Lifting the Lid on Academic Laboratory Automation

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Hit and Lead Discovery in an Academic Setting

• Julie Frearson
• Iain Collie
• University of Dundee

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Introduction

• The facility
• Current approaches technologies
• Progress

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A unique, fully integrated drug discovery team
within a world class research environment





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Sir James Black Centre
The recently constructed James Black
Centre housing, on level 1, the Drug Discovery
Unit
www.drugdiscovery.dundee.ac.uk
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Vision, Mission, Key Objectives

• Vision
• To be an internationally recognised drug
  discovery group and partner of repute for the
  biopharmaceutical sector in developing new
  therapeutics for unmet need
• Mission
• To increase the efficiency and success of
  translating life sciences research into
  therapeutic application
• Key Objectives
• To translate basic biomedical research into
  candidate medicines for neglected diseases
• and
• To vastly improve intellectual property positions
  on novel therapeutic targets through small
  molecule discovery

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Primary Activities
Novel Targets Pathways
Neglected Diseases
DDU
Hits Leads
Leads preclinical candidates
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Drug Discovery _at_ Dundee

• Total Funding over 5 years 12m
• 67 Wellcome Trust
• Remainder from
• Scottish Funding Council
• Scottish Enterprise
• ITI Life Sciences

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Key Successes

• Created a small Biotech style drug discovery
  group
• Operational within 4 months of opening

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Key Successes

• Created a small Biotech style drug discovery
  group
• Operational within 4 months of opening
• Creation of purpose designed compound libraries
• gt97,000 compounds diversity, bioactives, gene
  family focused
• Prosecution of 18 target or phenotypic screens
  through hit discovery
• 7 projects through hit to lead

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DDU Portfolio Nov 2008
Hit Discovery
Hits to Leads
Lead Optimization
Target validation
Assay Development
Hit Validation
N-Myristoyl- transferase
Sugar dehydrogenase
De-N-acetylase
PS-Q series
Focussed kinase set phenotypic screen hits
Phenotypic screening hit
Kinase
Kinase
PK4
Chemical target validation
KPS Series 01
Kinase
Chemical target validation
RNA Ligase
GSK
GSK 07
Kinase
PK50
Viral protease
Trypanothione synthetase
Chemical target validation
Replication licensing inhibitors
Chemical target validation
NF?B repressor RNAi screen
Fungal targets 2,3,4
Phosphatase inhibitors
SUMO repressor RNAi screen
miRNA regulator RNAi screen
Studies on hold and project returned to
originating lab to address no go issues
PTR1/DHFR
UDP-Glc-4- epimerase
CRK3
Trypanothione reductase
UDP-GlcNAc diphosphorylase
Fungal Target 1
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Key Successes

• Created a small Biotech style drug discovery
  group
• Operational within 4 months of opening
• Creation of purpose designed compound libraries
• gt97,000 compounds diversity, bioactives, gene
  family focused
• Prosecution of 18 targets/phenotypics screens
  through hit discovery
• 7 projects through hit to lead
• Advanced leads developed
• Series curing trypanosomiasis in mice
• Series under investigation in cancer xenograft
  models
• First group to be routinely screening in high
  throughput using hES cells

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Balanced Approach to Hit Discovery
P. falciparum
T. brucei
Molecular Target
hES cells
keratinocytes

• Diverse and flexible approach to lead discovery
• Not limited to any one technology or target bias
• Polypharmacology required for good sustained
  efficacy?

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Data Assurance at Dundee

• Our Policy
• Dedicated Database Informatics Manager
• Quality responsibilities throughout group
• Compound selection for libraries rigorous
• Naming conventions
• Barcode tracking of compounds plates
• Verify instrument performance (QC) routinely
• Standardised plate formats layouts
• QC plates reference standard per plate

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Drug Discovery for Tropical Diseases initiative
(DDTDi)- Goals

• To deliver at least 1 drug candidate for human
  African trypanosomiasis (HAT) for entry into
  formal pre-clinical development by March 2011
• To deliver high quality lead series for neglected
  diseases suitable for progression into lead
  optimisation in-house or by partner organisations

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Tropical Infectious Disease Burden

• 35,000 deaths per day in developing world
• Many of the deaths preventable
• Many current drugs need to be replaced due to
  toxicity, cost, emerging resistance, suitability
  to use in the developing world
• Health care budget in Sub-Saharan Africa
• 13 per capita per year
• No commercial market to offset 800m cost and
  risk to develop new drugs
• The solution does not lie solely with the
  commercial sector

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DDTDi Strategy

• Work in collaboration with academic, industrial
  and PDP partners
• Engaging in small focussed consortia to enable
  Drug Discovery for kinetoplastids from target to
  clinic
• Co-ordination with DNDi and SGC for lead
  generation
• Aligning ourselves with major downstream partners
• DNDi
• MMV

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DDTDi Strategy

• Portfolio approach
• Genetically validated parasite drug targets
• Chemical validation of targets with drug
  discovery precedence
• Phenotypic screening

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Chemical Validation

• Not sufficient number of genetically validated
  targets to support a drug discovery pipeline
• Many genetically validated targets are not
  druggable
• Traditional method of research will not address
  this issue going forward
• A large scale RNAi project not supported US/UK
• Need to identify other ways of identifying
  potential drug targets
• Seeking to chemically validate known drug
  discovery targets developed for other indications
• Potential targets across a wide range of
  parasites
• Seeking inhibitors from literature, but more
  importantly from companies with DD Projects

