Molecular modeling of CPT-11 metabolism by carboxylesterases (CEs): use of pnb CE as a model

CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.     

Camptothecin analog (CPT-11)-sensitive human pancreatic tumor cell line QGP-1N shows resistance to SN-38, an active metabolite of CPT-11.

In the course of our study to determine the cross-sensitivity between CPT-11 and its active metabolite, SN-38, we found a SN-38-resistant human pancreatic tumor cell line, QGP-1N, which shows sensitivity to CPT-11. The IC50 of SN-38 was 152 times greater for QGP-1N than for SUIT-2, also a human pancreatic tumor cell line, whose IC50 of CPT-11 was similar to that for QGP-1N. The uptakes of CPT-11 and SN-38 and the intracellular conversion of CPT-11 to SN-38 could not explain the difference in sensitivity. DNA synthesis of QGP-1N cells was inhibited by CPT-11 which did not affect that of SUIT-2, while SN-38 inhibited the DNA synthesis of SUIT-2 at lower concentrations than that of QGP-1N. The inhibition test of topoisomerase I catalytic activity by CPT-11 or SN-38 revealed no difference in the biochemical properties of the topoisomerase I enzymes to the compounds between these two cell lines. These results indicate that CPT-11 should have its own inhibitory effect on DNA synthesis through a yet unknown mechanism in QGP-1N cells, although SN-38 plays an essential role in the antitumor activity of CPT-11 in SUIT-2 cells. In some cases, the antitumor effect of CPT-11 might be consequent not only on SN-38 but also on CPT-11 itself.     

Identification of aspirinase with one of the carboxylesterases requiring a thiol group.

Aspirin-hydrolysing activity in guinea-pig liver is located mainly in the microsomal fraction. This activity was found by electrophoresis to be due to a single carboxylesterase band, out of 12 bands revealed with alpha-naphthyl acetate as substrate. The activity is inhibited completely and irreversibly by the carboxylesterase inhibitor bis-(-4-nitrophenyl) hydrogen phosphate, and also by thiol-blocking reagents.     

Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.     

Cytotoxicity of the anticancer drug doxorubicin for human embryonic stem cells

The cytotoxic effect of the anticancer drug doxorubicin (DR) on human embryonic stem cells (ESCs) C910 and fibroblasts spontaneously differentiated from these cells has been examined. The fibroblasts retained a diploid karyotype. It was found that ESCs are more sensitive to DR than fibroblasts: the DR dose killing 20% of cells was 0.01 and 0.1 μg/mL, respectively. DR induced ESC apoptotic death and reduced both ESC and fibroblast proliferation. DR reversibly inhibited ESC, but not fibroblast, proliferation. Thus, we demonstrated that ESCs and differentiated derivatives thereof are distinguished by sensitivity and response to the genotoxic agent.     

Kinetic properties of human thymidylate synthase,an anticancer drug target

We have determined the kinetic parameters of human recombinant thymidylate synthase (hrTS) with its natural substrate, dUMP, and E-5-(2-bromovinyl)-2(')-deoxyuridine monophosphate (BVdUMP), a nucleotide derivative believed to be the active species of the novel anticancer drug NB1011. NB1011 is activated by hrTS and is selectively toxic to high thymidylate synthase expressing tumor cells. BVdUMP undergoes hrTS-catalyzed thiol-dependent transformation. dUMP and BVdUMP act as competitive hrTS substrates. The natural folate cofactor, CH(2)-THF, inhibits the TS-catalyzed reaction with BVdUMP. We suggest that lower folate levels found in tumor cells favor TS-catalyzed BVdUMP transformation, which, in addition to higher levels of TS expression in tumor cells, contributes to the favorable therapeutic index of the drug NB1011.     

