1. 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), pp
Presented By: Cecilia Ramirez
And his associates

2. 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

3. Proteasome: a multiprotein complex in cells that breaks down proteins that are tagged with ubiquitin.
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.

4. 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

5. 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.

6. 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.

7. 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.

8. 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

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

10. 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

11. 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

12. 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.

13. 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.

14. 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

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

16. Questions?

17. 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)

18. Mechanism of SylA-GlbA Synthesis

19. 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.

20. 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.
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.

22. 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?

23. 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