Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a Chemical Genetics Approach

10.26686/wgtn.16969795 ◽

2021 ◽

Author(s):

◽

Reem Hanna

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Amino Acids ◽

Flow Cytometry ◽

Binding Site ◽

Mammalian Cells ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Peloruside A ◽

Microtubule Function

Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.

Download Full-text

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity

Biochemical Journal ◽

10.1042/bj20141223 ◽

2015 ◽

Vol 466 (1) ◽

pp. 177-188 ◽

Cited By ~ 22

Author(s):

Bethany Nancolas ◽

Richard B. Sessions ◽

Andrew P. Halestrap

Keyword(s):

Molecular Dynamics ◽

Amino Acids ◽

Binding Site ◽

Molecular Basis ◽

Specific Inhibitor ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Isoform Specificity ◽

Isoform Selectivity ◽

Dynamics Simulations

A combination of molecular modelling, site-directed mutagenesis and molecular dynamics simulations define the binding site of MCT1 for AR-C155858, a potent and specific inhibitor. Key amino acids within the binding site differ between MCT1 and MCT4 accounting for isoform specificity.

Download Full-text

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Biochemical Journal ◽

10.1042/bj20031832 ◽

2004 ◽

Vol 379 (3) ◽

pp. 795-803 ◽

Cited By ~ 6

Author(s):

Chung-Chun WU ◽

Min-Che CHEN ◽

Ya-Ru CHANG ◽

Tsuey-Ying HSU ◽

Jen-Yang CHEN

Keyword(s):

Amino Acids ◽

Binding Site ◽

Thymidine Kinase ◽

Crucial Role ◽

Metal Ion ◽

Epstein Barr Virus ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Barr Virus ◽

Epstein Barr

Thymidine kinase (TK), encoded by EBV (Epstein–Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure–function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392→Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3´-deoxy-2´,3´-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3´-azido-3´-deoxythymidine, iododeoxyuridine and β-l-5-iododioxolane uracil (l-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

Download Full-text

Identification of conserved amino acids in murine B7-1IgV domain critical for CTLA4\CD28:B7 interaction by site-directed mutagenesis: a novel structural model of the binding site

Molecular Immunology ◽

10.1016/s0161-5890(98)00041-8 ◽

1998 ◽

Vol 35 (4) ◽

pp. 215-225 ◽

Cited By ~ 12

Author(s):

Y Guo ◽

Y Wu ◽

X Kong ◽

Y Liu

Keyword(s):

Amino Acids ◽

Binding Site ◽

Structural Model ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Conserved Amino Acids

Download Full-text

Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: Functional analysis of conserved aromatic amino acids in Region 1

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2005.08.022 ◽

2006 ◽

Vol 448 (1-2) ◽

pp. 23-30 ◽

Cited By ~ 7

Author(s):

W. David Nes ◽

Archana Sinha ◽

Pruthvi Jayasimha ◽

Wenxu Zhou ◽

Zhihong Song ◽

...

Keyword(s):

Amino Acids ◽

Functional Analysis ◽

Binding Site ◽

Aromatic Amino Acids ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Sterol Binding ◽

Sterol Methyltransferase

Download Full-text

Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

10.26686/wgtn.17061701 ◽

2021 ◽

Author(s):

◽

Benjamin Jones

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Cell Proliferation ◽

Amino Acid ◽

Binding Site ◽

Drug Class ◽

Efflux Pumps ◽

Peloruside A ◽

Mutant Cells ◽

Β Tubulin

Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer. The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted. The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation. HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin. No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.

Download Full-text

Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.8.2063 ◽

1999 ◽

Vol 43 (8) ◽

pp. 2063-2065 ◽

Cited By ~ 16

Author(s):

Kazuo Taniguchi ◽

Akio Nakamura ◽

Kazue Tsurubuchi ◽

Aki Ishii ◽

Koji O’Hara ◽

...

Keyword(s):

Amino Acids ◽

Binding Site ◽

Site Directed Mutagenesis ◽

Macrolide Antibiotics ◽

Directed Mutagenesis ◽

Atp Binding ◽

Atp Binding Site ◽

Mutant Strains

ABSTRACT Macrolide 2′-phosphotransferase [MPH(2′)] transfers the γ phosphate of ATP to the 2′-OH group of macrolide antibiotics. The role of aspartic acids in the putative ATP-binding site of MPH(2′)II was investigated through the substitution of alanine for aspartate by site-directed mutagenesis. D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme.

Download Full-text

Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

10.26686/wgtn.17061701.v1 ◽

2021 ◽

Author(s):

◽

Benjamin Jones

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Cell Proliferation ◽

Amino Acid ◽

Binding Site ◽

Drug Class ◽

Efflux Pumps ◽

Peloruside A ◽

Mutant Cells ◽

Β Tubulin

Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer. The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted. The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation. HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin. No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.

Download Full-text