WCMCW
Weste r n C anad i an Me d i c i na l
C hem ist r y Work shop
S e ptemb er 2 6 - 2 8 , 2 0 1 4
S ask ato on , S a sk atc he w an
C hair
E d S. Krol
Vi c e C hair
D av id R . J. Pa lmer
Sp on s or s
Saskatchewan Health Research Foundation NSERC-ROF GlaxoSmithKline Gilead Merck Biological
and Medicinal Chemistry Division Canadian Society for Chemistry K Prime University of Saskatchewan
PRISM College of Pharmacy and Nutrition, Department of Chemistry
Contents
MESSAGE FROM THE ORGANIZERS
5
WCMCW SCHEDULE OF EVENTS
6
ABSTRACTS OF ORAL PRESENTATIONS
8
ABSTRACTS OF POSTER PRESENTATIONS
17
LIST OF AT TENDEES
25
CAMPUS MAP
27
4
Message from the organizers
Message from the
organizers
We wou l d l i ke to wel c ome you to Saskato on and t he Unive rsit y of Saskatche wan for t he four t h
We ste r n C ana di an Me di c i na l C he m ist r y Workshop. The WCMCW st r ive s to b e an ac cessible,
re g i ona l me e t i ng t hat c on ne c ts re s e arche rs f rom a v ar i e t y of dis cipl i ne s w ho are i ntereste d
i n di f fe re nt asp e c t s of phar mace ut i c a l s ci e nce s. The maj or go a ls of t he WCMCW are:
1 . an i ne x p e ns ive g at he r i ng to fo ste r re s e arch conne c t i ons b e t we e n re s e arche rs w ho wou ld
ot he r w is e ne ve r me e t at more t radit i ona l dis cipl i ne -sp e ci f i c me e t i ng s
2 . ma k i ng re s e arch at We ste r n C anadi an i nst itut i ons t he fo c a l p oi nt
3 . an opp or tu n it y to prov i de e x p o sure for stude nts and p o stdo c tora l re s e arche rs to fac u lt y
f rom ot he r i nst itut i ons and t he phar mace ut i c a l i ndust r y
It is ou r hop e t hat we have b e e n succe ssf u l i n ach i e v i ng t he s e go a ls.
Anyone w ho has org an i z e d a s ci e nt i f i c me e t i ng k nows t hat t he s e e ve nts wou l d b e i mp ossible
w it hout f i nanc i a l supp or t and we are g rate f u l to our many sp ons ors for helpi ng w it h t his
i n it i at ive. We have l iste d our sp ons ors on our website, i n t h is pro g ram b o ok and t h roug hout
t he work shop, pl e as e j oi n us i n t han k i ng t he s e ge ne rous donors.
We hop e t hat ou r e f for t s to put to ge t he r t he four t h WCMCW me e t i ng w i l l b e b ot h st i mu l at ing
and pro du c t ive. We are for tunate to have s e ve n e xcel l e nt i nv ite d sp e a ke rs, e ach coming
f rom di f fe re nt dis c ipl i nar y b ackg rounds, but hav i ng com mon go a ls of i mprov i ng t he qu a lit y,
e f f i c a c y and s afe t y of phar mace ut i c a ls, w h i ch u lt i mately ge ts to t he he ar t of t h is me et ing .
F i na l ly, we wou l d l i ke to t han k you for atte ndi ng and helpi ng to ma ke t h is me e t i ng a success.
Si nc e rely,
E d S . Krol, C hai r,
WC MC W
D av i d R . J. Pa lm er , Vi ce C hai r,
WCMCW
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
5
Schedule of Events
FRIDAY, SEPTEMBER 26, 2014
TIME
EVENT
LO CATION
6 :3 0 – 9 pm
Re g i st r at i on
6 :3 0 – 9 pm O p en i ng re c e pt i on and m ixer
Stude nt l ounge, E duc at i on bui l di ng ,
Unive rsit y of Saskatche wan
NSE RC - ROF Pre s ent ati on
SATURDAY, SEPTEMBER 27, 2014
Re g i str ati on an d or a l p o ster pre s ent ati on s to b e hel d i n E duc at i on Bui l di ng , ro om 1 0 0 4
Po ster pres ent ati on s to b e hel d i n t he stu dent l ounge of t he E duc at i on Bui l di ng
TIME
EVENT
LO CATION
8 am
Re g i st r at i on an d p o ster s e t up
E duc at i on ro om 1 004
8 :5 0 am
O p en i ng rem ar k s
9 am
Fr as er Hof ( Un i ver sit y of Vi c tori a )
Pe pt i di c and supr amol e c u l ar to ols for probi ng e pi ge ne t i c me t hy l at i on p at hway s.
9 :4 0 am
Man a l Al Ma l k i ( Un i ver sit y of Winnip e g )
Ac e t y l chol i ne ste r as e In hibit i ng Ste roi d a l A l ka l oi ds f rom Bux us macowani i
1 0 :0 0 am
Brent Pol l o ck ( Pr ai r i e Pl ant Systems , S a sk . )
Phy to c an nabi noi d dr u g de vel opme nt
1 0 :2 0 am
C of fe e bre a k and p o ste r v i e w ing
1 0 :5 0 am
G e of f Tr an m er ( Un i ver sit y of Manitob a )
Appl i c at i on of f l ow che m ist r y i n me di ci na l che m ist r y pro g rams
1 1 :3 0 am
Bren d an Re di ng ( NRC IR AP )
1 1 :5 0 am
C h r i s B ow m an ( Mit ac s )
1 2 :1 0 pm
E nd of mor n i ng s e ss i on . WCMCW lunch i n E duc at i on Bui l di ng
1 :2 0 pm
R i ck Wag n er ( Un i ver sit y of Minne s ota )
C he m i c a l ly s el f - ass e mbl e d nanor i ng s ( CSA NS) : From monitor i ng dr ug del ive r y to
t arge te d c el l t he r apy.
2 :0 0 pm
S an i y a Al w an i ( Un i ver sit y of S a sk atche wan)
Am i no a c i d f u nc t i ona l i z e d nano di amonds as p ote nt i a l ge ne del ive r y ve c tors
2 :2 0 pm
Han an Aw ad ( Un i ver sit y of S a sk atche wan)
Prop o s e d me chan is m for t he une x p e c te d for mat i on of [M+H]+ dur i ng MS ana lysis
of novel ant i ne opl ast i c c u rc u m i n ana l o g ue s and e st abl ish me nt of a ge ne ra l ESI-MS/ MS
f r ag me nt at i on b ehav i ou r
2 :4 0 pm
Ki sh or Was an ( Un i ver sit y of S a sk atche wan)
D e vel opme nt of an or a l Amphote r i ci n B For mu l at i on to t re at v is ce ra l
le ishman i as is
3 :0 0 pm
C of fe e bre a k and p o ste r v i e w ing
6
Schedule of events
SATURDAY, SEPTEMBER 27, 2014 cont’d
TIME
EVENT
3 :3 0 pm
S c ott Mu r phy ( Un ive rs it y of R e g i na)
Photore sp ons ive l ipi d-b as e d nanop ar t i cl e s: Towards on-de mand dr ug del ive r y
4 :1 0 pm
Nat ash a Mi l o s e v i ch ( Un i ver sity of Vi c tori a )
D e s i g n and s y nt he s is of p e pt i di c prob e s for p olycomb g roup prote i ns upre g u l ate d
i n ste m c el ls and pro st ate c ance r
4 :3 0 pm
S ai fu r K h an ( Un i ver sit y of Alb er ta )
Is on i a z i d: is it c y toprote c t ive ?
