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THEME 2 ORAL PRESENTATION ABSTRACTS

Mr. Xavier Chee Wezen

Department of Pharmacology, University of Cambridge

Email : [email protected]

The Voodoo of Virtual Screening – Not As Black As You Think

Experimental biologists have traditionally been skeptical on the application of computational methods in

drug discovery. Such notion is due to the misconception that computational scientists use computer

algorithms as a form of black box to make predictions about biological systems. However, with the

drying up of drugs in the developmental pipeline of pharmaceutical companies, the application of in

silico methods for drug predictions i.e. virtual screening may hold the very key to discovering new drugs.

Here, we attempt to show that using appropriate validation protocols and proper understanding of

biological systems, computational methods can be a formidable arsenal in discovering novel lead

compounds against various targets. We exemplify out belief by giving two examples on the application

of computational tools for drug discovery in two very distinct biological systems: antibacterial proteins

and human calcium signaling channel.

It was estimated that by 2050, 10 million individuals would die annually due to antimicrobial resistance.

Coupled with the decline of approved antibiotics by the FDA, the antimicrobial research field is thrown

into a dire need of discovering new and novel antimicrobial targets and drugs. Lipoteichoic acid synthase

(LtaS) is one such biologically attractive targets in Gram Positive bacteria. Using computational

modelling, we discovered two lead compounds that may potentially act on LtaS. Incubation of the lead

compounds at 100𝜇M against S. aureus and E. coli has shown phenotypical effects of an LtaS inhibitor –

reduction in growth at 42˚C and inhibition of biofilm formation. Further studies using immuno-blotting

assay and thermal shift binding assy are required to confirm mode of action.

Inositol-triphosphate receptor (IP3R) is a ubiquitous calcium signaling channel that is important in the

human physiology. Defects in the IP3R calcium signaling pathway leads to neurodegenerative diseases

such a Huntington’s disease and Alzheimer’s disease. Nevertheless, the lack of a selective, cell-

permeable IP3R inhibitor has hampered the progress in understanding the role of calcium signaling in

these disease till today. Using similar computational techniques, we show two lead compounds that are

able to reduce Carbachol-induced calcium release in human celled at 100𝜇M by conducting intact cell

calcium imaging studies. We aim to perform permeabilized cell calcium imaging assay in the near future

for further confirmation of direct IP3R inhibition.

Keywords: Virtual Screening; Drug Discovery; Computational Pharmacology

mailto:[email protected]


Mr. Izzat Fahimuddin Mohamed Suffian

Institute of Pharmaceutical Science, King’s College London


HER2-Targeted Hepatitis B Virus Core Particle as Nanocarriers to Treat Cancer

INTRODUCTION

Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer

therapy. HBc particles are hollow nano-particles of 30-34 nm diameter and 7 nm thick envelopes, consisting of 180-

240 units of 21 kDa core monomers. They have the capacity to non-specifically bind various cell types via the action

of arginine-rich domain. This study focussed on the development of functional nano-assemblies for therapeutic

applications. Herein we developed a genetically modified HBc particles to specifically recognise and target human

epidermal growth factor receptor-related 2 (HER2)-expressing cancer cells. Our recombinant HBc particles are

designed in such a way that non-specific binding property is reduced, via deleting C-terminal 150-183 aa part of the

core protein that encodes arginine-rich domain (ΔHBc). HER2 target-cell-specific recognition was acquired

genetically by inserting a ZHER2 affibody sequence into the 78-81 aa position of the core protein (ZHER2-ΔHBc).

METHODS

Wild type HBc, recombinant ΔHBc and ZHER2-ΔHBc particles were expressed and purified in E.coli expression system

and Urea-DTT dialysis method. To check the specificity of HBc particles, Western blot analysis was performed using

anti-His6 antibody. Nanodrop UV spectroscopy was used to measure the protein concentration. Atomic Force

Microscopy (AFM) were used to characterise HBc particles morphology and size, respectively. To evaluate the

binding affinity to HER2-expressing breast cancer cells, cancer cells were treated with fluorescently labelled Alexa

Fluor™ 488 HBc, ΔHBc and ZHER2-ΔHBc particles at different concentration (10, 20 & 40 µg/ml).

