Business from technology

TK4017 Mitosis and Drug Discovery Team: Cell Cycle and Mitosis Research at VTT

Marko Kallio, PhDVice Technology Manager and Team Leader VTT Biotechnology for Health & Wellbeing

224/08/2012Cell Cycle Research at VTT

Service focus on drug target discovery and validation- We perform compound screens, hit-to-lead confirmation, mechanism-of-action

studies, and functional protein studies in cells using VTT’s proprietary technologies- Cell cycle progression (FACS, IF, live cell monitoring, etc)- Cell growth (suppression) analyses (apoptosis, colony formation assays, etc)- Dissection of the target protein / pathway effects (ptw activity analyses, etc)- Gene and protein expression analysis (qPCR, affimetrix, HTS WB, etc) - In-silico data mining with VTT bio-informatics toolbox (REX, IST, etc)

Differentiating competences- Utilisation of proprietary VTT technologies such as 3-D cell culture models and HTS RNAi- In-depth knowledge on mitotic and cancer cell signalling- Packaging of offering into large entities (technologies + know-how)

324/08/2012Cell Division Research at VTT

Research focus on

2. Mechanisms of cell division events

1. Discovery of novel targets for cancer intervention

- Study of the mitotic checkpoint and functional analysis of mitotic proteins- Kinetochore biochemistry- Tubulin in vitro assays

- HTS cell-based assays for identification of novel anti-mitotic factors (siRNA/miRNA)- Discovery and target validation of LMW compounds

424/08/2012Gene Expression Studies at VTT

Work-flow of a typical project The project is planned together with the customer to meet the needs

1. Cells in culture (2-D, 3-D) are treated with a panel of compounds (negative and positive controls)2. Stress factors (UV, H2O2, heat shock) are added prior or after the compounds3. The fate of the cells is monitored using live cell imaging 4. At desired time points samples are collected for RNA / protein extraction, biomarker analysis (IF, WB)5. Target gene (or whole genome) gene expression analysis is performed6. Additional analyses at the protein level7. Bio-informatics data mining 8. Reporting to the customer including scientific insights9. Follow up

524/08/2012

Highly expressed in cancer 1 (Hec1)high expression levels in cancer are linked to poor prognosis

Key roles inmicrotubule-kinetochore attachmentchromosome segregation

Chemical and genetic perturbation of Hec1 result inchromosome misalignmentdefects in spindle checkpoint signallingarrest in mitosis and cell deathreduction in tumour growth in mice

High expression of Hec1 in dividing cells mayincrease the cancer cell selectivity of anti-Hec1therapeutics and cause less side effects.

Hec1 is a potential target for cancer intervention by small molecules

www.cbs.dtu.dk/staff/dave/roanoke/bio101ch09.htm www.cbs.dtu.dk/staff/dave/roanoke/bio101ch09.htm

Hec1

Ndc80 complex

Cell Division Research at VTT Featured Project #1

Discovery of novel anti-mitotic compounds targeting Hec1

624/08/2012

2. Cell-based HT functional screen=> 5 compounds forwarded to in cells analyses

BonsaiBuffer

Aurora B Buffer

Ndc6 + Bonsai

Ndc6 + Aurora B Time (s)

0 200 400 600 800 1000

Ani

sotro

py

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10Ndc6 + Bonsai Ndc6 + AurB Ndc6 + Plk1

Anisotropy measurement

Cell line Ndc106 Ndc106 a1 Ndc106 a2

HeLa 2,0 1,3 0,6

MCF7 4,4 2,0 0,5

MCF10A no effect no effect 2,4MDA-MB-231 2,6 0,9 0,4

LnCaP 2,5 0,5 0,2

HCT116 2,5 1,4 2,4

A549 3,9 1,0 1,5

Ovcar-3 3,6 2,8 2,7

3. Hit compound phenotyping=> 3 compounds to binding assays

4. Hit compound binding to Hec1 => 2 compounds positive

Hec1 project work flow and current status

1. Structure-based in-silico screen (4 M compounds) => 150 best scoring compounds purchased

5. Next steps: - chemical optimisation - animal experiments

724/08/2012

Work-flowTarget library: Dharmacon miRIDIAN mimic library (810 miRNAs) for gain-of-function miRNA studiesCell lines: HeLa H2B-GFP, MCF7, MCF10ATwo endpoints: mitosis arresting miRNAs and forced mitotic exit inducing miRNAs (override of taxol block)

1. HTS for mitosis arresting miRNA

72 hlive cell imaging +Image analysis

M-phase arresting miRNAs (n=3)

Forced exit inducingmiRNAs (n=3)

transient transfections

no apparent cell cycle effect

High MI (>20%)

2. HTS for SAC overriding miRNA

100 nM taxol for 12 h

forced exit from mitosis (NucF 60%)

Validation of the primary hits

Discovery of anti-mitotic miRNAs

Cell Division Research at VTT Featured Project #2

824/08/2012

miR-378*control miR

***

New anti-mitotic miRNAs cause genomic imbalance

miRNA-378* perturbs normal cell division and causes

chromosome missegregation

mir-378 overrides chemically hyperactivated mitotic checkpoint and causes massive genomic imbalance and cell death

miRNA-378*

miRNA-378* hyperactivates VEGFR2 and PDGFpathways leading to activation of MAPK cascade

miR

-378

*co

ntro

l miR

DNA pCenp-A

miRNA-378* targets indirectly AurBvia MAPK and therefore causes

the escape from mitosis

924/08/2012

Mitosis and Drug Discovery Team VTT – TK4017

Marko

JenniLeena

Adel

Anna-Leena

ElliMahesh

Sebastian

Cell Cycle and Mitosis Research at VTT

References:

Several commercial projects performedfor pharmaceutical and cosmetics industry(2007-ongoing); study of MoA of companiesexperimental drugs and bioactives

Discovery of anti-mitotic compounds- 9 notification of invention- Identification of 3 new Aurora B inhibitors- Hec1 pharmacophore

Recent publications- Salmela et al Carcinogenesis, 2009- Kukkonen-Macchi et al JCS, 2011- Vuoriluoto et al. Mol Oncol, 2011- Salmela et al ECR, 2012