4 :5 0 pm
Yu l i a Skov p en ( Un i ver sit y of S a sk atche wan)
Stu dy of a l l o ste r i c i n hibitors of di hydro dipi col i nate sy nt has e, a v a l i d ate d dr ug t arge t
5 :1 0 pm
E nd of af te r no on s e ss i on , p oste r v i e w i ng and judg i ng
6 :4 0 pm
Po ste r s e ss i on e nds
7 :3 0 pm
WC MC W B an qu e t
St . Trop e z , 238 2nd Avenu e S outh
SUNDAY, SEPTEMBER 28, 2014
Re g i str ati on an d or a l p o ster pre s ent ati on s to b e hel d i n E duc at i on Bui l di ng , ro om 1 0 0 4
8 :0 0 am
Bre a k fast wor k sh op for t r ai n e e s
Pre s e nt at i on on opp or tu n it i es i n t he phar mace ut i c a l i ndust r y, Ste ve Ar ns ( C e nt re for Dr ug
R e s e arch and D e vel opme nt , Vancouve r, B C)
8 :5 5 am
Int ro du c tor y rem ar k s
9 :0 0 am
St ac e y We t m ore ( L e t hbr i d ge University )
Us i ng c omput at i ona l che m ist r y to study DNA d amage and re p ai r
9 :4 0 am
Mas o om eh Po org h or b an ( University of S a sk atche wan)
Phy s i c o che m i c a l char a c te r i z at i on of t he ho st-g ue st compl e x of a c urc um i n ana l o g w it h
β -c ycl o de x t r i n and β -c ycl o dex t r i n-ge m i ni sur f ac t ant
1 0 :0 0 am
Mays Al - D u l ay m i ( Un i ver sity of S a sk atche wan)
Pe pt i de - mo di f i e d ge m i n i su r f ac t ant-b as e d ge ne del ive r y sy ste ms: i n v it ro e v a lu at i on and
phy s i c o che m i c a l char a c te r i z at i ons
1 0 :2 0 am
C of fe e bre a k
1 0 :5 0 am
C h r i s Ph en i x ( L a keh e ad Un iversity )
T he de s i g n and e v a lu at i on of prob e c andi d ate s for i mag i ng e nz y me ac t iv it y i n
l iv i ng s y ste ms
1 1 :3 0 am
Ke v i n Al l en ( Un i ver sit y of S a sk atche wan)
D e vel opme nt of 18 F l i g nan ana l o g ue s as prob e s for e nte rol ac tone upt a ke i n combi nat i on
t he r apy for pro st ate c anc e r
1 1 :5 0 am
E d Krol ( Un i ver sit y of S ask atche wan)
D e s i g n of n i c ot i ne and c af fe i ne prob e s to pre ve nt α -sy nucl e i n m isfol di ng
1 2 :1 0 pm
C l o s i ng re mark s , WCMCW lunch i n Marquis Ha l l
1 :3 0 pm
C on clu si on of WC MC W
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
7
Abstracts
Oral presentations / session 1
Peptidic and supramolecular tools for probing
epigenetic methylation pathways
Acetylcholinesterase Inhibiting Steroidal
Alkaloids from Buxus macowanii
Fraser Hof
Manal AlMalki1, Athar Ata1* and Robert M. Gengan2
Department of Chemistry and Centre for Biomedical
Research, University of Victoria, Victoria, BC
Post-translational methylations play central roles in epigenetic
gene regulation, but there remain few synthetic agents that
can help us to study or antagonize these pathways. Lysine
methylations are turn-on switches for hundreds of distinct
protein-protein interactions. We have been developing new
approaches to making chemicals that can mimic, sense,
or antagonize the lysine-methylation-driven biochemistry
important to healthy and disease-linked cellular processes.
We have created organic macrocycles that can recognize and
bind to methylated sites on proteins, including examples
that disrupt methylation-driven protein-protein interactions
and others that can provide a readout of a protein’s posttranslational modifications. We have also targeted a family
of methyllysine reader proteins called chromodomains,
having created antagonists of the epigenetic master controller
Chromobox homolog 7 (CBX7) that are the first inhibitors of
any chromodomain. We characterize all of our systems using
a wide array of biochemical, biophysical, physical organic,
and cell-based analytical techniques. In doing so we have
both made useful tool compounds by design, and discovered
molecules with completely unexpected properties.
8
1. Department of Chemistry, Richardson College for the
Environmental and Science Complex, University of
Winnipeg, Winnipeg, MB
2. Department of Chemistry, Durban University of
Technology, Durban, South Africa
The genus Buxus has provided a plethora of novel
biochemically active alkaloids over the years. Buxus alkaloids
have unique steroid-triterpenoid type structures and these
alkaloids exhibit various biological activities including, antiHIV, anti-malarial, anti-TB and anti-acetylcholinesterase.
Buxus macowanii, native to Eastern Cape of South Africa, is
used by the traditional healers to enhance memory in elderly
people and treat wounds. These ethnomedicinal use of this
plant promoted us to screen the crude methanolic extract of
this plant in our acetylcholinesterase (AChE) inhibition assay
and was found to be active with an IC50 value of 32 ug/ml.
Based on the observed bioactivity, we decided to carry out a
detailed phytochemical investigation of the crude methanolic
extract of this plant in order to isolate pure natural products
and evaluate them in our AChE inhibition assay. These efforts
resulted in the isolation of a few pure compounds having a
novel carbon skeleton. In this presentation, isolation, structure
elucidation of these novel compounds, their bioactivity data
and structure-activity relationships will be presented.
Abstracts / Oral presentations
Phytocannabinoid drug development
W. Brent R. Pollock
Purification Development Lab, Prairie Plant Systems,
Saskatoon, SK
Prairie Plant Systems Inc. has been producing medical
marijuana according to Good Manufacturing Practices for 13
years, first as the sole contract supplier for Health Canada’s
programme started under the Marihuana Medical Access
Regulations but now as an independent Licensed Producer
under the superseding Marihuana for Medical Purposes
Regulations. The single marijuana product supplied to Health
Canada under the MMAR has been used in three clinical trials,
all in the Phase II stage; PPS was the supplier but not a direct
sponsor of any of those trials. PPS is sponsoring a Phase II
clinical trial which is slated to commence this year. In addition
to cannabis cultivation facilities, we also have a lab tasked with
developing protocols for isolating cannabinoids from the plant
material. This also involves identification and quantification of
impurities. This lab has internal support for HPLC and UV-Vis
but needs external support for other assays (e.g. NMRs & GCMS), molecular modelling & physical chemistry expertise. We
are also looking to collaborate with university faculty who wish
to integrate these purified materials into their own experiments
(e.g. animal tests, drug formulation).
Application of Flow Chemistry in Medicinal
Chemistry Programs
Geoffrey K. Tranmer1,2
1. Faculty of Pharmacy, University of Manitoba, Winnipeg, MB
2. Department of Chemistry (Adjunct), University of
Manitoba, Winnipeg, MB
Synthetic organic chemistry has already had a significant impact
on the field of medicinal chemistry, with major advancements
being applied towards the development of new pharmaceutical
therapies for the study and manipulation of biological systems.
With this in mind, the future of synthetic medicinal chemistry
will involve the development and application of innovative
technologies in the field of organic synthesis, and in particular,
the burgeoning field of flow chemistry. My research interests
are largely focused on drug discovery, and have an end goal of
improving the process of drug development and facilitating
innovation in medicinal chemistry. Specifically, my group is
interested in developing novel flow chemistry techniques that
will aim at enhancing current synthetic organic methodologies,
and focus on lead generation and optimization in drug
discovery. New developments in synthetic methodologies
using flow chemistry will replace existing techniques and
avoid the need for the work-up, purification and manipulation
required by current methods, making syntheses shorter and
greener. My research is involved in the development and
application of flow chemistry techniques and solving some of
the current limitations of organic synthesis, and also applying
this technique towards enhancing and accelerating medicinal
chemistry programs.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
9
Abstracts
Oral presentations / session 2
Chemically Self-Assembled Nanorings
(CSANS): From Drug Delivery to Targeted Cell
Therapy
Carston R. Wagner
Department of Medicinal Chemistry, College of Pharmacy,
University of Minnesota, Minneapolis, MN
Based on our development of a highly efficient protocol for
the chemically controlled self-assembling of protein nanorings,
we have sought to exploit our methodology for the controlled
assembly of polyvalent antibody and peptide-nanorings for
tissue imaging and drug delivery. Tissue targeted rings have
been shown to be an effective means of delivering anti-cancer
drugs, oligonucleotides and proteins in vitro. We have also
shown that they are stable in vivo and capable of tissue targeted
imaging and drug delivery. In addition, we have used our
approach to design CSANs capable of reversibly re-programing
cellular surfaces and, for example, directing T-cells to selectively
kill tumor cells. The lecture will cover our latest developments
in the design and use of CSANs for therapeutic imaging and
anti-cancer therapy.
