Activity Enhancement of the Synthetic Syrbactin Proteasome Inhibitor Hybrid and Biological Evaluation in Tumor Cells Crystal R. Archer, Michael Groll, et al. Biochemistry, 2012, 51 (34), pp6880-6888 Presented By: Cecilia Ramirez And his associates

Syrbactins A class of bacterial natural product small molecules Syringolin A (SyIA): from the plant pathogen Pseudomonas syringae pv. Syringae (Pss) Glidobactin A (GIbA): from an unknown species of the order Burkholderiales (order within proteobacteria) Focus on the two predominant forms of syrbactins SylA comes from a bacterium that attacks plants as seen here Proteobacteria is the phylum pv: is a subspecies of a particular strain http://agdev.anr.udel.edu/weeklycropupdate/wp-content/uploads/2009/07/bacterialbrownspotlima.jpg

Proteasome: a multiprotein complex in cells that breaks down proteins that are tagged with ubiquitin. http://www.weizmann.ac.il/Biological_Chemistry/scientist/MichalSharon/images/HomeImage3.jpg A multisubunit complex It is a three-step process that has 3 different enzymes needed to attach ubiquitin to the protein. Found in cytoplasm and nucleus of eukaryotic cells. Talk about 7 polypeptides in the alpha and beta subunit. Necessary to maintain cellular processes Talk about how ubiquitin works: ubiquitin binds defective proteins through an ATP-dependent pathway. It has three different enzymes that exhibit different specificities for target proteins and thus regulation of different cellular processes. http://biochimej.univ-angers.fr/Page2/COURS/Zsuite/3BiochMetab/7Ubiquitinylation/3Figures/6StructProteasome/0ProteasomeCHIMERA/1Proteasome.png http://www.nature.com/nrm/journal/v10/n2/images/nrm2630-f2.jpg

p53 A gene that codes for a protein that regulates the cell cycle and functions as a tumor suppression In normal cells, p53 protein level is low. Stress such as DNA damage causes p53 accumulation P53 build up is desirable Leads to apoptosis p53 are regulated by the proteasome Blocking the proteasome will lead to accumulation of p53 Apoptosis is a programmed cell death. We want p53 build up in tumor cells: it is desirable (more is desirable for cancer) The next one is the opposite. Bottom line: proteasome blockage helps p53 to work and apoptosis to happen MDM2 is a ubiquitin ligase E3 for p53 (catalyzes the connection from E2 to the target protein). MDM2 ubiquitinates p53 ARF is a tumor suppressor that serves as a signal of cellular stress. ARF destroys MDM2 Found ARF through retina blastoma. Apoptosis in tumor cells

NF-kB Nuclear Factor kappa-light-chain-enhancer of activated B cells A protein complex that controls transcription of DNA Inhibiting the proteasome stabilizes the inhibitor protein IkB  no transcription Less is desirable NF-kB inhibitor NF-kB is a transcription factor that is normally bound to the inhibitor protein IkB. If block the proteasome, then you stabilize the IkB, hence no transcription, allowing it to be destroyed by chemotherapeutic agents or other cellular stresses.

Background In 2006, Syringolin (SyIA) induced massive p53 accumulation  apoptosis in neuroblastoma and ovarian tumor cells. Two years later, discovered SyIA irreversibly inhibits the eukaryotic proteasome by a novel mechanism. GlbA: highly potent proteasome inhibitors Bortezomib inhibits NF-KB through proteasome inhibition Prompted the design of synthetic syrbactin-inspired analogues: SylA-GlbA Hybrid Proteasome inhibition is good because it leads to cell breakdown and decrease in cell proliferation. macrocyclic residue critically influences subsite selectivity while the N-terminal lipophilic tail strongly enhances inhibition potency.

SylA-GlbA: Synthetic Hybrid Macrolactam portion of the SylA. And the hydrocarbon chain of GlbA. Peptide bond-like form: Proteasomes hydrolyze peptide bonds. Hydrocarbon part: nonpolar pocket These are the two most important parts (because of the next slide) of the chymotrypsin like activity Has the two components that make it desirable for binding to the CT-L activity Chymotrypsin is a protease that is specific to the carboxyl side of aromatic (nonpolar residues) These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Key Features: SyIA macrocycle connected to the lipophilic GlbA side chain.

