Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Two enzymatic reaction pathways in the formation of pyropheophorbide a

The demethoxycarbonyl reaction of pheophorbide a in plants and algae was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. One enzyme, designated `pheophorbidase (Phedase)', was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus). This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-13<sup>2</sup>-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. The activity of Phedase was inhibited by the reaction product, methanol. The other enzyme, termed `pheophorbide demethoxycarbonylase (PDC)', was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as shown in the Phedase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Phedase and PDC consisted of both senescence-induced and constitutive enzymes. The molecular weight of both Phedases was 113 000 and of senescence-induced PDC was 170 000. The K _m values against pheophorbide a for both Phedases were 14–15 μM and 283 μM for senescence-induced PDC. The activity of both Phedases was inhibited by the reaction product, methanol, whereas methanol had no specific effect on senescence-induced PDC. Phenylmethylsulfonic fluoride and N-ethylmaleimide inhibited the senescence-induced Phedase and PDC, respectively. Among the 23 species from 15 different families tested, Phedase activity was found in 10 species from three families. PDC activity was not detected in plants lacking Phedase activity, except for Chlamydomonas. Based on these findings, a likely conclusion is that at least two alternative pathways that are catalyzed by two different enzymes, Phedase and PDC, exist for the formation of pyropheophorbide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Design,synthesis, and biological activities of novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists with improved membrane permeability across MDR1 expressing cells

We previously reported octahydropyrrolo[1,2-a]pyrazine derivative 2 (T-3256336) as a potent antagonist for inhibitors of apoptosis (IAP) proteins. Because compound 2 was susceptible to MDR1 mediated efflux, we developed another scaffold, hexahydropyrazino[1,2-a]indole, using structure-based drug design. The fused benzene ring of this scaffold was aimed at increasing the lipophilicity and decreasing the basicity of the scaffold to improve the membrane permeability across MDR1 expressing cells. We established a chiral pool synthetic route to yield the desired tricyclic chiral isomers. Chemical modification of the core scaffold led to a representative compound 50, which showed strong inhibition of IAP binding (X chromosome-linked IAP [XIAP]: IC₅₀ 23 nM and cellular IAP [cIAP]: IC₅₀ 1.1 nM) and cell growth inhibition (MDA-MB-231 cells: GI₅₀ 2.8 nM) with high permeability and low potential of MDR1 substrate.     

Reversed isoniazids: Design,synthesis and evaluation against Mycobacterium tuberculosis

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a–c, 7 and 8; H37Rv-MIC₉₉ ≤1.25?µM, R5401-MIC₉₉ ≤2.5?µM, X_61-MIC₉₉ ≤5?µM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a–c and 3b (MIC₉₀ ≤13.42?µM) and inhibition of EB efflux demonstrated by these compounds are encouraging.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

Substrate specificity of chlorophyllase

Apparent Km and V_max values were obtained for hydrolysis of methyl and ethyl chlorophyllides a, methyl and ethyl pheophorbide a, and 9-hydroxymethyl pheophorbide a by chlorophyllase from Ailanthus altissima. Analysis of substrate specificity data for chlorophyllase indicates that the presence of a 9-keto group and a methyl alcohol group esterified at the 7-position in chlorophyll derivatives results in maximum binding affinity for substrates. Data on maximum reaction rates indicate that the rate-controlling step of hydrolysis occurs after release of the alcohol from the ester. Probable high affinity chlorophyllase inhibitors can be predicted on the basis of these specificity studies.     

N-Substituted piperazine derivatives as potential multitarget agents acting on histamine H3 receptor and cancer resistance proteins

Taking into account that multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment, the ability of novel histamine H₃ receptor ligands to reverse the cancer MDR was evaluated, using the ABCB1 efflux pump inhibition assay in mouse MDR T-lymphoma cells. The most active compounds displayed significant cytotoxic and antiproliferative effects as well as a very potent MDR efflux pump inhibitory action, 3–5-fold stronger than that of reference inhibitor verapamil. Although these compounds possess weak antagonistic properties against histamine H₃ receptors, they are valuable pharmacological tools in the search for novel anticancer molecules. Furthermore, for the most active compounds, an insight into mechanisms of action using either, the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp, was performed.     