RESULTS

Western blotting results confirmed the presence of specific protein bands at the desired positions (HBc, 21 kDA;

ΔHBc, 17 kDa and ZHER2-ΔHBc, 24 kDa). AFM confirmed the spherical structures of all assembled HBc particles

(HBc, 33.77 ± 4.58 nm; ΔHBc, 30.24 ± 2.87 nm and ZHER2-ΔHBc, 32.41 ± 2.33 nm). Flow cytometry confirmed the

non-specific uptake of wild type HBc by cancer cell lines in dose-dependent manner. ΔHBc particles showed

reduced median fluorescence intensity (MFI) of all cells, supporting the hypothesis that arginine-rich domain

deletion reduces the non-specific binding ability of the wild type HBc Specific uptake in HER2 positive cells treated

of ZHER2-ΔHBc particles was confirmed indicated by the increase in MFI by flow cytometry.

Keywords: Hepatitis B Virus core particles, HER2 targeting, HBc



Mr. Alvin Teo

School of Life Sciences, University of Warwick


Detergent-free approach to the studies of bacterial cell division membrane proteins

Membrane proteins represent a subset of proteins embedded in or associated with the biological

membrane. Despite accounting for 30% of the natural gene transcripts, and 50% of current

pharmaceutical targets, structural and functional studies of membrane proteins still lag behind soluble

proteins, largely due to the various challenges in isolation and purification of proteins from their native

lipidic environment. Conventionally, detergents have been actively employed for the extraction of these

proteins from the lipid membrane, with their subsequent solubilisation in mixed micelles. However, the

surrounding lipids could be sequestered during this process, which is potentially denaturing to the

proteins.

A novel method exploiting the styrene maleic acid (SMA) copolymer for synthetic or biological membrane

solubilisation (in absence of detergents) results in the generation of SMA/lipid particles (SMALP) or ‘native

nanodiscs’ of membrane proteins with surrounding lipid moieties. Besides facilitating the encapsulation

of target membrane proteins in their native environment, direct biochemical analysis of native protein-

lipid interaction and the profiling of lipid composition can be implemented. This technique has been

successfully applied to several membrane proteins in the divisome of the Gram-negative model bacteria

– Escherichia coli. Among the membrane phospholipids in the E. coli, the anioninc phosholipids like

phosphatidylglycerol and cardiolipin are implicated for the contractile (Fts)Z-ring formation during cell

division. Cardiolipin enrichment at the division site has also been reported, potentially providing the

localisation cues for specific cell division proteins due its biophysical properties, by inducing negative

curvature and lowering the energetic barrier, for constrictive to occur.

A robust lipidomics method utilising liquid chromatography-tandem mass spectrometry (LC-MS/MS) have

been developed to elucidate the lipid composition of key bacterial cell division proteins. We report the

first lipid profiling of cell division proteins ZipA and FtsA, both of which are essential in E. coli, to anchor

the FtsZ proteins to the membrane, forming the initial proto-ring at the middle of actively dividing cells.

This versatile lipidomics method facilitated by the SMA copolymer extraction of membrane proteins can

be a useful bioanalytical tool for protein-lipid interaction studies to better understand the structure and

function of the membrane proteins of interest in their native environment, which may provide vital

information to underpin the development of the next generation antimicrobial drugs.

Keywords: Bacterial cell division; SMALP/native nanodisc; Antimicrobial resistance



Dr. Adli Ali

Nuffield Department of Surgical Sciences, University of Oxford

Email : [email protected], [email protected]

Assessing the validity of Bilispect®, a non-invasive bilirubinometer in estimating total serum bilirubin

in neonates

Background : Neonatal hyperbilirubinaemia is a common problem affecting more than 75% of Malaysian

babies by the first week of life. Although estimation of serum bilirubin by visual inspection of the skin or

sclera is rapid and cost-free yet it is not accurate. A number of non-invasive devices to measure

transcutaneous bilirubin have been extensively studied over the years; nevertheless there have been various

findings in relation to previous generation of transcutaneous bilirubinometers with limited data in multi-

ethnic Asian newborns. Bilispect® (MBR Optical Systems GmbH & Co KG, Germany) is a new device that has

been recently developed for non-invasive bilirubin measurement. It offers quantitative bilirubin

measurement both in the blood as well as in the skin by the use of reflection spectroscopy technology.

