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Exploiting interactions between suitably functionalised conf

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Sara Fahs

on 29 August 2013

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Transcript of Exploiting interactions between suitably functionalised conf

Exploiting interactions between suitably functionalised conformationally distinct aromatic amides and phospholipid monolayers
Calcein Leakage Assays
Pyridinamide 2 induced the highest calcein leakage from all phospholipid composition monolayers
It also showed the best interaction with DMPS monolayer, which is negatively charged in comparison to DMPC and DMPE

Molecular Dynamic Simulations
Monomer 2 inserted in DMPC bilayer during the first 50 ns, but it was stuck on the surface of DMPS, probably due to the strong coulomb interactions between the monomer and the negatively charged pyridinamide
Monomer 2 required more energy to internalize and externalize from DMPS than DMPC after 200 ns  better interaction, more time for disruption mode of action
But: not enough info to make assumption.
Need more monomers, longer simulations, for more “realistic” results

Cell Culture Assays
Not enough information

Hurdles met:
Compound 3 poor solubility in DMSO: precipitated out of solution (media), and might have reacted with MTS reagent
Absorbance in the control wells was not consistent  interference with the viability calculation
No control which consists of only media+drug+MTS to account for any absorbance due to the drug-mts reaction

However: encouraging results for compound 2, which pushes to repeat these assays
Synthesize novel compounds with enhanced functionalization: charge and hydrophobicity, using the mentioned features of ACP SAR
Study the interactions of these potential monomers with phospholipid monolayers of different composition, to record any selectivity to cancer membrane composition
Try those compounds on cancer cell cultures and normal cell cultures to account for any selectivity in vitro

Conventional chemotherapy and radiotherapy
The most used option for cancer management
Multiple dowsides:
Failure of chemotherapy
Severe side effects
Vaccines for prevention or treatment
Encouraging, yet slowed down because of challenges such as
Reverse autoimmunity
Poor stability of immunotherapeutic agents
(Copier et al., 2009)
Novel therapies
Evolving biomedical engineering technologies
Drug delivery and targeting
Cancer heterogeneity and complexity
More extensive research needed
The most evolved forms of eukaryotic defense against bacteria, protozoa, fungi and even viruses!
Small peptides, ranging from 12 and 50 amino acids
Secondary structure follows 4 themes, the most studied of which is the alpha-helix
Most of them known as antimicrobial peptides (AMP)
Naturally occurring antibiotics as potential therapeutic applications
(Wang et al., 2009; Diamond et al., 2009)
Research and knowledge getting broader: more than 300 members paneled in the AMSDb database
Mechanism of action: mostly MEMBRANE interactive
Antimicrobial Peptides
New reports on the anticancer activities of some AMPs
General cationic architechtural structure
Most widely studied are the alpha-helices (more than 160 paneled in the database
mechanism of action mirrors antibiotic activity of AMPs
(Bhutia and Maiti, 2008; Hoskin and Ramamoorthy, 2008)
Anticancer Peptides
- Until today  cancer remains a global burden that no single universal treatment has been able to eradicate

- 26 M new cases and 17 M cancer deaths predicted on an annual basis within 20 years from now (Thun et al., 2010)

- It is important to keep developing knowledge and updated expertise on treatment approaches
Identification and development of peptides with anticancer properties!
Synthesis of pyridinamides and benzanilides
Yields: 90%, 88% and 60%, respectively for 1, 2, and 3
Full characterization with 1H NMR, 13C NMR, IR and MS

What make ACPs selective to cancer cells?
Membrane-based factors
Cancer cell membranes differ from normal cell membranes
Larger surface area
Higher fluidity
Net negative charge due to anionic components like glycolipids, proteoglycans and phospholipids
Peptide-based Factors
Secondary structure (alpha-helix)
Sequence length
Molecular weight
ACPs as anticancer therapeutics: what is the best strategy?
Raw ACPs as therapeutics
Peptides are inferior drug candidates
Low oral bioavailability
Poor metabolic stability
Potential toxicity

Peptide Modification
Addition of D-amino acids
Example: [D]-K6L9 from K6L9, which stopped primary and metastatic growth of human breast and prostate xenografts (Bhutia and Maiti, 2008)
Peptide "mimics"/peptidomimetics
Example: TSP-1 peptide mimic, called ABT-510 (Ebbinghaus et al., 2007)
COMPLEX folding process

Bioinspired foldamers
Examples: peptoids, beta-peptides, aminoxy acids, cyclic peptides...
Top-down design approach, BUT
Limited control over the structure and the folding

Abiotic, synthetic foldamers
Work from scratch/ de novo design
Determine novel backbones that can cooperate in the desired secondary structure
Strong grip on the synthesis of ACP mimics with desired properties such as folding and stability
Aromatic oligoamides are the most widely studied due to the privileges they offer

