Collaborative Drug Repositioning: Case Studies

between Universities, SMEs and Charities

SMEs

University

Charities

UNITE to search for cures for diseases

Dr Farid Khan, Chairman Protein Technologies

People

Paul Goddard

CEO

Dr. Farid Khan

CSO

Paul Goddard over 30 experience in high technology

manufacturing, including 15 years at Ajinomoto &

Commercial Director of CoEBio3 at the MIB, raising

over £6.8 M in IB.

Dr. Khan comes from industry (GSK +OGS) 8 years PhD in

FPs at Cambridge + academic positions (6 years). Winner

of LIFT award for future innovation 2010 (£0.5M). Previous

successful consultancy Lumophore Ltd (£1.8 M).

Took the opportunity of the closure of NeuTech by Novartis-

negotiated hard to obtain a state of the art £multi-million

protein production laboratory-saved the costs of

transportation to Switzerland, decontamination &

decommissioning of instruments.

The Team

4

Supported by a team of world-leading

biotech experts

Dr. Gareth DeBoos

Sorso Ltd.

Dr. Oleg Werbitzky

Lonza Ag

Professor Sir Alan Fersht

University of Cambridge

Dr. Sophie Jackson

University of Cambridge

Professor Roy Goodacre

University of Manchester

Dr. Anil Day

University of Manchester 5

Manchester Institute of

Biotechnology (MIB)

Where ?

PharmaKurePharmaKure

In the heart of

Manchester’s

Knowledge Quarter

• The largest clinical academic campus in Europe

• Engine for city-region’s economy – generates one

quarter of the city’s economic output

• Significant investment and transformation - £2.5bn

capital investment programme committed by 2020

• MSP offers unique proximity and access to world

class knowledge and research base – University of

Manchester, Manchester Metropolitan University

• 72,000 students and largest international student

population in the UK

• Tightly networked with all the leading innovators

and entrepreneur investors

Key Projects

Affordable Drug Discovery

1. Rare Diseases: Pre-clinical studies (Drug Discovery + Protein expression)’

‘The most affordable in the World’.

No charges for instrumentation or lab overheads usage-only staff and consumables.

A New National Centre for Drug Repositioning.

2. New facility for cGMP manufacture of biologics and drugs-partnerships

3. Development of new fluorescent probes for preclinical research

4. Development of new therapeutic probes for cancer

5. Development of biosimilars

6. Partnerships, partnerships, partnerships.

7. Delivered more that £4 million (2011-2014) in services + grants.

Proteins:

New materials, medicines and diagnosis of

disease

Proteins act as the machines of life,

they drive essentially all the

physical and chemical processes of life

(as we know it …)

Proteins are composed of

Just 20 amino acids (encoded

by DNA)

Structure: wool, silk, muscle, skin

Function: blood, enzymes etc.

Classical example of Revolutionary Biotechnology

Insulin is a hormone that regulates the amount of glucose (sugar)

in the blood and is required for the body to function normally.

Animal Insulin

Purified from pancreas of animals

(low yield, high demand and side-effects)

1950-1979

Recombinant Insulin 1980’s to present

Yeast and Bacteria

Fermentation technologyHuman gene Vector

The Drug Discovery Process

Why repositioning?

Many of the steps in drug development, such as determining ADMET properties and phase I

clinical trials, have already been done, hence reducing drug development costs, accelerating

drug development and improving chances of success

•Existing drugs are already known to exert a biological response in humans e.g. For

example, studies of patients taking cholesterol lowering statin drugs revealed an unexpected link

between cholesterol levels and AD, with patients on statins having a lower risk of AD.

Such molecules are already usually known to be bioactive, safe, bioavailable and have FDA

approval for use in humans. In addition, some can cross the blood–brain barrier, which is often

problematic for a drug for neurodegeneration diseases.

Rare Diseases

Challenges and Opportunities for Social Entrepreneurs

Edited by Nicolas Sireau

Chapter 6 Drug repositioning strategies for rare and orphan

diseases: A cost-effective approach of new uses for existing drugs.

