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

Rap1 Rif1/Rif2

Sir2/3/4

MRX(Mre11/Rad50/Xrs2)

Ku

Cdc13

Stn1 Ten1

Tel1 Tel2

Ndj1

Est1/2/3/Tlc1

Stn1

Ten1

X

survived starved

X

X

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 Non-derepressed 5 (GCN5) proteins

•  Bridges between specific activation domains and transcriptional activators

•  3. chromosome; 3p25.3, hAda3p ≈ 432 aas

pGBKT7 hAda3

hAda3

GAL4 BD

   _                            +                              +      _                            _                              +  

75  kDa  

25  kDa  

1                        2                          3                4    

Expression of Bait in Yeast

GAL4 BD mouse monoclonal antibody

Toxicity Test

SD/-Trp  

AH109[Bait]  

AH109[pGBKT7]

Transformation   Selection   Distinct 2 mm Colonies  

AH109[pGBKT7]   SD/-Trp   Yes  

AH109[Bait]   SD/-Trp   Yes

AH109[Bait]  

SD/-Trp/ X-α-Gal  

SD/-Ade/-His/-Trp/ X-α-Gal  

Autoactivation Test

Transformation   Selection   Distinct 2 mm Colonies   Color of Colonies  

AH109[Bait]   SD/-Trp/ X-α-Gal   Yes   White  

AH109[Bait]   SD/-Ade/-His//-Trp/ X-α-Gal   No distinct colonies   White/Light blue  

Control Test

Mating Strain [plasmid] SD Plates Selects for Phenotype

AH109[pGBKT7-53] x Y187[pTD1-1]

-Leu -Trp -Leu/-Trp -Ade/-His/-Leu/-Trp/X-α-Gal

pTD1-1 pGBKT7-53 Diploids containing pGBKT7-53 and pTD1-1 Diploids expressing the ADE2 and HIS3 reporters

White White White Blue

HindIII HaeIII

Controlling cDNA Inserts with Restriction Enzymes

Negative Control Positive Control

7

Mapping of Interaction

136  bp  

278  bp  

400  bp  

638  bp  

N-­‐Terminal  Region  

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