Yeast hybrid technology as a tool for interactomics in relation to drug development; What else can we do in collaboration with industry?
TURKEY-MICHIGAN FORUM: UNIVERSITY - INDUSTRY COLLABORATION and R&D TRENDS Istanbul June 05 2012 Zeki Topcu Ph. D Ege University Faculty of Pharmacy Department of Pharmaceutical Biotechnology, Izmir,Turkey
• Yeast 2 Hybrid (Y2H) in conjuction with MAPPIT is the workhorse of interactome mapping
Ndj1
Ten1 Tel2
Tel1
Stn1
Sir2/3/4 Ku Rap1
Est1/2/3/Tlc1 Ten1 Rif1/Rif2 Stn1 Cdc13
MRX(Mre11/ Rad50/Xrs2)
X X
X
survived
starved
Cloning of bait protein into yeast GAL4 BD vector Expression of bait in AH109 strain of Sacchromyces cerevisiae Toxicity, autoactivation, control tests Screening of human fetal brain cDNA library Resquing of library AD plasmids Rectrictional analysis of cDNA inserts in yeast AD plasmids Co-transformation of partners in yeast DNA sequencing and identification of interacting partners Bioinformatic analysis Verification
Glutaminase Interacting Protein (GIP) •  A protein with a single PDZ domain
Human Alteration/Deficiency in Activation 3 (hADA3) • Composed of ADA2, ADA3, and General Control Nonderepressed 5 (GCN5) proteins • Bridges between specific activation domains and transcriptional activators • 3. chromosome; 3p25.3, hAda3p ≈ 432 aas
Expression of Bait in Yeast 1 2 3 4 pGBKT7 hAda3
_ + + _ _ +
75 kDa
25 kDa
GAL4 BD mouse monoclonal antibody
hAda3
GAL4 BD
Toxicity Test Transformation
Selection
Distinct 2 mm Colonies
AH109[pGBKT7]
SD/-Trp
Yes
AH109[Bait]
SD/-Trp
Yes
SD/-Trp
AH109[pGBKT7]
AH109[Bait]
Autoactivation Test Transformation
Selection
Distinct 2 mm Colonies
AH109[Bait]
SD/-Trp/ X-α-Gal
AH109[Bait]
SD/-Ade/-His//-Trp/ X-α-Gal
Yes
White
No distinct colonies
White/Light blue
AH109[Bait]
SD/-Trp/ X-α-Gal
Color of Colonies
SD/-Ade/-His/-Trp/ X-α-Gal
Control Test Mating Strain [plasmid]
AH109[pGBKT7-53] x Y187[pTD1-1]
SD Plates
Selects for
Phenotype
-Leu
pTD1-1
White
-Trp
pGBKT7-53
White
-Leu/-Trp
Diploids containing pGBKT7-53 and pTD1-1
White
-Ade/-His/-Leu/-Trp/X-Îą-Gal
Diploids expressing the ADE2 and HIS3 reporters
Blue
Controlling cDNA Inserts with Restriction Enzymes HindIII
HaeIII
Negative Control
Positive Control
7
Mapping of Interaction N-‐Terminal Region 136 bp 278 bp 400 bp
638 bp
NOBODY IS PERFECT PROs: • Easy to modify • Sensitive • Economic • In vivo • Eukaryotic CONs: • Unicellular • Over-expression/Toxicity • Excludes non-nuclear interactions • Excludes certain proteins • Post-translational modifications • Y2H may not reflect physiological significance
FAILURE • Single biological targets not sufficient to achieve efficacy • Screens that do not reflect efficacy in man • Poor therapeutic indices leading to suboptimal dosing • Animal disease models misleading • The continued search for the universal cure-all blockbuster with high/broad market penetration • Lack of translation of the promise of the genomic revolution Mooney KG, 2001
• Critical skills shortfalls detected in the pharmaceutical industry • Academia should develop a stronger partnership with industry • Government funding should be prioritised, taking industry’s needs into account • While disciplinary excellence is vital, multi-disciplinary programmes are going to be increasingly important • The structure of the industry’s R&D has changed dramatically
• Understanding what industry now comprises and what it needs • Dealing with the balance between education in basic science and training in emerging areas of science and technology • Relative roles of Academia, Industry and Government in providing the optimal graduate and postgraduate education and training • Industry s role in continuously training and developing graduates and postgraduates in their career
FINANCIAL • Cost of labs and equipment • Cost of Legislation around the provision of scientific facilities (Health & Safety, Animal welfare, etc) • Government funding availability OTHER • Industry s image not being attractive to students • Science not being attractive to good students • Fascination with in-vitro biological sciences • Lack of qualified staff to teach courses & run PhD programmes • The change in industrial R & D expectations of students driven by competition and re-structuring
WHAT CAN ACADEMIA DO? • Provide more industrial training positions & post-docs • Provide more extensive mentoring and scientist time to both students and staff • Provide more visiting Professorships in Industry to engage in pre-competitive research • Help set up with Government agencies, a stronger case for multidisciplinary research in universities • Engage in multi-company sponsorship/consortia in research programmes and teaching aids • Help develop efficient centres of excellence
• Hard Skills - Scientific Creativity - Problem Solving capability - Multidisciplinary knowledge - Good publication/patents - Academic tract –record - Demonstrated practical skill
• Softer Skills - Ability to work in teams - Leadership potential - Willingness to learn - Presentation skills - Ability to think strategically - Ability to handle multiple projects and prioritise
REMARKS • cDNA library screening is a reliable approach to interrogate a large pool of proteins • Interactome network is essential developing new drug targets • Genomics and proteomics added a new dimension to Y2H • High-throughput technologies extended the potential applications of Y2H.
The partnerships provide greater encouragement for new entrants to participate, greatly increasing the overall research output in drug industry.
Acknowledgement • Sevil Zencir, Ph.D. • TUBITAK TBAG108T945