Maria P. del Castillo-Frias, Andrew J. Doig and Farid Khan

Clinical features:

Hepatic phenotype, abnormal brain development and function - reduced higher-brain

abilities (executive functions), neuropsychological dysfunction (imbalance of

neurotransmitters) - emotional disturbance and behavioral problems (clinical

depression)

Case Study 1

Phenylketonuria

• BH4 (Tetrahydrobiopterin) (synthetic version) as an alternative treatment for

mild phenylketonuria (N Engl J Med. 2002 Dec 26;347(26):2122-32) .

•Out of 38 with PAH deficiency, 87% showed responsiveness to BH4 (i.e. had

lower blood phenylalanine levels).

•No response in 7 patients with classic PKU • long-term treatment with BH4 in

5 patients increased daily phenylalanine tolerance enough to discontinue

Phe-restricted diet.

• Mutations connected to BH4 responsiveness predominantly in the catalytic

domain of the protein and were not directly involved in cofactor binding.

•60% of PKU mutations are missence mutations i.e. is a point mutation in

which a single nucleotide change results in a codon that codes for a different

amino acid.

Molecular Restoration

Phenylalanine Hydroxylase

Phenylketonuria Blau, Nenad et al.

The Lancet , Volume 376 , Issue 9750 ,

1417 – 1427, 2010

Inactive Active

Tetrahydrobiopterin

Developed Sapropterin (Kuvan)

Biomarin Pharmaceuticals

New Pharmacological Chaperones for PKU

Human Molecular Genetics, 2012, Vol. 21, No. 8 1877–188 2012

Enzyme binding assays= 6 compounds restored unstable PAHV106A variant

Cell based assays = increased its stability against proteolytic degradation.

Mice = During a 3-day treatment study, two compounds (benzylhydantoin

and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe

concentrations of PKU mice (Pahenu1). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin.

Identified two PCs with high in vivo efficacy that may be further developed

into a more effective drug treatment of PKU

Case Study 2

Alkaptonuria

Homogentisic Acid

(HGA)

Build up of HGA- polymerises and oxidises to produce black pigmentation

Eliminated in the urine

•Black urine disease

Deposited of pigment in body tissues

•the knees, shoulders, hips, spine and major joints

•Cause stones in the prostate

•the larynx, trachea and the bronchi

•Eyes, Ear, Nose, Cheeks, Hands and skin tendons

Cause valves to calcify or harden

•the aortic and mitral heart valves

Tyrosine transaminase

-

acid

Phenylalanine hydrolase

Phenylalanine

Tyrosine

p- hydroxyphenyl pyruvic acid-

Homogentisic acid

Maleylacetoacetic acid

p-hydroxyphenylpyruvic

acid oxidase

Homogentisic

acid dioxygenase X

MRC Wellcome Trust

rHuman HGO (in E.coli) is a 280-

residue N-terminal domain & 140-

residue C-terminal domain

Homogentisate 1,2-dioxygenase

(HGO)

Titus et al (2000). Nature Structural Biology 7 (7): 542–546

Associates as a hexamer and is

arranged as a dimer of trimers.

The active site binds an iron atom

which is coordinated near the interface

between subunits in the HGO trimer by a

Glu and two His side chains.

HGO represents a new structural class

of dioxygenases. The largest group of

AKU associated missense mutations

affect residues located in regions of

contact between subunits.