10
Amino acid functionalized nanodiamonds as
potential gene delivery vectors
Saniya Alwani1, Jackson M. Chitanda*2, Deborah Michel*1,
Ronald E. Verrall*2 and Ildiko Badea*1.
1. Drug Discovery and Development Research Group, College
of Pharmacy and Nutrition, Saskatoon, SK
2. Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
Purpose: This study synthesizes amino acid (AA) functionalized
nanodiamonds (NDs) as potential gene nanocarriers. LysineNDs have primary amines on the surface capable of binding
with genetic materials and interacting with the cell membrane
to facilitate gene delivery. Lysyl-histidine-NDs additionally
induce pH sensitivity causing endosomal escape to protect
therapeutic genes against enzymatic degradation.
Methods: NDs were covalently conjugated with lysine and
histidine. Functionalized NDs (fNDs) were characterized
by size and zeta potentials, infrared spectroscopy (IR) and
thermogravimetry (TGA). Surface loadings of fNDs were
calculated by thermograms. Physicochemical stability in
aqueous and biologically relevant media was characterized by
changes in size and zeta potentials. Binding with pDNA and
siRNA was analysed by gel electrophoresis. Finally, cellular
uptake of fNDs as diamoplex with siRNA was evaluated using
flow cytometry and scanning transmission X-ray microscopy
(STXM).
Results: IR spectra of fNDs shows distinct amide peaks of N-H
(stretch) at ~ 3000 cm-1, N-H (bend) at ~1490 cm-1 and C=O
(stretch) at ~1650 cm-1 evidencing functionalization. Surface
loading of lys-NDs is higher than the bare NDs (1.0 mmoles/
gm), i.e. 1.97 mmoles/gm. High surface loading refers to high
binding capacity. Lysine functionalization created a relatively
stable ND dispersion (PDI: 0.14) having highest volume % of
particles sized ~50 nm and zeta potential of +26.5 mV. Lys-NDs
dispersed well in aqueous medium and remained stable over a
period of 25 days. Lys-NDs were capable of binding pDNA at
1:1 and siRNA at 1:20 weight ratios. Flow cytometry results
showed a positive shift corresponding to FITC fluorescence
for the diamoplex treated cells compared to untreated cells,
indicating successful cellular association of lys-NDs/FITC
labelled siRNA diamoplexes. C-1 edge STXM spectra of lysNDs treated cells reveal absorption peaks corresponding to NDs
overlapping with proteins in the cells.
Conclusion: AA functionalization of NDs facilitates stability,
complexation, protection and delivery of therapeutic genes in
mammalian cells.
Abstracts / Oral presentations
Proposed mechanisms for the unexpected
formation of [M-H]+ during MS analysis of
novel antineoplastic curcumin analogues
and establishment of a general ESI-MS/MS
fragmentation behavior
H. Awad and A. El-Aneed
Development of an oral Amphotericin B
Formulation to treat visceral leishmaniasis
Kishor M. Wasan1,2* and Ellen K. Wasan1,2
1. Faculty of Pharmaceutical Sciences, University of British
Columbia, Vancouver, BC
2. College of Pharmacy and Nutrition, University of
Saskatchewan, Saskatoon, SK
College of Pharmacy and Nutrition, University of
Saskatchewan, Saskatoon, SK
Curcumin analogues are novel antineoplastic agents designed
by structural modifications of the natural product curcumin
to enhance its therapeutic effects. The ionization behavior of
thirteen curcumin analogues was investigated to reveal the
possible mechanisms for the unusual formation of the positively
charged [M-H]+ ions during single stage positive ion mode
MALDI-MS analysis. Different ionization techniques (i.e.,
ESI, APCI, APPI, and MALDI) were used to evaluate this
phenomenon. The results showed that curcumin analogues
ionize into [M-H]+ along with the expected [M+H]+ species
during MALDI and no dopant APPI-MS. In contrast, ESI,
APCI and the dopant mediated APPI showed only the expected
[M+H]+ peak. In addition, the tandem mass spectrometric
(MS/MS) fragmentation behavior of curcumin analogues was
investigated showing similar dissociation pathways that centered
on the piperidone ring of the 3,5-bis(benzylidene)-4-piperidone
moiety. The fragmentation patterns were established to confirm
the chemical structure of the tested compounds and identify the
diagnostic product ions of each compound. Common fragment
ions were identified allowing for the establishment of a general
MS/MS behavior for the tested curcumin analogues.
Visceral leishmaniasis is a deadly parasitic disease caused
by obligate intramacrophage protozoans of the Leishmania
genus. The World Health Organization estimates the annual
death toll to be 50,000, with 500,000 new cases each year.
Without treatment, visceral leishmaniasis is inevitably
fatal. For the last 70 years, the first line of defense has been
pentavalent antimonials; however, increased resistance has
brought amphotericin B to the forefront of treatment options.
Unfortunately, the difficult route of drug administration,
toxicity issues, and cost prevent amphotericin B from reaching
the infected population, and mortality continues to rise. Our
reformulation of amphotericin B for oral administration has
resulted in a highly efficacious antileishmanial treatment
that significantly reduces or eradicates liver parasitemia in
a murine model of visceral leishmaniasis. This formulation
has overcome amphotericin B’s significant physicochemical
barriers to absorption and holds promise for the development
of a self-administered oral therapy for the treatment of visceral
leishmaniasis.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
11
Abstracts
Oral presentations / session 2
Photoresponsive lipid-based nanoparticles:
Towards on-demand drug delivery
Yamuna S. Kandasamy, Jianxin Cai, Alisha Beler, M.-S.
Jemeli Sang, and R. Scott Murphy*
Department of Chemistry and Biochemistry, University of
Regina, Regina, SK
We are developing photoresponsive lipid-based nanoparticles
by integrating photochromic molecules with supramolecular
assemblies such as lipid vesicles. These biocompatible
nanoparticles will have potential application in ‘on-demand’
drug delivery where the delivered dose will be regulated with
light. We have synthesized and characterized the photochromic
properties of three new amphiphilic dithienylethenes (DTEs).
We have also examined their inclusion in lipid vesicles and
photocontrol of ion permeability. Three pure lipid systems and
one mixed lipid system were investigated using a fluorescencebased assay that reports on proton transport. Our proton
permeation studies show that the open-ring isomers of these
DTEs are better membrane disruptors than the closed-ring
isomers in both pure and mixed lipid systems. Notably, a
steric effect is clearly observed as the comparatively smaller
methyl derivative exhibits lower rates of ion permeation
than the bulkier phenylethynyl derivatives. Overall, UV
photoirradiation leads to a reduction in ion permeability. Our
most recent developments regarding the functionality of these
photoresponsive nanoparticles will be presented.
12
Design and Synthesis of Peptidic Probes for
Polycomb Group Proteins Upregulated in Stem
Cells and Prostate Cancer
N. Milosevich, C. Simhadri, M. Gignac, A. Dev, K. Daze, B.
Kilpatrick, F. Hof*
Department of Chemistry and Centre for Biomedical
Research, University of Victoria, Victoria, BC
Epigenetics is the study of heritable changes in gene expression
and function, which are caused by mechanisms that do not
involve a change in DNA. Polycomb group (PcG) proteins are
an epigenetic family of proteins, which constitutes the primary
switch that turns prostate cancer into castrate-resistant prostate
cancer (CRPC), the most highly aggressive and deadly form of
the disease. Post-translational methylation events (PTM’s) are
the marks which PcG proteins read leading to gene silencing.
