FK506-binding protein 2 (FKBP13) inhibit Bax-induced apoptosis in Saccharomyces cerevisiae (yeast).

FK506-binding protein 2 (FKBP13) is a part of the immunophilin protein family involved in immunoregulation. It is also believed to operate as a factor in membrane cytoskeletal framework and as an ER chaperone. FKBP2 (FKBP13) and FKBP1 (FKBP12), known as immunophilins, are binding proteins for rapamycin and FK506, which are immunosuppressive drugs. It was suggested that immunophilin-like and immunophilin proteins play significant roles in regulating intracellular calcium and protein folding/sorting, acting as molecular chaperones. Within the 15 mammalian FKBPs known, FKBP1 is merely the only one proven to form complexes with rapamycin and FK506 in the cytosol and facilitate their T cells immunosuppressive effects, FKBP2 is a luminal protein of the endoplasmic reticulum (ER) and is reported to take part in protein folding in the ER. However, little is known about FKBP2 link with apoptosis (either as a pro or anti-apoptotic protein). In this study, FKPB2 protein was co-expressed with the pro-apoptotic protein Bax after a yeast-based human hippocampal cDNA library screening. The yeast strain carrying the Bax gene was transformed with an episomal 2-micron plasmid that encodes the HA-tagged FKBP2 gene. The resultant strain would allow co-expression of Bax and FKBP2 in yeast cells. The results presented here show that a protein involved in protein folding can play a role in protecting yeast cell from Bax-induced apoptosis.

Human VAMP3 Suppresses or Negatively Regulates Bax Induced Apoptosis in Yeast.

Apoptosis is an essential process that is regulated genetically and could lead to a serious disease condition if not well controlled. Bax is one of the main proapoptotic proteins and actively involved in programmed cell death. It has been suggested that Bax induced apoptosis in yeast could be obstructed by enhancing vesicular membrane trafficking. Plasma membrane proteins and lipid oxidation were reduced by a vesicle-associated membrane protein (VAMP) when expressed in yeast, suggesting its potential role in repairing membranes. Membrane integrity is crucial, as the loss of membrane integrity will result in the leakage of ions from mitochondria, and ultimately cell death due to overproduction of reactive oxygen species (ROS). Expression of Arabidopsis' VAMP has been linked to antiapoptosis activity. Since plant VAMP has been associated with antiapoptotic activities, this study investigates the possible participation of human VAMP3 in blocking human Bax mediated apoptosis. Some novel genes were identified to rescue Bax's proapoptotic effects, in a yeast-based human hippocampal cDNA library screen. VAMP3 (a gene code for proteins involved in protein secretion) gene was chosen for further study to confirm its role in inhibiting apoptosis. VAMP3 was coexpressed with a chromosomally integrated Bax gene expression cassette driven by the GAL1 promoter. The antiapoptotic proteins of the Bcl-2 family (Bcl xL) were known to negate the proapoptotic properties of Bax. However, the new gene (VAMP3) results show that novel antiapoptotic proteins can be identified using a yeast-based assay. The findings presented here show that human VAMP3 protein has antiapoptotic property and could abrogate Bax induced apoptosis (cell death).

Apoptosis, Induced by Human α-Synuclein in Yeast, Can Occur Independent of Functional Mitochondria.

Human α-synuclein expression in baker's yeast reportedly induces mitochondria-dependent apoptosis. Surprisingly, we find that, under de-repressing conditions of the inducible MET25/GAL1 promoters, yeast cells expressing chromosomally-integrated copies of the human α-synuclein gene are not killed, but spontaneously form respiration-deficient rho-minus (ρ-) petites. Although yeast cells can undergo cell death (apoptosis) from loss of mitochondrial function, they can also survive without functional mitochondria. Such cells are referred to as ρ0 or ρ- petites. This study reports that minimal expression of human α-synuclein in yeast, from MET25/GAL1 promoter, gives rise to ρ- petites. Interestingly, the full expression of α-synuclein, from the same promoters, in α-synuclein-triggered ρ- petites and also in ρ0 petites (produced by treating ρ+ cells with the mutagen ethidium bromide) initiates apoptosis. The percentages of petites increase with increasing α-synuclein gene copy-number. ρ- petites expressing α-synuclein from fully-induced MET25/GAL1 promoters exhibit increased ROS levels, loss of mitochondrial membrane potential, and nuclear DNA fragmentation, with increasing copies of α-synuclein. Our results indicate that, for the first time in yeast, α-synuclein-triggered apoptosis can occur independently of functional mitochondria. The observation that α-synuclein naturally forms petites and that they can undergo apoptosis may have important implications in understanding the pathogenesis of Parkinson's disease.