Objectives : The aim of this study was to determine the correlation between the estimates of serum bilirubin

using the Bilispect® and venous bilirubin measurements using diazo-based method among neonates with

unconjugated hyperbilirubinaemia. We also compared the estimates of Bilispect® transcutaneous bilirubin

between covered and uncovered skin sites with venous bilirubin after commencement of phototherapy.

Methods :This was a prospective observational study carried out in the post-natal wards, neonatal intensive

care unit and emergency department of Universiti Kebangsaan Malaysia Medical Center. Each neonate

indicated for bilirubin measurement will be approached for consent and transcutaneous bilirubin

measurement will be performed simultaneously within 30 minutes of venous bilirubin measurement.

Results: Three hundred and twenty neonates were recruited of which, 58.4% were males. The mean birth

weight was 2988g and the mean gestation was 38 weeks. From this pool, a total of 138 neonates were

recruited for estimates of bilirubin on covered and uncovered sites after initiation of phototherapy. There

was a strong correlation between transcutaneous and bilirubin (r = 0.67, r2=0.44, p < 0.0001). However,

moderate correlation was observed between transcutaneous and venous bilirubin on both covered and

uncovered sites after phototherapy. The correlation between transcutaneous bilirubin on covered site and

venous bilirubin was better (r = 0.490, r2=0.27, p < 0.0001) as compared to between uncovered site and

venous bilirubin (r = 0.337, r2=0.09, p < 0.0001). Conclusion : Bilispect® is a reliable non-invasive screening

device in estimating venous bilirubin level. However, the reliability of the device is reduced after

phototherapy is initiated.

Keywords: Neonatal hyperbilirubinaemia, Non-invasive bilirubinometer, Phototherapy




Ms. Nor I A Muhsin

Department of Biochemistry, University of Oxford


gemuk – A Novel Transgenic Mouse Model with Pcsk1 Gene Mutation Causing Early Onset Monogenic

Obesity

Background

Statistically, 45% of Malaysian men and almost half of Malaysian women are overweight or obese (BMI>25) and

the national love for nasi lemak is not the only predisposition to the problem. It is widely documented that

genetic factors play a major role in determining adiposity. An autosomal recessive mutation in proprotein

convertase subtilisin/kexin type 1 (PCSK1) gene causes extreme childhood obesity, severe congenital

malabsorptive diarrhea and endocrine abnormalities in human. gemuk, a transgenic PCSK1 mutant mouse is

generated to help us to elucidate the functionality of the gene and the causal pathways of the disorder.

Objectives

The aims of this study are to fully characterise the gemuk mouse and establish a potential mechanism on how

mutation in PCSK1 gene results in obesity as observed in human patients.

Methods

A comprehensive phenotyping pipelines was used to characterise the phenotypes according to EMPReSS

standard protocols while genetic analysis utilises the Next Generation Sequencing (NGS) and pyrosequencing

technologies for high-throughput genotyping procedures. Subsequently, genetically engineered mutant PCSK1

protein was generated for in-vitro studies in order to understand its functionalities.

Results

Mutant gemuk mice are significantly obese from 8 weeks of age as a result of hyperphagia and metabolically

less efficient. This is due to improper processing of POMC into α-MSH by PCSK1 protein causing a disruption in

appetite regulation. Mutant gemuk mice also have incomplete processing of other downstream endocrine

prohormones such as proinsulin and proglucagon into active insulin and GLP-1. Mutant PCSK1 protein was found

to be retained in the endoplasmic reticulum which eventually been degraded by the proteasome. We also

observed that approximately 25% of the mutant gemuk mice develop diarrhea between 6 to 8 weeks of age,

which resolved within two weeks.

Conclusion

Mutant gemuk mice phenotype mimic human disease with mutation in PCSK1 gene. This mouse model will be a

valuable resource in understanding prohormone processing defects that caused monogenic obesity in human.

With full understanding of these mechanisms, it is possible to develop novel therapeutic strategy in the

management of obesity.

