Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules

Cell Chemical Biology

Review Mutant-Specific Targeting of Ras G12C Activity by Covalently Reacting Small Molecules € ller,2,3 and Daniel Rauh2,3 Roger S. Goody,1,* Matthias P. Mu 1Department

of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany 3Drug Discovery Hub Dortmund (DDHD) am Zentrum fu €r integrierte Wirkstoffforschung (ZIW), Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2019.07.005 2Faculty

In this review we discuss and compare recently introduced molecules that are able to react covalently with an oncogenic mutant of KRas, KRas G12C. Two different classes of compounds in question have been developed, both leading to the mutant being locked in the inactive (guanosine diphosphate [GDP]-bound) state. The first are compounds that interact reversibly with the switch-II pocket (S-IIP) before covalent interaction. The second class interact in a competitive manner with the GDP/guanosine triphosphate (GTP) binding site. The fundamental physico-chemical principles of the two inhibitor classes are evaluated. For GDP/GTPcompeting molecules, we show that special attention must be paid to the influence of guanine nucleotide exchange factors (GEFs) and their elevated activity in cells harboring abnormally activated Ras mutants. A new approach is suggested involving compounds that interact with the guanine binding site of the GTPase, but in a manner that is independent of the interaction of the GTPase with its cognate GEF. Introduction Ras GTPases were identified in the mid-1980s as proto-oncogenes, and the high prevalence of Ras mutations in human tumors was the reason for intense interest in these proteins, making Ras one of the most studied objects in fundamental biological research in the meantime (Simanshu et al., 2017). By the early 1990s, the basic properties of the so-called Ras superfamily, which encompasses the Ras, Rho, Rab, Arf, and Ran proteins, had been established, and these are summarized in Figure 1. Thus, Ras or any of the other members of the family exist in two different states that may be referred to as ‘‘off’’ when guanosine diphosphate (GDP) is bound or ‘‘on’’ (activated) when guanosine triphosphate (GTP) is bound. When the tightly bound GDP is released, generally with the help of a guanine nucleotide exchange factor (GEF), GTP can bind, generating the activated state. The term ‘‘active’’ refers to the ability to interact with so-called effector proteins that associate preferentially with the GTP-bound state. The life-time of this state is limited by GTP hydrolysis, normally with the help of a GAP (GTPase-activating protein) that accelerates the otherwise (for most GTPases) very slow intrinsic GTP hydrolysis reaction. In many oncogenic mutants of Ras, both the basic GTPase rate and, more dramatically, the GAP-activated rate, are significantly lower than for the wild-type proteins. This leads to abnormally high activation levels of Ras and loss of regulation of cell growth and division, which are characteristics of oncogenically transformed cells. Basic Structural and Kinetic Properties of Ras Proteins Ras proteins (HRas, NRas, and KRas4a/4b in humans) are lowmolecular-weight (21 kDa) GTPases consisting of a GTPase domain with almost identical sequences and structure up until

residue 165, followed by a variable region of approximately 25 amino acids (only
8% sequence identity; generally referred to as the hypervariable region in Ras superfamily proteins) until the C-terminal residue (Figures 1A and 1B). The 3D structures of the GTPase domains in the four Ras proteins are virtually identical and consist of six b strands and five a helices. Several motifs that are conserved in the whole RasGTPase superfamily are involved in GTP/GDP binding (G-motifs G1-G5). G1 is also called the P loop (phosphate binding loop) and has the generic sequence GxxxxGKS/T (GAGGVGKS in Ras), G2 is essentially a single threonine that interacts with the Mg2+ ion bound with GTP, G3 is DxxG (DTAG in Ras, with an essential Q (Q61 in Ras) after G; interacts mainly with the g-phosphate of GTP and is necessary for hydrolysis of GTP to GDP), G4 is NKxD (NKCD in Ras; interacts with the guanine base) and G5 is TSAK in Ras (also interacts with the base). It was noticed quite early that Ras and related GTPases have a very high affinity for guanine nucleotides. In fact, this affinity is so high (Kd in the 10 mM range) that initial estimates were erroneous, partly owing to the fact that Ras proteins are isolated after expression in bacteria as stable 1:1 complexes with GDP (Goody et al., 1991). Accurate measurements of nucleotide affinities could only be made after methods for the generation of the relatively unstable nucleotide-free protein were developed (Feuerstein et al., 1987; John et al., 1990). Owing to the high affinity of GDP/GTP, kinetic methods to determine association and dissociation rate constants were used (John et al., 1990). Recently determined values (Jeganathan et al., 2018) for these constants are for KRas and are shown in Scheme S1. The extremely high nucleotide affinity arises from a very small dissociation rate constant, with the effective association rate constant in the normal range for

1338 Cell Chemical Biology 26, October 17, 2019 ª 2019 Published by Elsevier Ltd.

Cell Chemical Biology

Review

Figure 1. Ras Proteins (A) Overview of the structure of KRas:GDP that illustrates the common fold of small GTPases of the Ras superfamily (PDB: 4lpk). The QR code allows visualization of the structure in augmented reality (Wolle et al., 2018). (B) Sequence alignment of the four isoforms of Ras (HRas, UniProt: P01112; NRas, UniProt: P01111; KRas4a, UniProt: P01116; KRas4b, UniProt: P01116-2). Note that the color code of important sequences (G1-G5 motifs and switch regions as explained in the main text) within the Ras proteins is the same as in (A). (C) GTPase cycle illustrating the regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), allowing tight spatial and temporal control of Ras activity in healthy cells. Mutations within the Ras proteins that cause a change in the relative abundance of Ras:GDP and Ras:GTP play a prominent causal role in many human cancers.

nucleotide-protein association reactions. These are often twostep processes with a rapid weak initial binding step followed by a second step with a large equilibrium constant in the forward direction (Bagshaw et al., 1974; John et al., 1990). A fundamental aspect of the manner in which Ras and other GTPases function is the release of GDP under the influence of a GEF. Their mechanism of action can be described as allosteric competitive with respect to guanine nucleotides (Goody and Hofmann-Goody, 2002). In the ternary complex between Ras, GDP, or GTP and the GEF Sos, both entities (GDP/GTP and Sos) are bound orders of magnitude more weakly to Ras than in their corresponding binary complexes. This is in contrast to classical competition, in which only one or the other entity is bound. This kinetic mechanism, which was first analyzed quantitatively for the interaction between the GTPase Ran and its exchange factor RCC1 and has been confirmed for a number of other GTPase/GEF pairs including Ras/Sos (Esters et al., 2001; Guo et al., 2005; Itzen et al., 2007; Klebe et al., 1995; Lenzen et al., 1998) is shown in Figure 2A. The structural mechanisms of action are understood in a number of cases and have been reviewed several times (Itzen and Goody, 2011; Lee et al., 2009; Muller and Goody, 2018). An important aspect of the mechanism is that there is no preferential exchange of GTP for GDP, but that GEFs act similarly on Ras:GDP and Ras:GTP, and that this is in fact a thermodynamic requirement of the mechanism (Goody and Hofmann-Goody, 2002). Under cellular conditions, the reaction is only driven toward production of Ras:GTP by the significantly higher concentration of GTP than GDP in the cytoplasm (Traut, 1994).