Identification of proteins involved in transcription/translation (eEF 1A1) as an inhibitor of Bax induced apoptosis.

Eukaryotic elongation factor 1A1 (eEF1A1) is central to translational activity. It is involved in complexes that form signal transduction with protein kinase C, as well as being a signal transducer and activator of transcription 3. eEF1A1 and eEF1A2 are isoforms of the alpha subunit of elongating factor 1 complex. It has been reported that eEF1A1 is expressed in most human tissues but the brain, skeletal muscle and heart. eEF1A1 has been linked to both apoptosis and anti-apoptotic activities. In this study, eEF1A1 was co-expressed with Bax, a proapoptotic protein via heterologous expression of recombinant DNA in yeast cells. Assays were carried out to monitor the fate and state of yeast cells when eEF1A1 was co-expressed with Bax. The yeast strain (bearing an integrated copy of the Bax gene) was transformed with an episomal 2-micron plasmid that encodes HA-tagged eEF1A1 gene. The resultant strain would allow co-expression of Bax and eEF1A1 in yeast cells, Bax being under the control of the GAL1 promoter, while the PGK1 promoter drives eEF1A1 expression. Bcl 2A1, a known anti-apoptotic protein, was also co-expressed with Bax in yeast cells as a positive control, to study the anti-apoptotic characteristic of eEF-1A1. The part eEF1A1 plays in apoptosis has been contentious, amidst the pro and anti-apoptotic properties of eEF1A1, it was shown clearly, in this study that eEF1A1 portrays only anti-apoptotic property in the presence of pro-apoptotic protein, Bax.

The effect of copy number on α-synuclein's toxicity and its protective role in Bax-induced apoptosis, in yeast.

Apoptosis is a form of programmed cell death which is essential for the growth of dividing human cells whereas, in contrast, it is deleterious for post-mitotic cells such as neurons. Bax and α-synuclein are two human proteins which play a role in the induction of neuronal apoptosis in neurodegenerative diseases like Alzheimer's and Parkinson's. Human Bax and α-synuclein also induce cell death when expressed in baker's yeast, Saccharomyces cerevisiae. Quite unexpectedly, the human α-synuclein gene had been identified as an inhibitor of pro-apoptotic Bax using a yeast-based screen of a human hippocampal cDNA library. Plasmids were constructed with different promoters, which allow expression of wildtype and Parkinson's disease (PD)-related mutant α-synuclein genes, from (i) multi-copy 2µ (episomal) plasmids and (ii) integrative plasmids that compel expression of genes from chromosomal sites in varying copy numbers (1-3). All α-synuclein-containing plasmids were introduced, through transformation, into a yeast strain which already contained a chromosomally integrated copy of Bax. It is for the first time that it was observed that, depending on gene dosage, only wildtype α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wildtype α-synuclein has a remarkable ability to manifest two contrasting effects depending on its level of expression: (i) normally, it would negate apoptosis but (ii) when overexpressed, it tends to induce apoptosis which is probably what happens in PD.

Sensing the Generation of Intracellular Free Electrons Using the Inactive Catalytic Subunit of Cytochrome P450s as a Sink.

