Wuyuan Lu et al. JBC, 398, 200-213 (2010)

The E3 ubiquitin ligase, MDM2, negatively regulates the activity of p53 via two main mechanisms: (1) reduces transcriptional activity by sequestering the N-terminal transactivation domain of p53 and (2) ubiquitylates lysines in the C-terminal domain of p53, thus promoting p53 degradation by the proteosome. The structural basis for the first mechanism has been well characterized and consists of a minimum binding sequence within 19-26p53 that forms an amphiphilc α-helix containing three critical residues; Phe19, Trp23, and Leu26. These residues are located within a hydrophobic cavity of MDM2. Previous in vitro studies by Bernal and colleagues have demonstrated reactivation of the p53 tumor suppressor cascade following treatment with a small peptide bearing the key interacting residues. We plan to use a solid phase peptide synthesis method to generate a 14 amino acid length peptide (p53i) containing the minimum binding sequence necessary for MDM2 interaction. In recent years, the advent of small peptide therapeutic agents has resulted in the ability to enhance target specificity and blunt toxicity compared to small molecule drugs. Despite this, serum instability and rapid renal clearance have plagued their widespread usage. Lipidation and enhanced plasma protein binding are two strategies capable of extending the half life of prodrugs. Our plan is to combine these methods by synthesizing p53i conjugated to FA (FA-p53i); creating a species capable of being incorporated and transported by HSA. The application of the synthesis described above is to develop an efficient drug carrier system utilizing HSA for the targeted co-delivery of methotrexate (MTX) and FA-p53i. Such a system offers the unique ability for one drug formulation to simultaneously deliver two anti-cancer agents to a single cell and thus, promote a supra-additive effect on apoptosis. MTX is a well described and widely used chemotherapeutic agent whose mechanism of action relies on the inhibition of key enzymatic activities required for DNA synthesis. It will thus serve as a model drug for the development of this platform technology.

Introduction

Reactivating the p53 Pathway Using Human Serum Albumin to Co-Deliver Methotrexate and a p53-Derived Peptide

Michelle R. Joshi1, Nianhuan Yao1, Zhiyu Li1 1Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA 19104

Caspase-3 Acl I p53i Spe I Nhe I

DEVDG ETF SDLWKLLPETAA TS AS Amino acid sequence

DNA sequence

Caspase-3 PMI ScaI SacII Nhe I

DEVDG TSFAETWALLSP PR AS Amino acid sequence

DNA sequence

Cargo: FA-Methotrexate and FA-p53i1) Methotrexate

MTX has been conjugated to:• Albumin Clin. Cancer Res. 9, 1917-26 (2003).• Gelatin Pharm. Res. 17, 1309-15 (2000).• Fibrinogen Cancer Lett. 148, 189-95 (2000).• Polyethylene glycol (PEG) Bioconjug Chem. 13, 773-85 (2002).• Tumor-targeting Ab Nat. Biotechnol. 23, 1137-46 (2005).

Conjugation has been shown to increase plasma retention and enhance accumulation in tumor tissue

2) p53-Derived Peptide Designed to inhibit native p53-MDM2

interaction

3) Design and construct a recombinant HSA C-terminal fusion protein

2

HSAp53iMTX

alternative strategy

wt p53

high affinity peptide

PMI

Methods

ResultsFigure 2

1 2 3 4 5 6 7 8HSA:FA-FITC-p53i (1:4)

unlabeled FA-FITC (pmol) 120 240 480360 960720- -

FA-bound HSA

Free FA

Figure 2: FA-FITC-p53i forms a stable complex with human serum albumin that is not displaced by the presence of unlabeled FA. HSA/FA-FITC-p53i complexes were allowed to form at a 1:4 molar ratio (HSA:FA-FITC-p53i; 120 pmol:480 pmol) as described in Methods. Next, samples were incubated with increasing

concentrations of unlabeled FA. The absence of a lower band indicates FA-FITC-p53i was not displaced by the unlabeled FA even at the highest concentration. The upper band corresponds to the less mobile HSA/FA complex, while the lower band contains unbound free FA-FITC or free FA-FITC-p53i.

