RAS Initiative: Progress and Working Group UpdateRAS Resources • 1,121 clones generated and...
Transcript of RAS Initiative: Progress and Working Group UpdateRAS Resources • 1,121 clones generated and...
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DEPARTMENT OF HEALTH AND HUMAN SERVICES • National Institutes of Health • National Cancer InstituteThe Frederick National Laboratory is a Federally Funded Research and Development Center operated by Leidos Biomedical Research, Inc., for the National Cancer Institute
RAS Initiative: Progress and Working Group UpdateDwight Nissley, Director, Cancer Research Technology Program, FNLCR
October 29, 2018
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Ras Initiative: Major Goals• Discover small molecules that bind to RAS directly or disrupt RAS/effector interactions
– RAS activity Assays• Cell-based / biochemical
– Biophysical screens• Tethering / covalent inhibitors
– In silico screens– Partner through cCRADAs
• Molecular description of RAS/RAF signaling complexes in membranes– Biochemistry, biophysics and structural biology
– RAS/RAF dynamics in cells
– Molecular dynamic (MD) simulations with DOE National Labs
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RAS Initiative Collaborations: Hub and Spokes
cCRADAs
Strategic Collaborations Academic CollaborationsIndustry Collaborations
RAS Initiative
“The Hub”
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RAS Resources
• 1,121 clones generated and sequence verified• 647 large-scale protein productions (24 grams of protein produced)• 412 small-scale protein production scouting experiments performed• 427 liters of insect cell protein expression culture grown, 100 liters of baculovirus produced
• 1,750 liters E. coli protein expression culture grown• 230 cell lines generated
• 7,057 plasmids distributed through Addgene
- 314 universities and NPOs, 38 states, 36 countries, 6 continents
• 119 MTAs for direct distribution (323 plasmids, 476 cell lines)
• RAS-dependent MEFs licensed to 5 companies
• KRAS-FMe materials licensed to 4 companies
In FY2018:
Overall since 2014:
Dom Esposito and RAS Reagents Core
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Structural Biology Reveals Potential Therapeutic Opportunities
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Pockets in oncogenic KRAS mutants
WT-KRAS bound to GMPPNP and Mg
G12C in complex with GMPPNP and Mg
Switch-1Switch-2
Q61L in complex with GMPPNP and Mg
G13D in complex with GMPPNP and Mg
Dhirendra Simanshu
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In silico screening • In silico screening - in collaboration with Nir London, Weizmann Institute of Science, Israel.
• Hits validated using SPR and NMR (Andy Stephen, RAS Biophysics)
G12C in complex with GMPPNP and Mg
• 6 out of 14 compounds showed weak binding to G12C mutant by SPR
• Test these 6 compounds for binding by NMR
• Testing additional 44 analogs for SPR binding Nir London (Weizmann)L. Bindu, Andy Stephen (FNLCR)
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cCRADAs Amplify RAS Initiative Efforts
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RAS cCRADAs
Contractor Cooperative Research and Development Agreement (cCRADA)
Agreement between FNLCR and a private company or university to work together on research and development.
Provides IP protections to the collaborator.
SanofiAll efforts are in-kind, Chemistry supported by Sanofi, Biology shared between Sanofi and FNLCRProject team: 8 FNLCR, 24 SanofiProposal to carry out fragment screen against Oncogenic KRAS allele under negotiation
KyrasFunds to support 1 FTE equivalent over 2 yearsProject team: 4 FNLCR, 5 Kyras
BeatsonFunds to support 5 FTE equivalents over 2 yearsHired 3 new employees, additional funds are to support existing FNLCR staff
PharmaAravaFunds to support 2 FTE equivalents over 2 yearsChemistry supported by PharmaAarava, Biology supported by FNLCR
TheRasExpanded to support 6 FTE equivalents over 2 years (chemistry and in silico modeling and design)Chemistry shared between TheRas and FNLCR, Biology supported by FNLCRTheras engaged CROs for additional chemistry and LLNL for CADD
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Anna MaciagDavid TurnerVandana KumariMarcin DybaChris BrassardJoseph SaavedraBrian Smith
In–house discovery efforts transition to cCRADA with Theras
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Small Molecules that prevent Kras4b membrane localization
1 10 1000
50
100
150
% A
ctiv
ity
FOV
Mea
n PM
GFP
48 hr induction
994566 (µM)
Vinculin
KRas
KRas4b G12D(membrane fraction)
Myr-KrasG12D/C185S
(membrane fraction)
994566 (μM)
0 20 30 0 20 30
CTRL 994566, 30µM
farnesyltransferase
geranylgeranyltransferase
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Covalent derivatives of tethering hits modify C185
DIHHYRE(93-99)+adduct
KMSKDGKKKKKKSKTKCVIM(170-189)+adduct
GKKKKKKSKTKCVIM(174-189)+adduct
2157
1608
970
2675
2085
1320 23241735
350350
351
0
1000
2000
3000
4000
Inte
nsity
(AU
)
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
m/z
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MD simulation of HVR dynamics in KRAS4b
blue – GDP-loaded KRAS G-domain orange – GDP- loaded KRAS HVRcyan – GTP-loaded KRAS G-domainred – GTP-loaded KRAS HVR
Yue Yang and Felice Lightstone, LLNL