Approaches to Ras Inhibition Approaches that target general Ras activity or a particular Ras interaction might be thought not to be very promising because there will not be a preferential effect on cells in which Ras activity is higher than ‘‘normal’’ or is not regulated by the usual mechanisms. Despite this, there have been several attempts to do exactly this, that is, to inhibit Ras activity in a general manner, with no selection either for specific Ras mutants or other situations leading to inappropriately high or unregulated Ras activity. An early example was the use of prenylation inhibitors to prevent C-terminal farnesylation and therefore membrane localization, because Ras must be membrane attached to play its role in signaling. Highly potent inhibitors of prenylation were developed (Kohl et al., 1994), which have, however, mainly failed in the development to a clinical setting, partly because of alternative prenylation by other prenyl transferases (Berndt et al., 2011). A more recent example is the use of a molecule that modulates the distribution of KRas between the plasma membrane and endomembranes by inhibiting the interaction of KRas with PDEd, a protein involved in distributing the GTPase between these membrane systems (Zimmermann et al., 2013). Despite the perceived weakness of approaches that target Ras activity in a generic fashion, there is still the possibility of selective effects on tumor cells because of specific susceptibilities, as also seen for example in the selective induction of apoptosis in tumor cells by inhibitors of Rab geranylgeranyl transferase (Lackner et al., 2005). It is therefore important to pursue such approaches. A more promising approach, in principle at least, would be the targeting of specific Ras mutants that have oncogenic Cell Chemical Biology 26, October 17, 2019 1339

Cell Chemical Biology

Review Figure 2. The Interaction of Ras with GEFs (A) The general scheme shows the interaction of a GEF (orange) with a small GTPase (blue) to form the ternary low-affinity GTPase:nucleotide:GEF complex. Either the nucleotide (abbreviated as GXP) or the GEF can dissociate to form the binary highaffinity Ras:nucleotide or Ras:GEF complexes, respectively. Thus, the exchange factor facilitates release of the otherwise strongly bound nucleotide and stabilizes the GTPase in the nucleotide-free form while opening the nucleotide binding pocket (note that the nucleotide-binding site is depicted in a more open conformation in the Ras:GEF complex). (B) Structure of the complex between Sos (rainbow colored and orange surface) and Ras (switch I and II and the P loop are colored as in Figure 1; PDB: 1nvw). In Sos, an additional allosteric binding site for Ras:GTP, or the non-hydrolysable analog of GTP, GppNHp (guanosine 50 -[b,g-imido]triphosphate), can further increase the rate of nucleotide exchange and provides the basis for a positive feedback loop (Margarit et al., 2003). The QR code allows visualization of the structure in augmented reality (Wolle et al., 2018).

properties. However, it is not immediately obvious how to achieve this aim. For example, a potent reversible inhibitor of GTP binding, even if it could be found, would not have intrinsic selectivity against Ras mutants, and it is difficult to imagine a modification that could provide selectivity against known mutants if the interaction is reversible. The examples discussed below are therefore concentrated on the concept of exploiting the chemical nature of an oncogenic mutation in the context of its exact position in the protein sequence, and taking advantage of the chemical reactivity of this specific residue. In the two approaches described, the cysteine at position 12 in the oncogenic variant KRas G12C is targeted. This mutation occurs in a large proportion of lung tumors (Cancer Genome Atlas Research Network, 2014; Prior et al., 2012). Covalently Reacting Inhibitors that Do Not Compete Directly with Nucleotide Binding As is the case for other P loop mutants, replacement of glycine at position 12 by cysteine leads to inhibition of the intrinsic GTPase reaction (in this particular case only moderate inhibition; Hunter et al., 2015), and a dramatic inhibition of the important GAPactivated GTP hydrolysis (Hunter et al., 2015). Interestingly, such oncogenic variants result not only in enhanced levels of the activated mutant Ras protein, but additionally also of other unmutated Ras isoforms (H- and NRas) via the positive feedback loop described in Figure 2 (Jeng et al., 2012). Because of the nucleophilic activity of the side chain of the cysteine at position 12 in the KRas sequence, several attempts have been made to address it, specifically by reaction with an appropriate electrophile. A promising approach to identifying potential covalent 1340 Cell Chemical Biology 26, October 17, 2019

modifiers at this position was that of Ostrem et al. (2013), who used a library of disulfides for initial identification of lead structures. Since disulfides are not likely to be useful in the intracellular context because of the reducing nature of the cytoplasm, the information gained from the initial screening was used to generate inhibitors with electrophilic warheads of several types, in particular acrylamide residues. Acrylamide residues are known to be biocompatible and have been successfully used in kinase inhibitors (Lategahn et al., 2018). The expectation here is that there would be low general (unspecific) reactivity toward thiol groups, but nevertheless adequate reactivity if there is prior non-covalent interaction with the target (KRas G12C), and if this places the warhead close to Cys12. Compounds of this type were indeed identified (see Figure 3 for an optimized example), and crystallographic analysis showed that the bulk of such molecules was located in a previously postulated (Ostrem et al., 2013), but not well characterized, pocket referred to as the switch-II pocket (S-IIP) (Figure 3), since it consists mainly of residues from the switch-II region. This site is distinct from the GTP binding site, so there is no direct competition between occupants of it and GDP. Interestingly, the identified inhibitors are specific for the GDP state of KRas G12C and furthermore have the property of rendering KRas:GDP insensitive to GEFs, presumably because of steric hindrance of GTP binding (Figure 3A). Although the detailed mechanism of this effect is not fully understood, the compounds keep Ras trapped in the inactive state (Hansen et al., 2018; Janes et al., 2018; Lito et al., 2016; Ostrem et al., 2013; Patricelli et al., 2016). Several further studies describe new S-IIP inhibitors binding in the same pocket (Figure 3B), but with improved pharmacokinetics and target engagement compared with earlier examples (Fell et al., 2018; Zeng et al., 2017). Overall, it is apparent that members of the above-mentioned inhibitors specifically target KRas G12C and also affect KRas G12C tumor cell lines and induce tumor regression in mouse

Cell Chemical Biology

Review Figure 3. Selected Switch-II Pocket Inhibitors (A) An optimized compound that reacts covalently with KRas G12C (Janes et al., 2018). The compound referred to as ARS-1620 binds into the switch-II pocket (S-IIP) in close proximity to the nucleotide binding pocket and is covalently linked to Cys12 in KRas G12C (left; PDB: 5v9u). Note that compound binding is incompatible with GTP binding due to steric clashes with the g-phosphate of GTP (the QR code allows visualization of the structure in augmented reality; Wolle et al., 2018). The structure of ARS-1620 is shown on the right, with the ‘‘warhead’’ (acrylamide group) in the upper part of the drawing. The molecule exists as atropisomers because of restricted rotation around the red bond connecting the aryl ring systems (the restricted rotation is also indicated by red arrows here and in the corresponding structure on the left). The Sa-atropisomer shown here is
1,000-fold more reactive than the corresponding Ra-atropisomer with KRas G12C. (B) Further examples of compounds that bind into the S-IIP include compound 1_AM (Zeng et al., 2017), compound 4, and compound 12 (Fell et al., 2018), which have been co-crystallized with KRas G12C (PDB: 5v9l, 6n2j, and 6n2k, respectively).