The methylated proteins are recognized and bound by members
of the Chromobox family of ‘methyllysine reader’ proteins,
with each member playing a different role in the progression
of the disease from early stages through to the most aggressive,
and ultimately untreatable forms of the disease. We will report
on our synthesis of targeted libraries of chromobox inhibitors
and on the behaviours of these inhibitors in preliminary cell
based assays. We will also report a set of fluorescently tagged
lead compounds and demonstrate their use in the improvement
of fluorescence polarization assays, array-based studies of target
selectivity, and in live cell imaging. These are the first reported
inhibitors of any methyllysine reading chromodomain, and
represent a generation of tool compounds needed to test this
particular epigenetic therapeutic hypothesis.
Abstracts / Oral presentations
Isoniazid: Is it cytoprotective?
Saifur R. Khan1, Argishti Baghdasarian1, Richard P.
Fahlman2, and Arno G. Siraki1*
1. Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, AB.
2. Department of Biochemistry, Faculty of Medicine &
Dentistry, University of Alberta, Edmonton, AB
Isoniazid (INH), an anti-tuberculosis drug, carries a risk
of idiosyncratic drug reactions which are suspected as INH
reactive metabolites induced adaptive immune response;
however, there has yet to be an experimental model to
establish the fact. The objective of our study was to explore
the role of INH and its reactive metabolites in cell death.
Contrary to our expectations, cell viability experiments found
concentration dependent cytoprotection by INH against
glucose/glucose oxidase (G/GO)-produced H2O2 in HL60 cells. We tested whether INH-induced cytoprotection
depended on its capacity to enhance ATP biosynthesis which
could inhibit the release and translocation of AIF. ATP assays
showed a significant increase in ATP production upon INH
treatment in G/GO treated cells. Immunoblots showed that
INH at pharmacological concentrations sufficiently prevented
AIF release from mitochondria. Additionally, quantitative
proteomics discovered highly significant changes in 51 proteins,
among which 18 were upregulated and 33 were downregulated.
Pathway analysis (String 9.1) revealed cellular pathways for
enhanced ATP biosynthesis. Moreover, we found significant
increase of covalently bound proteins in anti-INH immunoblots
that correlated with cytoprotection. In conclusion, INH was
shown to have cytoprotective ability against G/GO in HL-60
cells, and INH reactive metabolites may have a beneficial role in
this circumstance.
Study of allosteric inhibitors of
dihydrodipicolinate synthase, a validated drug
target
Yulia V. Skovpen, Cuylar J. T. Conly, David A.R. Sanders,
and David R.J. Palmer*
Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
Dihydrodipicolinate synthase (DHDPS) is an enzyme of the
meso-diaminopimelate biosynthesis pathway and is allosterically
regulated by L-lysine. DHDPS is considered an essential
enzyme for bacterial growth, and the development of inhibitors
of DHDPS is part of the discovery of new antibiotics. Here
we report our progress on design, synthesis, kinetics, and
crystallographic studies of allosteric inhibitors of DHDPS from
Campylobacter jejuni. The inhibitory activity of the inhibitors
was also tested on mutants of C. jejuni DHDPS that are
insensitive to lysine inhibition. The results of the study should
lead to the development of novel antibiotics.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
13
Abstracts
Oral presentations / session 3
Using Computational Chemistry to Study DNA
Damage and Repair
Stacey D Wetmore
Department of Chemistry, University of Lethbridge,
Lethbridge, AB
The overall objective of research performed in the Wetmore
laboratory is to better understand the implications of DNA
damage and the mechanism of DNA repair. Despite its
relatively simplistic structure, DNA contains all information
vital to life. However, this crucial information can become
damaged by exposure to external agents (X-rays, UV sunlight)
or by natural processes (errors when DNA is copied). Since
DNA repair has been correlated with the prevention of lifethreatening illnesses, it is important to understand DNA
damage and repair pathways, including how enzymes repair
the damage in our bodies. The research in the Wetmore
group uses calculations on computers to gain chemical
information about these important processes. This talk will
provide a survey of some recent topics of interest including
the structural implications of damage to the guanine DNA
nucleobase that leads to bulky lesions, and the repair of
non-bulky nucleobase damage by the DNA glycosylase repair
enzymes.
14
Physicochemical characterization of the hostguest complex of a curcumin analog with
β-cyclodextrin and β-cyclodextrin-gemini
surfactant
Masoomeh Poorghorban1, Abdalla Karoyo2 , Ronald
Verrall2, Pawel Grochulski1,3, Lee Wilson2, Ildiko Badea1*
1. Drug Discovery and Development Research Group, College
of Pharmacy and Nutrition, University of Saskatchewan,
Saskatoon, SK
2. Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
3. Canadian Light Source, Saskatoon, SK
Curcumin analogs are promising anticancer agents with high
logP value, suffer from poor solubility and bioavailability.
Cyclodextrin-based nanocarriers are able to encapsulate
lipophilic drugs and enhance their solubility. Understanding
the geometry of the interaction of curcumin analogs with
β-cyclodextrin and β-cyclodextrin-gemini surfactant in hostguest inclusion complex will help to develop more efficient
anticancer drug delivery systems.
Method: Inclusion of a curcumin analog, NC 2067, in
β-cyclodextrin and β-cyclodextrin-gemini surfactant has been
studied using 1H NMR and 2D rotating frame Overhauser
effect spectroscopy (ROESY) at 500 MHz. Moreover,
synchrotron-based small and wide-angle X-ray scattering have
been used to characterize the supramolecular morphology.
Result: 1H NMR spectrum of free β-cyclodextrin-gemini
surfactant showed significant upfield shift of β-cyclodextrin
internal protons. 2D ROESY showed cross-peaks corresponding
to interaction of the interior cavity protons with Gemini
hydrocarbon tail protons. Moreover, ROESY spectrum of NC
2067 inclusion in β-cyclodextrin confirmed the interaction
of β-cyclodextrin interior protons with alkene conjugated
aromatic rings of NC 2067. Based on the continuous variation
method the stoichiometry of the inclusion of NC 2067 in
β-cyclodextrin calculated as 1 to 2.
Conclusion: The host-guest complexes of NC 2067 with
β-cyclodextrin and β-cyclodextrin-gemini surfactant have been
characterized.
Abstracts / Oral presentations
Peptide-Modified Gemini Surfactant-Based
Gene Delivery Systems: In Vitro Evaluation and
Physicochemical Characterizations
Mays Al-Dulaymi ,Waleed Mohammed-Saeid , Jackson
Chitanda3, Anas El-Aneed1, Ronald Verrall2, Ildiko Badea1*
1
1
The design and evaluation of probe candidates
for imaging enzyme activity in living systems
Christopher P. Phenix*1,2,3, Morshed Chowhury1, Ignace
Moya1, Benjamin Adams1,2, Brady Vigliarolo2, Shardul
Bhilocha3, Sarah Nicolli2 and Simon Lees2
1. College of Pharmacy and Nutrition, University of
Saskatchewan, Saskatoon, SK
2. Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
3. Department of Chemical and Biological Engineering,
University of Saskatchewan, Saskatoon, SK
1. Biomarker Discovery, Thunder Bay Regional Research
Institute, Thunder Bay, ON
2. Medical Sciences Division, Northern Ontario School of
Medicine, Sudbury, ON
3. Department of Chemistry, Lakehead University, Thunder
Bay, ON
Cationic gemini surfactants hold a promising future in the
development of non-viral gene delivery. In order to capitalize
on the potential of these agents, there is a need to design
gemini surfactants with higher transfection efficiency and lower
toxicity. Recently, we have developed a series of novel peptidemodified gemini surfactant-based gene delivery nanoparticles.