Keywords: Monogenic obesity, mouse model, PCSK1 mutation



Mr. Elijah Mak

University of Cambridge

Charting the trajectory of brain changes in Parkinson’s disease before dementia

Cognitive impairment is often associated with Parkinson’s disease (PD) and many PD patients will

eventually develop mild cognitive impairment (MCI) and dementia (PDD). It is therefore important to

determine early brain changes that are associated with progression to dementia as this may allow us to

identify PD patients who are at risk. In this study, I present recently published data from a longitudinal

study where we followed 105 PD patients and 37 healthy controls over 18 months and compared the

trajectory of structural brain atrophy. An extensive neuropsychological assessment allowed the

stratification of the PD sample into subgroups of PD patients with no cognitive impairment (PD-NC) and

PD patients with mild cognitive impairment (PD-MCI). At baseline, severe thinning of the cortex was found

in PD-MCI although there were no differences between PD-NC compared to PD-MCI and healthy controls.

Over 18 months, we also found increased rates of cortical thinning in both PD-MCI (frontal, temporal and

parietal regions) and PD-NC (frontal regions) groups compared to healthy controls. In conclusion, an

extension of cortical thinning in the temporo-parietal regions in addition to frontal atrophy could be a

biomarker in therapeutic studies of PD-MCI for progression towards dementia.


Ms. Melissa Suet Tyng Tiong

Department of Health Psychology, King’s College London


Cross-cultural Comparison between Illness Perceptions in Type 2 Diabetes between British and

Malaysian using the IPQ-RH: A Cross-Sectional Study

Type 2 diabetes is an illness that is becoming more prevalent in this day and age due to increase in rich

diets and sedentary lifestyles. This is an ongoing cross-sectional study which aims to study the

perceptions of healthy people towards Type 2 diabetes and the cross-cultural differences between

Malaysian and British non-diabetics, using the Revised Illness Perception Questionnaire for Heathy

People (IPQ-RH), which is based on Leventhal’s Self-Regulatory Mode of illness perceptions. As the IPQ-

RH has not been validated in Malay or in Mandarin communities, this study will be the first to test the

validity and robustness of the questionnaire in the diabetes context. Participants perform the study

online using the Bristol Online Survey (BOS) tool, and are allowed to choose to do it either English or

Bahasa Malaysia. Participants are asked to enter demographic information before performing the

questionnaire. Quantitative analyses in the form of confirmatory and exploratory factor analyses will be

done to assess the factor structure of the IPQ-RH in Malaysian communities, and to compare it with the

current factor structures of the IPQ-RH. If the analysis do not find significant differences between

current and previous factor structures, it shows that the IPQ-RH is potentially applicable to the

Malaysian population as a tool to tap into healthy people’s illness perceptions. More replication of the

study looking at the use of the IPQ-RH in other illnesses is required. Due to the quantitative nature of

the study, the held perception is Malaysian and British non-diabetics towards diabetes cannot be

studied in detail, and the cross-sectional nature only shows perceptions helps at a certain time point.

Therefore, the results will need careful interpretation. This is an area that needs further study with more

time and resources before the results can be applied to health services (e.g. in the form of

interventions).

Keywords: Diabetes, perceptions, IPQ-R



Ms. Natasha Hui Jin Ng

Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford


Translating genetic association signals for type 2 diabetes-related traits into biological mechanisms

The understanding of the genetics of common complex disease such as type 2 diabetes (T2D) has

improved over the past decade with advances in sequencing technologies and access to large sample

sizes through international collaboration. More than 100 regions in the human genome have now been

associated with T2D and its related traits. However, the rate at which we learn about novel biological

mechanisms is comparatively slower. In particular, the G6PC2 locus harbours the strongest association

signals for fasting plasma glucose (FG) levels in healthy adults. We therefore aim to gain further insight

into the role of G6PC2 by establishing clearer links between genetic variation and protein function at

this locus.

By combining whole exome sequencing (of 12,940 individuals across multiple ancestries) and previously

published exome array data (from 33,407 non-diabetic Europeans), we identified a catalogue of coding

variants in G6PC2. Non-synonymous G6PC2 variants were assessed for protein expression and enzymatic

activity.

