Andrographolide derivatives inhibit guanine nucleotideexchange and abrogate oncogenic Ras functionHarrison J. Hockera,1, Kwang-Jin Choa,1, Chung-Ying K. Chena, Nandini Rambahala, Sreenivasa Rao Sagineedub,2,Khozirah Shaarib, Johnson Stanslasb,c,3, John F. Hancocka,3, and Alemayehu A. Gorfea,3

aDepartment of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX 77030; and bLaboratory of Natural Products,Institute of Bioscience and cPharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang,Selangor 43400, Malaysia

Edited by Michael Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved May 8, 2013 (received for review January 8, 2013)

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteinsoccurs in ∼15% of all human tumors, yet direct inhibition of Ras bysmall molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras andinhibit its function by interfering with exchange factor binding.However, it is unclear whether, or how, these ligands could leadto drugs that act against constitutively active oncogenic mutantRas. Using a dynamics-based pocket identification scheme, ensem-ble docking, and innovative cell-based assays, here we show thatandrographolide (AGP)—a bicyclic diterpenoid lactone isolatedfrom Andrographis paniculata—and its benzylidene derivativesbind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP–GTP exchange. As expected for inhibitors of exchange factor bind-ing, AGP derivatives reduced GTP loading of wild-type K-Ras inresponse to acute EGF stimulation with a concomitant reductionin MAPK activation. Remarkably, however, prolonged treatmentwith AGP derivatives also reduced GTP loading of, and signaltransmission by, oncogenic mutant K-RasG12V. In sum, the com-bined analysis of our computational and cell biology results showthat AGP derivatives directly bind Ras, block GDP–GTP exchange,and inhibit both wild-type and oncogenic K-Ras signaling. Impor-tantly, our findings not only show that nucleotide exchange fac-tors are required for oncogenic Ras signaling but also demonstratethat inhibiting nucleotide exchange is a valid approach to abro-gating the function of oncogenic mutant Ras.

cancer | molecular dynamics | allosteric site | drug design

Monomeric rat sarcoma (Ras) proteins are molecular switchesthat cycle between inactive GDP-bound and active GTP-

bound conformational states and regulate multiple cell signalingpathways that include the MAPK cascade (1). Activation of Ras isfacilitated by guanine nucleotide exchange factors (GEFs) and in-activation by GTPase-activating proteins (GAPs) (2, 3). About 15%of all human cancers are associated with somatic Ras mutations atamino acid positions 12, 13, or 61 that impair GAP-catalyzed GTPhydrolysis. Of the three most common human Ras isoforms N-,H-, and Kirsten-Ras4B (K-Ras4B), mutations on K-Ras4B(hereafter K-Ras) are most prevalent in cancers, including in upto 90% of cases of pancreatic cancer (4). However, decades ofefforts to inhibit oncogenic Ras by small molecules have to datebeen unsuccessful (5). Attempts to abrogate the plasmamembrane binding of Ras, which is required for biologicalactivity, by inhibiting farnesyl transferase (6, 7) have failedbecause N-Ras and K-Ras are also good substrates for geranyl-geranyl transferase 1 in cells treated with farnesyl transferaseinhibitors (8, 9). Other efforts along this line include the de-velopment of farnesyl analogs, currently in clinical trials (10),and other compounds that dislodge Ras from the plasma mem-brane (11, 12). Although the potential therapeutic value andmechanism of action of these compounds are still under in-vestigation, it is clear that they do not directly bind to Ras.Recent efforts by us (13) and others (14–16) toward directinhibition of Ras have yielded promising initial results. Forinstance, using fragment screening, crystallography, and other

methods, two groups reported ligands that directly bind Ras andinhibit GEF-dependent nucleotide exchange (15, 16). However, itis unclear whether, or how, these ligands could lead to drugsthat act against constitutively active, GTP-loaded mutant Ras.GTP-Ras can exist in at least two conformational states (17, 18).