The aim of this study was to investigate the changes in the
activity and toxicity as a function of carbon tail length and to
compare with the conventional parent gemini surfactants.
Three novel third-generation peptide-substituted gemini
surfactants were used to engineer DNA nanoparticles at
phosphate to nitrogen charge ratios of 1:1, 1:2.5, 1:5, 1:10,
1:15 and 1:20 in the presence of a helper lipid, DOPE. The in
vitro gene expression and cell toxicity of gemini nanoparticles
were evaluated in Cos-7 cells. Characterization of the delivery
systems was accomplished by using dynamic light scattering
(DLS), zeta (ζ) potential analysis and small angle X-ray
scattering (SAXS) measurements.
The highest activity of peptide-modified gemini surfactant
was observed with the 16 carbon tail compound compared to
the 12 and 18:1 carbon tail compounds and the most efficient
phosphate to nitrogen charge ratios were 1:2.5-1:5. All lipids
exhibited significantly lower toxicity compared to commercially
available transfection agents. The DNA/gemini lipid/ helper
lipid (P/G/L) nanoparticles showed a wide range of particle
size, 50-800 nm. The zeta potential value also varied depending
on the charge ratios. SAXS results indicated structural diversity
in the morphology of the P/G/L nanoparticles.
Two major hurdles prevent the production of high quality
medical images reflecting enzyme activity in vivo; the rapid
diffusion of the contrast agent from the site of enzyme activity
and low signal intensity resulting from slow reaction with
the target enzyme. We have designed probe candidates that
are either activatable substrates of the target enzyme OR
potent mechanism-based inhibitors that quickly alkylate only
active enzyme. In either case, it is critical that the contrast
agent is specifically and efficiently processed by the target
enzyme and retained at the site of enzyme activity. To evaluate
these approaches for PET imaging, we have synthesized
profluorophore probes for the cancer biomarker Cathepsin B.
These activatable substrates release an immobilizing reporter
molecule that covalently attach to nearby nucleophilic proteins
thus preserving enzyme activity while locking the contrast agent
the site of enzyme activity. We have also developed a new class
of N-akylated conduritol aziridines that potently, efficiently
and specifically label the active site of Glucocerebrosidase,
the dysfunctional enzyme found in Gaucher disease and
recently linked to Parksinson’s disease. Initial data suggests
that bothclasses of compounds, once modified with a
radiolabel, may be useful to molecular image Cathepsin B or
Glucocerebrosidase activity in vivo.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
15
Abstracts
Oral presentations / session 3
Development of 18F Lignan Analogues as Probes
for Enterolactone Uptake in Combination
Therapy for Prostate Cancer
Kevin Allen, and Ed S. Krol*
Drug Discovery and Development Research Group, College
of Pharmacy and Nutrition, University of Saskatchewan,
Saskatoon, SK
Flaxseed is a rich source of the lignan secoisolariciresinol
(SECO), which is metabolized in the human gut to
enterodiol (END) and enterolactone (ENL). ENL is believed
to be responsible for many of the pharmacological properties
attributed to flaxseed such as reduction of cholesterol,
diabetes, colon, breast, and prostate cancer. Though these
lignans are rapidly cleared from the body there is evidence
using a rat model that chronic administration of flaxseed
results in ENL accumulation in specific tissues including the
prostate. Our group is studying the anti-cancer properties of
ENL in vitro and one objective is to determine ENL uptake
into prostate cancer. In order to accomplish this objective we
are developing an 18F analogue of ENL (18F-ENL) as a novel
probe for positron emission tomography (PET) in a mouse
xenograft model. We have successfully prepared the nonradioactive fluorinated ENL analogue (F-ENL) for control
experiments and are currently developing a method to quickly
incorporate 18F to compensate for the short half-life of the
radioisotope.
16
Design of nicotine and caffeine probes to prevent
α-synuclein misfolding
Robin Wu1, Omid Tavassoly2, Jeremy Lee2 and Ed S. Krol1*
1. Drug Discovery and Development Research Group, College
of Pharmacy & Nutrition, University of Saskatchewan,
Saskatoon, SK
2. Department of Biochemistry, College of Medicine,
University of Saskatchewan, Saskatoon, SK
One of the pathogenic features of Parkinson’s disease is the
formation of insoluble fibrils composed of misfolded alphasynuclein. Alpha-synuclein is a disordered protein in its native
state and is sensitive to environment. Aggregation of alphasynuclein in the early stages of misfolding has hampered study
of this system. Nanopore analysis has provided some insight
however into misfolding of alpha-synuclein. Nanopore analysis
discriminates between different protein conformations in
solution at low concentrations. We will use nanopore analysis
to identify small molecules which bind to alpha-synuclein and
determine their influence on misfolding. We have demonstrated
that small molecules associated with high incidence of
Parkinson’s disease produce misfolded alpha-synuclein and small
molecules associated with low incidence of Parkinson’s protect
against misfolding. We are investigating the synthesis of linked,
bifunctional molecules derived from the small molecules that
protect against alpha-synuclein misfolding and will describe our
progress thus far.
Abstracts / Oral presentations
Abstracts
Poster presentations
1.
2.
Targeted Mass Spectrometric Metabolomic
Analysis of Urine; a Promising Approach for
Improving the Diagnosis of Asthma and COPD
A validated simple flow injection analysistandem mass spectrometry (FIA-MS/MS) for the
quantification of metformin in dog serum Mona M Khamis 1,2, Hanan Awad1, Darryl J Adamko3,4,5 and
Anas El-Aneed1*
Deborah Michel1, Matthew Gaunt2, Terra Arnason3 and
1. College of Pharmacy and Nutrition, University of Saskatchewan,
Saskatoon, SK
2. Faculty of Pharmacy, Alexandria University, Egypt
3. The Department of Pediatrics, University of Alberta, Edmonton
4. The Department of Medicine, University of Alberta, Edmonton
5. The Department of Pediatrics, University of Saskatchewan
Asthma and Chronic Obstructive Pulmonary Disease (COPD)
are complex respiratory diseases with overlapping clinical
presentations. Their differential diagnosis is difficult and is
usually biased by the patient’s history. Currently available
diagnostic tests are laboursome, unachievable in young children
and/or insensitive to the inflammatory status differences
between these diseases. Briefly, a simple and reliable test that
can be routinely applied in regular outpatient clinic is lacking.
Recent reports using nuclear magnetic resonance (1H-NMR)
analysis identified 50 differentially expressed metabolites among
asthma and COPD patients.
Compared to 1H-NMR, Mass Spectrometry (MS) is
more sensitive and selective. We are developing liquid
chromatographic-MS methods for the targeted quantification
of these metabolites. Based on structural features, metabolites
are divided into three groups that share common functional
groups. Standards and urine solutions are derivatized
with isotope-labeled dansyl chloride (23 compounds) or
dimethylaminophenacyl bromide (13 compounds). The 12C/13C
isotope pair of each metabolite is analyzed by tandem mass
spectrometric (MS/MS) analysis, allowing for the development
of selective LC-MS/MS methods. The ultimate goal is to
identify diagnostic urinary biomarkers for these overlapping
diseases.
Anas El-Aneed1*
1. College of Pharmacy and Nutrition, University of
Saskatchewan, Saskatoon, SK
2. Western College of Veterinary Medicine, University of
Saskatchewan, Saskatoon, SK
3. College of Medicine, University of Saskatchewan, SK
A simple, fast and sensitive quantification method for the
drug metformin in dog serum was developed using flow
injection analysis (FIA)- tandem mass spectrometry (MS/
MS). The method was fully validated according to industry
standards. It is the first time that FIA-MS/MS for metformin
was developed surpassing all existing methods in terms of time
of analysis. The quantification method was dependent on the
formation of [M+H]+ using electrospray (ESI) and employing
multiple reaction monitoring (MRM) using quadrupole- linear
ion trap (4000 QTRAP®) instrument. Deuterated internal
standard (IS) of metformin bearing six deuterium atoms was
used to compensate for matrix effects and variation in ion
current within the ESI source. The ion transitions that were
monitored were 136.4 71.0 and 136.460.1 for metformin
and 136.077.0 for the internal standard. A linear response
(r = 0.9966) was established for a range of concentrations
of 5- 2340 ng/ml. The inter- and intra-day variations were
within the acceptable criteria for all quality control samples.