SylA-GlbA Path of Action (for proteasome) Chymotrypsin-like (CT-L) catalytic active site: best fit for inhibitor Tumor cell viability decreases: multiple myeloma Accumulation of p53  apoptosis Inhibition of NF-KB  decrease in cell proliferation

Determining Potency of SylA-GlbA Method #1: Cell culture-based Proteasome Activity Assay Method #2: Protein Crystallization

Method #1: Cellular culture-based Proteasome Activity Assay A panel of 5 human tumor cell lines Proteasome with chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) catalytic activities SylA-GlbA added Incubated treated cells with the bioluminescent substrates (Suc-LLVY-aminoluciferin, Z-LRR-aminoluciferin, and Z-nLPnLD-aminoluciferin) Measure proteasome inhibition by the CT-L, T-L, and C-L proteasome activities Performed twice. If cleaved substrate then will show luminescence (proteasome not inhibited) CT-L cleaves the aminoluciferin off and becomes fluorescent. 100%: Fluorescence caused by fluorescence activity. So no inhibitor is at 100%. As add more of the compound (proteasome inhibitor) CT-L is not working as well, so not releasing much aminoluciferin So, if inhibited, then no light. Fluorescence caused by CT-L activity

Panel of Human tumor cell lines SylA-GlbA Inhibits Best CT-L Catalytic Activity of the Proteasome from Cell Cultures Panel of Human tumor cell lines Multiple myeloma Neuroblastoma Ovarian cancer The hybrid inhibited all. The syrbactin GlbA inhibited the proteasomal activities in a similar fashion. (maybe because its nonpolar backbone is stabilized by the nonpolar pocket.) CT-L was the most inhibited. There are three protease activities. The cell lines were treated individually over a period of 2 hours with SylA-GlbA at various drug concentrations (0-10 micromolar). Two controls: SylA (natural product wild type) and bortezomib (FDA-approved proteasome inhibitor) tested in two cell lines. Bortezomib has a lot of negative side effects

Method #2: Protein Crystallization Vapor Diffusion Initially, the droplet of protein solution contains low precipitant, buffer, and protein concentrations As the drop and reservoir equilibrate, water moves to the reservoir, then the precipitant and protein concentrations increase in the drop. Crystal growth occurs in the drop Salting Out: adding high amount of sodium ions takes the water away from the protein and the protein crystallizes and falls out of solution. Removes the water of hydration Droplet: purified protein, buffer, and precipitant larger reservoir: similar buffers and precipitants in higher concentrations as the droplet the reservoir slowly diffusing into the first This method is used because it allows for gentle and gradual changes in concentration of protein and precipitant concentration, which aid in the growth of large and well-ordered crystals. There is a vapor pressure greater on the top in comparison to that of the bottom. Salt is present in the reservoir. http://www.xtal.iqfr.csic.es/Cristalografia/archivos_07/gota-colgante-en.jpg

Crystal Structure of SylA-GlbA with the Yeast 20S Proteasome Ether covalent bond Hydrogen bond formation at active sites Aliphatic fatty chains of GlbA and SylA-GlbA in a nonpolar pocket Use X-Ray Crystallography and use MAIN software to visualize the molecule. The electron density map (A, B) shows the macrolactam ring of the SylA-GlbA ligand covalently bound to the Thr1Ogamma atom by formation of an irreversible ether bond. At the CT-L and T-L active sites, the Peptide backbone is stabilized by hydrogen bond formation with Asp (D) 114 located on the adjacent subunit Beta 6. This is absent at the substrate binding channel forming the C-L active site explaining the reduced affinity of syrbactins. Large variations of the binding probability of the 3 different active sites is due to molecular flexibility to the distinct pockets with respect to entropic and enthalpic ligand stabilization. Hence explaining a preference for syrbactins mostly blocking the CT-L site. GlbA and SylA-GlbA fatty chains backbone are stabilized equally but differ in their orientation. (strong binding affinity expressed) Increases stabilization of the molecule to the proteasome by the irreversible ether bond with the Thr1Oɣ atom.