When in state 1, Ras has reduced affinity for effectors and harborsopen pockets (19, 20), whereas in state 2, Ras is able to effectivelybind effectors (21, 22). In principle, small-molecule inhibitors thatcan selectively stabilize the state 1 conformation have the potentialto inhibit Ras signaling by interfering with either effector or ex-change factor binding. Along this line, a recent study founda compound that binds to an open switch 1 conformation of state1 Ras (23). In this work, we use ensembles of K-Ras obtained frommolecular dynamics (MD) simulations that sample state 1 andother intermediate structures (24) to address two major questions:first, to evaluate whether the reported anticancer activity (25) ofandrographolide (AGP), a diterpene from the medicinal plantAndrographis paniculata (26), and its benzylidene derivativesinvolves direct inhibition of Ras; and second, to use these com-pounds to test a unique hypothesis that prolonged inhibition ofnucleotide exchange can abrogate the function of oncogenic mu-tant Ras. Combining data from ensemble docking, simulations, andexperiments in intact cells, we show that AGP and its derivativesinhibit Ras function by preventing GEF-induced nucleotide ex-change. We further show that prolonged treatment with AGPderivatives significantly impairs oncogenic K-RasG12V signaling,and highlight how inhibiting nucleotide exchange can be a validapproach to abrogating the function of oncogenic mutant Ras.

Results and DiscussionAGP and Benzylidene Derivatives Target the Switch Regions of K-Ras.AGP has oxidative, antiviral, and anticancer properties, and itsbenzylidene derivatives (Fig. 1) exhibit an enhanced ability to in-duce apoptosis and G1 cell-cycle arrest in breast and colon cancercells (25, 27). Other studies have shown that AGP interferes withMAPK activation, increases sensitivity of Ras-transformed cellsto radiation treatment in vitro and in vivo (27–30), and is not toxic(31). The drug-like (32) AGP has three hydrogen-bond donors andfive acceptors and a LogP of 2.6. Its slightly larger SRJ series

Author contributions: J.S., J.F.H., and A.A.G. designed research; H.J.H., K.-J.C., C.-Y.K.C.,S.R.S., K.S., J.S., J.F.H., and A.A.G. performed research; H.J.H., K.-J.C., N.R., S.R.S., K.S., J.S.,J.F.H., and A.A.G. contributed new reagents/analytic tools; H.J.H., K.-J.C., C.-Y.K.C., N.R.,J.S., J.F.H., and A.A.G. analyzed data; and H.J.H., K.-J.C., J.F.H., and A.A.G. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1H.J.H. and K.-J.C. contributed equally to this work.2Present address: Department of Pharmaceutical Chemistry, School of Pharmacy, Interna-tional Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.

3To whom correspondence may be addressed. E-mail: jstanslas@yahoo.co.uk, john.f.hancock@uth.tmc.edu, or alemayehu.g.abebe@uth.tmc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1300016110/-/DCSupplemental.

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of derivatives each has one donor, five acceptors, and an estimatedLogP of 5.6.We docked these ligands onto a diverse set of 75 K-Ras con-

formers and ranked them by their preference for a given site andreceptor conformation as described inMaterials and Methods andSI Materials and Methods, with appropriate controls (Fig. S1).We found that the ligands preferentially target three distinctpockets: p1, p2, and p3. The residues defining these pockets arelisted in Table 1 and their location in the 3D structure is shownin Fig. 2. Pocket 1 comprises the effector binding loop (residues30–40), β2 (residues 55 and 57), and several residues in α-helix1. Pocket 2 involves the core β-strands 1 and 2, part of the ef-fector loop, and switch 2. The C-terminal pocket 3 is bounded byα-helix 5 plus the N-terminal and preceding loop residues of β5and β6. Remarkably, each of these pockets is very similar tothose we have previously characterized using a different ap-proach and a different set of probes (13). Mapping the energeticpreference of small-molecule fragments by FTMap (33) identi-fied the same binding hotspots, with binding preference for eachsite modulated by protein motion (Fig. S2). Opening of pocket 1is primarily a function of the displacement of Y40 that occursduring the simulations (Figs. S3 and S4). Relative to the refer-ence X-ray structure Protein Data Bank (PDB) ID code 3GFT,the Cα of Y40 is displaced by 4 Å and the side chain is orientedaway from D38 (Fig. S3). Similarly, expansion of a narrow sur-face groove between switches 1 and 2 (Fig. S4 B and D) opens p2,a pocket surrounded by hydrophobic (I36 and M67) and polarresidues (Y64 and E37) with V7 at the bottom (Fig. 3B). Tocheck whether structures within each cluster have reasonablysimilar binding-site topologies, we calculated the SD of the meansolvent-accessible surface area of p1 residues, which was foundto be 33% smaller for structures within clusters (averaged overall 75 clusters) than the corresponding value across clusters.Whereas the parent compound AGP displays some preference

