PDZ Binding peptides - PowerPoint Presentation

Slide1

PDZ Binding peptides

Characterisation of NeuroprotectivePDZ binding peptidesby Jamie Al-NasirProject supervisor: Prof. Brian Austen

Presented as part of M.Pharm project at:

Kingston university and St. Georges Hospital medical school, 24/01/2011Slide2

What are PDZ domains?Recall that domains are regions of a protein with a particular sequence

PDZ domains are important regions of intracellular signaling proteins They function as modular protein-protein signalling complexes in Eukaryotic cellsPDZ acquires its name from the first proteins found to contain these regionsP

SD-95 (Post-Synaptic-Density, 95 kDa),DLG (Drasophila melangolaster Discs Large Protein) andZO-1 (Zonula occludens 1)

Some proteins contain more than one PDZ regionPDZ domains have been found in over 250proteins across a wide range of organisms fromDrasophila to HumansPDZs bind to complementary sequences at C-termini

Implicated in a wide range of cellular processes– therefore an extremely useful drug target

IntroductionSlide3

PDZ domains and Neuroprotective bindingPDZ Domains are found in the NMDA receptor complex where they function as signaling scaffolds that mediate protein-protein interactions

(1 and 2, figure below)In normal physiological function Glutamate is a neurotransmitter that acts on NMDA receptors resulting in the activation of intracellular processesExcesses of Glutamate however can cause downstream toxicity, termed Excitotoxicity - a mechanism implicated in a variety of neurologically degenerative disorders such as Alzheimers, Parkinsons, epilepsy and stroke.

This pathological process results indamage to the neurons!

PSD-95 is a PDZ containing protein whichforms part of the NMDA receptor complexdisruption of which can mitigate and reduceglutamate mediated neuro-degeneration.Prof. Austen and his team at St. GeorgesAcademic Neuroscience department are

working to develop PDZ binding ligands toachieve this therapeutic objective.

PDZs in NeuroscienceSlide4

The NMDA receptor complex

Glutamate binds on NR2A sub-unitCa2+ pumped into intracellular spaceIntracellular signaling pathwaysare activated

This is coupled by PSD-95 (pink) containingtwo PDZ domains (1 and 2).

Normal responseOR Pathological response if prolongedactivation (in presence of excess glutamate)

Evidence favoring this approachImmunopreciptation studies by Prof. Austen et al confirm that the NR2A subunit interacts with PMCA2b via PSD-95.

Spaller, Pizerchio et al have demonstrated that disrupting this interaction using peptides generated from PDZ binding proteins decreases the vulnerability of neurons to

excitotoxicity

Mechanism & Evidence of ApproachSlide5

Objectives“To alter the function of the NMDA receptor complex by targeting PDZ1 domain of PSD-95 thereby disrupting it’s interaction with PMCA-2b

…to develop a PDZ binding ligand with improved binding characteristics over an existing PDZ binding peptide ligand, R2……the putative ligand should therefore possess a smaller Kd dissociation constant binding at nano-molar concentrations – R2 currently binds at micromolar concentrations.” Structure and Function

Pizerchio et al proposed a cyclic peptide structure based on the straight chainC-Termini of PMCA2b but with a cyclic linker – This led to development of R2 ligand by St. Georges, Royal Holloway, university of Otago, New Zealand and uni. of London

- R2 presented at Association International Conference on Alzheimer's Disease, 2010Later studies by Li, Spaller et alhave yielded importantthermodynamic data relatingto the effects of alterations inthe ring length and residues on

binding stability

Ligand designSlide6

New Ligand J1 (Jamie-1) based on R2 (Ruth-2)

IncorporatesHexa-Arginine sequence – Cell penetration enhancerBackbone - based on C-Terminus of PMCA2bCyclic ring - with β-Aspartyl linker (previously β-Alanine)Biotin Marker - to aid in microscopy and assess how much drug penetrates neurones

J1 modeledIn Silico

Ligand design & Molecular modeling

Left : J1 shown without

Hexa-Arg or Biotin

Above : J1, note shorthand

Hexa-Arginine on left of structureSlide7

Molecular docking

A methodology by which a putative ligand, stored as a 3d data model is docked to a target protein’s active site (also stored as a 3d model).The software re-arranges the atoms to compute the lowest overall energy by means of energy minimisation algorithms such as GROMOS or docking scores such as PMF

Ligand

design & Molecular modeling

Argus Lab: PSD-95 protein

with J1 Ligand (yellow)