xenograft models. They are therefore considered to be promising agents for further development. A puzzling aspect of the developed compounds is their high degree of specificity for KRas G12C compared with other thiolcontaining molecules. Initially, they were regarded as affinitydriven irreversible inhibitors (formerly referred to as affinity labels), but it has become clear that their reversible affinity is actually very low (Ki values in the hundred micromolar range; Hansen et al., 2018), which does not fully explain their high specificity. It has recently been suggested that the high reactivity of the acrylamide residue in the context of the KRas active site is a critical factor. Crystallographic analysis of the covalent adducts leads to the interpretation that Lys16, which interacts with the b-phosphate of GDP, plays an important role by interacting via a hydrogen bond between the protonated side chain amino group and the carbonyl function of the acrylamide residue. This presumably helps to correctly position the warhead for the reaction with Cys12 (Hansen et al., 2018), and also contributes to catalysis by the electron-withdrawing effect of this interaction. Structural evidence also suggests an additional mechanism for discrimination of the inhibitors between the GDP and the GTP states of KRas G12C. Thus, the g-phosphate group of GTP might not only prevent correct positioning of the acrylamide group for the proposed mechanism, but also compete with the inhibitor for interaction with Lys16, because an oxygen of the g-phosphate group also interacts with Lys16. A further initially puzzling aspect of the success of these compounds at the cellular level stems from the fact that they react preferentially with the GDP-bound form of KRas G12C. However, such KRas mutants are considered to be present mainly in the GTP-bound form in the cell, which is the reason for their oncogenic properties. Since an effect at

the cellular level is only to be expected if all or a substantial amount of KRas G12C is effectively trapped by the covalent reaction, there must be a mechanism for generation of the GDP- from the GTP-bound form of KRas G12C. The only possibilities here are the intrinsic GTPase rate (since activation by GAP is strongly inhibited) and the exchange of GDP for GTP on Ras catalyzed by GEFs, in particular Sos. The latter effect could be important, since GEFs operate essentially symmetrically on GTPase:GDP and GTPase:GTP complexes (Goody and Hofmann-Goody, 2002), so that cellular levels of GDP (
10% of GTP levels) could generate significant concentrations of KRas:GDP (
10% if one neglects the stabilizing effect of effector proteins on the GTP state). This could allow reaction with the compounds and gradual but complete depletion of KRas G12C:GTP. Similarly, the GTPase reaction, while slow (
5 3 104 s1, or 1.8 h1), could also lead to depletion of KRas G12C:GTP, because generated KRas G12C:GDP could react with the compounds. In one study, the conclusion is reached that the slow intrinsic GTPase reaction is responsible for the inactivation seen in cells (Lito et al., 2016). One important piece of evidence is that introduction of a second mutation into KRas G12C, which further reduces the already slow GTPase rate, leads to a slower inactivation rate. This is an expected effect if the GTPase reaction is the source of the GDP form that reacts with inhibitors. However, measures that lead to increased GEF (Sos) activity in the cell lead to a decrease of inhibitory potency of the compounds, while inhibition of GEF activity leads to potentiation of inhibition. This is surprising, because, in a simple system consisting only of nucleotides, KRas G12C, and inhibitor, increasing GEF activity should potentiate inhibition due to increased availability of KRas G12C:GDP. These effects are illustrated in Figure 4, in which we have used known values of rate constants (Scheme S1 and Figure 4) to predict the time course of reaction with Cell Chemical Biology 26, October 17, 2019 1341

Cell Chemical Biology

Review Figure 4. Simulated Time Courses of Inactivation of KRas G12C by Switch-II Pocket Inhibitors Simulated time course of inactivation of KRas G12C and decay of the activated (GTP) state in the presence of 10 mM ARS-1620 using the rate constants given in Scheme S1 and concentrations given in Table S1. A value not shown in Scheme S1 is the effective second-order rate constant for the covalent reaction of KRas G12C with ARS-1620, which has been measured to be 1.1 3 103 M1 s1 (Hansen et al., 2018).The inhibited state is designated as RasInh, but also harbors reversibly bound GDP, while the active state is referred to as Ras:GTP. (A) In the absence (solid lines) or presence (dashed lines) of GEF activity. (B) As in (A), but in the presence of effectors such as Raf.

ARS-1620 (all simulations shown in this publication were performed with KinTek Explorer; Johnson et al., 2009). In Figure 4A, we see that the inactivation of KRas G12C should take place over several thousand seconds at the chosen inhibitor concentrations (10 mM) in the absence of GEF activity (t1/2 =
23 min). This is in qualitative agreement with the observation that onset of inhibition is relatively slow in cells (Lito et al., 2016). As shown by the dashed line in in Figure 4A, addition of relatively low GEF activity to the simulation leads to more rapid reaction (t1/2 =
18 min). Reaction would still occur (t1/2 =
45 min; not shown) when the rate of the GTPase reaction is reduced to zero, whereas essentially no reaction would occur in the absence of GEF. This is in contrast to the experimental results discussed above. Reduction of the rate of inactivation is only seen at very high GEF concentrations in simulations of the type shown in Figure 4A, and is simply due to extensive complex formation with the KRas:GDP complex (under the assumption that the Sos:KRas:GDP complex cannot react with the inhibitor). This is actually an unlikely scenario, because Sos is known to be significantly less abundant than KRas (Shi et al., 2016). What could the reason for the competing effects of inhibitor and Sos seen in the reported experiments be? A possible factor that was not included in the simulations discussed is the presence and interaction of molecules interacting specifically with KRas:GTP, that is, effectors. Activated Ras interacts with high affinity with Raf, which is present at similar abundance to Ras (Shi et al., 2016). It is therefore conceivable that KRas:GTP at the plasma membrane is largely complexed to Raf (and other effectors) and therefore not free to interact with Sos. Under these conditions, there will probably be only a small effect of Sos on Ras:GTP (in the sense of generation of Ras:GDP), so that very little Ras:GDP will be produced in this manner because of competition between GEF and Raf binding to Ras:GTP. The inhibitor will then only be able to interact with Ras:GDP produced by the intrinsic GTPase reaction in the Raf:KRas G12C:GTP complex, which is known to be similar in rate to the wild-type reaction (Herrmann et al., 1996). Moreover, increasing the GEF activity is expected to reduce the rate of inhibition via this route because it will decrease the life-time of the KRas G12C:GDP complex. This situation is simulated in Figure 4B, showing the qualitatively expected reduction of reaction rate on increasing GEF activity in the presence of substantial concentrations of Raf. This is in agreement with the results reported by Lito et al. (2016). 1342 Cell Chemical Biology 26, October 17, 2019