for p3 in addition to p1 and p2, the derivatives have much lesspreference for p3, as can be seen from the binding frequencyhistograms in Fig. S5 for the top five ligand clusters. In fact, theSRJ ligands consistently hit p3 only when docked onto the crystalstructure. When the structure is relaxed but still somewhat closeto the starting crystal structure PDB ID code 3GFT (Cα rmsd of2.8 and 2.9 Å, respectively, for switches 1 and 2), the top-rankedligand cluster targets p2 with a probability of 5.6%. When itadopts a more open switch conformation (3.6 and 4.3 Å), SRJ23[3,19-(3-chloro-4-fluorobenzylidene)andrographolide] targets p1with a probability of 3.1% (Table 2). To probe whether the li-gand might prefer a region proximal to p2, such as the site oc-cupied by the ligands benzimidazole (BZIM) and its halogenatedderivative 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI)

(15), we docked SRJ23 onto the K-Ras–BZIM structure (PDBID code 4DSU). We found that SRJ23 does not recognize thisregion, because four of the top five ligand clusters (predictedaffinity 0.04–1.2 μM) targeted p3. We conclude that pockets nearthe highly dynamic canonical switches, which became accessibleduring the simulations, represent the most probable binding sitesfor AGP and its derivatives.

MD Simulations of K-Ras–SRJ23 Complexes Suggest Stable Binding atp1 but Not p2. To further evaluate the viability of ligand bindingat p1 or p2, we conducted multiple MD simulations of K-Ras incomplex with SRJ23 with different initial velocity assignments.The ligand dissociated within 60 ns in four out of five of the runswith SRJ23 at p2 and after an additional 40 ns in the fifth run(Fig. S6A). By contrast, the K-Ras–SRJ23 complex remainedstable (with 45–80% of the ligand surface area buried) duringfour out of five simulations with SRJ23 bound at p1 (Fig. S6 Aand C). In the remaining run, switch 1 moved away from theGTP and the pose of SRJ23 was altered, but it did not dissociate.These results suggest that p1 is the preferred pocket for ourligands, despite the fact that p2 showed up repeatedly duringdocking (Fig. S6). We conclude that switch 1 is a viable targetwhose dynamics leads to the opening of a pocket that canaccommodate even comparatively large ligands such as SRJ23.During the simulations with p1-bound SRJ23, the ligand forms

a hydrogen bond with the α-phosphate of GTP (Fig. 3D), but ittargets the same pocket when docked onto a nucleotide-free–likeconformer (Fig. 4B). This indicates that the nucleotide is notrequired by SRJ23 for binding but that it might stabilize thebound ligand. As noted earlier, SRJ ligands target an openswitch 1 conformation in which, for instance, the distance be-tween D33 and D38 Cα atoms is 14 Å instead of 11 Å, as in theclassic (i.e., closed) GTP-bound structures (Fig. S3). Relaxingthe K-Ras–SRJ23 complex by MD further expanded the pocketto better accommodate the halogenated phenyl ring (Fig. 3 Aand C). This led us to the question of what might constitutea minimal scaffold to target this pocket. To address this question,we fragmented SRJ23 into 16 substructures and docked themonto the K-Ras conformer that served as the starting point forthe K-Ras–SRJ23 simulations. A fragment common to all threeSRJ ligands has the highest preference for this site (256/256poses) (Fig. S7A). We predict that this three-membered ringrepresents the chemical signature of AGP derivatives to target p1,but the role of the halogenations for affinity, if any, is not obvious(SI Materials and Methods). To test whether compounds thatshare structural similarity with SRJ bind to p1, we carried outvirtual screening of about 1,000 ligands from the ZINC database(http://zinc.docking.org) selected based on a Tanimoto similarityindex of 0.6 (SI Materials and Methods). The results suggest thatthese ligands would bind to p1 with a micro- to nanomolar affinity(AutoDock Vina energy score of −6 to −10 kcal/mol) whenswitch 1 is open (Fig. S7B). Their affinity for closed switch 1conformations was about three orders of magnitude weaker. Thisresult not only reemphasizes the correlation between Ras dy-namics and ligand binding but also suggests that it may be pos-sible to find other small-molecule Ras binders that preserve thekey features of SRJ but bind with better affinity (Fig. S7C).