We identified 69 G6PC2 coding variants in our sequence data, mostly rare (minor allele frequency <

0.5%). G6PC2 encodes the glucose-6-phosphatase catalytic subunit specific to the pancreatic islets. We

prioritised 14 variants for follow-up and found that 79% of them exhibited markedly reduced protein

levels (P<0.001) in HEK293 cells due to enhanced proteasomal degradation. Replication in the rat

insulinoma cell line INS-1 revealed that many of the variants had substantial reduction of the

glycosylated form of the protein. One variant (I171T-G6PC2), which was expressed at levels similar to

wild type protein, had decreased enzyme reaction rate (Vmax) by ~40% (P=0.01). These results confirmed

that the 5 variants with evidence for reducing FG levels were indeed loss-of-function variants.

Overall, by combining genetic analyses with validation in molecular and cellular systems, we have

identified multiple coding variants in the G6PC2 gene that affect protein function through altered

protein stability or activity. This was consistent with its role in glucose homeostasis, and established

G6PC2 as a key regulator of pancreatic beta cell function. Improved understanding of such effector

transcripts will open up opportunities for the exploration of new therapeutic targets for T2D.

Keywords: type 2 diabetes, exomes, variant characterisation



Mr. Muhammad Kaiser Abdul Karim

Clinical Neurosciences, University of Cambridge


High-content screen using human pluripotent stem cell-derived forward-programmed

oligodendrocyte precursor cells

Myelin disorders like Multiple Sclerosis and transverse myelitis remain a subject of great interest to

researchers because current clinical treatment do not target remyelination, a process where

oligodendrocytes in the brain reinsulate axons that lost myelin due to disease. This requires the

production of new oligodendrocyte precursor cells (OPCs) within the brain. They are recruited to regions

of demyelination and undergo differentiae into mature oligodendrocytes; however, in a disease this

process, from recruitment to differentiation, becomes inhibited or inadequate. Naturally, OPCs have

become a promising target for cell- and drug- based therapies. The advancements in stem cell

technology has paved the way for numerous strategies for producing stem cell-derived OPCs and their

subsequent mature state. Previously, these protocol required extended periods of cell culture and in

some, produced mixed populations. Moreover, most of them involved animal stem cells. One possible

solution is to reprogram pluripotent stem cells (PSCs) directly into OPCs, a technique called forward

programming. By inducing the right selection of transcription factors, it is possible to drive the formation

of OPCs directly from PSCs in a much shorter time period. With this protocol, large scale generation of

homogenous population of human-derived OPCs becomes possible, along with any high-throughput

goals. One such goal would be to screen a library of drugs on the cells, which has never been achieved

with human0derived OPCs. It will be a quick and effective way of uncovering new pharmacological

therapies for enhancing remyelination and potentially open-up new insights into its biology.

Keywords: Oligodendrocytes; oligodendrocyte precursor cells; remyelination; pluripotent stem cell;

neural stem cell; Glial progenitor cell; Cell reprogramming; High-content screening; Multiple Sclerosis;

Demyelinating disease



Dr. Hui Yee Chee

Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia

Email : [email protected] ; [email protected]

Clinical predictors of dengue fever co-infected with leptospirosis in patients admitted for dengue fever

Dengue and leptospirosis infections are two infectious diseases present in Malaysia with dengue causing

major endemic. Overlapping clinical symptoms for both diseases often causes misdiagnosis and

confusion of treatment. Therefore, we investigated the incidence of leptospirosis co-infection among

dengue confirmed patients and identified significant parameters to predict occurrence of co-infection.

268 serum specimens from patients diagnosed for dengue fever were subjected for dengue virus

serotyping by real-time PCR. Clinical, laboratory and demographic data were extracted from the hospital

database to identify patients with confirmed leptospirosis among the dengue patients. Frequency of co-

infection was calculated and association of the dataset with dengue-leptospirosis co-infection was

statistically determined. DENV 1 was found to be the predominant circulating serotype from August

2014-August 2015. The frequency of dengue co-infection with leptospirosis was 4.1%. Male has higher

preponderance to developing the co-infection and shock as clinical symptom has the predictive value.

The increasing incidence of leptospirosis among dengue infected patients has posed the need to

precisely identify the co-infection without mistakenly ruling out either one of the diseases. Laboratory

result for leptospirosis confirmation takes longer time, therefore the preliminary predicting value

identified in this study may be able to provide some clue for co-infection. However, future study involve

more centers and patients with co-infection need to be performed to assess the usefulness of the

predictive symptom.

Keywords: Dengue fever, leptospirosis, Malaysia



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