Conversely, reducing GEF activity will potentiate the activity of the inhibitors. This analysis is in harmony with the idea that activated Ras is mainly complexed to effectors, a situation that could apply to normal physiological conditions as well. However, more direct evidence would be needed to establish this point unequivocally. The question of how inactivation of activated Ras occurs by interaction with GAP is not cast into doubt by this conclusion, because the relatively rapid dissociation kinetics of Ras-Raf complexes allows equilibration and interaction with GAP leading to GTP hydrolysis (Sydor et al., 1998). The main conclusion based on the arguments presented is that the major route for the action of the inhibitors discussed involves generation of KRas:GDP via hydrolysis of GTP, but it seems likely that there will also be some generation by GEF activity, because the affinity of Ras for Raf, while high, is not high enough to reduce the concentration of free Ras:GTP to zero. Presently available results on the irreversible S-IIP pocket inhibitors discussed here are highly encouraging, although it is not yet finally established that their pharmacokinetics and specificity will be commensurate with their use as drugs. In this context, reaction with a number of other targets has been shown for one representative of this class of inhibitor (Wijeratne et al., 2018), but there is no evidence that this will constitute a problem. Perhaps of more concern are observations of possible resistance mechanisms, although evidence is presented that these can be alleviated in some cases by combination with PI3K inhibitors (Misale et al., 2019). Considerations Concerning the Possibility of Modulation of Ras Activity by Agents that Compete with Nucleotide Binding The finding that GTP and GDP bind to Ras with extraordinarily high affinity, combined with the high prevailing concentrations of GTP in cells, has discouraged scientists from searching for compounds that compete with their binding as an approach to inhibition of Ras activity. Re-examining this question, it is worth comparing it with the situation with protein kinase inhibitors, many of which bind competitively with ATP. A common feature of the two scenarios is the high prevailing concentration of the natural ligand of the protein targets, that is , GTP and ATP. Any approach involving competition with nucleotide binding will necessarily be limited by the fact that the concentrations of these nucleotides is high in cells, with estimates for GTP being in the

Cell Chemical Biology

Review several hundred micromolar range and about an order of magnitude higher for ATP (Traut, 1994). Thus, based on cellular concentrations of GTP or ATP alone, it might actually be considered more feasible to inhibit GTPases than kinases, if it were not for the fact that GTPases bind GTP many orders of magnitude more strongly than kinases bind ATP. This might be thought to have a potentially negative impact in the case of GTPases, because the high affinities are the result of extremely slow dissociation rates (
2.5 3 105 s1, or 0.09 h1 from recent estimates; Jeganathan et al., 2018), meaning that access to the active site will be limited by this. We will return to this point later and show that this conclusion might not be correct under the conditions pertaining in the cell if activating mutations of Ras play a role. Possibly of more significance is the already extremely high affinity of GDP/GTP, making it unlikely that compounds with significantly higher affinity for the binding site can be generated. This is quite different to the situation with kinase inhibitors, with Km values for ATP in the micromolar range, and there are indeed numerous examples of ATP competitive inhibitors with low nanomolar affinity, giving them a significant competitive advantage. There is perhaps no theoretical upper limit to the affinity of a modified GDP, but the prospect of generating a ligand with sub-picomolar affinity is daunting. However, there are reasons to believe that both of the points mentioned might not in fact be prohibitive in the development of inhibitors that operate under conditions that prevail in cells harboring activated Ras mutants. The main factor to be considered here is the influence of GEF activity. The Influence of GEF Activity on the Effective Affinity of Ras for GTP Since the high affinity of Ras for GTP is perceived as a major problem in treating cancer by any approach that involves competition with GTP, it is worth examining the prevailing situation in cells harboring activating mutations in Ras. In these cells, there is a high concentration of the Ras mutant in the activated form (Ras:GTP). The Ras GEF Sos is known to have two Ras binding sites, one of which is the site that is catalytic for the nucleotide exchange reaction, and another which binds specifically to Ras:GTP (Figure 2) (Margarit et al., 2003; Sondermann et al., 2004). This second interaction has two consequences. On the one hand, it can recruit Sos to the plasma membrane, where it can then act on other Ras:GDP complexes. In addition to this, it also allosterically activates the exchange activity of the other Ras binding site. These factors appear to generate a positive feedback loop augmenting Ras activation, as confirmed by the observation that, in cells harboring an activating KRas mutation, increased levels of activation of other Ras isoforms (HRas, NRas) that share the same exchange factor are seen despite the fact that they do not have activating mutations themselves (Jeng et al., 2012). This will presumably also apply to wild-type KRas in cells that are heterozygotic for an activated mutant. The evidence reviewed suggests that we should consider potential inhibition of Ras by inhibitors that act in a GTP-competitive manner solely against the background of the presence of Sos activity. This activity will catalyze the otherwise extremely slow replacement of GDP by GTP, but also the replacement of either GDP or GTP by an agent that competes with them. If this reagent has GDP/GTP-like properties toward Ras, as is highly likely for GDP-derivative competitive inhibitors, Sos will

have the property of catalyzing equilibration of the various species according to the relative affinities of the partners. Looking at this situation in a more quantitative manner, we could describe the effect of Sos as reducing the effective affinity between Ras and GTP, making Ras potentially more available for a GTP-competitive inhibitor. The Kd value for the interaction of GDP/GTP with the binary complex of Ras and Sos is in the micromolar range (Guo et al., 2005), but this would only apply at very high (saturating) concentrations of Sos. Because the Kd value for Sos binding to Ras:GXP is
50 mM (Guo et al., 2005), Sos concentrations in the several hundred micromolar range would be needed to reduce the GTP/GDP binding affinity to the micromolar range. This would be unrealistically high at prevailing cellular concentrations, although it should be borne in mind that local effective concentrations at the membrane could be quite high. However, even at much lower Sos concentrations, there will be a pronounced effect of Sos on the effective affinity of GTP. This is illustrated in Figure S1, which is not intended to mimic the in vivo situation with respect to concentrations, but to show the dramatic effect of Sos on the effective affinity of GTP in a familiar type of experiment. To simulate the curves shown, we have chosen a low concentration of Ras (1 nM) to which GTP is titrated first in the absence of GEF and then in its presence. As expected from the high affinity of Ras for GTP, there is essentially a straight line response of the amount of GTP bound as a function of added GTP, reaching the maximum amount bound abruptly (at
1 nM added [GTP], the concentration of Ras used in the simulation). If it were possible to produce genuine data of this kind (which it is not in practice due to the very slow equilibration rates at the low nucleotide concentrations used combined with the very low thermal stability of nucleotide-free Ras), it would hardly be possible to extract a Kd value from the data, perhaps an upper limit. In the presence of 0.1 mM Sos, the binding curve looks more familiar and has an approximately hyperbolic shape with an apparent Kd (concentration for 50% saturation) of
4 nM, which is 3 orders of magnitude higher than in the absence of GEF, and further increasing the GEF concentration leads to an approximately linear increase in Kd. Thus, the effective affinity of GTP to Ras is dramatically reduced in the presence of GEF, obviously dependent on the prevailing Sos concentration. Does this lower effective affinity of Ras for GTP/GDP offer advantages when searching for reversible competitive inhibitors of Ras? The simple answer is no, because the governing factor will still be the relative affinity of an inhibitor binding to Ras to that of GTP/GDP if the inhibitor binds in a GDP/GTP-like manner. However, there are two possible properties of inhibitors that might change this situation, one of which is the covalent reaction of the inhibitors with Ras, the other of which is GEF-insensitive binding to Ras. These scenarios and their potential combination are considered below. Covalent Interaction of GDP Derivatives with Ras The idea of adding an electrophilic group to a GDP-like molecule in such a manner that it can react with, for example, the KRas G12C mutant is immediately an attractive one, and the first reported attempt to do this initially appeared to be successful. In this work, the electrophilic ‘‘warhead’’ was a chloroacetamide group attached to the b-phosphate of GDP to give the substance Cell Chemical Biology 26, October 17, 2019 1343