There is 67% predominance of missense HGD mutations in the AKU

patient population

Name Mutation Nucleotide changeAmino acid change/predicted consequence

Patient chromosome code

R53W Missense C324C→T Arg53Trp 45a

V181F Missense C708G→T Val181Phe 44a,b

T196fs Frameshift C754delCFrameshift after Thr198 46a,b

H292R Missense C1042A→G His292Arg 33a

R321X Nonsense C1128C→T Arg321Stop 19b

W322R Missense C1131T→C Trp322Arg 47a,b

INV13+1G→T Splice site C1355+1G→T Aberrant splicing? 22b

There is 67% predominance of missense HGD mutations in the

AKU patient population

GFP-HGO

Design of Human recombinant HGO for expression in E.coli

•N-Terminal His and GFP for quick visualization of the protein

product for expression trials

•Fluorescent tag for tracking the protein in cells (e.g. cellular

uptake)

•Comparison with the WT non-GFP tagged enzyme (e.g. enzyme

kinetics, hexameric structure)

•WB probe for detection of small amounts of proteins in samples

•potential imaging/ diagnostic reagent

WT-HGO

•N-Terminal His6-tag-for purification-cleavage site.

•Immobilization tag for polymers for the slow release

and administration of stable HGO conjugates

• Used to screen drug activation of HGO

•Used to generate monoclonal or polyclonal

antibodies for highly sensitive and specific use in AKU

diagnosis (e.g. ELISA based assays).

Titus et al (2000). Nature Structural Biology 7 (7): 542–546

Design of WT-HGO Gene constructs

WT-HGO DNA Sequence WT-HGO Protein Sequence

atgcatcatcatcatcatcatagcagcggcctggtgccgcgcggcagcgcggaactgaaa

tatattagcggctttggcaacgaatgcagcagcgaagatccgcgctgcccgggcagcctg

ccggaaggccagaacaacccgcaggtgtgcccgtataacctgtatgcggaacagctgagc

ggcagcgcgtttacctgcccgcgcagcaccaacaaacgcagctggctgtatcgcattctg

ccgagcgtgagccataaaccgtttgaaagcattgatgaaggccatgtgacccataactgg

gatgaagtggatccggatccgaaccagctgcgctggaaaccgtttgaaattccgaaagcg

agccagaaaaaagtggattttgtgagcggcctgcataccctgtgcggcgcgggcgatatt

aaaagcaacaacggcctggcgattcatatttttctgtgcaacaccagcatggaaaaccgc

tgcttttataacagcgatggcgattttctgattgtgccgcagaaaggcaacctgctgatt

tataccgaatttggcaaaatgctggtgcagccgaacgaaatttgcgtgattcagcgcggc

atgcgctttagcattgatgtgtttgaagaaacccgcggctatattctggaagtgtatggc

gtgcattttgaactgccggatctgggcccgattggcgcgaacggcctggcgaacccgcgc

gattttctgattccgattgcgtggtatgaagatcgccaggtgccgggcggctataccgtg

attaacaaatatcagggcaaactgtttgcggcgaaacaggatgtgagcccgtttaacgtg

gtggcgtggcatggcaactataccccgtataaatataacctgaaaaactttatggtgatt

aacagcgtggcgtttgatcatgcggatccgagcatttttaccgtgctgaccgcgaaaagc

gtgcgcccgggcgtggcgattgcggattttgtgatttttccgccgcgctggggcgtggcg

gataaaacctttcgcccgccgtattatcatcgcaactgcatgagcgaatttatgggcctg

attcgcggccattatgaagcgaaacagggcggctttctgccgggcggcggcagcctgcat

agcaccatgaccccgcatggcccggatgcggattgctttgaaaaagcgagcaaagtgaaa

ctggcgccggaacgcattgcggatggcaccatggcgtttatgtttgaaagcagcctgagc

ctggcggtgaccaaatggggcctgaaagcgagccgctgcctggatgaaaactatcataaa

tgctgggaaccgctgaaaagccattttaccccgaacagccgcaacccggcggaaccgaac

tga

Histag/Thrombin/HGO2(1-460) [Wild-Type] amino acid sequence Theoretical pI/6.85 Mw / 51636.5

Start codon (green), His-tag (grey), thrombin site (purple), HDO2 (yellow) and stop

codon (red).