Scigress: PSD-95 proteinwith J1 Ligand (red/blue/grey)Slide8

Molecular docking

β-Aspartyl was chosen as a linker as we want to facilitate the bonding between theβ-Asp negative COO- side-chain and a nearby Lysine-98 which has a positive NH3

+ side-chain.This bonding will be ionic providing extra stability to the bound complex

Ligand

design & Molecular modeling

Zeus-PDB: PSD-95 protein with J1 Ligand (green)

DeepView: PSD-95 protein

with J1 Ligand (red/blue/white)Lysine 98 (green) – (Zoomed in)Slide9

I s

imulatated the docking of a selection of L- and D- linker ligands in both alpha and beta- configurations, the results determined what was synthesised

In Silico

results

L- and D- amino acids in alpha and beta

config

Some standard amino acids in L-a- configSlide10

SPPS, Solid Phase Peptide Synthesis

Original method pioneered by Robert Bruce MerrifieldEssentially synthesis of peptide from C-terminus to the N-terminus, stepwiseFirst residue is bound to solid beads and is protectedBeads are washed with a solution of the subsequent residue, which is then washed again to deprotect it. The cycle is repeated with each further residue and finally washed with TFA

(TriFluoracetic Acid to cleave the peptide from the beads).

Straight chain of J1 can be automated using St. George’s Peptide synthesiserLinker (ring with β-Asp) will be added through a separate reaction to connect theside-chains (Lysine-2 and Glutamate-4 to the linker, β-Aspartyl

).Synthesis of J1

Synthesis will be on a Leu Peg-PS resin using temporary α-amino Fmoc protection and orthogonally protected residuesResidues will be

deprotected and reacted with the bridging residue before finally being deprotected.

The synthesised peptide will be purified using HPLCThe product is then verified using mass spectrometry (MS) and MS2 by Maldi

Synthesis of J1

ligandSlide11

Orthogonal Protecting group strategy

(Figure 30) - Schematic of the J-1 (Jamie-1) backbone – precursor to cyclic J-1 based on R-2 (Ruth-2).

(From right to left) blue region shows the initial

Leucine, Leu-1 bound to the Resin (Fmoc-Leu-PEG-PS

)followed by Orthogonally protected groups (shown in red) on Lys-2(IvDde) and Glu-4(PhiPr

) which areseparated by Thr-3. Two conventionally Fmoc coupled residues Leu-5 and Ser-6 precede BiotinylatedLysine, Lys-7 (shown in Green). Gly-8 and Gly-9 are proceeded by

Pbf protected Hexa-Arginine unit (Purple

with Pbf shown in Magenta) which has been acetylated. Two t-Butyl groups are shown in Orange and areused to protect the hydroxyl of Threonine and Serine.Slide12

FMoc

deprotection and cleavageFmoc protection strategy is stable in acidic conditions and therefore required Basic condition for cleavage of temporary protection (Piperidine 20% in DMF)Slide13

HATU activation of acyl

groups during peptide constructionSlide14

Characterisation of

synthesised peptidesHPLC (High performance Liquid Chromatography)

MALDI mass

spectrometrySlide15

Determine the Kd binding constantGST (Glutathione-S-Transferase) Pull-down assay used to attain Kd dissociation constant

GST is a marker-tag that can be attached to proteins under studyIf binding occurs then the complex will contain marked protein+bound proteinIn the presence of J1 we can quantify the potency of binding between J1 and PSD95-PDZ1Quantifying the effect of J1 on altering excitotoxicity

Utilises a cell line, SHSY-5Y cells derived from 30 year old blastoma stored atSt. Georges, refrigerated at a specific temp. & under CO2

to prevent alkalinisation.J1 will be added to the SHSY-5Y neuronal cells in presence of excess glutamate (2mmol) and quantifying the amount of cells that survive.Immunohistological analysisUsed to study the extent to which J1 internalises withinSHSY-5Y cells and to PSD-95Makes use of the integrated biotin marker within the structure as a

target for anti-biotin antibody - visualised by microscopy

Testing J1: Binding assays

Biotin+Ab. marked neuronal cellsSlide16

Cell culturing: SHSY-5Y line

SHSY-5Y cells cultured in DMEM-F12 bufferSlide17

Immunohistochemistry

Fluorophores (three Flurophores were used)DAPI –

to label the A-T rich regions of DNA in the Nuclei (for contrasting and locating cells) FITC-Avidin conjugate

to label Biotin incorporated in PDZ peptides (labels the peptide) TRITC-anti-rabbit antibody for PSD-95 (labels the PSD-95 target on the membrane)