Cytochrome P450 reductase (CPR) abstracts electrons from Nicotinamide adenine dinucleotide phosphate H (NADPH), transferring them to an active Cytochrome P450 (CYP) site to provide a functional CYP. In the present study, a yeast strain was genetically engineered to delete the endogenous CPR gene. A human CYP expressed in a CPR-null (yRD-) strain was inactive. It was queried if Bax-which induces apoptosis in yeast and human cells by generating reactive oxygen species (ROS)-substituted for the absence of CPR. Since Bax-generated ROS stems from an initial release of electrons, is it possible for these released electrons to be captured by an inactive CYP to make it active once again? In this study, yeast cells that did not contain any CPR activity (i.e., because the yeasts' CPR gene was completely deleted) were used to show that (a) human CYPs produced within CPR-null (yRD-) yeast cells were inactive and (b) low levels of the pro-apoptotic human Bax protein could activate inactive human CYPs within this yeast cells. Surprisingly, Bax activated three inactive CYP proteins, confirming that it could compensate for CPR's absence within yeast cells. These findings could be useful in research, development of bioassays, bioreactors, biosensors, and disease diagnosis, among others.

Conversion of amino acids to aryl/heteroaryl ethanol metabolites using human CYP2D6-expressing live baker's yeast.

Different natural aromatic/heterocyclic l-amino acids were biotransformed into aryl/heteroaryl ethanol metabolites via oxidative deamination, decarboxylation and reduction cascades using live baker's yeast cells producing intracellular human CYP2D6 enzyme. Among the three yeast strains expressing 3 different CYP2D6 variants, CYP2D6(2) (i.e. CYP2D6 wild-type) provided the best result under neutral pH conditions at RT. We have successfully converted six natural amino acids into their corresponding alcohols, having one carbon atom less, with moderate yields. Some of the downstream products like tryptophol and tyrosol induced the pTrKB (Tropomyosin receptor kinase B) activation pattern similar to that of BDNF (brain-derived neurotrophic factor), thereby depicting potential antidepressant activity. Control experiments and molecular modelling studies revealed that this tandem bio-transformation probably happens via a pyruvate intermediate. This study establishes that CYP2D6-expressing live yeast cells can be a powerful tool for the enzymatic C-N, C-C bond cleavage of amino-acids.

CYP enzymes, expressed within live human suspension cells, are superior to widely-used microsomal enzymes in identifying potent CYP1A1/CYP1B1 inhibitors: Identification of quinazolinones as CYP1A1/CYP1B1 inhibitors that efficiently reverse B[a]P toxicity and cisplatin resistance.

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in 'suspension' for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.

Radicicol rescues yeast cell death triggered by expression of human α-synuclein and its A53T mutant, but not by human ßA4 peptide and proapoptotic protein bax.

Aggregation/misfolding of α-synuclein and ßA4 proteins cause neuronal cell death (NCD) associated with Parkinson's and Alzheimer's disease. It has been suggested that a heat shock protein-90 (Hsp90) inhibitor can prevent NCD by activating the heat shock transcription factor-1 which, in turn, upregulates molecular chaperones such as Hsp70 that targets aggregated/misfolded proteins for refolding/degradation. We have isolated radicicol, an Hsp90 inhibitor, from a fungus occurring in the crevices of marble rocks of Central India. Radicicol, which was found to be a strong antioxidant, was tested for its ability to rescue yeast cells from death induced by expression of wild-type α-synuclein, its more toxic A53T mutant, and ßA4. It effectively overcomes wild-type/mutant α-synuclein mediated yeast cell death, concomitantly diminishes ROS levels, reverses mitochondrial dysfunction and prevents nuclear DNA-fragmentation, a hallmark of apoptosis. Surprisingly however, radicicol is unable to rescue yeast cells from death triggered by expression of secreted ßA4. Moreover, although radicicol acts as an antioxidant it fails to prevent yeast cell death inflicted by the proapoptotic protein, Bax. Our results indicate that radicicol specifically targets aggregated/misfolded α-synuclein's toxicity and opens up the possibility of using multiple yeast assays to screen natural product libraries for compounds that would unambiguously target α-synuclein aggregation/misfolding.