Stabilization of a Unique K-Ras Conformation. The docking resultsindicate that our ligands favor state 1-like conformers with anopen switch 1 (Fig. 4B). To test whether the ligands stabilize

Fig. 1. Chemical structures of AGP and its benzylidene derivatives SRJ09,SRJ10, and SRJ23.

Table 1. Pockets on K-RasQ61H targeted by AGP and derivatives SRJ09, SRJ10, and SRJ23

Pocket Residues Locations

1 17, 20, 21, 24, 25, 29, 30–40, 55, and 57 α1, switch 1, β22 4–7, 35–39, 54–59, 61–78 β1, β2, switch 23 97, 101, 107–111, 137–140, 162, 163, 166 α5, β5, β6, loops α3/β5 and α4/β6

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a particular Ras conformation, we used a principal component(PC)-based analysis developed previously (13, 34–36) to mapconformers from K-Ras–SRJ23 trajectories onto a PC plane de-fined by crystal structures. Fig. 4B shows that p1-bound SRJ23stabilizes conformations that are different from the canonical GTP/GDP or nucleotide-free states (see Fig. S8 for full PC data).Alignment of the simulated K-Ras structures with p1-bound SRJ23onto those from a control ligand-free simulation further showsstabilization of D38 in an orientation that allows for an opening ofa pore behind switch 1 (Fig. S3). Given their structural similarity,we expect SRJ09 and SRJ10 will have a similar effect.

Proposed Mechanism of Action. Recent reports revealed that ligandbinding at a pocket between the core β-sheet and helix 2 of K-Rasstabilizes alternate side-chain conformations at or around switch 2and thereby affects exchange factor binding (15, 16). For instance,the side chains of bothY64 andY71were displaced in these ligand-bound structures relative to an SOS-bound H-Ras structure(15, 16). We therefore compared the orientation of these side chainsin our K-Ras–SRJ23 conformers with those in K-Ras–DCAI (PDBID code 4DST),H-Ras–SOS [PDBID codes 1BKDand 1NVV(37,38)], and other K-Ras–ligand complexes (PDB ID code 4EPV)(Fig. 4A). The orientation of Y71 in K-Ras–SRJ23 mimics that inK-Ras–DCAI andPDB IDcode 4EPV.Moreover, Y64 is displacedby >5 Å (Cα atom) in K-Ras–SRJ23 relative to its position in theH-Ras–SOS complex (Movie S1). Although other modes of actioncannot be ruled out, these observations suggest that our ligandsmaystabilize Ras in a conformation that is not conducive to GEFbinding. AGP and its derivatives could thus inhibit GEF-inducednucleotide exchange in a similar manner as those reported byMaurer et al. (15) and Sun et al. (16).

In Vitro Assays Indicate That SRJ Compounds Inhibit Ras GTP Loadingand Cancer Cell Growth. To explore the effects of AGP and itsderivatives on Ras function in intact cells, we first measured acti-vation of the Ras/MAPK cascade in response to EGF stimulation.Serum-starved BHK cells were incubated with AGP or derivativesfor 6 h and stimulated with EGF. Fig. 5A shows that SRJ09 andSRJ23 significantly reducedRasGTP loading, asmeasured inRas-binding domain (RBD) pull-down assays. The reduction in Rasactivation correlated closely with a concomitant reduction in