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Gene Family Approach

• Kinases

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Kinases as Drug Targets for T. brucei

• Known druggable targets
• Increasing genetic evidence of essentiality of
  kinases in trypanosomes and other parasites
• Evidence of inhibitor selectivity between T.
  brucei and human homologues
• Non-selectivity within tryp kinases an advantage
• Acute dosing regimen for HAT could limit toxicity
  concerns

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DDTDi Strategy

• Developed focused kinase library
• 5000 compounds
• gt150 scaffolds with even representation
• Tackle known genetically validated kinases in
  parallel
• Collaborate with expert biology groups
• Multiple kinase enzyme assays in parallel
• Phenotypic screening against T. brucei, malaria
  and TB in vitro

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Kinase Targets Under Investigation
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Kinase Target Selectivity
PK4 70 confirmation rate Final hit rate 5.4
PK50 91 confirmation rate Final hit rate
12.8 TbGSK3 91 confirmation rate Final hit
rate 12.6
GSK3 project advanced
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Target vs. parasite proliferation - correlation?
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Lack of trypanosome activity
T. brucei potency pEC50
T. brucei potency pEC50
CRK3 potency pIC50
CRK3 potency pIC50
No evidence of Leishmania in vitro activity
either (Mottram et al.)
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Kinase Set Phenotypic Screen

• Screen
• Kinase Set
• 3,885 compounds
• 121 compounds progressed to potency
• Multiple series
• lt1µM EC50
• gt10-fold selectivity
• Best
• T. brucei EC50 20nM
• MRC-5 EC50 gt50?M
• Following up in chemistry and DMPK
• Investigating Serenex, Cellzome technology to
  identify potential targets

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N-Myristoyl Transferase Project Leaders
Stephen Brand, Stuart McElroy Debbie Smith
(York) Parasitology, Mode of action studies
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NMT Introduction

• Catalyses the transfer of myristate from MyCoA to
  the N-terminal glycine NH2 of potentially gt60
  target proteins
• Myristoylation important for localisation at
  membranes and/or activation
• Strong biological chemical evidence for NMT
  being a viable target

• Screened 63,362 compounds within DDU
• Identified one key compound series (5 examples
  lt20µM)
• Hit validation DDD00064558 resynthesised and
  retested

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Summary so far

• Identified sub nanomolar inhibitors of T. brucei
  NMT and T. brucei proliferation in culture
• Good correlation - NMT IC50 and T. brucei
  activity
• Selectivity over human enzyme (gt100-fold)
  reflected in selectivity vs. MRC5 growth assay
  (gt1000-fold)
• Enzyme kinetics in line with proposed NMT Bi Bi
  mechanism
• Cidal mode of action
• Reduction of activity vs. T. brucei on 20-fold
  over- expression of TbNMT
• Inhibition of protein myristoylation without
  inhibition of protein synthesis
• Binding mode confirmed by X-ray structure

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Good affinity achieved and early selectivity
driven by varying off-rates
HsNMT1
TbNMT
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Pharmacokinetics of DDD00085646
Oral pharmacokinetics of DDD00085646 good
exposure at 50mg/kg
Cmax 6712 ng/ml Tmax 2 hours T1/2 2.0
hours AUC0-T 22 µg.h/ml
EC90 (free)
EC50 2nM (1ng/ml) Fraction unbound 0.11 (89 ppb)
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Efficacy in mouse model of acute African sleeping
sickness
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Conclusions

• Balanced approach of molecular target and
  phenotypic screening vital in a drug
  discovery-immature area
• Need robust assays systems to support both
  approaches
• Series of secondary assays to support target
  deconvolution
• Particular success experienced with a molecular
  target leading to pleiotropic effects
• Toxicity issues manageable short term
  treatment?
• Phenotypic hits may well be affecting multiple
  targets and therefore less susceptible to rapid
  resistance development
• Efficient way forward for Neglected Disease Drug
  Discovery
• Build up chemistries for druggable gene families
  and cross organism molecular targets to allow
  therapeutic switching
• Leishmaniasis, Chagas, malaria etc.

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Interactions

• CRK3 - Jeremy Mottram (Univ. of Glasgow)
• NMT - Debbie Smith (Univ. of York)
• PK4, PK50, PLK - Tansy Hammarton (Univ. of
  Glasgow)
• GSK3 - University of Washington GSK
• Quinols - Malcolm Stevens (Nottingham) Pharminox
• Kinase Phenotypic Screening - Swiss Tropical
  Institute
• Compound sets Imperial College London, MRCT,
  WEHI, Max Planck (Dresden)
• Companies GlaxoSmithKline, Pfizer, Pharminox,
  Genzyme, Schering-Plough Scotland, BioFocus DPI,
  Vernalis, iTi Life Sciences
• PDPs DNDi, MMV
• Drug Discovery Consortium MRCT, CRT, ICR

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Acknowledgements
Core Group Daan van Aalten, Ruth Brenk, Alan
Fairlamb, Mike Ferguson, Julie Frearson, Ian
Gilbert, Andrew Hopkins, Bill Hunter, Kevin Read,
Paul Wyatt
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Automation brings with it
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NMT inhibition correlates with activity in
trypanosomes in vitro
Selectivity over human enzyme (gt100-fold)
reflected in selectivity vs. MRC5 growth assay
(gt1000-fold)