10 20 30 40 50 60

MHHHHHHSSG LVPRGSAELK YISGFGNECS SEDPRCPGSL PEGQNNPQVC PYNLYAEQLS

70 80 90 100 110 120

GSAFTCPRST NKRSWLYRIL PSVSHKPFES IDEGHVTHNW DEVDPDPNQL RWKPFEIPKA

130 140 150 160 170 180

SQKKVDFVSG LHTLCGAGDI KSNNGLAIHI FLCNTSMENR CFYNSDGDFL IVPQKGNLLI

190 200 210 220 230 240

YTEFGKMLVQ PNEICVIQRG MRFSIDVFEE TRGYILEVYG VHFELPDLGP IGANGLANPR

250 260 270 280 290 300

DFLIPIAWYE DRQVPGGYTV INKYQGKLFA AKQDVSPFNV VAWHGNYTPY KYNLKNFMVI

310 320 330 340 350 360

NSVAFDHADP SIFTVLTAKS VRPGVAIADF VIFPPRWGVA DKTFRPPYYH RNCMSEFMGL

370 380 390 400 410 420

IRGHYEAKQG GFLPGGGSLH STMTPHGPDA DCFEKASKVK LAPERIADGT MAFMFESSLS

430 440 450 460 50 60

LAVTKWGLKA SRCLDENYHK CWEPLKSHFT PNSRNPAEPN*

Note WT sequence has 12 cysteines!Codon Optimisation:

Welch et al. (2009) Design Parameters to Control Synthetic Gene

Expression in Escherichia coli. PLoS ONE 4(9): e7002.

Plasmid Vector pJExpress414 was used to insert the HGO genes

for recombinant expression in E.coli.

Protein extracted

and purified

Inserted and grown in bacteria

(E. coli)

HGO Protein Variants

SDS-PAGE Analysis

WT-HGO(50 kDa)

GFP-HGO(80 kDa)

UV excitationFluorescence

Yields 5mg/litre

Circular dichroism (CD)

Differential absorption of left and right

circularly polarized light

CD spectra can be used to estimate

the fraction of a protein that is in the

alpha-helix conformation and the

beta-sheet conformation.

Analysis of WT-HGO shows a characteristic

U-shape between 200-250nm.

This indicates presence of secondary structure

i.e. alpha-helix and the beta-sheet conformation

Enzymatic Reaction of

Homogentisate 1,2-dioxygenase

(HGO)

Homogentisic Acid(HGA)

Maleylacetoacetic Acid(absorbance at 330 nm)

HGO KINETICS

WT-HGO

GFP-HGO

High HGA Low HGA

High HGA Low HGA

WT-HGO

[Substrate]0 0.2 0.4 0.6 0.8 1 1.2 1.4

Rate

0

0.02

0.04

Parameter Value Std. Error

Vmax 0.0439 0.0018

Km 0.0331 0.0064

WT-HGO Enzyme Kinetics

Data

1 / [Substrate]

0 20 40 60

1 /

Rate

0

20

40

60

[Substrate]0 0.2 0.4 0.6 0.8 1 1.2 1.4

Rate

0

0.02

0.04

Parameter Value Std. Error

Vmax 0.0411 0.0013

Km 0.0241 0.0040

WT-HGO Enzyme Kinetics

Data

1 / [Substrate]

0 20 40 60

1 /

Rate

0

20

40

60

Vmax is a measure of how fast the enzyme can go at full speed (40 uM/min)

Km is a measure of roughly how much substrate is required to get

to full speed (30μM)

mM mM

Screening of ~ 1000 known drugs

Discovery of a three candidate

repositioned drugs (Oral) for AKU

Next Steps

1. Test the compounds in mutant enzymes and measure activity.

2. Test the compounds in cell based models or clinical samples for increase in activity

3. Trial the compounds in mice models

4. Go straight to human trials (if drug toxicity is known, prescribe off label)

Funds needed to fast track these drugs

Antimalarial Drug Repositioning

Case Study 3

Malaria New figures from Public Health England (PHE) on World Malaria Day show an overall

decrease of 18% in imported malaria infections reported in the UK in 2012 (1,378)

compared with 2011 (1,677).

