQC experiments a -CH 2 (11)-CH 2 OH(12) spin system was identified, and the NMR data further indicated the presence of three methyl groups and an isolated CHOR group. From methyl-8 an HMBC NMR correlation to the isolated CHOR group (C-1) as well as to two sp 2 carbons (C-2 and C-3) were detected. H-3 correlated to C-1 and to three sp 2 carbons C-3a, C-4 and C-7a. H-1 showed correlation to C-3a and C-7a together forming a cyclopentadiene moiety ( Figure 2). C-7a correlated also to methyl-13, which in turn correlated to two additional sp 2 carbons (C-6 and C-7). Methyl-10 correlated to C-4, C-5 and C-6, of which the latter also correlated to the H 2 -11, rendering in the proposed structure of compound 1, which seems to be of sesquiterpenoid origin but has lost one carbon during the biosynthesis. Due to the indene structural backbone of the compound the name granuloinden A was suggested. Mosher's test was preformed to be able to determine the configuration of the compound [12], but since compound 1 has three hydroxyl groups of which all might react with the chiral reagent α-methoxy(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) used for the test, compound 1 was selectively acetylated on the hydroxyl groups on carbons 4 and 12 to produce compound 1a ( Figure S19). Treatment of 1a with MTPA-Cl yielded the S-MTPA and R-MTPA monoesters of 1a, which respective 1 H-NMR shifts were assigned (Table S1). From the comparison of the shift differences between the S-and R-MTPA monoesters of 1a a 7S configuration was determined ( Figure 3). HRMS determined the molecular formula of compound 2 to be C 15 H 20 O 3 and it had consequently a degree of unsaturation of six. According to the 13 C-NMR spectrum the structure contained one carbonyl group and six other sp 2 carbons (Table 1), leading to the conclusion that the compound must be bicyclic. From COSY and HSQC NMR data one -CH 2 (11)-CH 2 OH(12) spin system was identified. HMBC NMR showed correlations from the CH 2 end of the spin system to three sp 2 carbons (C-5, C-6 and C-7) (Figure 2). Methyl-13 had also HMBC correlations to C-6 and C-7 as well as an additional sp 2 carbon (C-7a), the latter correlated in turn to an isolated CH 2 group (H 2 -1). H 2 -1 had HMBC cross peaks to a quaternary carbon (C-2), to the sp 2 -carbon C-3a, to the geminal methyl groups 8 and 9 as well as to a carbonyl group (C-3), which in turn had a cross peak to a proton linked to a sp 2 carbon (H-4). H-4 had HMBC correlations to C-6 and C-7a and to an isolated CH 2 OH group (C-10). The latter of which also had correlations to C-5 and C-6, thus concluding the structure elucidation of compound 2, for which the name dihydrogranuloinden was proposed.
Compound 3 was obtained as a strongly orange colored powder and had molecular formula of C 14 H 16 O 3 according to HRMS analysis. The 1 H-and 13 C-NMR data displayed substantial similarities with those of compound 1 (Table 1) and from 2D NMR data it could be deduced that the only difference between the two was that C-1 had been oxidized to a carbonyl in compound 3 ( Figure 2) and it was consequently named granuloinden B. Compound 3 was isolated from the liquid culture but was also formed in small amounts by auto-oxidation of compound 1, making it uncertain whether the formation of compound 3 actually is an enzymatic process or if it is formed as a degradation product of 1.
Compound 3 showed strong cytotoxic activity against both the Huh7 and MT4 cell lines, with CC 50 values of 6.7 and 0.15 µM, respectively, whereas compounds 1 and 2 showed no cytotoxicity against Huh7 cells at concentrations up to 400 µM and only moderate cytotoxic activity against MT4 cells ( Table 2). This result might be due to the fact that compound 3, unlike compounds 1 and 2, has an α,β-unsaturated carbonyl group which may facilitate a Michael type reaction. The cytotoxic effect of the known alkylating agents illudin M and S has been partially explained by invoking a similar mechanism, involving a Michael type addition of thiols in e.g. amino acids to an α,β-unsaturated carbonyl moiety [13,14]. To test if 3 would react with cysteine in a similar way as described for illudin M and S [14], compound 3 was dissolved in acetate buffer (pH 5.4, 50 mM) and reacted with a large excess of cysteine to show pseudo first order kinetics. Indeed formation of a product arising from a Michael type addition of cysteine to 3 occured rapidly (Figure 4), indicating that 3 may have a similar mechanism of action in the cell as illudin M and S.  Figure 4. Suggested mechanism of the reaction between L-cysteine and compound 3.

