1. Human Serum Albumin: A Vehicle to Co-Deliver Fatty Acid-Modified MethotrexateHuman Serum Albumin: A Vehicle to Co-Deliver Fatty Acid-Modified Methotrexate
and a p53-Derived Peptide to Enhance Apoptosis in Cancer Therapyand a p53-Derived Peptide to Enhance Apoptosis in Cancer Therapy
Michelle R. Joshi, Nianhuan Yao, Song Chae, Zhiyu Li
Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA 19104
INTRODUCTIONINTRODUCTION RESULTSRESULTS
CONCLUSIONSCONCLUSIONS
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.
BACKGROUNDBACKGROUNDBACKGROUNDBACKGROUND
Co-Delivery
Combination Therapy
Vs.
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 tissue2Nat Struct Biol. 5(9), 827-35 (1998).
1
METHODSMETHODS
2
1) 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 (p53i)
 Designed to inhibit native p53-MDM2
interaction
3) Design and construct a recombinant HSA C-terminal fusion protein
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
wt p53
high
affinity
peptide
PMI
Wuyuan Lu et al. JBC, 398: 200-213 (2010)
alternative strategyalternative strategy
N-protected C-terminal amino acid residueN-protected C-terminal amino acid residue was anchored via its COOH group to the hydroxyl group of Wang resin. Sidewas 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 waschain 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 wasprotected 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 groupsrepeated 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.were removed. Resulting peptide was detected by ESI/MS and purified by HPLC.
Solid phase peptide synthesis
Synthesis of FA-Modified FITC, FA-Modified p53i, & 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.
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
Albumin/Fatty Acid Mobility Shift Assay
For experiments designed to detect albumin/FA-p53i complex formation, 120 pmol albumin (dissolved in 1X PBS) wasFor 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-p53iincubated +/- 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 molarby 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).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 usingReactions 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.Tris-Boric polyacrylamide gel 12 mA for 20 minutes. The gel was visualized under 305 nm UV.
Cytotoxicity Assays
Cells were plated in standard growth media at approximately 40% confluence and allowed to attach overnight. On day 2,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 concentrationsMTX, 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%COand allowed to incubate at 37ºC/5%CO22 for the indicated time period. Cell proliferation was then measured by CyQuantfor the indicated time period. Cell proliferation was then measured by CyQuant
assay (Invitrogen). Data represented three replicas at indicated concentrations.assay (Invitrogen). Data represented three replicas at indicated concentrations.
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 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 free unbound FA-FITC.
Figure 1
Carrier: Human Serum Albumin
Cargo: FA-Methotrexate & FA-p53i/PMI
Figure 2 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.
1 2 3 4 5 6 7 8
FA-bound HSA
Free FA
HSA:FA-FITC-p53i (1:4)
unlabeled FA-FITC (pmol) 120 240 480360 960720--
120 pmol
FA-FITC-p53i
Benefits of Co-deliveryBenefits of Co-delivery
Both anti-cancer agents reach
target
Can use 2 species with
complimentary mechanisms to
promote robust apoptotic
response
Ease of formulation and
administration
One agent → multiple effects
Enhanced therapeutic effect at
lower doses
Targeted co-delivery reduces side
effects due to toxicity in normal
tissues
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rHSA-p53i rHSA-PMI
Treatment Conditions
%CellDeath(RelativetoControl)
5 µM
10 µM
Figure 3
Figure 3: Recombinant HSA fusion proteins (A), FA-p53, FA-PMI and HSA-WT/FA-peptides (B) promote
cytotoxicity in SJSA-1 cells. SJSA-1 cells were plated in 96-well plates in standard growth media and allowed
to attach overnight. rHSA fusion proteins were added at the indicated concentrations in RPMI media containing
1% FBS + 0.05% DMSO and allowed to incubate x 24 hrs. DNA content was quantitated using the CyQuant
Assay (Invitrogen).
0
10
20
30
40
50
60
p53 PMI
Peptide
DNAcontent(as%ofControl)
FA-peptide (50 uM)
HSA-WT + FA-
peptide (50 uM)
AA BB
Figure 5
IP: MDM2
WB: MDM2
H
SA-W
T
HSA-p53i
HSA-PM
I
Figure 5: Biotinylated rHSA-p53i and rHSA-PMI bind MDM2 as
determined by co-immunoprecipitation using an SJSA-1 whole cell
lysate. To detect MDM2/rHSA-p53i &MDM2/rHSA-PMI interaction,
biotinylated HSA-WT, rHSA-p53i, or rHSA-PMI were added to an SJSA-1
lysate and incubated at RT x 1 hr. MDM2 antibody (Santa Cruz) was added
to each sample and allowed to incubate O/N at 4°C. Following a 1 hr RT
incubation with protein A/G-coupled agarose beads, immune complexes
were eluted and analyzed by SDS-PAGE followed by western blot analysis
to verify the identity of the antigen.
Figure 4: 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
O/N. Recombinant HSA
fusion proteins were
Figure 4
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rHSA-p53i rHSA-PMI
Treatment Conditions
FoldChangeRelativetoControl
5 µM
10 µM
AA BB CC DD
EE FF
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)(A). Phase micrographs of 10 µM-treated wells: (B) untreated (C) nutlin
(D) FA-p53i (E) rHSA-p53i (F) rHSA-PMI.
Figure 7: 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 repeated twice. 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 7
p53p53
MDM2MDM2
GAPDHGAPDH
untreateduntreated
NutlinNutlin
rHSA-p53irHSA-p53i
rHSA-PMIrHSA-PMI
FA-p53iFA-p53i
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 6
Figure 8: 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.
Figure 8
HSA
p53iMTX
FA-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.
p53-GFP
p53
GAPDH
GAPDH
MDM2
31 4 5 76 98 11102
31 4 5 76 98 11102
1: PMI 5 µM
2: FA-p53i 50 µM
3: FA-p53i 5 µM
4: Nutlin 10 µM
5: untreated
6: PMI 50 µM
7: PMI 5 µM
8: FA-p53i 50 µM
9: FA-p53i 5 µM
10: Nutlin 10 µM
11: untreated
p53-GFP
transfected
untransfected