Jamie-3-Asn

Jamie-3-Glu

Slide#

1A

2A

3A

4A

1B

2B

3B

4B

2.7uM PDZ peptide

X

X

X

X

X

X

X

X

DAPI (Blue)

X

X

X

X

X

X

X

X

FITC-Avidin(Green)

X

X

X

X

X

X

Anti-PSD95

X

X

X

X

X

X

TRITC Anti-rabbit (Red)

X

X

X

X

X

XSlide18

Immunohistochemistry

DAPI / FITC / TRITC stained SHSY-5Y cells, Jamie-3 peptide (x40)

Jamie 3-Glu

(DAPI+FITC)

Jamie 3-Glu

(DAPI+FITC)

(

Slide #1B)

Jamie 3-Glu (DAPI+TRITC)

Negative control slides ( lacking either FITC {peptide} or TRITC {psd95 target} )Slide19

Immunohistochemistry

DAPI / FITC / TRITC stained SHSY-5Y cells, Jamie-3-Asn (x63)

Jamie 3-Asn

(DAPI+FITC+TRITC)

Top left = DAPI

Top right =FITC (peptide)Bottom left = TRITC

Bottom right =

Composite image (DAPI+FITC+TRITC)showing good

co-localisation(orange and yellow) ofJamie-3-Asn (green)

and target PSD-95

(red)Slide20

Immunohistochemistry

FITC / TRITC stained SHSY-5Y cells, Jamie-3 Asn (x63)

Composite image of

Jamie 3-AsnFITC+TRITC but excluding DAPI.

It shows co-localisation (orangeand yellow) of the Jamie-3-Asn

peptide with its PSD-95 target.Jamie-3-Asn was stained with

FITC and the PSD-95 target withAnti-PSD95 TRITC

the co-localisation is the combination of the two colours

Slide #3ASlide21

Immunohistochemistry

FITC / TRITC stained SHSY-5Y cells, Jamie-3 Asn (x63)

Composite image of

Jamie 3-Asn FITC+TRITC+DAPI

It shows co-localisation (orangeand yellow) of the Jamie-3-Asn peptide with its PSD-95 target.

Jamie-3-Asn was stained with FITC

and the PSD-95 target withAnti-PSD95 TRITC the

co-localisation is the combination of the two colours

Slide #3ASlide22

PDZ domainsPDZ domains are important protein-protein cell signaling mediators

Implicated in a variety of cellular processes not just neurologicalImportant drug target for novel drugsMolecular modeling and In Silico methods

Molecular modeling and docking is extremely useful method of simulating thermodynamic stability and binding between ligands and proteinsData can be used to select a putative ligand prior synthesis vs the traditional combinatorial approach

Immunohistochemical analysisJamie-3-Asn pdz binding peptide showed good co-localisation of peptide with PSD-95Jamie-3-Glu showed some co-localisation but was less consistent across repeated imagingResults consistent with MTT assay, in which Jamie-3-Asn pdz binding peptide performed better at mitigating excitotoxic damage than Jamie-3-GluImplications

More favorable binding characteristics than R2 - further development may yield a useful pharmacological agent that could be administered to slow down neurological degeneration in diseases such as Alzeimers, Parkinsons, epilepsy and stroke.

ConclusionsSlide23

References used

Harris BZ, Lim WA. Mechanism and role of PDZ domains in signaling complex assembly. Journal of Cell Science, 2010; 114: 3219-3231Cui H, Hayashi A, Sun HS, Belmares MP, Cobey C, Phan T et al. PDZ Protein Interactions Underlying NMDA Receptor-Mediated Excitotoxicity and Neuroprotection by PSD-95 Inhibitors. The Journal of Neuroscience, 2007; 27(37): 9901-9915Li T, Saro D, Spaller MR. Thermodynamic profiling of conformationally constrained cyclic ligands for the PDZ domain. Bioorganic & Medicinal Chemistry Letters, 2004; 14(6): 1385-1388

Austen BM, Duberley K, Turner P, Empson R. Cyclic hexa-arg PDZ-binding peptides that bind PSD95 inhibit glutamate-mediated toxicity. Alzheimer's Association International Conference on Alzheimer's Disease 2010, 6(4): Supp. 562

Software used (in order of images shown)Argus Lab by Mark ThompsonScigress explorer by Fujitsu Siemens

Zeus PDB viewer by Jamie al-nasirDeepView/Swiss-PDB viewer

References