The method was successfully applied for measuring metformin
serum concentration in dogs after intravenous injection.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
17
Abstracts
Poster presentations
3.
4.
Serotonin transporter clustering in blood
lymphocytes from animals exposed to repeated
corticosterone parallels the alterations found in
depressed patients Development of a new in vitro brain slice culture
method to screen bioactive agents for CNS repair
Kendra Furber1,3, Rhonda Sobchishin1,3, J. Ronald
Doucette2,3 and Adil J. Nazarali1,3
Erin Y. Fenton , Katherina Lebedeva , Lisa E. Kalynchuk ,
and Hector J. Caruncho 1*
1
2
3
1. College of Pharmacy and Nutrition, University of Saskatchewan,
Saskatoon, SK
2. Department of Psychology, University of Saskatchewan,
Saskatoon, SK
3. Division of Neurology, College of Medicine, University of
Saskatchewan, Saskatoon, SK
Clustering of proteins into specific membrane domains is
important for their functional regulation. Recently, we have
shown alterations in the clustering of the serotonin transporter
(SERT) in blood lymphocytes of depressed patients (RiveraBaltanas et al., 2012, J. Affect. Disord. 137:46-55). In the
present work our aim was to identify whether similar alterations
in SERT clustering occur in a well-established rodent model
of depression. To examine this, 20 male rats were administered
the stress hormone corticosterone for 21 days and depressivelike behaviour was assessed. Blood was collected via cardiac
puncture and immunocytochemistry was used to examine
SERT protein clusters in lymphocytes. As expected, CORTtreated animals showed a significant increase in depressivelike behaviours. Interestingly, the size of SERT clusters was
increased by 13% in CORT-treated rats but the number was
unchanged, much like what we found in depressed patients.
Moreover, a significant positive correlation was found between
depressive-like behaviour and SERT cluster size and a negative
correlation was found between depressive-behaviour and
cluster number. Together with our previous findings, these data
suggest that SERT clustering may be an important biomarker
of depression, and that our rodent model of the disorder is a
reliable way for us to examine this relationship more closely.
18
1. Laboratory of Molecular Cell Biology, College of Pharmacy and
Nutrition, University of Saskatchewan, Saskatoon, SK
2. Department of Anatomy and Cell Biology, College of Medicine,
University of Saskatchewan, Saskatoon, SK
3. Neuroscience Research Cluster, University of Saskatchewan,
Saskatoon, SK
A major challenge in evaluating novel therapeutic agents
is having appropriate models of human disease states. The
cuprizone (copper chelator) diet is a common model for
central nervous system (CNS) demyelination / remyelination;
however, in vivo rodent studies can be expensive and require
long-timelines (≥9 weeks). We have recently developed a novel
in vitro brain slice model to screen bioactive molecules for
neuroprotective properties. Unlike typical in vitro preparations
using immature animals, our new slice culture from mature
mice allow for the study of myelination pathways after the
period of intense neurodevelopment. The brain slice culture
maintains the cytotypic organization observed in the intact
brain, yet reagents have direct access to tissue through the
media and assessments have shorter time intervals. We have
designed a cuprizone treatment that rapidly demyelinates slice
cultures (24-36h) with effects comparable to the cuprizone
diet in vivo, in both the amount of demyelination observed
and the brain structures impacted. Initial studies using this
model indicate that long-chain polyunsaturated fatty acids
(PUFAs) and antioxidants (ie. ascorbic acid) are effective agents
at preventing demyelination. This validates the feasibility of
this approach to screen bioactive molecules for neuroprotective
properties prior to undertaking intensive in vivo studies.
(Funded by CIHR)
Abstracts / Poster presentations
5.
6.
Allosteric Signaling in Dihydrodipicolinate
Synthase: Dead End Crystallization
pH dependent stability and oxidative metabolism
of secoisolariciresinol analogues
Cuylar Conly, Yulia Skovpen, David Palmer, David
Leah McGurn1,2, Ed S. Krol1*
Sanders*
Department of Chemistry, University of Saskatchewan
Dihydrodipicolinate synthase (DHDPS) is an enzyme found in
most bacterial species and regulates the production of cell wall
precursors necessary for the life of the organism. Specifically,
DHDPS catalyzes the condensation of pyruvate and aspartateβ-semialdehyde (ASA) to produce dihydrodipicolinic acid
leading to the synthesis of cell wall precursors lysine and
mesodiaminopimelate. DHDPS is regulated through feedback
inhibition when lysine binds at a location distinct from the
reactive site. The mechanism by which lysine remotely disrupts
catalysis is not well understood. A clear understanding of how
the natural inhibitor, lysine, binds to DHDPS and what effects
this has on the machinery of the enzyme will be invaluable
for development of novel antibiotic leads. Here we present
a strategy for the crystallization of DHDPS with its second
substrate ASA, by formation of a DHDPS:pyruvate dead end
complex.
1. Drug Discovery and Development Research Group, College
of Pharmacy and Nutrition, University of Saskatchewan
2. Toxicology Graduate Program, University of Saskatchewan,
Saskatoon, SK
Several analogues of secoisolariciresinol (SECO), a lignan
from flax seed with potential pharmaceutical properties,
were synthesized. The analogues were used to probe the
effect of structure on SECO metabolism. High performance
liquid chromatography (HPLC) methods for the detection
and separation of SECO analogues, autoxidation products,
metabolites and glutathione (GSH) adducts were developed.
The stability of SECO analogues (1 mM) in 50 mM Na2HPO4
buffer at pH 6.0 and 7.4 were quantified with and without
a 5:1, GSH:substrate, trapping system. Enzymatic oxidation
experiments using mushroom tyrosinase (MT) on the SECO
analogues with and without the GSH trapping system were
preformed. Mass spectroscopy analysis was used to identify
the products. All SECO analogues were stable at pH 6.0.
SECO-2 was the only stable analogue at pH 7.4. SECO-1,3
and 4 are unstable at pH 7.4 with t1/2 of 30 h, 3.5 h and 4
h respectively. SECO-1,3 and 4 are oxidized by MT while
SECO-2 is not. The results of oxidation and subsequent GSH
conjugation are consistent with a linear aromatic conjugate.
Possible mechanisms for the oxidation of the SECO analogues
are proposed.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
19
Abstracts
Poster presentations
7.
8.
Generation and Screening of Lariat Peptide
Libraries Using Yeast Surface Display
Structural modification of PLGA based
nanoparticulate system using antibody ligand: An
approach to design a targeted delivery model
VVM Bharathikumar1, Kris Barreto2, John DeCoteau3, Yu
Luo1, and C. Ronald Geyer3*
1. Department of Biochemistry, University of Saskatchewan,
Saskatoon, SK
2. Department of Biochemistry and Molecular Biology, University of
British Columbia, Vancouver, BC
3. Department of Pathology, University of Saskatchewan, Saskatoon,
SK
Previously, we developed a yeast two-hybrid system to generate
and screen libraries of lactone-cyclized peptides, referred to as
‘lariat peptides’, against proteins of interest. Here, we report
a new method to generate and isolate lariat peptides using
the yeast surface display technology. Yeast display expresses
lariat peptides on the outer cell membrane thus allowing lariat
peptide libraries to be screened against a wide range of targets,
including recombinant proteins, peptides and small molecules.