MAPK activation (Fig. 5B). The concentrations of AGP requiredto inhibit EGF-stimulated extracellular-signal–regulated kinase(ERK) activation were ∼10-fold higher than the active concen-trations of SRJ09 and SRJ23, indicating that the SRJ compoundswere considerablymore potent than the parent compound (Fig. 5Bcompared with Fig. S9B). We then immunoblotted the RBD pull-down assays with isoform-specific antisera to determine whetherall Ras isoforms were equally sensitive to SRJ09 and SRJ23. Fig.5C shows that K-Ras GTP loading was significantly suppressed bySRJ09 and SRJ23, whereas H-Ras and N-Ras GTP loading weremuch less sensitive. For example, SRJ23 reduced K-Ras, H-Ras,and N-Ras GTP levels by 47%, 28% and 13%, respectively. Thestructural basis for K-Ras selectivity is not immediately clear, but itis consistent with previous suggestions (34, 36) that K-Rasmight bemore dynamic than H-Ras and samples open switch 1 con-formations more frequently. This is supported by results from MDsimulations of wild-type K- and H-Ras (Fig. S10). Importantly, noneof the compounds suppressed activation of the EGF receptor, asmeasured byY1068 phosphorylation. Furthermore, 5 μMSRJ09 didnot inhibitCRaf-mediatedMAPKactivation (Fig. S11), showing thatthe andrographolides do not inhibit any of the kinases in the Raf/MEK/ERK signaling cascade. These results strongly suggest thatAGP, SRJ09, and SRJ23 directly target Ras to block the exchange ofGDP for GTP and thus prevent Ras activation. Consistent with thismechanism of action, a 6-h incubation in SRJ09 and SRJ23 (5μM) had no measurable effect on the extent of GTP loading of

Fig. 2. Overview of the K-RasQ61H structures derived fromMD simulations forensemble docking. From among the 75 unique K-Ras conformers used fordocking (Materials and Methods), 5 representative cluster centroids are shownalong with the percentage of the total conformers they represent. Pockets mostfrequently targeted by SRJ23 are highlighted in red van der Waals spheres.Notice the major conformational changes in switch 1 (cyan) and switch 2 (green).

Fig. 3. Overview of transiently opening pockets 1 and 2 on K-RasQ61H withrelevant residues colored according to their electrostatic potential. (A)Docking pose of SRJ23 at pocket 1, where its phenyl group occupies the spacepreviously occupied by Y40 and is stabilized by the residues I21 and T20. (B)Docking pose of SRJ23 at pocket 2 opened by the movement of Y71 and linedby hydrophobic residues I36 and M67. (C) MD-optimized complex of SRJ23 atpocket 1. (D) The conformation in C is modified to visualize the proximity ofSRJ23 to Mg2+ and GTP (switch 1 is now shown as a transparent surface). Thehydroxyl group on the lactone ring of SRJ23 forms a hydrogen bond with theα-phosphate of GTP as well as an electrostatic contact with Mg2+ similar tothat made by the hydroxyl of T35 on Ras (20). Electrostatic potentials werecalculated using the Adaptive Poisson-Boltzmann Solver (50).

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oncogenic mutant K-, H-, and N-RasG12V, or on the extent ofMAPK activation in Ras-transformed cell lines (Fig. 5D).Oncogenic mutant Ras is constitutively GTP-loaded because the

oncogenic mutation blocks the ability of GAP to stimulate GTP hy-drolysis and returnRas to the inactive GDP-bound ground state.Wereasoned, however, that the intrinsic GTPase activity of oncogenicmutant Ras must eventually return Ras to the ground state eventhough the expected rate would be very slow (∼8–17 h, with kcat of1–2× 10−3min−1) (39, 40).Once in theGDPground state, oncogenicRas would then need to interact with an exchange factor to bereloaded with GTP. If the SRJ compounds are present at this pointandbind toRasG12V–GDP(or other substateswith anopen switch 1conformation), GTP loading may be abrogated. We tested thishypothesis by incubating oncogenic mutant K-Ras–transformedcells in SRJ09 and SRJ23 (5 μM) for 3 d to give sufficient timefor GTP hydrolysis and thus reveal a requirement for GDP–GTPexchange. Remarkably, after this prolonged incubation period,SRJ09 and SRJ23 reduced K-RasG12V GTP levels by ∼50%(Fig. 5E), which resulted in a concomitant reduction in phos-phorylated ERK (ppERK) levels in K-RasG12V–transformedcells (Fig. 5F). Moreover, the ability of AGP, SRJ09, and SRJ23to induce growth inhibition against PC-3 (prostate), HCT-116(colon), and MDA-231 (breast) cancer cells was assessed asdescribed previously (25). PC-3 cancer cells harbor wild-type Rasfor all of the three different isoforms (H-, K-, and N-Ras), whereasHCT-116 and MDA-231 cancer cells express G13D mutant K-Ras