Cell Chemical Biology

Review

Figure 5. A Modified GDP Derivative for Covalent Targeting of KRas G12C Compound referred to as SML-8-73-1 bound in the nucleotide binding pocket and covalently to Cys12 in KRas G12C (upper panel; PDB: 4nmm), and the original compound harboring an electrophilic group (chloroacetamide) on the b-phosphate of GDP (lower panel) (Lim et al., 2014). The QR code allows visualization of the structure in augmented reality (Wolle et al., 2018).

referred to as SML-8-73-1 (shown in Figure 5) (Hunter et al., 2014; Lim et al., 2014). In a further study, several additional derivatives of this type were synthesized and tested as KRas G12C inhibitors (Xiong et al., 2017). SML-8-73-1 was successful in the sense that it reacts specifically with the KRas G12C mutant, and not with wild-type KRas, to produce the expected product. However, despite the quite rapid reaction reported in the first publication on this subject, even in the presence of excess GTP, it was later shown that, at near physiological GTP/GDP and, crucially, Mg2+ concentrations, the rate of reaction with the mutant was extremely slow at realistically obtainable inhibitor concentrations, and this is due to the quite low affinity of the analog for Ras (
104-fold lower than GDP) and the extremely slow dissociation rate of GDP or GTP (Muller et al., 2017). Figure 6A shows a simulation of the kinetics of the covalent reaction at nucleotide and Mg2+ concentrations in the physiological range using the kinetic parameters derived in that study (Muller et al., 2017). The predicted half-life for the inactivation of hundreds of hours was confirmed semiquantitatively using mass spectrometry (Muller et al., 2017). This is despite a relatively rapid covalent reaction once the analog is reversibly bound (
0.1 s1; t1/2 =
70 ms; Muller et al., 2017), and is due to the
104-fold loss of reversible affinity to Ras caused by attachment of the warhead to the b-phosphate group. If the low affinity of SML-8-73-1 for Ras is the reason for its slow rate of covalent reaction at physiological concentrations of nucleotides and Mg2+, what would be expected of an irreversible inhibitor that could bind reversibly with similar affinity and kinetics to GDP or GTP? This question has been examined by kinetic simulation, and, as shown in Figure 6B, the effective rate of covalent reaction with Ras is significantly faster than with SML-8-73-1, even though the same intrinsic rate constant of the covalent reaction was assumed (Muller et al., 2017). How1344 Cell Chemical Biology 26, October 17, 2019

ever, the rate is still very slow and unlikely to be adequate for inactivation of mutant KRas in the cell. The simulations of potential rates of inactivation under physiological conditions have so far neglected the effect of GEF activity. As argued above, GEF activity in cells harboring persistently activated Ras mutants is likely to be constitutively high, independent of activating signals from outside the cell. Further simulations were therefore performed assuming the presence of GEF activity (Figures 6C and 6D) (Muller et al., 2017). Although there is now more rapid inactivation by SML-8-73-1, the effect of GEF activity is much more dramatic, with a putative covalent inhibitor that has GDP-like reversible binding characteristics. The reason for this difference is the much longer residence time of a strongly binding inhibitor in the ternary inhibitor:Ras:GEF as well as the binary Ras:inhibitor complex, allowing more time for the covalent reaction to take place. These arguments suggest that a covalent inhibitor that has GTP/GDP-like reversible binding characteristics might have the potential to specifically inactivate Ras mutants they are designed to interact with. However, their efficacy will still be limited by the fact that they will have to compete with high prevailing cellular GTP concentrations. We have so far considered putative covalently reacting inhibitors of KRas G12C that have GDP/GTP-like affinity, but without discussing the conceivable structures of such derivatives. Covalently reacting GDP/GTP derivatives with GDP/GTP-like affinity to GTPases have recently been described, and they have been used to react with strategically placed cysteines in Rab proteins (Wiegandt et al., 2015). In these derivatives, the electrophilic amide group is attached to the N2 position of the guanine base. Even though this position of the guanine base is too far remote from position 12 of KRas to allow covalent interaction in this case, alternative points of attachment of a linker and electrophilic group can be envisaged that could allow the warhead to approach the desired location, but without affecting the reversible binding affinity of this nucleotide analog compared with GDP/GTP. We therefore conclude that the approach is highly interesting and further nucleotide derivatives with warheads attached at varying positions should be tested in the future. The Possibility of Generating GEF-Insensitive Covalent Inhibitors Competing with GTP In this section, we would like to introduce a novel idea of nucleotide-competing molecules that bind to Ras and react covalently, but are insensitive to exchange by GEFs. Even though such molecules have not yet been synthesized, they would have several putative advantages compared with molecules discussed above as they would not only covalently react with Ras, but also sequester and thereby inactivate GEF activity (as outlined below). We would like to emphasize that this is distinct from the approach of attempting to inhibit the Ras-Sos interaction, about which there is also a substantial literature (see Hillig et al., 2019 and references therein). The extensive available knowledge of GTPase:nucleotide and GTPase:GEF structures and of nucleotide exchange mechanisms suggests that much of the effect of GEFs on the interaction of GTPase with GDP/GTP arises from disturbance of interactions of Ras with the b- and g-phosphate groups and ejection of the Mg2+ ion. It therefore seems likely that guanosine or guanosine derivatives that do not harbor a b-phosphate group might bind

Cell Chemical Biology

Review Figure 6. Simulations of the Kinetics of Generation of the Covalent Adducts between KRas G12C and GDP Derivatives Harboring an Electrophilic Group Able to React with Cysteine at Position 12 Rate constants used are shown in Scheme S1, and concentrations in Table S1. (A) With a weak inhibitor such as SML-8-73-1. In addition to those rate constants included in Scheme S1, the rate constants for SML-8-73-1 derived in an earlier study are used (koff = 1 s1, kinact = 0.1 s1) (Muller et al., 2017). Note that the rapid loss of Ras:GTP occurs because GDP cannot be replaced by GTP because of the lack of GEF activity, and this, of course, does not reflect the situation in cells. (B) As in (A) but with the rate constants for a hypothetical strong inhibitor (Scheme S1). The inset allows an easy comparison of kinetics of covalent reaction with the weak and strong inhibitors on a Log2 timescale. (C) As in (A), but with 0.1 mM GEF. (D) As in (B), but with 0.1 mM GEF. Inset as for (B).