Cink4T, a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-based virtual screening, for efficient anticancer therapy.

Inhibition of cyclin dependent kinase 4 (Cdk4) prevents cancer cells from entering the early G0/G1 phase of the cell division cycle whereas inhibiting tubulin polymerization blocks cancer cells' ability to undergo mitosis (M) late in the cell cycle. We had reported earlier that two non-planar and relatively non-toxic fascaplysin derivatives, an indole and a tryptoline, inhibit Cdk4 with IC50 values of 6.2 and 10 µM, respectively. Serendipitously, we had also found that they inhibited tubulin polymerization. The molecules were efficacious in mouse tumor models. We have now identified Cink4T in a 59-compound quinazolinone library, designed on the basis of ligand-based virtual screening, as a compound that inhibits Cdk4 and tubulin. Its IC50 value for Cdk4 inhibition is 0.47 µM and >50 µM for inhibition of Cdk1, Cdk2, Cdk6, Cdk9. Cink4T inhibits tubulin polymerization with an IC50 of 0.6 µM. Molecular modelling studies on Cink4T with Cdk4 and tubulin crystal structures lend support to these observations. Cancer cell cycle analyses confirm that Cink4T blocks cells at both G0/G1 and M phases as it should if it were to inhibit both Cdk4 and tubulin polymerization. Our results show, for the very first time, that virtual screening can be used to design novel inhibitors that can potently block two crucial phases of the cell division cycle.

Aegeline, a natural product from the plant Aegle marmelos, mimics the yeast SNARE protein Sec22p in suppressing α-synuclein and Bax toxicity in yeast.

Herein, we have identified yeast Sec22p (ySec22p), a SNARE protein essential for endoplasmic reticulum to Golgi trafficking, as a suppressor of Bax-induced yeast apoptosis and corroborated published observations that ySec22p suppresses α-synuclein's toxicity in yeast. It has been suggested that compounds which enhance expression, in neurons, of human homologues of ySec22p (Sec22Bp/Sec22p/Sec22A) would prevent synucleinopathies, such as Parkinson's disease. With the aim of finding a small molecule that would mimic ySec22p, a library of natural products consisting of 394-compounds was screened using yeast cells that express either human α-synuclein or human Bax. The antioxidant aegeline, an alkaloid-amide occurring in the leaves of the plant Aegle marmelos Correa, was the only molecule that overcame apoptosis induced by both α-synuclein and Bax in yeast. Besides, aegeline also prevented growth block in cells expressing the more toxic A53T α-synuclein mutant. Restoration of cell growth occurred through inhibition of increased ROS levels, mitochondrial membrane potential loss and nuclear DNA fragmentation, characteristics of apoptosis manifested in α-synuclein or Bax-expressing cells. These results highlight the importance of yeast systems to identify rapidly molecules that may prevent the onset of apoptosis that occurs in Parkinson's disease.

Inhibitors of Aß42-induced endoplasmic reticular unfolded protein response (UPRER), in yeast, also rescue yeast cells from Aß42-mediated apoptosis.

Aggregated Aß peptides which cause amyloid deposits, a characteristic of Alzheimer's disease (AD), activate a stress response in the endoplasmic reticulum (ER), known as the unfolded protein response, UPRER. Nascent UPRER induction helps in reducing ER stress by eliminating accumulated misfolded/aggregated secretory proteins. However, prolonged UPRER induction may trigger apoptosis. Here we show that, when expressed in yeast with an NH2-terminal secretory signal sequence (ss), the 42-amino acid human Aß42 (h_Aß42), but not the mouse/ratAß42 (m_Aß42) which reportedly does not misfold/aggregate, induces UPRER as monitored via an eGFP reporter. We also show that expression of ss-h_Aß42, not ss-m_Aß42, blocks yeast cell growth, with cells expressing ss-h_Aß42 manifesting distinctive features of apoptosis such as loss of mitochondrial membrane potential, increase in ROS levels and DNA fragmentation. Screening for suppressors of ss-h_Aß42-activated UPRER-eGFP induction, in a computationally-designed 29-compound methoxy-stilbene library, revealed three compounds that reduce >95% of UPRER-eGFP induction at 5 µM concentration, with EC50 values of 40-50 nM. Surprisingly, the compounds also rescue yeast cells from ss-h_Aß42-mediated apoptosis, with EC50-s of 50-60 nM. These results provide direct evidence, probably for the first time, that there is a direct correlation between deactivation of UPRER and attenuation of apoptosis.

