Inhibition of prenylated KRAS in a lipid environment

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.


Plasmid Construct 244-35 (=BV3095) Avi-tag-(Gly) 8 -KRas G12V (2-188)
Using a full length construct (KRas FL (G12V) in pBB45 (Thermo-Fisher Scientific) as a template, a BamHI plus Avi-tag-(Gly) 8 sequence (Avidity©) was added to the 5' end and HindIII to the 3'end in one PCR reaction using overlapping primers. After gel purification of the PCR product, the PCR fragment was cut with BamHI and HindIII restriction enzymes, and ligated into pFastBac1 vector (Thermo Fisher Scientific) cut with the same enzymes and transformed into chemically competent Top10 cells (Thermo-Fisher). Resultant colonies were screened for the fragment insert and sequence confirmed.

Plasmid Constructs 151-18 bRaf FL (BV837) and 151-19 bRaf FL V600E (BV836)
The full length bRaf (wild type and V600E) coding sequence (amino-acids 1-765) was PCR amplified using previously obtained cDNAs as template. The PCR primers contained an XbaI site at the 5' end and a (His) 6 -stop-SalI at the 3' end. The PCR fragment was gel purified and ligated into the XbaI and SalI sites of phosphatase-treated baculovirus transfer vector pBlueBac4.5 (Thermo Fisher Scientific). The ligation reaction was transformed into E. coli as above. Colonies were screened for insert and sequence verified.

Dual Plasmid Construct with MEK1 K97R-cAvi tag and BirA (BV2950)
A dual expression of BirA and N-terminal (His) 6 -HRV3C-MEK1 K97R-(Gly) 4 -C terminal Avi-tag (Avidity©) in the pFastBac Dual vector (Thermo Fisher Scientific) was generated. For convenient coexpression of biotin ligase with an AviTagged protein, the E. coli BirA gene was cloned into pFastBac Dual (Invitrogen) behind the p10 promoter. Using a 5' PCR primer containing a SmaI site and a 3' primer containing an XhoI site, the BirA coding sequence was amplified from the pBirAcm plasmid. The resulting PCR fragment was sequentially digested with SmaI and XhoI, spin column purified and ligated to pFastBac dual cut with the same enzymes. The AviTagged gene of interest was then cloned behind the Polyhedrin (PH) promoter. The human MEK1 K97R gene with a N-terminal (His) 6 followed by a HRV3C cleavage sequence and a C-terminal (Gly) 4 -linker followed by an Avi-tag (Avidity©) was driven by the polyhedrin promoter.

Expression of plasmid Construct 244-34 = (His) 6 -TEV-Avi-tag-(Gly) 8 -KRas (1-180)_G12V
Plasmid 244-34 and a BirA (biotin ligase) plasmid were co-transformed into E.coli expression strain BL21 (DE3) (Stratagene/Agilent). The transformation was plated on dual LB plates with 100g/mL carbenicillin and 20g/mL of chloramphenicol. A single colony was used to grow an overnight starter culture in LB with 100g/mL of carbenicillin and 20 g of chloramphenicol at 37 o C and this used to inoculate 2 x 1L cultures to a starting OD 600 = 0.1 OD/mL. These pre-induction cultures were grown at 37 o C, at 240rpm to an OD 600 = 1.9 OD/mL. The temperature was turned down to 18 o C and culture flasks were chilled without shaking and when chilled, D-biotin to 50 uM was added and then induced with IPTG to 0.5 mM. Flasks were grown overnight at 18 o C at 240 rpm. Cells were harvested at OD 600 = 3.9OD/mL, pelleted by centrifugation, supernatant poured off and the pellet stored at -80 o C.