Isolation of 10-Hydroxypheophorbide a as a Photosensitizing Pigment from Alcohol-treated Chlorella Cells

A new pigment causing intense photosensitivity in rats was isolated from alcohol-treated Chlorella cells and identified as 10-hydroxypheophorbide a by chemical analysis, chromatography, and visible, infrared, nuclear magnetic resonance and mass spectroscopy methods.

When rats administered orally with this pigment were exposed immediately to the visible light, signs of the intense photosensitivity including death occurred after a few hours of photoirradiation. The photosensitizing activity of this pigment in rats was markedly higher than those of pheophorbide a from Chlorella cells and pyropheophorbide a from pickled greens. Chlorophylls a and b, pheophytin a, and methyl and ethyl pheophorbides a were inactive under the same experimental conditions     

Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a

Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD⁺ complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD⁺ as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.     

Design,synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors – Part 3

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein 1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We observed that the MDR1 efflux ratio was correlated with the pK_a value of the acidic moiety. The resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic acid hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral administration, increased urethral resistance in a dose-dependent manner.     

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India

ObjectivesThe present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India.Methods76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed.ResultsOut of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35^th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump.ConclusionsThis study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.     

HPLC analysis of chlorophyll a breakdown products to interpret microalgae dynamics in a shallow bay

Phytoplankton production is determined by growth, senescence, sinking and zooplankton grazing. In an attempt to follow algal senescence and grazing, some authors have used HPLC fluorescence detection of chlorophyll a breakdown products. Laboratory grazing experiments have shown that copepods reduce chlorophyll a from diatoms leading to an increase in pheophytin a rather than pheophorbide a. However, field measurements only indicated a slight increase of pheopigment concentrations in summer. During this period, high heterotrophic activities (zooplankton and bacteria) seemed to be responsible for rapid pheopigment disappearance. On the other hand, highest chlorophyllide a levels appeared to be related to spring accumulation of nutrient-limited senescent algae. While increases in pheophytin a accounted for chlorophyll a consumption, changes in pheophorbide a concentrations could be linked to chlorophyllide a abundance. These results suggest that laboratory studies cannot be uncritically extrapolated to the field.     

Synthesis,spectroscopic and molecular docking studies on new schiff bases,nucleosides and α-aminophosphonate derivatives as antibacterial agents

New Nucleosides, analogues derived from 1, 3, 4-oxadiazole, arylidene analogues and α-aminophosphonate were prepared. Infrared (IR), elemental analysis and ¹HNMR elucidated nucleosides; arylidines and phosphonate derivatives. The prepared derivatives were purified and allowed to test against bacteria strains. Phosphonate derivative 12a showed the higher antibacterial against E. coli with inhibition zone 35 mm, P. aeruginosa with inhibition zone 30 and S. aureus with inhibition zone 22 while compounds 4, 6d, 9a, 9c and 12c showed moderate to weak activity against these bacteria species with inhibition zones ranged from 12 mm to 24 mm. The molecular docking studies was applied on compound 12a, which showed the binding at the active DNA Gyrase.     

Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles

The ability of cells to acquire resistance to multiple pharmaceuticals, namely multidrug resistance (MDR), is often mediated by the over-expression of efflux transporters of the ATP-binding cassette (ABC) superfamily; for example P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP or ABCG2), and multidrug resistance-associated protein MRP1. ABCs pump drug molecules out of cells against a concentration gradient, reducing their intracellular concentration. The ability of polymeric amphiphiles to inhibit ABCs as well as the cellular pathways involved in the inhibition has been extensively investigated. This work investigated for the first time the effect of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines) on the levels of mRNA encoding for MDR1, BCRP and MRP1, in a human hepatoma cell line (Huh7). Copolymers with a broad range of molecular weights and hydrophilic-lipophilic balances were assayed. Results confirmed the down-regulation of mdr1 and abcg2 genes. Conversely, the mrp1 gene was not affected. These findings further support the versatility of these temperature- and pH-responsive copolymers to overcome drug resistance in cancer and infectious diseases.     