in a GEF-independent manner, or that their binding might not be so strongly affected by GEFs. We have therefore examined the theoretical potential of hypothetic covalent guanosine derivatives that bind to Ras in a less GEF-sensitive manner. In the first example, we have used a Kd value of 10 nM for the putative inhibitor, and assumed that the interaction with Ras is competitive with GDP/GTP, but that this is not influenced by GEF binding. As shown in Figure 7A, this leads initially to relatively rapid generation of the covalent complex (in this case mainly in the GEFbound form) to an extent that depends on the starting concentration of GEF, followed by a much slower rate. The reason for this retardation is that essentially all the GEF is sequestered in a complex with the Ras-covalent inhibitor species owing to its high affinity in this complex, so that free GEF is not available to catalyze further rounds of displacement of nucleotide to allow binding of more inhibitor. However, GEF is also not available to catalyze replacement of GDP by GTP, and the GTPase reaction leads to relatively rapid reduction of activated Ras (red curve in Figure 7A; t1/2 =
40 min). This suggests that the principle of inhibiting GEF dissociation from its complex with Ras would be an interesting avenue to follow, because it would lead to depletion of Ras:GTP, at least in mutants with a relatively rapid GTPase reaction, which is the case with KRas G12C. What types of compounds are potential candidates as GEFinsensitive inhibitors that also fulfill the condition of blocking GTP/GDP binding? One possibility is to start with GMP, which is known to bind with low affinity (Kd =
30 mM) to Ras (John et al., 1990). In the absence of the b- and g-phosphate groups, it seems highly unlikely that GMP is able to dissociate Sos from its complex with Ras, which in turn would mean that Sos cannot dissociate GMP from Ras, which is the definition of GEF (Sos)-insensitive binding of the nucleotide. If the nucleotide were now modified to have higher affinity to Ras (and Ras:Sos) without additional phosphate groups and to have a

warhead added on some kind of linker structure that can approach the cysteine at position 12 in KRas G12C, it is possible that the inhibitor would react covalently with Ras in the Ras:Sos complex and prevent release of Sos, thus inhibiting the cycle of GTP hydrolysis and replacement of GDP by GTP to maintain a high concentration of activated Ras. There is not enough experimental evidence available to judge whether compounds harboring a guanine group, but without phosphates equivalent to the b- and g-phosphates of GTP could really bind to Ras in a manner that is GEF insensitive. However, it seems highly likely, on the basis of the numerous interactions of the b- and g-phosphate groups with the protein and Mg2+ ion, that the absence of these groups will lead not only to a dramatic loss of affinity to Ras, as already shown experimentally, but also to a loss of sensitivity to GEF binding. In Figure 7B, we have examined the potential use of inhibitors that are partially sensitive to GEF (we have chosen a factor of 100 difference in the affinity of a nucleoside or nucleotide analog between their complexes with Ras and Ras:GEF). Since it is unlikely that compounds of this type (i.e., with a guanosine or guanine residue but without a b-phosphate group) with GDP-like affinity can be generated, we have chosen the more modest value of 109 M for the Kd of interaction of the derivative with Ras, while realizing that even this might not be easily achievable. This leads to moderately rapid inactivation and depletion of Ras:GTP, and suggests that such efforts could be productive. Targeted Inhibition of GEF Activity toward KRas Mutants as an Approach to Therapy Comparison of aspects of the two types of inhibitor discussed so far leads to the realization that the end effect in two of the cases examined is an inhibition of Sos (or more generally GEF) activity. In the case of the S-IIP inhibitors, this is the result of inhibition of the action of Sos on KRas G12C after covalent binding of the inhibitor (Patricelli et al., 2016). In the case of the as yet only notional inhibitors that would not weaken the Ras:Sos interaction, or only partially weaken it, the effect on KRas G12C:GTP Cell Chemical Biology 26, October 17, 2019 1345

Cell Chemical Biology

Review Figure 7. Simulation of Inactivation Kinetics by Covalent Inhibitors that Show No or Little GEF Sensitivity in Binding to KRas (A) Time course for a completely GEF-independent inhibitor with a Kd value of 10 nM. Rate constants and concentrations are as shown in Scheme S1 and Table S1. (B) Time course for an inhibitor that binds 100-fold less strongly in the presence of GEF. The Kd value for the inhibitor is 1 nM.

levels would arise because free Sos is depleted by becoming essentially irreversibly (or very strongly) bound to the covalent complex between KRas G12C and the inhibitor. Because of this, Ras:GTP cannot be generated from Ras:GDP owing to the extremely slow spontaneous GDP dissociation rate. The end result, in both cases, is the depletion of KRas G12C:GTP levels. Interestingly, in the second case this even occurs without extensive covalent modification of Ras, or at least only to an extent which corresponds to the relative abundancies of Sos and KRas in the cell, which appears to be much less than 1 (Shi et al., 2016). Does this mean that schemes aimed at identification of Sos inhibitors, or strategies that will lead to depletion of Sos activity, is a good approach to obtain agents that might have a therapeutic effect against Ras-dependent tumors? In the general case, the answer to this question is likely to be negative, since untargeted powerful inhibition of Sos is likely to have general cytotoxic effects. However, in the cases discussed, selective targeting to tumor cells by virtue of covalent reaction with KRas G12C is (or would be) achieved. There is also the possibility of taking a completely different approach and learning from a known example of inhibition of GEF activity of a GTPase by a small molecule. This is the natural product, Brefeldin, which is able to bind to and stabilize the complex between Arf proteins, GDP, and their exchange factors (Renault et al., 2003). GDP dissociation from this complex is inhibited, so that GTP binding and activation of the Arf protein is prevented. Compounds with such activity against Ras:Sos complexes could be of great interest, especially if they have the potential to react covalently with specific oncogenic mutants of Ras. Conclusions At present, of the two types of covalent Ras inhibitors discussed here, those attacking Cys12 of KRas G12C via the S-IIP look more promising. For mechanistic reasons that were not initially suspected, they show a high selectivity toward reacting with this particular residue when compared with other SH group containing species in the cell, including small molecules and proteins. They also have the advantage of being cell membrane permeable, have been shown to function in the desired manner in cell culture, and have even shown activity against tumors in model systems (Hansen et al., 2018). Some representatives of this type of inhibitor are in early clinical trials (Halford, 2019). In contrast, covalent inhibitors that compete with GDP/GTP are at an earlier stage of development and are faced with signif1346 Cell Chemical Biology 26, October 17, 2019