Construction of recombinant HSA fusion proteins• Amplified rHSA-p53i DNA by PCR• Ligated rHSA-p53i into pPICZα A vector (Invitrogen)• Transformed competent E.Coli cells using plasmid DNA & select for zeocin resistance• Isolated single colonies from overnight culture & grow up in selection media• Purified plasmid DNA & confirmed DNA sequence by restriction digest• Linearized plasmid DNA & transformed Pichia yeast cells• Expressed protein in Pichia strain, isolated and purified recombinant protein

Negative control

742 bp

4608 bp

1763 bp3587 bp

23K bp9.4K bp6.6K bp4.4K bp2.3K bp 2K bp

KasI/XhoI digest SpeI/XbaI digest

Caspase-3 Acl I p53i Spe I Nhe IDEVDG ETF SDLWKLLPETAA TS AS Amino acid sequence

DNA sequenceKasI

Background

Co-Delivery

Combination Therapy

Vs.

Both anti-cancer agents reach target

Can use two species with complimentary mechanisms to promote a robust apoptotic response

Ease of formulation & administration

One agent → multiple effects

Enhanced therapeutic effect at lower doses

Targeted co-delivery reduces side effects due to toxicity in normal tissues

Benefits of Co-Delivery

Co-Delivery Approach

• Most abundant plasma protein• Molecular weight = 66.5 kDa• Long half-life• Solublizing agent for long chain FA’s• Binds a number of drugs • Proven lack of toxicity and immunogenicity1

• Accumulates in malignant and inflamed tissue2 Nat Struct Biol. 5(9), 827-35 (1998).

Carrier: Human Serum Albumin1

Background

N-protected C-terminal amino acid residue was anchored via its COOH group to the hydroxyl group of Wang resin. Side chain functional groups of amino acids were masked with permanent protection groups. The N-terminal amino group was protected by a temporary moiety that can be removed for coupling to the next residue. Deprotection/coupling process was repeated until desired sequence was complete. Peptide was then released from resin and side chain protecting groups were removed. Resulting peptide was detected by ESI/MS and purified by HPLC.

Mol. Biotechnology. 33, 242-254 (2006).

Solid phase peptide synthesis

Methods

Synthesis of FA-Modified FITC, FA-Modified p53i, and FA-Modified MTX

Fmoc-Lys(Alloc) was first coupled to Wang resin. After Fmoc deprotection, palmitic acid was coupled to the a- amino group of Lys. After Alloc deprotection, Fmoc-SS-linker and NHS-Fluorescein were coupled sequentially. The final product was cleaved using TFA.

Albumin/Fatty Acid Mobility Shift AssayFor experiments designed to detect albumin/FA-p53i complex formation, 120 pmol albumin (dissolved in 1X PBS) was incubated +/- FITC-labeled FA-p53i at desired molar ratios. Experiments aimed at detecting displacement of FA-FITC-p53i by unlabeled FA included an initial incubation carried out as described above, but at a fixed albumin:FA-FITC-p53i molar ratio. Following this incubation, unlabeled FA was added at increasing molar ratios up to 1:8 (albumin:unlabeled FA). Reactions were conducted in 20 µl of PBS under room temperature for 30 minutes. The products were separated using Tris-Boric polyacrylamide gel 12 mA for 20 minutes. The gel was visualized under 305 nm UV.

Cells were plated in standard growth media at approximately 40% confluence and allowed to attach overnight. On day 2, MTX, FA-MTX, HSA/MTX, HSA/FA-MTX complexes or HSA fusion proteins were added at the indicated concentrations and allowed to incubate at 37ºC/5%CO2 for the indicated time period. Cell proliferation was then measured by CyQuant assay (Invitrogen). Data represented three replicas at indicated concentrations.

Cytotoxicity Assays

1 2 3 4 5 6 7 8 9 10

Figure 1: Recombinant HSA fusion proteins are able to form complexes with FA-FITC. Recombinant HSA fusion proteins as well as wild type HSA were incubated at the indicated molar ratios

Figure 1

with FA-FITC. The speed at which molecules move through the gel is dependent on size and charge. The upper band corresponds to the less mobile HSA/FA complex, while the lower band contains unbound free FA-FITC.