Aromatic Oligoamides
Easy of synthesize
“Predictability” of folding (Huc, 2004)
Example 1 : benzene/pyridine
Example 2: The amide bond and conformation (Dennison et al., 2012)
Benzene/pyridine substitution
Conformation switching
Results and discussion
Synthesis of benzanilides and pyridinamides
Calcein Leakage Assays
Phospholipid monolayers
6 different phospholipid compositions: DMPC, DMPG, DMPS, DMPE, E. coli and S. aureus lipid extracts
5 concentrations of the compounds

Molecular Dynamic Simulations
To get more insight on compound interaction with phospholipid BILAYERS, theoretically.
Monomer 2 assembled using ATB database, and MD simulations using GROMACS software were undertaken
2 simulations, one with DMPS bilayers, and one with DMPC bilayers

in vitro cell culture assays
3 cell culture lines: SVGP12, 1321N1, and U87
2 compounds: 2 and 3
5 different concentrations: 1, 10, 100, 500 and 1000 μM
MTS assay for viability
Vehicle control: DMSO (5% in the initial concentration)

Significantly high calcein leakage from DMPS monolayer using compound 2, which calls for a better interaction and potential disruption
E coli and S aureus extract showed consistency of results with DMPG and DMPE (good reference for results analysis)

Optimizing a potential monomer in ACP mimic foldamers
This study aimed at optimizing a functionalized oligamide monomer, made in previous studies
Synthesized novel pyridinamides with/without positive charge and alkyl chains to enhance interaction with negatively charged cancer cell membranes and compared it to benzanilide
Charged pyridinamide interacted best with all the phospholipid monolayers, with significant selectivity for DMPS, which was backed up by energy calculation on MD simulations

Carry out more cell culture assays to get insight on the selectivity of these monomers in vitro
Incorporating this novel pyridinamide in the larger synthetic oligoamides, in an attempt to optimize the mimickry of the 3D structural folding of defense peptides (ACPs) as much as possible, thus mirroring their anticancer activity

Future Work
Bhutia, SK and Maiti, TK. Targeting tumors with peptides from natural sources. Trends in Biotechnology, 26:210{217, 2008.
Dennison, S; Akbar, Z; Phoenix, D, and Snape, T. Interactions between suitably functionalised conformationally distinct benzanilides and phospholipid monolayers. Soft matter, 8:3258{3264, 2012a.
Dennison, S; Snape, T, and Phoenix, D. Thermodynamic interactions of a cis and trans-benzanilide with escherichia coli bacterial membranes. European Biophysics Journal, 41(8):687{693, 2012b.
Dennison, S; Phoenix, D, and Snape, T. Synthetic oligoureas of metaphenylenediamine mimic host defence peptides in their antimicrobial behaviour. Bioorganic & Medicinal Chemistry Letters, 23:2518{2521, 2013.
Dennison, SR; Whittaker, M; Harris, F, and Phoenix, DA. Anticancer alpha-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Current Protein and Peptide Science, 7:487{499, 2006.
Dennison, SR; Harris, F, and Phoenix, DA. The interactions of aurein 1.2 with cancer cell membranes. Biophysical Chemistry, 127:78{83, 2007.
Hammami, R and Fliss, I. Current trends in antimicrobial agent research: chemo- and bioinformatics approaches. Drug Discovery Today, 15:540{546, 2010.
Hanahan, D and Weinberg, RA. Hallmarks of cancer: the next generation. Cell, 144: 646{674, 2011
Harris, F; Dennison, SR; Singh, J, and Phoenix, DA. On the selectivity and efficacy of defense peptides with respect to cancer cells. Medicinal Research Reviews, 33:190{234, 2013
Hoskin, DW and Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. Biochimica et Biophysica Acta, 1778:357{375, 2008.
Huc, I. Aromatic oligoamide foldamers. European Journal of Organic Chemistry, 2004: 17{29, 2004.
Owen, DR. Short bioactive peptides, 2005.
Riedl, S; Rinner, B; Asslaber, M; Schaider, H; Walzer, S; Novak, A; Lohner, K, and Zweytick, D. In search of a novel target|phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochimica et Biophysica Acta, 1808:2638{2645, 2011
Thun, MJ; DeLancey, JO; Center, MM; Jemal, A, and Ward, EM. The global burden of cancer: Priorities for prevention. Carcinogenesis, 31:100{110, 2010.

Special thanks to

Dr Tim Snape,
Dr Sarah Dennison,
Dr Manuela Mura,
Dr Marco Pinna,
Dr Laura McShane,

And all those who made this work possible
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