To demonstrate the feasibility of our approach, we constructed
a yeast-displayed lariat peptide library with 10E8 members, and
isolated lariat peptides that bind to streptavidin. Selections were
performed using magnetic-activated and fluorescence-activated
cell sorting techniques, and selection outputs were analysed
using deep sequencing. Clustering analysis revealed the presence
of novel streptavidin-binding motifs in addition to a previously
identified HPQ-motif. We synthesized lariat and linear peptides
that occur frequently in these clusters, and characterized
their binding to streptavidin using bio-layer interferometry.
In summary, we have developed and validated a yeast display
system to easily and rapidly isolate lariat peptides against a wide
variety of targets. These reagents can be helpful in validating the
function and therapeutic potential of proteins.
20
Sheikh Tasnim Jahan and Azita Haddadi*
College of Pharmacy and Nutrition, University of
Saskatchewan, Saskatoon, SK
FDA approved poly-(D, L-lactic-co-glycolide), PLGA is
particularly competent due to its low immunogenicity, low
toxicity, biocompatibility and biodegradability. Utilizing
this polymer, a nanoparticulate vaccine delivery system was
developed to treat cancer through packaging of therapeutic
cargoes and delivering them to target immune cells (dendritic
cells, DCs). Structurally modified nanoparticles will be better
able to identify the CD-205 receptors on DCs. Our aim is to
investigate the advantage of introducing the targeting ligand on
the nanoparticle system. Targeting efficiency of nanoparticles
will be confirmed from the DC uptake studies. PLGA
nanoparticles were formulated by O/W emulsification-solvent
evaporation technique. The DC targeting ligand was attached
on the nanoparticle surface through covalently binding in the
presence of spacer molecule (bis sulfosuccinimidyl suberate)
and physical adsorption method. The PLGA nanoparticles had
suitable physicochemical characteristics for in-vitro biological
experiments. Infra-red (IR) studies confirmed the presence of
antibody in the targeted formulation when compared with the
ligand free formulation. DC uptake study showed that when
ligand is covalently attached, higher uptake of nanoparticles was
observed compared to ligands that are adsorbed. This indicates
the strong bond between activated nanoparticle and ligand
selective for CD-205 receptor. Therefore, our target is that the
structurally concealed nanoparticle vaccine delivery system will
be able to perform targeted delivery when administered in-vivo.
Abstracts / Poster presentations
9.
10.
Antimicrobial Compounds from an Endophytic
Fungus of Forest Coniferous Trees of Manitoba
Isolation and Characterization of Anti-Diabetic
Compounds from Tinospora Cordifolia
Tasnim H. Beacon1, Lindsay Bowman2, Paul Holloway2 and
Athar Ata1*
Tharmini Jenny Rathinagopal, Madelyn Attia, and Athar
Ata*
1. Department of Chemistry, University of Winnipeg,
Winnipeg, MB
2. Department of Biology, University of Winnipeg, Winnipeg,
MB
Endophytes are highly diverse microorganisms (primarily fungi)
that inhabit plant tissue in a mutualistic symbiosis with their
host causing asymptotic infections. In fact, endophytes use
plant nutrients for their survival and in turn produce natural
products that help plants in their growth and competitiveness
in nature. These microorganisms are found in all plants that
have been tested for their presence suggesting their ubiquitous
nature. It has been reported in the literature that endophytes
produce natural products with different bioactivities. We
have recently initiated a project dealing with the isolation of
bioactive natural products from an unidentified endophytic
fungus, sample no.-1b12p042. The aim of the project is to
isolate useful metabolites. Pure culture of the sample was
prepared and methanolic extraction was performed to obtain
methanolic crude extract. The crude extract exhibited activity
against Gram-positive bacteria Staphylococcus aureus and
Corynebacterium xerosis. In this poster, chemical and biological
studies on the crude methanolic extract of an unidentified
fungus, sample no.-1b12p042, will be presented.
Department of Chemistry, University of Winnipeg, Winnipeg,
MB
Diabetes mellitus is a common glucose metabolic disorder
throughout the world. A person with this disorder is unable to
produce or maintain proper level of glucose. Elevated glucose
levels in the bloodstream (hyperglycemia), can lead to a host
of chronic complications, including cardiovascular diseases,
blindness, renal failure, and arthritis disease. Presently there
is no proper cure for diabetes. However, one of the most
effective approaches for combatting diabetes is to reduce
alpha-glucosidase activities. Alpha-glucosidase is one of the
enzymes responsible for breaking down disaccharides into
monosaccharides. Inhibitors of alpha-glucosidase have potential
use in the treatment of diabetes mellitus. The stem extract of
Tinospora cordifolia was evaluated for inhibition of the enzyme.
This stem extract can lower blood glucose by influencing the
body’s sensitivity to insulin and is used for treating type 2
diabetes. The reported IC50 value of this methanolic extract
is 3 mg/mL. Our recent phytochemical investigation of this
methanolic extract resulted in the isolation of a triterpenoid,
3-acetylaleuritolic acids. In this presentation, alpha-glucosidase
inhibitory activity of Tinospora cordifolia and structure
elucidation of an isolated natural product will be presented.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
21
Abstracts
Poster presentations
11.
12.
Characterization of the kab operon for
kanosamine biosynthesis from Bacillus cereus
UW85
Designing novel inhibitors of dihydrodipicolinate
synthase
Aarti Bhagwat , Yulia V. Skovpen, and David R. J. Palmer *
Theerawat Prasertanan, Natasha D. Vetter, Rajendra
Jagdhane, and David R.J. Palmer*
Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
Recently, our laboratory discovered a novel kanosamine
biosynthesis pathway in Bacillus subtilis. These enzymes are
encoded in the ntd operon, which was originally proposed
to be synthesize of 3-3’-neotrehalosadiamine (NTD). NTD
is an unusual nonreducing disaccharide antibiotic made up
of 3-amino-3-deoxy-D-glucose, or kanosamine, residues.
Kanosamine itself is known to have antibiotic and antifungal
activity. We now know the ntd operon encodes enzymes making
kanosamine, and the complete NTD pathway is unknown. B.
subtilis is not the only organism containing these enzymes.
The kab operon from Bacillus cereus contains genes encoding
homologs of these enzymes, KabA, KabB, and KabC, although
different functions have been proposed for these enzymes. As
part of an exploration of the biosynthesis of aminoglycoside
antibiotics, we now report on functional studies of all three
enzymes encoded in the kab operon.
22
Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
Dihydrodipicolinate synthase (DHDPS) is an enzyme found
in the meso-diaminopimelate pathway required for lysine
biosynthesis in bacteria and plants. Diaminopimelate is an
essential component of the bacterial cell wall and its absence
results in cell death. This makes DHDPS an essential enzyme
for bacterial cell survival and a target for novel antibiotics, but
to date no potent inhibitors have been reported. Our group has
studied the allosteric inhibition of DHDPS from Campylobacter
jejuni by its natural regulator, lysine. Our plan for developing
novel inhibitors and our progress is described.
Abstracts / Poster presentations
13.
14.
Enzymatic generation of 13C-labelled inositol
isotopomers
Targeted chemotherapy: An innovative approach
against HER2 positive breast cancer
Joseph Smith, Rajendra Jagdhane, Hari Aamudalapalli, and
David R.J. Palmer*
Sams Mohammad Anowar Sadat and Azita Haddadi*
Department of Chemistry, University of Saskatchewan,
Saskatoon, SK
Biosynthesis of inositol is an evolutionarily conserved pathway
throughout the biological kingdom, and the synthesis of
myo-inositol from D-glucose-6-phosphate substrate is a wellunderstood reaction pathway. In this research project however,
13
C-labelled D-glucose-6-phosphate was used as a substrate to
prepare 13C-labelled L-myo-Inositol. This was accomplished
using purified recombinant 1L-myo-inositol-1-phosphate
synthase (MIPS) and myo-inositol-1-monophosphatase
(IMPase) enzyme activity on glucose-6-phosphate on a 50
mg scale. Using 1-13C-, 2-13C-, 3-13C- and 4-13C-D-glucose6-phosphate, we were able to generate 4-13C-, 5-13C-, 6-13C-,
and 1-13C-myo-inositol respectively, as demonstrated by NMR
spectroscopy.
