Transcript A knotty problem: Synthesis of the trypsin inhibitor MCoTiII

BBSRC David Phillips Research Fellowship
an interdisciplinary perspective
Dr. Ed Tate, Department of Chemistry, ICL
• Context:
• Current research
• Career
• Fellowship Applications:
•
•
Personal perspective
Hints and Tips
• Research fellowship:
• Advantages, challenges
Faculty of Medicine Fellowships Day
July 2009
Dr. Ed Tate, Department of Chemistry
BBSRC David Phillips Research Fellow (since 2006)
[email protected]; http://www3.imperial.ac.uk/people/e.tate/research
1. Development & application of new technologies in chemical biology
 New chemical approaches for site-specific protein labelling
 Chemical tools for exploring post-translational modification of proteins
 Reactive probes for in vitro & in vivo enzyme activity profiling & imaging
2. Design and synthesis of small molecule inhibitors of:
 Protein-protein interactions: site-specific disruption of multi-protein complexes
• Martin Buck (CBC), Xiaodong Zhang (CBC), Tony Holder (NIMR)
 Protein-ligand interactions: transferase and protease inhibitors
• Neil Fairweather (CMMI), Miguel Seabra (NHLI), Debbie Smith (York)
What is chemical proteomics?
Transcription
DNA
Translation
RNA
Protein
Post-translational
modification
 Applied organic chemistry of living systems
 Targets complex post-translational
phenomena: PTMs, enzyme activity in living
systems, protein-protein interactions…
 Proteomic complement of chemical genetics
 Protein labelling, imaging, biomarker
analysis…
>300 types: Phosphorylation,
glycosylation, lipidation,
acetylation, proteolysis…
Post-translational Modifications
Phosphorylation
Signalling
Sulfenic acids
Redox response
Prenylation
Trafficking
Chemical
Proteomics
Ubiquitination
Protein
degradation
Origin
Function
Site
Acetylation
Transcriptional
regulation
Acylation
Membrane
association
Dynamics
Proteolysis
Apoptotic cascade
Glycosylation
Immune system
Cell adhesion
Site-specific labelling of proteins
-
+
++
Metabolic
tagging
Target proteins
tagged at site of PTM
Tagged PTM
applied to
cells/animal
Bioorthogonal
ligation chemistry
Drug mode-ofaction studies
ID proteins and
site of PTM
Chemical or
enzymatic tagging
Protein bearing sitespecific chemical feature
(active site, PTM)
Proteins labelled
site-specifically
Live- and fixedcell imaging
A platform for Chemical Myristomics
OH
N3
O
(‘Azido-myristic’
acid)
H
N
OH
N3
N3
O
O
H
N
N3
O
S CoA
N3
H
N
O
N3
H
N
Secondary
Labels
MudPIT
analysis
O
N3
O
Bioorthogonal
ligation
H
N
2D-DIGE
On-bead
purification
Secondary
Labels
N
N
N
O
Activity-based probes
Surface layer formation in C. difficile
 C. difficile




Spore-forming anaerobe
Most lethal hospital superbug
Resistant to most antibiotics
Lack of genetic tools
 Excretes a crystalline S-layer
 Post-translational cleavage
 Unknown cysteine protease
O
H
N
LysTyrGlu
PEG3 Biotin
HOOC
O
Warhead
Linker
Specificity
element
Label
Feed to C. difficile
Affinity purify, ID
Future perspectives
 New chemical labelling
technologies
 New chemistries
 Extend to other PTMs
 ABPs for new enzyme activities
 Discovery of new PTMs
 Pull out after metabolic labelling
 Nucleotide tagging
 Protein-DNA interactions
 Transcriptional activation
+ Chemical genetics
 Drug screening
 Biomarker analysis
+ Synthetic biology
 Introducing new chemistries
+ Live cell & in vivo imaging
 Trafficking due to PTM
 Cell-surface display
+ Systems biology
 Dissect pathways
 Probes for protein-protein &
protein-membrane interactions
Career Overview
PhD and Postdoctoral Work
1993
1996
BSc in Chemistry
(Durham)
1996
1999
PhD in Organic
Chemistry
(Cambridge)
New C-glycosidation reactions
Total synthesis of natural products
2000
2001
1851 Research
Fellowship
(Ecole Polytechnique)
Radical cyclisation chemistry
Total synthesis of natural products
2002
2003
Howard Trust
Research Fellowship
(Institut Pasteur)
Role of DNA helix stability & upstream
sequences in transcriptional regulation
2004
2006
BBSRC-funded PDRA
(ICL)
Protein/peptide synthesis and engineering
Library generation and screening techniques
Application Stage
Situation as of August 2005
 Prior Applications:
 WT fellowship application in 2005 rejected – insufficient
biochemical track record
 Never been involved in writing a standard grant application!
 Research Track Record:
 Good chemical research track record
 No biochem research track record at time of application
 Personal Situation:
 Current PDRA contract due to expire 1 month after expected
fellowship decision, no follow-on funding…
 Baby due 22nd November!
Application Stage
Plan of Action
 Novel proposal: chemical proteomics
 Topical and interdisciplinary
 Very limited literature precedent
 Minimal preliminary data, and no prior track record!
 Applied for multiple fellowships