and wild-type H- and N-Ras. Table 3 shows the 50% growth-inhibition (GI50) values of AGP, SRJ09, and SRJ23 in this panelof cancer cell lines.One can see that SRJ09 andSRJ23 are generallymore active than theparent compoundAGPand,more importantly,cancer cells with endogenous mutant K-Ras (HCT-116 and MDA-231) were found to be significantly more sensitive toward thecompounds. The mean GI50 value in these cells was approximatelyhalf comparedwith the cancer cell harboringwild-typeK-Ras (PC-3).We conclude that cancer cells with mutant Ras are more sensitivetoward the compounds, which is consistent with our results withectopically expressed mutant Ras.Taking the computational and cell biology results together, we

conclude that SRJ09 and SRJ23 bind to Ras and prevent GTPloading, thus effectively blocking GDP–GTP exchange in live cells.Using these compounds that directly target Ras, we also show thatexchange factors are required to maintain oncogenic mutant Ras inthe active GTP-bound state, and that inhibiting nucleotide ex-change is a valid approach to abrogating the function of oncogenicmutant Ras.

ConclusionUsing molecular docking and simulations, we show that AGPand its derivatives bind to the switch regions of Ras, preferen-tially targeting a transient pore behind switch 1 as well asa groove between switches 1 and 2. Binding of AGP derivativesto p1 stabilizes Ras in a unique conformation where key residues

Fig. 4. Comparison of known Ras structures boundto SOS and small-molecule ligands. (A) Overlay ofthe switch 2 region of a K-Ras–SRJ23 snapshot (or-ange), the crystal structure of K-RasG12D–DCAIfrom PDB ID code 4DST (purple, with only residues62–75 shown for clarity), K-Ras–0QX from PDB IDcode 4EVP (yellow, residues 62–75), and two struc-tures of H-Ras–SOS from PDB ID codes 1NVV (iceblue) and 1BKD (cyan). (B) Projection of simulatedK-Ras conformers onto a PC space defined by crys-tallographic structures, with the cluster of GDP–H-Rasstructures highlighted in blue and the cluster of loss-of-function mutants in orange. PDB ID code 4DST(purple) lies in the major GTP cluster, whereas PDB IDcode 4EPV (yellow) is intermediate to the GTP andGDP clusters. Two K-Ras–ligand conformations fromdocking (MD-Lf, green dots) are shown to illustratethe ability of MD to capture putative excited-statestructures with open p1 that are preferred by ourligands. An example of simulated K-Ras–SRJ23 (MD-Lb) lies between Ras–SOS (cyan/ice blue) and Ras–inhibitor (purple/yellow) conformations. The crystalstructure of nucleotide-free H-Ras (PDB ID code 1BKD) is also shown, with SRJ23 docked at switch 1. SRJ23 is shown as a yellow surface and switches 1 and 2 asblue and green surfaces, respectively. The Bio3D package (http://thegrantlab.org/bio3d) was used to generate the figure in B.

Table 2. Ranking ligand–receptor pairs by the joint probability of occurrence of a given receptor conformation andligand pose, followed by visual pocket identification

Receptor Ligand Receptor–ligand pair

Cluster Fraction total conformers Cluster Fraction total poses Ranking by joint probability, % Pocket ID

4 0.09 2 0.66 5.6 22 0.11 3 0.27 3.1 17 0.04 1 0.64 2.9 31 0.12 17 0.20 2.4 21 0.12 18 0.17 1.4 2

Shown here are data for a few preferred poses of SRJ23 on selected K-RasQ61H cluster representatives, highlighting the relativepreference of the ligand for a specific binding-site and receptor conformation. The joint probability is simply the product of thefraction of individual ligand and receptor clusters (column 2 times column 4). Note that these are just samples and do not reflectthe total probability of binding to a particular site; binding sites for the rest of the ligands and receptor conformations were de-termined in a similar way.