Furanoflavones pongapin and lanceolatin B blocks the cell cycle and induce senescence in CYP1A1-overexpressing breast cancer cells.

Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs) which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/lanceolatin B, inhibit CYP1A1 with IC50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ∼10-fold improved potency (IC50, 0.49 µM). Pongapin/lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G0-G1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.

Identification of karanjin isolated from the Indian beech tree as a potent CYP1 enzyme inhibitor with cellular efficacy via screening of a natural product repository.

CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 µM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40-97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC50 values of the 'hits', belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the 'hits' were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC50, 20 nM) and karanjin (IN-195; IC50, 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.

Khellinoflavanone, a Semisynthetic Derivative of Khellin, Overcomes Benzo[a]pyrene Toxicity in Human Normal and Cancer Cells That Express CYP1A1.

Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procarcinogens such as benzo[a]pyrene, B[a]P, into carcinogens, which initiates the process of carcinogenesis. Thus, stopping the metabolic activation of procarcinogens can possibly prevent the onset of cancer. Several natural products have been reported to show unique ability in inhibiting CYP1 enzymes. We found that khellin, a naturally occurring furanochromone from Ammi visnaga, inhibits CYP1A1 enzyme with an IC50 value of 4.02 µM in CYP1A1-overexpressing human HEK293 suspension cells. To further explore this natural product for discovery of more potent and selective CYP1A1 inhibitors, two sets of semisynthetic derivatives were prepared. Treatment of khellin with alkali results in opening of a pyrone ring, yielding khellinone (2). Claisen-Schmidt condensation of khellinone (2) with various aldehydes in presence of potassium hydroxide, at room temperature, provides a series of furanochalcones 3a-v (khellinochalcones). Treatment of khellinone (2) with aryl aldehydes in the presence of piperidine, under reflux, affords the flavanone series of compounds 4a-p (khellinoflavanones). The khellinoflavanone 4l potently inhibited CYP1A1 with an IC50 value of 140 nM in live cells, with 170-fold selectivity over CYP1B1 (IC50 for CYP1B1 = 23.8 µM). Compound 4l at 3× IC50 concentration for inhibition of CYP1A1 completely protected HEK293 cells from CYP1A1-mediated B[a]P toxicity. Lung cancer cells, A549 (p53+) and Calu-1 (p53-null), blocked in growth at the S-phase by B[a]P were restored into the cell cycle by compound 4l. The results presented herein strongly indicate the potential of these khellin derivatives for further development as cancer chemopreventive agents.

Biotransformation, Using Recombinant CYP450-Expressing Baker's Yeast Cells, Identifies a Novel CYP2D6.10A122V Variant Which Is a Superior Metabolizer of Codeine to Morphine Than the Wild-Type Enzyme.