icant hurdles, all of which could lead to failure at particular stages. Even under in vitro conditions, inhibitors with ideal properties have not yet been identified, although the steps that need to be taken are relatively clear, as discussed above. Even if the aims are achieved at the biochemical level, the problem of cell membrane permeability must be solved, and this is not trivial if, for example, the compounds harbor the phosphate groups of GDP. This problem is probably soluble, since prodrug approaches have been successfully applied in the development of antiviral nucleotide derivatives with masked phosphate groups that are unmasked in the cytosol (Weinschenk et al., 2015). Additional research and compound development will be key to address the question as to whether such obstacles can be overcome for clinical development. The ultimate advantage of such inhibitors lies in their potentially very high degree of specificity. The approach of mutant-specific targeting of oncogenic KRas offers great potential for the treatment of tumors that are currently intractable by other approaches. The approaches discussed here together with further variations on this theme, including targeting other mutations, including KRas G13C, which is also oncogenic, should therefore be pursued with high priority. Ideally, extension to other mutations, in particular to KRas G12D, would be desirable, since this is the most common variant seen in a large variety of tumors. This presents a significant challenge in terms of development of an appropriate warhead, but there is progress in this direction, as exemplified for the protein Pde6d, in which a glutamic acid residue was targeted covalently (MartinGago et al., 2017). In a study aimed at covalent labeling of the aspartate group in KRas G12D, an aziridine group was used as an electrophilic moiety on a scaffold of similar structure to that of ARS-1620 (McGregor et al., 2017). However, although it could be shown that this moiety is more reactive toward carboxyl groups than thiol groups, no covalent adduct with KRas G12D could be generated. The authors suggest that this is because of unfavorable steric factors, and that compounds might still be identified in which these constraints are alleviated. In principle, an extension of this approach could also include nucleotides equipped with such a warhead. In general, the possibility of covalent modification offers the unique advantage of irreversibility. In this context, an interesting approach to modifying Ras activity by interfering with its correct membrane localization via covalent modification of the cysteine in the CAAX box using a farnesyl transferase neo-substrate has recently been reported (Novotny et al., 2017). However, specificity for oncogenic mutants is not expected to result from this approach. In contrast, those approaches analyzed in

Cell Chemical Biology

Review detail in the present review illustrate the potential advantages of combination of covalent interaction and a high degree of specificity, thus justifying major efforts in this direction.

Hunter, J.C., Gurbani, D., Ficarro, S.B., Carrasco, M.A., Lim, S.M., Choi, H.G., Xie, T., Marto, J.A., Chen, Z., Gray, N.S., et al. (2014). In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C. Proc. Natl. Acad. Sci. U S A 111, 8895–8900.

SUPPLEMENTAL INFORMATION

Hunter, J.C., Manandhar, A., Carrasco, M.A., Gurbani, D., Gondi, S., and Westover, K.D. (2015). Biochemical and structural analysis of common cancer-associated KRAS mutations. Mol. Cancer Res. 13, 1325–1335.

Supplemental Information can be found online at https://doi.org/10.1016/j. chembiol.2019.07.005.

Itzen, A., and Goody, R.S. (2011). GTPases involved in vesicular trafficking: structures and mechanisms. Semin. Cell Dev. Biol. 22, 48–56.

ACKNOWLEDGMENTS

Itzen, A., Rak, A., and Goody, R.S. (2007). Sec2 is a highly efficient exchange factor for the Rab protein Sec4. J. Mol. Biol. 365, 1359–1367.

This work was funded by the Max Planck Society, the German Federal State North Rhine-Westphalia (NRW), the European Union (European Regional Development Fund: Investing In Your Future) (EFRE-800400) and the Drug Discovery Hub Dortmund (DDHD).

Janes, M.R., Zhang, J.C., Li, L.S., Hansen, R., Peters, U., Guo, X., Chen, Y.C., Babbar, A., Firdaus, S.J., Darjania, L., et al. (2018). Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor. Cell 172, 578–589.e17.

AUTHOR CONTRIBUTIONS All authors contributed to the writing of this review. REFERENCES Bagshaw, C.R., Eccleston, J.F., Eckstein, F., Goody, R.S., Gutfreund, H., and Trentham, D.R. (1974). Magnesium ion-dependent adenosine-triphosphatase of myosin. Two-step processes of adenosine-triphosphate association and adenosine-diphosphate dissociation. Biochem. J. 141, 351–364. Berndt, N., Hamilton, A.D., and Sebti, S.M. (2011). Targeting protein prenylation for cancer therapy. Nat. Rev. Cancer 11, 775–791.

Jeganathan, S., Muller, M.P., Ali, I., and Goody, R.S. (2018). Assays for nucleotide competitive reversible and irreversible inhibitors of Ras GTPases. Biochemistry 57, 4690–4699. Jeng, H.H., Taylor, L.J., and Bar-Sagi, D. (2012). Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis. Nat. Commun. 3, 1168. John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R.S. (1990). Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry 29, 6058–6065. Johnson, K.A., Simpson, Z.B., and Blom, T. (2009). Global kinetic explorer: a new computer program for dynamic simulation and fitting of kinetic data. Anal. Biochem. 387, 20–29.

Cancer Genome Atlas Research Network (2014). Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550.

Klebe, C., Prinz, H., Wittinghofer, A., and Goody, R.S. (1995). The kinetic mechanism of Ran-nucleotide exchange catalyzed by RCC1. Biochemistry 34, 12543–12552.

Esters, H., Alexandrov, K., Iakovenko, A., Ivanova, T., Thoma, N., Rybin, V., Zerial, M., Scheidig, A.J., and Goody, R.S. (2001). Vps9, Rabex-5 and DSS4: proteins with weak but distinct nucleotide-exchange activities for Rab proteins. J. Mol. Biol. 310, 141–156.

Kohl, N.E., Wilson, F.R., Mosser, S.D., Giuliani, E., deSolms, S.J., Conner, M.W., Anthony, N.J., Holtz, W.J., Gomez, R.P., Lee, T.J., et al. (1994). Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice. Proc. Natl. Acad. Sci. U S A 91, 9141–9145.

Fell, J.B., Fischer, J.P., Baer, B.R., Ballard, J., Blake, J.F., Bouhana, K., Brandhuber, B.J., Briere, D.M., Burgess, L.E., Burkard, M.R., et al. (2018). Discovery of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRASG12C with in vivo activity. ACS Med. Chem. Lett. 9, 1230–1234.

Lackner, M.R., Kindt, R.M., Carroll, P.M., Brown, K., Cancilla, M.R., Chen, C., de Silva, H., Franke, Y., Guan, B., Heuer, T., et al. (2005). Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 7, 325–336.

Feuerstein, J., Goody, R.S., and Wittinghofer, A. (1987). Preparation and characterization of nucleotide-free and metal ion-free P21 apoprotein. J. Biol. Chem. 262, 8455–8458.

Lategahn, J., Keul, M., and Rauh, D. (2018). Lessons to be learned: the molecular basis of kinase-targeted therapies and drug resistance in non-small cell lung cancer. Angew. Chem. Int. Ed. 57, 2307–2313.

Goody, R.S., Frech, M., and Wittinghofer, A. (1991). Affinity of guanine nucleotide binding proteins for their ligands: facts and artefacts. Trends Biochem. Sci. 16, 327–328.

Lee, M.T.G., Mishra, A., and Lambright, D.G. (2009). Structural mechanisms for regulation of membrane traffic by Rab GTPases. Traffic 10, 1377–1389.