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HVR interaction with G-domain may generate a binding pocket
KRAS4b (1-188) + C185 tethering compound
KRAS4b (1-169) DMSO control
KRAS4b (1-188) DMSO control
Lixin Fan, The Small-Angle X-ray Scattering Core Facility, NCI
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1 2 4 8 1 6 3 2 6 4 1 2 80
5 0
1 0 0
T S G -0 1 2 5 - - 9 /4 /2 0 1 8
C o n c e n tra tio n (µM )
% C
on
tro
l
K R a s 4 b G 1 2 DM y r-K R a s 4 b G 1 2 D /C 1 8 5 SK R a s 4 b G 1 2 V
1 2 4 8 1 6 3 2 6 4 1 2 80
5 0
1 0 0
T S G -0 1 2 3 - - 9 /4 /2 0 1 8
C o n c e n tra tio n (µM )
% C
on
tro
l
K R a s 4 b G 1 2 DM y r-K R a s 4 b G 1 2 D /C 1 8 5 SK R a s 4 b G 1 2 V
1 2 4 8 1 6 3 2 6 4 1 2 80
5 0
1 0 0
T S G -0 1 2 4 - - 9 /4 /2 0 1 8
C o n c e n tra tio n (µM )
% C
on
tro
l
K R a s 4 b G 1 2 DM y r-K R a s 4 b G 1 2 D /C 1 8 5 SK R a s 4 b G 1 2 V
Compound 1 Compound 2 Compound 3
Targeting C185 - Status
• C185 covalent binders are KRAS4b specific
• Prevent prenylation at C185 and membrane localization
• Inhibit proliferation of KRAS4b-dependent cell lines
• MD simulations suggest that the HVR interacts with the G-domain to form a pocket
• Potency/selectivity increased by CADD/Med Chem
• Design and synthesis of analogues in progress
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Covalent derivatives of tethering hits also modify H95
KRAS FAINNTKSFEDIHHYREQIKRVKDHRAS FAINNTKSFEDIHQYREQIKRVKDNRAS FAINNTKSFADINLYREQIKRVKD
DIHHYRE(93-99)+adduct
KMSKDGKKKKKKSKTKCVIM(170-189)+adduct
GKKKKKKSKTKCVIM(174-189)+adduct
2157
1608
970
2675
2085
1320 23241735
350350
351
0
1000
2000
3000
4000
Inte
nsity
(AU
)
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
m/z
H95
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Targeting H95 - status
• H95 binders are KRAS specific
• Docking/MD/NMR suggest a pocket between Helix3/Switch2
• Bind to KRAS, not RhoA (by SPR)
• Design and synthesis of analogues in progress
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RAS Inhibitor development
in silico docking
MD simulations
chemistryCRO synthesis
In-house synthesis
Biophysics, biochemistry cell biology
data analysissmall molecule design
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Fred Streitz, Director HPC, LLNL
DOE Lead for Pilot 2
Advanced Computation to better Understand RAS Biology
NCINational Cancer
InstituteDOEDepartment
of EnergyCancer driving
computing advances
Computingdriving cancer
advances
DOE-NCI partnership to advance exascaledevelopment through cancer research
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Experimental Gaps – Activation of RAF• RAS structure in context of
membrane• RAS dynamics
– states
• RAS monomer/dimer/multimer– Interfaces
• RAF structure• RAS:RAF engagement
– Order of addition– Activation/conformational
dynamics
• Local lipid environment
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RAS Initiative studies used as input to parameterize MD simulations: KRAS4b
KRAS4b
Debanjan Goswami, De Chen,Tommy Turbyville
0 1´1 0 -0 5 2´1 0 -0 5 3´1 0 -0 50 .0
0 .2
0 .4
0 .6
[K R A S 4 b ] (M )
RU
S/R
UL
L1
L2
L3
L4
L5
L6
L7
L8
L10
L11
L ip o s o m e s
Frantz Jean-Francois
+----HVR++
Fluorescence
2. Lipid-protein interaction
3. Orientation
1. Diffusion
FLCCSFCS
FRET Aniso-tropy
4. Rotational diffusion of KRAS4b on lipid bilayer
Rebika Shresta,Tommy Turbyville
Andy Stephen
Andy Stephen, Que Van
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Initial KRAS:membrane Simulations
Chris Neale and Angel Garcia, LANL
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CHARMM (all atom)MARTINI (coarse grain) DDFT +HyCoP simulations
c1, c2, h,R, State
c1, c2, h,R, State
A framework to couple macro and micro scale simulations
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New framework simulation of longer time and length scales
Micro Scale - Coarse Grain Bead Martini Model
1 µs into production simulation
30 nm
Back-mapping
8 lip
id ty
pes
Marco Scale - Continuum Phase Field Model
100 x 100 nm,10 KRas; 60 µs 8/6 lipids inner/outer leaflet
100 nm
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First-of-a-kind simulations will explore:• Dependence of KRAS mobility and and dynamics as a function of membrane environment• Aggregation of KRAS in context of realistic membrane• Effect of KRAS concentration on local membrane composition Sierra Supercomputer
First Run on Sierra: RAS Dynamics
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On-the-fly feedback from the macro simulations improve micro model parameters
• Online analysis of state dependent RAS lipid interactions• Micro model parameters are updated after unbiasing through the ML framework Tim Carpenter, LLNL
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Simulation predicted RAS-Lipid contacts
fraction of time contacting0 1
Microsecond timescale Millsecond timescale
Tim Carpenter, LLNL
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Summary
• RAS Initiative has consolidated to two primary focus areas– Directly Targeting KRAS– Understanding the biology of KRAS in the context of the plasma membrane
• Identified novel classes of compounds that specifically target KRAS
• Multiple screens to identify leads are ongoing
• Working with DOE National Labs to bridge experimental gaps using computation
• Partnering with Biotech, Pharma and NCI to develop leads and push towards clinic