College of Pharmacy and Nutrition, University of
Saskatchewan, Saskatoon, SK
Human epidermal growth factor receptor (HER2) overexpresses
in around 30% of breast cancers which is a suitable target
receptor for targeted chemotherapy. Main purpose of this study
is to formulate Trastuzumab modified HER2 specific drug
delivery system. Physicochemical characterization has been
applied to see the effect of the delivery system against HER2
specific breast cancer cells. Emulsification-solvent evaporation
technique was applied to prepare Docetaxel loaded Poly(D,Llactic-co-glycolide) (PLGA) nanoparticles which was preactivated with homo-bifunctional spacer, bis(sulfosuccinimidyl)
suberate (BS3) to bind covalently with Trastuzumab. The size
of drug loaded nanoparticles was found below 1000 nm after
freeze-drying using no cryoprotectant in the formulation. An
improvement in the particle size (200 to 400 nm), surface
charge (-5 to -25 mV) and polydispersity index (0.16 to 0.86)
was observed after freeze-drying using 0.1% to 10% sucrose
as cryoprotectant. Different BS3 amounts were also used to
prepare plain nanoparticles for measure Trastuzumab binding
efficiency. Size of the modified nanoparticles was found below
900 nm. The physicochemical characteristics of modified
nanoparticles were found within the desired range. 10%
cryoprotectant has been optimized in all the formulations to
preserve the nanoparticles for developing a targeted delivery
system suitable for receptor-mediated endocytosis.
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
23
Abstracts
Poster presentations
15.
Application of Quadrupole-Linear Ion Trap
Mass Spectrometry to the Analysis of Reactive
Metabolites of Nordihydroguaiaretic acid (NDGA)
and its Analogues
Isaac Asiamah and Ed S. Krol*
Drug Discovery and Development Research Group, College
of Pharmacy and Nutrition, University of Saskatchewan,
Saskatoon, SK
Reactive metabolites (RMs) have been implicated in many
drug-induced toxicities including hepatotoxicity. In order to
minimize bioactivation of new chemotypes during the drug
discovery process, a variety of experimental approaches have
been developed and incorporated into early optimization of
lead compounds including routine in vitro evaluation for
RMs formation. Nordihydroguaiaretic acid (NDGA) has
known pharmacological properties but its use is also associated
with toxicities possibly mediated by RMs. In our efforts to
better understand the toxicological mechanisms of NDGA,
we synthesized a number of NDGA analogues and evaluated
their metabolic activation potential with a goal of eliminating
RMs liability through rational structural modification. We
incubated our analogues in rat liver microsomes (RLM) in the
presence of glutathione as nucleophilic trapping agent. We also
investigated their potential to form para-quinone methides
using silver oxide. Glutathione conjugates were detected by
electrospray ionization-mass spectrometry (ESI-MS) scanning
for neutral loss (NL) 129 Da or 307 Da in positive ion mode
or precursor ion (PI) scanning for 272 Da in negative ion mode
and further characterized by liquid chromatography–tandem
mass spectrometry (LC–MS/MS) or in a single LC-MS run
using multiple reactions monitoring (MRM) as a survey scan
to trigger acquisition of enhanced product ion (EPI) data. Our
results show that catechol-type analogues were converted to
ortho-quinones by cytochrome P450s. We saw no evidence
of RMs when phenol-type analogues were incubated with
microsomes although analogue A2 formed a para-quinone
methide in silver oxide oxidation system. Significantly, our
MS techniques provide a simple and rapid approach to easily
distinguish between aromatic and benzylic conjugates which
could be routinely used for screening lignans and related
chemotypes. This is really important in early drug optimization
process for providing insight into the nature of RM without the
need for reference standards or the tedious and time-consuming
isolations for nuclear magnetic resonance (NMR) identification.
24
Abstracts / Poster presentations
3rd WCMCW Attendees
Manal AlMalki
University of Winnipeg
Mays Al-Dulaymi
University of Saskatchewan
mays.al-dulaymi@usask.ca
JKholud Algabbass
University of Saskatchewan
kaa653@mail.usask.ca
Kevin Allen
University of Saskatchewan
kja782@mail.usask.ca
Saniya Alwani
University of Saskatchewan
ssa930@mail.usask.ca
Steve Arns
Centre for Drug
Research&Development
sarns@cdrd.ca
Hanan Elsayed
University of Saskatchewan
hae875@mail.usask.ca
Erin Fenton
University of Saskatchewan
erin.fenton@usask.ca
Kendra Furber
University of Saskatchewan
klf615@mail.usask.ca
Mona Hamada
University of Saskatchewan
mmh882@mail.usask.ca
Fraser Hof
University of Victoria
fhof@uvic.ca
Tasnim Jahan
University of Saskatchewan
stj885@mail.usask.ca
JIsaac Asiamah
University of Saskatchewan
isa356@mail.usask.ca
Saifur Khan
University of Alberta
mdsaifur@ualberta.ca
Kuttiyatveetil, J Ayanath
University of Saskatchewan
jia482@mail.usask.ca
Ed Krol
University of Saskatchewan
ed.krol@usask.ca
Ildiko Badea
University of Saskatchewan
Ildiko.badea@usask.ca
Leah McGurn
University of Saskatchewan
ldm133@mail.usask.ca
TasnimBeacon
University of Winnipeg
Deborah Michel
University of Saskatchewan
dlm137@mail.usask.ca
Aarti Bhagwat
University of Saskatchewan
aarti.bhagwat@gmail.com
Hector Caruncho
University of Saskatchewan
hector.caruncho@usask.ca
Cuylar Conly
University of Saskatchewan
sapiensfuturus@gmail.com
Natasha Milosevich
University of Victoria
nmilosev@uvic.ca
R. Scott Murphy
University of Regina
scott.murphy.uregina@gmail.com
David Palmer
University of Saskatchewan
dave.palmer@usask.ca
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
Chris Phenix
Thunder Bay Regional Research
Institute
phenixc@tbh.net
Brent Pollock
Prairie Plant Systems
wbrp@prairieplant.com
Masoomeh Poorghorban
University of Saskatchewan
map521@mail.usask.ca
Theerawat Prasertanan
University of Saskatchewan
thp785@mail.usask.ca
Josseline Ramos-Figueroa
University of Saskatchewan
jsr303@mail.usask.ca
Tharmini Rathinagopal
University of Winnipeg
Sams Sadat
University of Saskatchewan
sms476@mail.usask.ca
Naheda Sahtout
University of Saskatchewan
nms267@mail.usask.ca
David Sanders
University of Saskatchewan
david.sanders@usask.ca
Yulia Skovpen
University of Saskatchewan
yulia.skovpen@usask.ca
Joseph Smith
University of Saskatchewan
jos425@mail.usask.ca
Geoffrey Tranmer
University of Manitoba
geoffrey.tranmer@umanitoba.ca
Maruthachalam B Vellalore
University of Saskatchewan
vmb512@mail.usask.ca
25
3rd WCMCW Attendees
con’t
Natasha Vetter
University of Saskatchewan
ndv857@mail.usask.ca
Carston Wagner
University of Minnesota
wagne003@umn.edu
Kishor Wasan
University of Saskatchewan
Kishor.Wasan@usask.ca
Stacey Wetmore
University of Lethbridge
stacey.wetmore@uleth.ca
Yazdi Mohi
University of Saskatchewan
mom983@mail.usask.ca
Zalatar Nataliya
University of Saskatchewan
nataliyazalatar@gmail.com
26
Attendees
University of Saskatchewan map
Where to find us
1004, Education building
Parking lot F
From the
Parktown
WCMCW / September 26-27, 2014 / Saskatoon, Saskatchewan
27