Overall SylA-GlbA: A potent proteasome inhibitor Inhibits proteasome  causes p53 accumulation  apoptosis Strongly blocks the activity of NF-KB like bortezomib Inhibits the proliferation of several cancer cell lines Blocks the CT-L subcatalytic activity the best in a dose-dependent manner and at concentrations comparable to that of bortezomib It’s orientation with the proteasome improved binding: irreversible

Acknowledgements Dr. Rebecca Crawford Department of Chemistry and Biochemistry Classes of 2015 and 2016

Questions?

Method #1: In Vivo Proteasome Activity Assay Clear-bottom, white-walled 96-well microtiter cell culture plates Seeded with human tumor cells and treated individually with syrbactin or bortezomib at different concentrations Incubated for 2 hours Incubated for 15 min. with 100 μL of proteasome Glo reagent (permeabilizes cells and contains bioluminescent substrates.) to measure CT-L, T-L, and C-L proteasome activities. The luminescence was measured with a Dynex MLX luminometer. Each experiment performed in duplicate (n=6)

Mechanism of SylA-GlbA Synthesis

Panel of Human tumor cell lines SylA-GlbA Inhibits All Three Catalytic Activities of the Proteasome from Cell Cultures Panel of Human tumor cell lines Multiple myeloma Neuroblastoma Ovarian cancer The hybrid inhibited all. The syrbactin GlbA inhibited the proteasomal activities in a similar fashion. (maybe because its nonpolar backbone is stabilized by the nonpolar pocket.) The cell lines were treated individually over a period of 2 hours with SylA-GlbA at various drug concentrations (0-10 micromolar). Two controls: SylA (natural product wild type) and bortezomib (FDA-approved proteasome inhibitor) tested in two cell lines.

Method #2 Crystals incubated with SylA-GlbA X-Ray Crystallography MAIN Identify atomic and molecular structure of a crystal Crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. Measuring angles and intensities of these diffracted beams, the mean positions of the atoms in the crystal can be determined MAIN visualization software for molecular graphics shows electron densities and its application for protein structure determination. http://media.tumblr.com/0f015d2b0f4f61d959edc8011f4b3497/tumblr_inline_n2ww5vWjfs1syyfyn.jpg Beam of x-rays produce a diffraction pattern. If they add constructively, then the reflection forms a diffraction. The two-dimensional images taken at different rotations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms, combined with chemical data known for the sample. X-rays are used to produce the diffraction pattern because their wavelength λ is typically the same order of magnitude (1–100 angstroms) as the spacing d between planes in the crystal. X-rays might have a wavelength comparable to the unit-cell spacing in crystals. http://www.assignmenthelp.net/assignment_help/images/chemistry/solid-state/x-ray-crystallography.gif

Method: NF-KB Activity Assay In well plates: Human embryonic kidney cells (HEK293/NF-KB-Luc cells) Tumor necrosis factor added to stimulate NF-KB activity Three syrbactin inhibitor compounds added Luciferase activity observed NF-kB inhibitors (positive controls) determined the IC50 values. Results expressed as a percentage relative to control samples. Luciferase activity?? Tells us what?

Syrbactins Inhibit NF-KB Activity SylA-GlbA strongly inhibited the NF-KB activity Inhibits cell proliferation Similar to bortezomib NF-kB is a transcription factor that is normally bound to the inhibitor protein IkB. If block the proteasome, then you stabilize the IkB, hence no transcription, allowing it to be destroyed by chemotherapeutic agents or other cellular stresses. Mention the IC50 values of the hybrid in comparison (sort of similar) to the controls (well-established NF-kB inhibitors). IC50 means the inhibitory concentration at which cell viability is reduced by 50%. Results similar to bortezomib of the inhibition of NF-kB