Expression of Plasmid Construct 244-26 = (His) 6 -TEV-KRas (1-180) G12V
Plasmid 244-26 was transformed into E. coli expression strain Rosetta2 DE3 (EMD-Millipore) and plated for isolated colonies. A 300ml starter culture was grown in LB with 100g/mL of carbenicillin and 20 g/mL of chloramphenicol to an OD 600 =0.95. The starter culture was used to inoculate a fermenter containing the following modified YEP medium (4% Casein Peptone, 2% Tastone 154, 1% Potassium Phosphate, (dibasic), 1% glycerin, 1% Glucose, 20g/mL chloramphenicol and 50g/mL carbenicillin) The culture was grown at 37 o C to an OD 600 =8-10. At this point the temperature was reduced to 18 o C and S-3 induced with 0.81mM IPTG and the culture continued to an approximate final OD 600 =30. The cells were harvested and frozen at -80 o C.

Expression of plasmid Construct 262-1 = GST-cRAF RBD (aa 51-131)
Plasmid 262-1 in the pGEX2T vector (GE Healthcare) containing a tac promoter, was transformed into E. coli BL21 expression cells (EMD-Millipore) and selected on LB plates containing 100g/mL carbenicillin. A single colony was used to grow an overnight starter culture at 37 o C in LB + carbenicillin. Used the starter culture to inoculate 4 x 1L cultures to a starting OD 600 = 0.1 OD/mL. These pre-induction cultures were grown at 37 o C, 240rpm to an OD 600 = 0.7 OD/mL. The cultures were induced by adding IPTG to 0.5mM final concentration. The cultures were incubated with shaking at 37C for 4hrs. Cells were harvested by centrifugation. Pellets were stored at -80 o C until processed.
Bacmid and baculovirus generation: The KRas-Avi-tag 244-35 (BV3095) and MEK1 K97R-Avi-tag/BirA (BV2950) plasmid DNAs were used to generate bacmid DNA as instructed in the Bac-to-Bac manual (Invitrogen), in particular the section headed "Generating the Recombinant Bacmid". The resultant plates of DH10Bac transformants contained a mix of small blue colonies and a few large white colonies. Two large white, well separated colonies were chosen for bacmid DNA preps. DNAs were prepared using the PowerPrep HP BAC Buffer Kit (OriGene, cat# NP10003/11448-100). The recombinant bacmid DNA was analyzed by PCR per the Bac-to-Bac manual, using a gene specific forward primer and the M13 Rev primer. Baculovirus expressing the recombinant proteins was generated using the Bac-To-Bac Expression System method (Invitrogen) following manufacturer's protocol. The virus generated from the transfected insect Sf9 cells was amplified using a standard low MOI infection method.

Expression of plasmid Construct 244-6 (=BV1689)-KRas FL, no tags, prenylated Plasmid Constructs 151-18 bRaf FL (BV837) and 151-19 bRaf FL V600E (BV836)
To generate recombinant proteins, suspension cultures of insect cells (Sf9, Sf21 or Tn5) were seeded at a density of 1.5x10 6 cells/ml and infected with virus at an MOI of 10 or a with a 3% volume. The infected insect cells were cultured for 48 hours at 27°C shaking at 120 rpm using 2L glass Erlenmeyer flasks and serum free media. Cells were harvested two days post-infection, frozen and stored at -80°C.

Expression of Plasmid Construct 244-35 (=BV3095) Avi tag-(Gly) 8 -KRas G12V (aa 2-188)
To generate biotinylated Avi-tagged protein, a suspension culture of insect cells (Tn5) were seeded at a density of 1.5x10 6 cells/ml and co-infected with virus at an MOI of 5:5 (BV3095 and BirA-expressing virus, BV1845, both at passage 2). D-biotin was added to 50M final concentration. The infected insect cells were cultured for 48 hours at 27°C shaking at120 rpm using 2L glass Erlenmeyer flask and serum free media. Cells were harvested two days post-infection, frozen and stored at -80°C.

Expression of Dual Plasmid Construct with MEK1 K97R and Bir1 (BV2950)
To generate biotinylated MEK1 K97R, a suspension culture of insect cells (Sf9) were seeded at a density of 1.5x10 6 cells/ml and infected with virus at an MOI of 10 or with a 3% volume. D-biotin was added to 50M final concentration. The infected insect cells were cultured for 48 hours at 27°C at 120 rpm using