Dozens of test tubes in a holder

SMART molecule Milestones

GROUP: Near-term milestone: Hire Post-docs! https://ejobs.umd.edu/postings/30999

SMART Molecule Milestones: December 9, 2014

Bryan lab

Near-term Milestones:

Engineer oncogenic RAS-specific proteases

·       Production of wild type and mutant/oncogenic RAS proteins in E. coli

·       Benchmark demonstration of catalysis with engineered protease

·       Work with Brian Pierce and John Moult to use Rosetta design software for computational design and production of 1st generation RAS binding proteins and Computation design and production of dual function inhibitor

·       Prototype bi-functional binding protein

Longer Term milestones:

Combine sequence-specific proteases and binding components into SMART Therapeutics

·       Determination of binding affinity vs. RAS mutants by ITC

·       Phage-display selection of 2nd and 3rd generation RAS binding proteins

·       Determination of binding mode by epitope mapping (NMR) and H-D exchange. 

·       Analysis of RAS binding and protease inhibition

·       In vitro analysis of bind-compute-respond properties SMART therapeutic

·       Testing kinetic properties of proteases vs. synthetic peptides and mutant RAS proteins

·       Design/selection of 2nd and 3rd generation proteases

·       Determination of x-ray structure of engineered protease with cognate sequence

Toth Lab:

Near-term Milestones:

Proteolysis of active HRAS-G12V

·       Express and purify 10-50 mg of post-translationally-modified HRAS-G12V

·       Baculovirus and mammalian expression close for RalGDA Ras-interacting domain

Longer Term milestones:

·       Solve crystal structure of HRAS-G12V complex with RalGDS RID domain

·       Validate SMART molecule activity in cells and solve crystal structure of SMART molecule alone and in complex with HRAS-G12V

Wilder Lab:

Near-term Milestones:

Cell Line Characterization and Engineering

  • CAL27 order from ATCC (week 2)
  • Confirm HRAS & other RAS status; CD44 variant levels (week 2-3)
  • Optimize transfection (week 4-6)
  • Create stable lines if needed (week 6-10)

•       HRAS G12V

•       CD44v3 high & low

Longer-term Milestones:

Assay Development

  • Fluorescence microscopy (weeks 3-4 after cells created)

•       Identify antibodies to protein or tag

  • Westerns (for expression and RAS activity) (weeks 3-4 after cells created)

•       Identify antibodies to protein or tag

•       Protease

•       GDP

•       RAS

•       Active RAS pulldown

•       Other?

  • AlphaLISA (weeks 3-6 after cells created)

•       Detect direct RAS-RAL or other RAS binding protein interaction or general RAS activation

•       Identify antibodies to protein or tag

•       Optimize buffers, growth conditions, and other assay parameters

  • Cell growth (weeks 2-3 after cells created)

•       SYBR Green, Alamar Blue, MTT, or other

•       Check +/- HRAS G12V

Other Longer Term Milestones:

·      Intracellular delivery techniques (Sasha)

·      Labelling for tracing intracellular delivery