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on the nucleotide-binding switches, such as Y64 and Y71, re-organize in a manner that would impair interaction with Rasmodulators. Combining these findings with earlier reports (15,16), we predict that AGP derivatives should inhibit Ras functionby preventing GEF-assisted nucleotide exchange. We confirmedthis prediction by experiments showing that SRJ treatmentblocks acute GTP loading of wild-type Ras. Even more in-triguing was the observation that extended incubation with SRJcompounds also reduced oncogenic mutant Ras-GTP levels

and hence signal output. The likely explanation for this observa-tion is that the SRJ ligands deplete the pool of GTP-Ras over timeby allowing intrinsic hydrolysis of bound GTP while preventingreactivation via GEF-catalyzed GDP–GTP exchange. This mecha-nism can operate because oncogenic mutations prevent the abilityof GAP to accelerate the intrinsic GTPase activity of Ras and onlymodestly reduce the actual intrinsic GTPase activity (41–44). Inshowing that GEF-mediated nucleotide exchange is critical foroncogenic Ras signaling, our findings represent an important proofof concept for anti-Ras drug discovery. Drug development need notbe limited to ligands that abrogate effector interactions of GTP-loadedRas or directly inhibit Ras effectors, butmay be expanded toexploit the requirement of GEF-mediated GTP loading for a sus-tained signaling through oncogenic Ras.We therefore propose thatfuture efforts toward Ras inhibition should additionally considerligands that abrogate GEF binding and/or prevent GDP–GTP ex-change; such ligandsmay potentially bemore effective and selectivegiven the fewer number of exchange factors than effectors and thepossibility that Ras isoforms may differ in dynamics and response.

Materials and MethodsEnsemble Docking. Expanding the concept of a relaxed complex scheme forstructure-based drug discovery (45, 46), we built an ensemble of K-Ras conformersthat contained infrequently sampled structures. Such structures could potentiallyharbor open binding sites that are invisible in crystal structures. To this end, weisolated an ensemble of 75 structures based on rmsd clustering of GTP-boundK-RasQ61H conformers derived from previously reported MD simulations (24).Taking advantage of the small number of related compounds that we were in-terested in, we deliberately used a small rmsd cutoff of 1.3 Å to generate a largenumber of clusters and used all of the cluster centroids to include infrequentlyvisited K-Ras conformers. Blind docking against these structures was carried outusing AutoDock 4.2 (47) and a search area that encompasses the entire Rasstructureplus abuffer spaceof 10Å ineachdirection.GTPwas retained toexcludenonspecific hits at the catalytic site (see SI Materials and Methods for detailsand controls).

Binding-Site Identification and Selection of Ligand Poses. To account for thejoint probability that K-Ras samples a given conformation and AutoDockconsistently places the ligand into a consensus site, binding sites were iden-tified based on the size of both the ligand and receptor clusters. Therefore,ligand–receptor pairs were ranked by the product of the fraction of Rasconformers within a cluster and the fraction of ligand poses within a ligandcluster (Table 2). Visual inspection of the high-ranked complexes and histo-grams of contact frequencies was then used to identify the most commonlytargeted pockets (SI Materials and Methods).

Molecular Dynamics Simulations. After docking and site identification, wetested whether binding at the predicted sites is viable by performing MD onselected protein–ligand complexes. The simulation details are similar to thosedescribed previously (24) and in SI Materials and Methods. By assigning dif-ferent initial velocities, we generated two sets of five separate trajectorieswith SRJ23 bound to K-Ras at either of two preferred sites (p1 and p2).

In Vitro Cell-Based Assays. Based on initial work that formed the basis of thisstudy (25, 48), AGP, SRJ09, and SRJ23 were prepared as reported earlier (48)

A B

C D

E F

Fig. 5. Mean fold increase in Ras-GTP (A) and ppERK (B) ± SEM from threeindependent experiments where wild-type BHK cells were treated withSRJ09 or SRJ23 for 6 h in the absence of serum, followed by 25 ng/mL EGFstimulation for 2 min. Ras-GTP levels were measured using an RBD pull-downassay, and ppERK levels were measured by quantitative immunoblotting. (C)Representative blots from three independent experiments in which wild-type BHK cells were treated with 5 μM SRJ23 for 6 h in the absence of serum,followed by 25 ng/mL EGF stimulation for 2 min. Levels of K-, H-, and N-Ras–GTP loadings were measured using an RBD pull-down assay, and phospho-EGF receptor (EGFR; Y1068) levels were measured by quantitative immu-noblotting. (D) Mean ± SEM from three independent experiments in whichBHK cells stably expressing oncogenic Ras isoforms were treated with 5 μMSRJ09 or SRJ23 for 6 h in the absence of growth serum. ppERK levels weremeasured by quantitative immunoblotting. Mean Ras-GTP (E) and ppERK(F) ± SEM from three independent experiments in which BHK cells stablyexpressing oncogenic K-Ras were treated with 5 μM SRJ09 or SRJ23 for 72 h.Growth media with the drug were replaced every 24 h. Ras-GTP levels weremeasured using an RBD pull-down assay, and ppERK levels were measuredby quantitative immunoblotting. Differences between DMSO- and drug-treated cells were assessed using one-way ANOVA tests. Significant differ-ences are indicated (*P < 0.05; **P < 0.01; ***P < 0.001).