CYP2D6, a cytochrome P450 (CYP) enzyme, metabolizes codeine to morphine. Within the human body, 0-15% of codeine undergoes O-demethylation by CYP2D6 to form morphine, a far stronger analgesic than codeine. Genetic polymorphisms in wild-type CYP2D6 (CYP2D6-wt) are known to cause poor-to-extensive metabolism of codeine and other CYP2D6 substrates. We have established a platform technology that allows stable expression of human CYP genes from chromosomal loci of baker's yeast cells. Four CYP2D6 alleles, (i) chemically synthesized CYP2D6.1, (ii) chemically synthesized CYP2D6-wt, (iii) chemically synthesized CYP2D6.10, and (iv) a novel CYP2D6.10 variant CYP2D6-C (i.e., CYP2D6.10A122V) isolated from a liver cDNA library, were cloned for chromosomal integration in yeast cells. When expressed in yeast, CYP2D6.10 enzyme shows weak activity compared with CYP2D6-wt and CYP2D6.1 which have moderate activity, as reported earlier. Surprisingly, however, the CYP2D6-C enzyme is far more active than CYP2D6.10. More surprisingly, although CYP2D6.10 is a known low metabolizer of codeine, yeast cells expressing CYP2D6-C transform >70% of codeine to morphine, which is more than twice that of cells expressing the extensive metabolizers, CYP2D6.1, and CYP2D6-wt. The latter two enzymes predominantly catalyze formation of codeine's N-demethylation product, norcodeine, with >55% yield. Molecular modeling studies explain the specificity of CYP2D6-C for O-demethylation, validating observed experimental results. The yeast-based CYP2D6 expression systems, described here, could find generic use in CYP2D6-mediated drug metabolism and also in high-yield chemical reactions that allow the formation of regio-specific dealkylation products.

Nonantioxidant Tetramethoxystilbene Abrogates α-Synuclein-Induced Yeast Cell Death but Not That Triggered by the Bax or ßA4 Peptide.

The overexpression of α-synuclein (α-syn) and its aggregation is the hallmark of Parkinson's disease. The α-syn aggregation results in the formation of Lewy bodies that causes neuronal cell death. Therefore, the small molecules that can protect neuronal cells from α-syn toxicity or inhibit the aggregation of α-syn could emerge as anti-Parkinson agents. Herein, a library of methoxy-stilbenes was screened for their ability to restore the cell growth from α-syn toxicity, using a yeast strain that stably expresses two copies of a chromosomally integrated human α-syn gene. Tetramethoxy-stilbene 4s, a nonantioxidant, was the most capable of restoring cell growth. It also rescues the more toxic cells that bear three copies of wild-type or A53T-mutant α-syn, from cell growth block. Its EC50 values for growth restoration of the 2-copy wild-type and the 3-copy mutant α-syn strains are 0.95 and 0.35 µM, respectively. Stilbene 4s mitigates mitochondrial membrane potential loss, negates ROS production, and prevents nuclear DNA-fragmentation, all hallmarks of apoptosis. However, 4s does not rescue cells from the death-inducing effects of Bax and ßA4, which suggest that 4s specifically inhibits α-syn-mediated toxicity in the yeast. Our results signify that simultaneous use of multiple yeast-cell-based screens can facilitate revelation of compounds that may have the potential for further investigation as anti-Parkinson's agents.

Glycyrrhiza glabra extract and quercetin reverses cisplatin resistance in triple-negative MDA-MB-468 breast cancer cells via inhibition of cytochrome P450 1B1 enzyme.

The development of multi-drug resistance to existing anticancer drugs is one of the major challenges in cancer treatment. The over-expression of cytochrome P450 1B1 enzyme has been reported to cause resistance to cisplatin. With an objective to discover cisplatin-resistance reversal agents, herein, we report the evaluation of Glycyrrhiza glabra (licorice) extracts and its twelve chemical constituents for inhibition of CYP1B1 (and CYP1A1) enzyme in Sacchrosomes and live human cells. The hydroalcoholic extract showed potent inhibition of CYP1B1 in both Sacchrosomes as well as in live cells with IC50 values of 21 and 16 µg/mL, respectively. Amongst the total of 12 constituents tested, quercetin and glabrol showed inhibition of CYP1B1 in live cell assay with IC50 values of 2.2 and 15 µM, respectively. Both these natural products were found to be selective inhibitors of CYP1B1, and does not inhibit CYP2 and CYP3 family of enzymes (IC50 > 20 µM). The hydroalcoholic extract of G. glabra and quercetin (4) showed complete reversal of cisplatin resistance in CYP1B1 overexpressing triple negative MDA-MB-468 breast cancer cells. The selective inhibition of CYP1B1 by quercetin and glabrol over CYP2 and CYP3 family of enzymes was studied by molecular modeling studies.