EPSRC Advanced Research Fellowship (ARF)
BBSRC David Phillips Fellowship
Royal Society URF
Wellcome Trust
MRC
 Plan B: other grant proposals
 Co-I on MRC Discipline-Hopping grant (related subject)
 ‘Researcher Co-I’ on BBSRC responsive mode grant (different subject)
Application Stage
Making Multiple Applications
 Advantages
 Spreads the risk of hitting a referee with strong negative bias
 Not always clear in advance of submission which funder is most
appropriate
 Address Remit
 Direct your proposal towards the funder’s remit





BBSRC: basic biology, complete independence (PI), 3-year postdoc
MRC: medical emphasis, strong training/career development component
Wellcome: technician for 5 years, previously had to have HoD as PI
EPSRC: steer clear of biological outputs (stick to technology only)
Royal Society: two FRS’s strongly supporting application, no support grant.
 Remit is strictly based on the research outputs (‘deliverables’) of
your project (not the methodology and techniques applied)
 Be flexible, rewrite application to take account of funding available,
in particular in justifying your costs
Application Stage
Writing Applications
 Writing the proposal
 Steep learning curve, very time-consuming (2-3 months)
 Sole PI, so responsible for all aspects of the proposal: financial,
planning, scientific
 Very good practice for later PI grant applications!
 Costing the proposal
 Costing may feel rather abstract on the application, but you will
appreciate careful costing if you get the fellowship.
 Acquire a working knowledge of Full Economic Costing (fEC)
 The headline cost can be big: >£1 million for BBSRC DP
Fellowship – but 30%+ goes direct to Faculty under fEC…
 Request costs at the upper limit allowed if you can justify them
for the proposed work.
 You can usually move cash around later (within limits!)
Application Stage
General Tips
 Create a career narrative
 Justify your career choices (in hindsight)
 Highlight why you are ready to go independent
 Salary level
 Most funders don’t fix the level arbitrarily
 You don’t usually need to justify your own salary level
 Fellowship proposals vs. grant proposals
 An original, cutting-edge idea can carry more weight than a
strong track record (esp. when compared to standard grants)
 You can get away with having (much) less preliminary data
 Interdisciplinary research may fare better in fellowship
applications than in standard grant applications.
Application Stage
Writing Applications

– external peer review (it is a grant proposal!)
– originality, fresh thinking
 Has to be
– you have to convince the referees that you
are capable of doing the work proposed, in the time available
 Show them that you have considered the riskier elements, and
propose contingency plans; don’t have the whole project
dependent on a risky proposition
 Mention
if you have it – I would say this is not
absolutely essential, but you do need to be able to convince the
referees that the riskier aspects could work
 Know your competition in the scientific field – cite the key work
of others in your background section
 Cheaper proposals do not mean more chance of success; but
high-cost proposals where costs are not justified will also not
impress.
Application Stage
Stay or Go?
• Stay…
 Access to existing equipment and collaborations
 Minimal start-up time
 Can you achieve independence?
• Or go?
 Start afresh on your own terms
 Will take time to get started
• May help to get out of the ‘golden triangle’…
• In either case…
•
•
•
Ensure your expectations (support, space, teaching etc.) are understood
by host institute
Be prepared to justify choice of institute in the application and at interview
Be prepared for questions regarding future independence if you decide to
stay
Interview Stage
Some suggestions
 Presentation
 Practise in front of a diverse audience
 Keep it simple!
 Interview
 BBSRC interview is very brief (20min), and (relatively)
friendly.
 Interviews for other funders can be more intense…
 Be prepared for questions on:
 Past career choices
 How will a fellowship benefit you and your research?
 Minimal teaching, independence, opportunity to apply for
further grants during fellowship.
 How will you achieve independence?
 Where do you see yourself in 5 years, 10 years…
 Permanent position in academia, leader of a vibrant
research group
Award Stage
Challenges and Benefits
 Challenges
 Making the transition from PDRA to PI 
 Managing & applying for grants, recruitment
 Supervising a group on your own
 Dealing with internal departmental politics
 You only have a few years to find a permanent position! 
 Start looking after 1-2 years
 May entail a move to another institute
 Consider whether fellows have routinely been taken on
permanently at the host department/institute
 Benefits
 Minimal teaching load (vs. lectureship)
 = Time to set up collaborations, supervise research, write new
grants…
 Support from BBSRC
 Once in post, be pro-active in applying for further funding:
 From October: 18 (4 PDRAs, 12 PhD + 2 UG research students)
 Over £1 million in PI research income (+ £1 million fellowship)
Acknowledgements
 Members of the Tate group
Dr. Will Heal, Dr. Sasala Wickramasinghe, Dr. Lucia de la Riva Perez, Martyna Snopek,
Kavita Ramji, Irvine Howson, Vlad Turek, Tam Dang, Jemima Thomas, Lucy Rayner, Alex
Berry, Neki Patel, Richard Bradshaw, Megan Wright, Pinar Tulay, Jenny Früh
 Collaborators
 Prof. Miguel Seabra (NHLI)
 Neil Fairweather (CMMI)
 David Mann (Biology)