Table 3. Growth-inhibitory effect of AGP, SRJ09, and SRJ23 onprostate (PC-3), colon (HCT-116), and breast MDA-231 cancer cells

GI50, μM

Compounds PC-3* HCT-116† MDA-231†

AGP 9.7 ± 1.3 4.5 ± 1.8 5.9 ± 1.4SRJ09 12.4 ± 1.3 3.1 ± 0.7 5.0 ± 0.4SRJ23 6.6 ± 0.5 4.0 ± 1.2 5.8 ± 0.7

Ras status was obtained from the Catalog of Somatic Mutations in Cancer,Wellcome Trust Sanger Institute. Values represent mean ± SD of three in-dependent experiments.*Wild-type for H-, K-, and N-Ras.†Mutant K-Ras (substitution, p.G13D) and wild-type for H- and N-Ras.

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(see Tables S1 and S2 for proof of purity). Antibodies against H-Ras (F235; sc-29) and N-Ras (F155; sc-31) were obtained from Santa Cruz Biotechnology.Monoclonal K-Ras antibody (R3400) was obtained from Sigma-Aldrich.Rabbit phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody (9101),mouse anti–phospho-EGF receptor (Y1068) antibody (2236), and rabbit totalEGF receptor antibody (2232) were purchased from Cell Signaling Technol-ogy. Monoclonal anti-Ras antibody (610001) was obtained from BD Trans-duction Laboratories. For cell culture, baby hamster kidney (BHK) cells weremaintained in Dulbecco’s modified Eagle medium (Gibco) supplementedwith 10% (vol/vol) donor calf serum and 2 mM L-glutamine. Ras-GTP levelswere measured in a GST–Ras-binding domain pull-down assay as describedpreviously (49). Samples were analyzed by quantitative Western immuno-blotting using pan-Ras or Ras isoform-specific antibodies.

The potential of AGP, SRJ09, and SRJ23 to induce growth inhibition againstPC-3 (prostate), HCT-116 (colon), and MDA-231 (breast) cancer cells was assessedaccording to Jada et al. (25). Briefly, exponentially growing cells were seeded inflat-bottom 96-well plates at a density of 2,000 cells in 0.18 mL per well andincubated overnight for cell attachment. The cells were then treated with

compounds at a final concentration range of 0.1–100 μM (n = 4). The controlwells were introduced with 0.1% of DMSO equivalent to the highest amount ofDMSO used as a vehicle in the compound-treated wells. After 96 h of incubationin a CO2 incubator, the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The 50% growth-inhibitionvalues were obtained from dose–response growth-inhibitory curves.

ACKNOWLEDGMENTS. We thank Dr. Rommie Amaro for kick-starting thecollaboration between the J.S. and A.A.G. laboratories. Wong CharngChoon and Wong Hui Chyn (Pharmacotherapeutics Unit, Departmentof Medicine, Faculty of Medicine and Health Sciences, Universiti PutraMalaysia) are gratefully acknowledged for performing the in vitro anticancertest. H.J.H. is supported by a fellowship from Keck Gulf Coast ConsortiaTraining in Pharmacological Sciences (National Institute of General MedicalSciences Grant T32GM089657). This work is supported in part by grantsfrom National Institutes of Health General Medical Sciences (R01GM10078to A.A.G.), Cancer Prevention and Research Institute of Texas (RP100483 toJ.F.H.), and Ministry of Higher Education, Malaysia (Research UniversityGrant Scheme Grant 04-02-12-2017RU to J.S.).

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