Results

p53

MDM2

GAPDH

untr

eate

d

Nut

lin

rHSA

-p53

i

rHSA

-PM

I

FA-p

53i

Figure 5 Figure 5: FA-p53i, rHSA-p53i, and rHSA-PMI increase p53, but not MDM2 protein expression. This is in contrast to the actions of the cis-imidazoline analog, nutlin. SJSA-1 cells were plated in standard growth media and allowed to attach overnight. On day 2, 10 µM nutlin, rHSA-p53i, rHSA-PMI or FA-p53i were added in RPMI media containing 1% FBS + 0.05% DMSO and allowed to incubate x 24 hrs. Cell monolayers were then washed, lysed and immunoblotted for p53 and MDM2 (Santa Cruz).

P53 protein expression increased by approximately 60% in rHSA-p53i-treated cells and 30% in both rHSA-PMI and FA-p53i treatments (as determined using Image J software), while MDM2 expression did not change relative to untreated wells.

Figure 3

Figure 3: Cytotoxicity of MTX, FA-MTX, HSA/MTX, and HSA/FA-MTX complexes. Cell proliferation was measured by CyQuant assay (Invitrogen). Data represented three replicas at indicated concentrations. Experiments were repeatedtwice. MTX and FA-MTX showed comparable cytotoxicity in MDA-MB-435 and SKBR-3 cells. In MCF-7 cell, cytotoxicity of FA-MTX was about 3 times lower than that of MTX. FA modification on MTX has no obvious effects on in vitro cytotoxicity.

Figure 4Figure 4: Effect of MTX and FA-MTX on H1299 cells xenografts. Fifteen mice were randomly split into 3 groups for USP saline, MTX, and FA-MTX treatment. Cancer cells were injected as described in Methods and tumor size was measured 2X per week. MTX (25 mg/kg) and FA-MTX (equal to MTX 4.15 mg/kg) were administered i.p. once a week for 3 weeks. Based on the relative tumor volume, FA-MTX with 1/6 dose of MTX showed comparable or slightly better efficacy.

ResultsFigure 6

Figure 6: Recombinant HSA fusion proteins promote apoptosis via caspase activation. SJSA-1 cells were plated in 96-well plates in standard growth media and allowed to attach overnight. Recombinant HSA fusion proteins, FA-p53i or nutlin were added at the indicated concentrations in RPMI media containing 1% FBS + 0.05% DMSO and allowed to incubate x 24 hrs. Cells were then washed and caspase activation was quantitated using a fluorimetric Homogeneous Caspase Assay (Roche). Phase micrographs of 10 µM-treated wells: (A) untreated (B) nutlin (C) FA-p53i (D) rHSA-p53i (E) rHSA-PMI.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

contr

ol

nutlin

10 µ

M

FA-p

53i 1

0 µM

rHSA-p5

3i 5 µ

M

rHSA-p5

3i 10

µM

rHSA-P

MI 5 µM

rHSA-P

MI 10 µ

M

Treatment Conditions

Fold

cha

nge

rela

tive

to c

ontr

ol

A

ED

CB

ConclusionFA-modification of MTX and p53i is a valid method to facilitate non-covalent

incorporation into HSA

Recombinant HSA fusion proteins already containing the integrated p53i and PMI peptides are capable of delivering FA-MTX.

The increase in p53 protein expression by FA-p53i, rHSA-p53i and rHSA-PMI confirms intracellular uptake and reactivation of p53.

rHSA fusion proteins already containing the integrated p53i and PMI peptides are capable of promoting apoptosis via caspase activation.

Studies are currently underway to determine if rHSA fusion proteins co-delivered with FA-MTX can enhance cytotoxicity compared to single agent administration.

Wuyuan Lu et al. JBC, 398: 200-213 (2010)

Recipient of the Joseph B. Schwartz Graduate Student Pharmaceutics Travel Fund Award

AM

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