General Procedures
Preparative HPLC was performed on a Gilson 305/306 system, equipped with a Gilson 118 UV/VIS detector (254 nm). The NMR data were recorded at 303 K on a Bruker Avance III 600 MHz NMR spectrometer (5 mm QXI probe, 5 mm CryoProbe or a 5 mm SmartProbe) or on a Bruker DRX400 NMR spectrometer (5 mm QNP probe). The chemical shifts are reported relative to the residual solvent signal of MeOH-d 4 (δ H 3.31; δ C 49.00). HRMS data were obtained on a Bruker maXis Impact ESI UHR Q-TOF with Na formate (positive) as calibrant. The optical rotation was measured with a Perkin Elmer 341 polarimeter (λ 589 nm, path length 10.0 cm, 20 °C). The UV-data was recorded on a Hitachi U-2001 spectrophotometer and the melting point was measured on an Electrothermal 9100, using Vitrex S80 (1.4 × 80 mm) capillary tubes.

Fungal Cultivation
Isolation and identification of fungal culture of G. vellereum strain olrim243 is described in a previous study [8]. For the production of metabolites, G. vellereum was grown in 500 mL Erlenmeyer flasks each containing 250 mL of liquid Hagem medium [15]. Five agar plugs 0.5 × 0.5 cm in size with established fungal mycelia from an actively growing colony were aseptically inoculated in each flask and incubated on a rotary shaker at 120 rpm at room temperature (ca. 21 °C) for average periods of four weeks. After cultivation, cultures were filtered to obtain mycelium-free samples.

Bioassay Procedure
MT4 cells (T-cell line, a kind gift from Prof. Yamamoto, Yamaguchi University, Japan) were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL). Huh7 cells (hepatocarcinoma cell line, ReBlikon GmbH, Schriesheim, Germany) were maintained in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL). Briefly, cells were passaged into 96 well microplates (2 × 10 4 cells/well) and the following day, test compounds in two-fold serial dilutions were added in DMSO. After six days the number of viable cells in each well was assessed by using a soluble formazan (XTT) assay [16] and the concentration causing 50% decrease in cell proliferation (CC 50 ) was determined.

Isolation of Compounds from Liquid Cultures
The cell free filtrates of G. vellereum were extracted on 10-g SPE columns (50 mL filtrate per 1 g packing material; C18 (EC), International Sorbent Technology, Hengoed, UK). The columns were washed with water to remove the non-bonding materials before eluting the more lipofilic substances with aqueous 95% MeCN. The combined lipofilic fractions were dried in a vacuum centrifuge and redissolved in aqueous 40% MeCN before fractionation by preparative reversed-phase HPLC (linear gradient 10%-95% MeCN in water in 10 minutes, followed by a hold at 95% MeCN in 10 min, at 10 mL/min, Reprosil-Pur ODS-3, C 18 , 5 μm, 100 × 20 mm and guard column 30 × 20 mm, Dr Maisch GmbH, Ammerbuch, Germany). The fractionation was monitored by a UV-detector at 254 nm and 2 mL fractions were collected in deep-well plates.

Formation of Compound 1a
To a solution of compound 1 (8.3 mg, 0.04 mmol) in pyridine (500 µL), acetic anhydride (8.4 µL, 0.09 mmol) was added at 0 °C. The reaction was stirred for 24 h, after which the crude mixture was washed with 3 × 0.5 mL of water. The solvent was then evaporated and the crude product was redissolved in 50% aqueous MeCN, before being purified by preparative reversed-phase HPLC (aqueous 10%-95% MeCN 1-10 min and then a hold at 95% MeCN for 10 min; column as above, 10.0 mL/min) yielding 1.3 mg (12%) of the desired product.

Formation of the (S)-MTPA Ester of Compound 1a
To a solution of compound 1a (0.62 mg, 0.0026 mmol) in pyridine-d 5 (500 µL) R-(-)-MTPA-Cl (4.6 µL, 0.027 mmol) was added in r.t., and the reaction mixture was left stirring for 72 h. The solvent was then evaporated and the crude product was redissolved in 50% aqueous MeCN, before being purified by preparative reversed-phase HPLC (aqueous 10%-95% MeCN 1-10 min and then a hold at 95% MeCN for 10 min; column as above, 10.0 mL/min) yielding the desired product, after which the solvent was evaporated and the product redissolved in acetone-d 6 and analysed by NMR.

Conclusions
The present study extends our knowledge of the secondary metabolites of G. vellereum, to also now include seven illudalane sesquiterpenes of which one, granuloindene B, showed potent cytotoxic activity against the Huh7 and MT4 tumor cell lines. The cytotoxic effect of granuloindene B is probably due to its chemical reactivity (Michael type addition) and not to enzymatic effects. It is possible that granuloindene B has a similar mechanism of action in the cell as the known alkylating agents illudin M and S.