Mutagenicity of the anticancer drug, caracemide, and related compounds for salmonella

Caracemide, MeCON(CONHMe)(OCONHMe) (I), is a novel anticancer drug. Since it was derived from acetohydroxamic acid (II), a known mutagen, its potential metabolites and related compounds were synthesized and tested for mutagenicities in S. typhimurium TA98 and TA100. These compounds were: MeNHCONH(OCONHMe) (III), MeCONH(OCONHMe) (IV), MeCONOH(CONHMe) (V), MeNHCOONH2 X HCl (VI), MeNHCONHOH (VII), MeNHCOON(CONHMe)2 (VIII), and NOH(CONHMe)2 (IX). The mutagenicities in the absence of rat liver homogenate were: (VI) much greater than (IV) greater than (II), (III), (V). The other compounds were not mutagenic. (I) was mutagenic only in the presence of rat liver homogenate. The doses required to demonstrate mutagenicities of these compounds were from 0.05 to 5 mumoles/plate. The major hydrolytic products at 25 degrees C, pH 7, were (III), (IV), and (V) from (I); (II) and (III) from (IV); and (II), (III), (VII) and MeNHCONH(OCOMe) (X) from (V). (III) was stable at pH 7. Treatment of (IV) with HCl yielded (VI). Hydrolysis of (III) or (V) with ammonia yielded (VII). These results suggest that caracemide may be activated enzymatically or nonenzymatically by deacetylation or decarbamoylation, and its anticancer activity may be related to the reactivity of its metabolites with DNA. The synthetic procedures and characterizations of new compounds (IV), (V) and (X) are described.     

Design,synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.     

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity.

Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages phi11, 80alpha, 187, Twort, and phiPVL harbor a central domain that displays sequence homology to known N-acetylmuramyl-L-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the phi11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the phi11 enzyme has D-alanyl-glycyl endopeptidase as well as N-acetylmuramyl-L-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, phi11(Delta181-381), revealed that the D-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.     

Identification of novel activation mechanisms for FLO11 regulation in Saccharomyces cerevisiae

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild "flor" strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.     

Identification of HUHS190, a human naftopidil metabolite,as a novel anti-bladder cancer drug

We carried out structure-activity relationship study on anti-cancer effects of naftopidil (1) and its metabolites, resulted in identification of 1-(4-hydroxy-2-methoxyphenyl)piperazin-1-yl)-3-(naphthalen-1-yloxy) propan-2-ol (2, HUHS190), a major human metabolite of 1, which exhibited the most selective toxicities between against normal and cancer cells (Table 1). 2 was more hydrophilic compared to 1, was enough to be prepared in high concentration solution of more than 100 μM in saline for an intravesical instillation drug. Moreover, serum concentration of 2 was comparable to that of 1, an oral preparation drug, after oral administration at 32 mg/kg (Fig. 3). Both of 1 and 2 showed broad-spectrum anti-cancer activities in vitro, for example, 1 and 2 showed inhibitory activity IC₅₀ = 21.1 μM and 17.2 μM for DU145, human prostate cancer cells, respectively, and IC₅₀ = 18.5 μM and 10.5 μM for T24 cells, human bladder cancer cells. In this study, we estimated anticancer effects of 2 in a bladder cancer model after intravesical administration similar to clinical cases. A single intravesical administration of 2 exhibited the most potent inhibitory activities among the clinical drugs for bladder cancers, BCG and Pirarubicin, without obvious side effects and toxicity (Fig. 4). Thus, HUHS190 (2) can be effective for patients after post-TURBT therapy of bladder cancer without side effects, unlike the currently available clinical drugs.     

In vitro genotoxic effects of the anticancer drug gemcitabine in human lymphocytes

This research was carried out to investigate in vitro genotoxic effects of the anticancer agent gemcitabine on the induction of chromosomal aberrations and sister-chromatid exchange in human lymphocytes. Three doses of gemcitabine (0.001, 0.002 and 0.004 microg/ml) were applied to lymphocyte cultures from 15 donors. There was a significant increase in the induction of chromosome aberrations and in the occurrence of sister-chromatid exchange in these cells. In addition, gemcitabine significantly decreased the mitotic index and replicative index for all doses. Dose-response regression lines were used to compare the individual susceptibilities to gemcitabine with respect to the chromosome aberration and sister-chromatid exchange frequencies. Our results indicate that gemcitabine is able to induce both cytotoxic and genotoxic effects in human lymphocyte cultures in vitro in a dose-dependent manner.     

Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.     

Molecular basis for the inhibition of human NMPRTase, a novel target for anticancer agents

Nicotinamide phosphoribosyltransferase (NMPRTase) has a crucial role in the salvage pathway of NAD+ biosynthesis, and a potent inhibitor of NMPRTase, FK866, can reduce cellular NAD+ levels and induce apoptosis in tumors. We have determined the crystal structures at up to 2.1-A resolution of human and murine NMPRTase, alone and in complex with the reaction product nicotinamide mononucleotide or the inhibitor FK866. The structures suggest that Asp219 is a determinant of substrate specificity of NMPRTase, which is confirmed by our mutagenesis studies. FK866 is bound in a tunnel at the interface of the NMPRTase dimer, and mutations in this binding site can abolish the inhibition by FK866. Contrary to current knowledge, the structures show that FK866 should compete directly with the nicotinamide substrate. Our structural and biochemical studies provide a starting point for the development of new anticancer agents.     

Discovery and development of sesquiterpenoid derived hydroxymethylacylfulvene: a new anticancer drug.

Hydroxymethylacylfulvene (HMAF, MGI 114) is derived from the sesquiterpene illudin S by treatment with dilute sulfuric acid and excess paraformaldehyde. It is less cytotoxic than illudin S but exhibits much greater selectivity in toxicity to malignant cells compared to normal cells. HMAF is believed to undergo bioreductive activation in vivo. It is now being tested in human clinical phase II trials against solid tumors.     

Identification, molecular cloning, and characterization of subunit 11 of the human 26S proteasome

We sequenced five peptides from subunit 11 (S11), a 43 kDa protein of the human 26S proteasome, and used this information to clone its cDNA. The S11 cDNA encodes a 376 amino acid protein with a pI of 5.6 and a molecular mass of 42.9 kDa. Translation of S11 RNA in the presence of [35S]methionine produces a radiolabeled protein that co-migrates with S11 of the human 26S proteasome on SDS-PAGE. Polyclonal antiserum made against recombinant S11 recognizes a protein of the same size in extracts of bacteria expressing S11 and in purified 26S proteasomes from human red blood cells or rabbit reticulocytes. The S11 sequence does not contain motifs that suggest a biological function. S11 is, however, the human homolog of Rpn9, a recently identified subunit of the yeast 26S proteasome.     

Chemical characterization,antidiabetic and anticancer activities of Santolina chamaecyparissus

Santolina chamaecyparissus is an important medicinal plant growing in the Mediterranean region and has been reported as a potent anti-inflammatory, antibacterial, antioxidant, and antifungal agent. The purpose of the current research is to identify the chemical constituents in ethyl acetate extract (EAE) from the leaves of S. chamaecyparissus, and to evaluate antidiabetic, and anticancer activity. Chemical constituents of EAE were identified by GC-MS, and the antidiabetic activity was evaluated by α-glucosidase inhibition assay. The anticancer activity was assessed by Epidermal Growth Factor Receptor (EGFR) expression in human breast cancer cell line (MCF7) by using quantitative RT-PCR method. GC-MS analysis of EAE of S. chamaecyparissus yielded 44 compounds. Tetrapentacontane (27.15%), eicosyl acetate (8.40%), 2-methylhexacosane (6.87%), and n-pentadecanol (5.44%) were found as major chemical constituents. The EAE of S. chamaecyparissus showed concentration dependant inhibition of α-glucosidase enzyme and the IC₅₀ value (IC₅₀ 110 ± 4.25 µg/mL) was found comparable with standard acarbose (IC₅₀ 105 ± 3.74 µg/mL). The real-time qRT-PCR results showed that the EGFR protein (bcl-2) in human breast cancer cell line (MCF7) was negatively expressed with a value of −0.69297105 after treatment with EAE (100 µg/mL). The study results are suggesting the possible use of S. chamaecyparissus in the management of diabetes, and human breast cancer.     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.