Goody, R.S., and Hofmann-Goody, W. (2002). Exchange factors, effectors, GAPs and motor proteins: common thermodynamic and kinetic principles for different functions. Eur. Biophys. J. 31, 268–274. Guo, Z., Ahmadian, M.R., and Goody, R.S. (2005). Guanine nucleotide exchange factors operate by a simple allosteric competitive mechanism. Biochemistry 44, 15423–15429. Halford, B. (2019). Amgen unveils its KRas covalent inhibitor AMG 510. Chem. Eng. News 9, 4. Hansen, R., Peters, U., Babbar, A., Chen, Y., Feng, J., Janes, M.R., Li, L.S., Ren, P., Liu, Y., and Zarrinkar, P.P. (2018). The reactivity-driven biochemical mechanism of covalent KRAS(G12C) inhibitors. Nat. Struct. Mol. Biol. 25, 454–462. Herrmann, C., Horn, G., Spaargaren, M., and Wittinghofer, A. (1996). Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and RRas with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J. Biol. Chem. 271, 6794–6800. Hillig, R.C., Sautier, B., Schroeder, J., Moosmayer, D., Hilpmann, A., Stegmann, C.M., Werbeck, N.D., Briem, H., Boemer, U., Weiske, J., et al. (2019). Discovery of potent SOS1 inhibitors that block RAS activation via disruption of the RAS-SOS1 interaction. Proc. Natl. Acad. Sci. U S A 116, 2551–2560.

Lenzen, C., Cool, R.H., Prinz, H., Kuhlmann, J., and Wittinghofer, A. (1998). Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm. Biochemistry 37, 7420–7430. Lim, S.M., Westover, K.D., Ficarro, S.B., Harrison, R.A., Choi, H.G., Pacold, M.E., Carrasco, M., Hunter, J., Kim, N.D., Xie, T., et al. (2014). Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor. Angew. Chem. Int. Ed. 53, 199–204. Lito, P., Solomon, M., Li, L.S., Hansen, R., and Rosen, N. (2016). Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science 351, 604–608. Margarit, S.M., Sondermann, H., Hall, B.E., Nagar, B., Hoelz, A., Pirruccello, M., Bar-Sagi, D., and Kuriyan, J. (2003). Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 112, 685–695. Martin-Gago, P., Fansa, E.K., Winzker, M., Murarka, S., Janning, P., SchultzFademrecht, C., Baumann, M., Wittinghofer, A., and Waldmann, H. (2017). Covalent protein labeling at glutamic acids. Cell Chem. Biol. 24, 589–597.e5. McGregor, L.M., Jenkins, M.L., Kerwin, C., Burke, J.E., and Shokat, K.M. (2017). Expanding the scope of electrophiles capable of targeting K-Ras oncogenes. Biochemistry 56, 3178–3183.

Cell Chemical Biology 26, October 17, 2019 1347

Cell Chemical Biology

Review Misale, S., Fatherree, J.P., Cortez, E., Li, C.D., Bilton, S., Timonina, D., Myers, D.T., Lee, D., Gomez-Caraballo, M., Greenberg, M., et al. (2019). KRAS G12C NSCLC models are sensitive to direct targeting of KRAS in combination with PI3K inhibition. Clin. Cancer Res. 25, 796–807.

Sondermann, H., Soisson, S.M., Boykevisch, S., Yang, S.S., Bar-Sagi, D., and Kuriyan, J. (2004). Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 119, 393–405.

Muller, M.P., and Goody, R.S. (2018). Molecular control of Rab activity by GEFs, GAPs and GDI. Small GTPases 9, 5–21.

Sydor, J.R., Engelhard, M., Wittinghofer, A., Goody, R.S., and Herrmann, C. (1998). Transient kinetic studies on the interaction of ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex. Biochemistry 37, 14292–14299.

Muller, M.P., Jeganathan, S., Heidrich, A., Campos, J., and Goody, R.S. (2017). Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity. Sci. Rep. 7, 3687.

Traut, T.W. (1994). Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22.

Novotny, C.J., Hamilton, G.L., McCormick, F., and Shokat, K.M. (2017). Farnesyltransferase-mediated delivery of a covalent inhibitor overcomes alternative prenylation to mislocalize K-Ras. ACS Chem. Biol. 12, 1956–1962. Ostrem, J.M., Peters, U., Sos, M.L., Wells, J.A., and Shokat, K.M. (2013). K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551. Patricelli, M.P., Janes, M.R., Li, L.S., Hansen, R., Peters, U., Kessler, L.V., Chen, Y., Kucharski, J.M., Feng, J., Ely, T., et al. (2016). Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state. Cancer Discov. 6, 316–329. Prior, I.A., Lewis, P.D., and Mattos, C. (2012). A comprehensive survey of Ras mutations in cancer. Cancer Res. 72, 2457–2467. Renault, L., Guibert, B., and Cherfils, J. (2003). Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor. Nature 426, 525–530. Shi, T., Niepel, M., McDermott, J.E., Gao, Y., Nicora, C.D., Chrisler, W.B., Markillie, L.M., Petyuk, V.A., Smith, R.D., Rodland, K.D., et al. (2016). Conservation of protein abundance patterns reveals the regulatory architecture of the EGFRMAPK pathway. Sci. Signal. 9, rs6. Simanshu, D.K., Nissley, D.V., and McCormick, F. (2017). RAS proteins and their regulators in human disease. Cell 170, 17–33.

1348 Cell Chemical Biology 26, October 17, 2019

Weinschenk, L., Schols, D., Balzarini, J., and Meier, C. (2015). Nucleoside diphosphate prodrugs: nonsymmetric DiPPro-nucleotides. J. Med. Chem. 58, 6114–6130. Wiegandt, D., Vieweg, S., Hofmann, F., Koch, D., Li, F., Wu, Y.W., Itzen, A., Muller, M.P., and Goody, R.S. (2015). Locking GTPases covalently in their functional states. Nat. Commun. 6, 7773. Wijeratne, A., Xiao, J., Reutter, C., Furness, K.W., Leon, R., Zia-Ebrahimi, M., Cavitt, R.N., Strelow, J.M., Van Horn, R.D., Peng, S.B., et al. (2018). Chemical proteomic characterization of a covalent KRASG12C inhibitor. ACS Med. Chem. Lett. 9, 557–562. €ller, M.P., and Rauh, D. (2018). Augmented reality in scientific Wolle, P., Mu publications-taking the visualization of 3D structures to the next level. ACS Chem. Biol. 13, 496–499. Xiong, Y., Lu, J., Hunter, J., Li, L., Scott, D., Choi, H.G., Lim, S.M., Manandhar, A., Gondi, S., Sim, T., et al. (2017). Covalent guanosine mimetic inhibitors of G12C KRAS. ACS Med. Chem. Lett. 8, 61–66. Zeng, M., Lu, J., Li, L., Feru, F., Quan, C., Gero, T.W., Ficarro, S.B., Xiong, Y., Ambrogio, C., Paranal, R.M., et al. (2017). Potent and selective covalent quinazoline inhibitors of KRAS G12C. Cell Chem. Biol. 24, 1005–1016.e3. Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S.A., Triola, G., Wittinghofer, A., Bastiaens, P.I., et al. (2013). Small molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS signalling. Nature 497, 638–642.