Rep1p negatively regulating MDR1 efflux pump involved in drug resistance in Candida albicans

Overexpression of MDR1 efflux pump is a major mechanism contributing to drug resistance in Candida albicans, the most common human fungal pathogen. To elucidate the regulatory pathway of drug resistance, we have identified a negative regulator of MDR1 and named it Regulator of Efflux Pump 1 (REP1). Overexpression of REP1 in Saccharomyces cerevisiae increased susceptibility to fluconazole. Furthermore, null mutations on REP1 decreased the susceptibility to antifungal drugs in C. albicans resulting from increased expression of MDR1 mRNA. Hence, Rep1p is involved in drug resistance by negatively regulating MDR1 in C. albicans.     

Evaluation of the Potential Antimicrobial Resistance Transfer from a Multi-Drug Resistant Escherichia coli to Salmonella in Dairy Calves

Previous research conducted in our laboratory found a significant prevalence of multi-drug resistant (MDR) Salmonella and MDR Escherichia coli (MDR EC) in dairy calves and suggests that the MDR EC population may be an important reservoir for resistance elements that could potentially transfer to Salmonella. Therefore, the objective of the current research was to determine if resistance transfers from MDR EC to susceptible strains of inoculated Salmonella. The experiment utilized Holstein calves (approximately 3 weeks old) naturally colonized with MDR EC and fecal culture negative for Salmonella. Fecal samples were collected for culture of Salmonella and MDR EC throughout the experiment following experimental inoculation with the susceptible Salmonella strains. Results initially suggested that resistance did transfer from the MDR E. coli to the inoculated strains of Salmonella, with these stains demonstrating resistance to multiple antibiotics following in vivo exposure to MDR EC. However, serogrouping and serotyping results from a portion of the Salmonella isolates recovered from the calves post-challenge, identified two new strains of Salmonella; therefore transfer of resistance was not demonstrated under these experimental conditions.     

Cellular drug efflux and reversal therapy of cancer

A prevalent form of multidrug resistance (MDR) in cancer cells is caused by an ATP-dependent drug efflux pump; this pump catalyzes the rapid exit of cytotoxic chemotherapy drugs from the cells. The Michaelis equation can be used to describe drug efflux through the MDR pump at a low drug substrate concentration [S]. The inhibition mechanism of an MDR reversal agent can be characterized when two different values of [S] are used to determine two values for the half-inhibition of efflux through the pump (I ₅₀). The reaction is noncompetitive when the two values ofI ₅₀ are identical; the reaction is competitive when an increase in [S] produces a significant increase in the value ofI ₅₀ TheI ₅₀ has been determined for several different reversal agents with the substrate rhodamine 123. The inhibition potency observed is: cyclosporin A >DMDP>amiodarone>verapamil>quinidine>quinine>propranolol. Chemotherapy drugs that are potent inhibitors of the MDR pump could be used for the treatment of MDR neoplasia.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot culture and antimicrobial activity of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.     

Discovery of a novel activator of 5-lipoxygenase from an anacardic acid derived compound collection

Lipoxygenases (LOXs) and cyclooxygenases (COXs) metabolize poly-unsaturated fatty acids into inflammatory signaling molecules. Modulation of the activity of these enzymes may provide new approaches for therapy of inflammatory diseases. In this study, we screened novel anacardic acid derivatives as modulators of human 5-LOX and COX-2 activity. Interestingly, a novel salicylate derivative 23a was identified as a surprisingly potent activator of human 5-LOX. This compound showed both non-competitive activation towards the human 5-LOX activator adenosine triphosphate (ATP) and non-essential mixed type activation against the substrate linoleic acid, while having no effect on the conversion of the substrate arachidonic acid. The kinetic analysis demonstrated a non-essential activation of the linoleic acid conversion with a K_A of 8.65 μM, αK_A of 0.38 μM and a β value of 1.76. It is also of interest that a comparable derivative 23d showed a mixed type inhibition for linoleic acid conversion. These observations indicate the presence of an allosteric binding site in human 5-LOX distinct from the ATP binding site. The activatory and inhibitory behavior of 23a and 23d on the conversion of linoleic compared to arachidonic acid are rationalized by docking studies, which suggest that the activator 23a stabilizes linoleic acid binding, whereas the larger inhibitor 23d blocks the enzyme active site.