Malaria killed at least 1.2 million people worldwide in 2010

Salford UniversityDrug Libraries

Need for a new approach

Malaria burden

1- 3 million deaths per year

Majority in children under 5

Problems

No effective vaccine available

Insecticide-resistant mosquitoes

Drug-resistant parasites.

Source: WHO, 2009

Artemisinins: the last hope for

malaria treatment

Drug resistance

WHO recommends: Artemisinin based combination therapy (ACT)

Source:(Ekland & Fidock, 2008)

Drug Libraries and assay development

Flow CytometerClass 3 SafetyAccess human blood &

plasmodium parasite culture

Human resources:

PI: Expertise in Malaria

Technicians + Students

Partnerships

Pre-optimisation of assay with DHA

Optimisation of SG Flow

cytometer assay (SG-FCM)

Comparability between SG-MTT, SG-FCM

and Giemsa assays

0

50

100

InfectedControl

1.25nMDHA

2.5nMDHA

5nMDHA

10nMDHA

20nMDHA

40nMDHA

Pa

ras

ite

mia

(%) Schizont

Trophozoite

Ring

0

50

100

Infectedblood

1.25nMDHA

2.5nMDHA

5nMDHA

10nMDHA

20nMDHA

40nMDHA

Flu

ore

sc

en

ce

(%

)

0

50

100

Bloodonly

Infectedblood

1.25nMDHA

2.5nMDHA

5nMDHA

10nMDHA

20nMDHA

40nMDHA

Pa

ras

ite

mia

(%

)

Multinuclear

Mononuclear

Methods adapted from Karl et al., 2009

0

1

2

3

4

5

6

7C

ontr

ol

0.6

3n

M D

HA

1.2

5n

M D

HA

2.5

nM

DH

A

5n

M L

um

065

1n

M L

um

067

1n

M L

um

066

10

nM

Lum

066

2n

M L

um

068

20

nM

Lum

068

1n

M L

um

069

10

nM

Lum

069

5n

M L

um

065 +

0.6

3 D

HA

5n

M L

um

065 +

1.2

5 D

HA

InM

Lu

m0

67 +

0.6

3 D

HA

1n

M L

um

067 +

1.2

5 D

HA

1n

M L

um

066 +

0.6

3 D

HA

1n

M L

um

066 +

1.2

5 D

HA

10

nM

Lum

066

+ 0

.63 D

HA

2n

M L

um

068 +

0.6

3 D

HA

2n

M L

um

068 +

1.2

5 D

HA

20

nM

Lum

068

+ 0

.63 D

HA

1n

M L

um

069 +

0.6

3 D

HA

1n

M L

um

069 +

1.2

5 D

HA

10

nM

Lum

069

+ 0

.63 D

HA

Para

sit

em

ia (

%)

Multinuclear

Mononuclear

Compound leads using FACS assay

DHA Single

Compounds

DHA Combinations

066069

068

066

0

1

2

3

4

5

6

Blo

od

on

ly

Infe

cte

dco

ntr

ol

10n

M

20n

M

30n

M

40n

M

50n

M

60n

M

70n

M

80n

M

90n

M

100

nM

Para

sit

em

ia (

%)

Lum067 dose response

Multinuclear

Monnuclear

Single dose Lum067 Dose Response

Emetine dihydrochloride hydrate, an anti-protozoal drug previously used for

intestinal and tissue amoebiasis was shown to have potent inhibitory properties

(IC50 doses of ~ 47nM) in the multidrug resistant K1 strain of P. falciparum.

Acknowledgements

Drug Libraries

Ms. Holly Matthews

Dr. Niroshini Nirmalan

Ms Maryam Usman-Idris

Dr. Martin Read

Mr. Santosh Dacha

Paul Goddard

Ms Emily Stott

Ms Lovely Dey

Funding and Support

Mr. Jeff Wang

Dr. Nick Sireau

Anne Dornan

Rowena Burns