Assays for Nucleotide Competitive Reversible and Irreversible

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Assays for nucleotide-competitive reversible and irreversible inhibitors of Ras GTPases Sadasivam Jeganathan, Matthias Philipp Müller, Ali Imtiaz, and Roger S Goody Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00234 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Biochemistry

Assays for nucleotide competitive reversible and irreversible inhibitors of Ras GTPases

Sadasivam Jeganathana, Matthias P. Müllerb, Imtiaz Alia and Roger S. Goodya* a

Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany

b

Faculty of Chemistry and Chemical Biology, Dortmund University of Technology, Otto-Hahn-Straße 4a, 44227, Dortmund, Germany

Abstract Although the Ras protein has been seen as a potential target for cancer therapy for the past 30 years, there was a tendency to consider it undruggable until recently. This has changed with the demonstration that small molecules with specifcity for (disease related mutants of) Ras can indeed be found, and some of these molecules form covalent adducts. A subgroup of these molecules can be characterized as competing with binding of the natural ligands GTP and GDP. Because of the distinct properties of Ras and related GTPases, in particular the very high nucleotide affinities and associated very low dissociation rates, assays for characterizing such molecules are not trivial. This is compounded by the fact that Ras family GTPases tend to be thermally unstable in the absence of bound nucleotide. Here, we show that instead of using the unstable nucleotide-free Ras, the protein can be isolated as a 1:1 complex with a modified nucleotide (GDPβ-methyl ester) with low affinity to Ras. With this nucleotide analogue bound to the protein, testing of inhibitors is made experimentally more convenient and we present assays that allow the rapid assessment of the kinetic constants describing the inhibition process.

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Introduction The idea of inhibiting the activity of the Ras GTPase by agents that compete directly with nucleotide binding is an approach that was essentially discarded shortly after the extremely high affinity of Ras for GTP and GDP was revealed in kinetic studies1, 2. However, there has been a recent renewal of interest in such approaches when competition with nucleotide binding is combined with covalent attachment to targeted Ras molecules, in particular oncogenic mutants with a cysteine near the nucleotide binding site. An example of this is the use of a GDP derivative harboring an electrophilic group on the β-phosphate to form a covalent adduct with the oncogenic mutant KRas G12C3. However, while this approach was successful under specific conditions in vitro, it was shown that the properties of the compound used (referred to as SML 8-73-1; Figure 1) rendered it highly unlikely to be of use under prevailing conditions in the cell4. This is due to the relatively low affinity of the reversible binding step of the compound for KRas (Kd = 0.14 µM4) compared to that of GTP and GDP (Kd for GDP = ca. 2.5 pM1, 5). This dramatic loss of affinity on attaching a group to the β-phosphate arises from loss of and/or weakening of a number of important interactions with the protein and a Mg2+ ion. In the cell, assuming strategies can be developed to mask phosphate groups to allow membrane penetration followed by spontaneous or enzymatic demasking6, 7, the feasible concentrations of electrophilic agent that could be reached (realistically in the micromolar to tens of micromolar range) would have to compete with several hundred micromolar GTP/GDP concentrations, making effective rates of the covalent reaction much too slow to allow any useful application4. Recent work has addressed structure-reactivity relationships of electrophilic guanosine derivatives modified in the phosphate region that can broadly be designated as GDP analogs8. They have dealt with important issues concerning stability and cellular delivery, but not with the basic problem of achieving a radical increase of the relatively low affinity of the reversible binding step to KRas, which will be required for a successful application of these compounds for the envisaged applications.


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Biochemistry

Figure 1: Structures of nucleotide analogues referred to in this work. Chemical modifications are highlighted in grey

A potential solution to the problems addressed here would be the generation of compounds (probably guanine derivatives) that retain the affinity of unmodified GDP because of an intact diphosphate structure, but carry an electrophilic group attached elsewhere. Already described reagents of this type have a linker and electrophilic group attached to the N2 position of the guanine base9, but this position is too remote from the P-loop cysteine of KRas G12C to allow the covalent reaction to occur. There are no reports that such nucleotide-competitive reagents have yet been tested, but there are several feasible approaches to the synthetic challenges that would have to be overcome to generate such derivatives. It is therefore important to establish methods for the characterization of such compounds. Determination of affinities and kinetic parameters for interaction of nucleotides with Ras superfamily GTPases is complicated by the extremely high affinity of the GTPases for GDP, which is invariably bound to the protein after expression and purification. A number of approaches have been developed for the generation of GDP-free preparations of Ras, but they suffer from certain disadvantages. The classical approach uses a 2-step procedure (replacement of GDP by GppCH2p, followed by degradation of the relatively weakly bound GppCH2P by phosphodiesterase in a relatively time-consuming procedure)1. A more recent method involves addition of a stoichiometric amount of the exchange factor SOS, followed by removal of GDP by gel filtration and ACS Paragon Plus Environment

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disruption of the Ras:SOS complex at high salt concentration followed by gel filtration in the presence of a high concentration of the weakly bound GMP to stabilize the protein8. Here, we present a new method based on the use of a GDP derivative that binds with moderate affinity to Ras but is stable to alkaline phosphatase, allowing a one-step procedure to generate GDP-free Ras. We outline methods based on the use of this weakly bound GDP derivative to avoid the practical problems caused by the very high nucleotide affinity and the accompanying extremely low off-rate of GDP present in preparations derived from overexpression in bacteria. In the first part, we describe quick and potentially high-throughput compatible procedures for the initial characterization of putative inhibitors without a detailed kinetic examination of their properties. In the second part, we move on to estimating the kinetics of reversible and irreversible inhibition. The reasoning behind this strategy is that the initial reversible binding step is crucial for efficient inhibitory action of irreversible inhibitors.

Materials and Methods Chemicals: Nucleotide analogues such as mantdGDP, GDP-β-methyl ester (GDPβMe), mantdGDPβMe, acyclovir triphosphate and SML 8-73-1 (custom synthesis) were purchased from Jena Bioscience GmbH (Jena, Germany). Mant versions of deoxy nucleotides were generally used to avoid potential complications (i.e. biphasic kinetics) arising from mixed 2’- and 3’-esters in the ribo derivatives. 2’-amino GTP was obtained from Trilink Biotechnologies (California, USA). Phosphodiesterase (PDE), alkaline phosphatase (AP) and streptavidin conjugated AP were obtained from Roche (Germany). Protein purification: KRas1-188 and KRas1–169 G12C were expressed in the BL21 (DE3) E. coli strain. Purification was achieved as published previously4. The protein was typically stored in a final buffer that contains 25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 20 µM GDP. Protein concentration was determined by absorption at 280 nm using molar extinction coefficients of the protein (calculated from http://web.expasy.org/protparam/) and, whenever necessary, of guanine nucleotide (7720 M-1 cm-1)10. Nucleotide exchange: In general, protein-bound GDP nucleotide was digested in the presence of a nonhydrolysable analogue, EDTA and alkaline phosphatase. Weaker affinity analogues, for example GDPβMe, were added in 5–fold molar excess in order to increase the stability of the Ras protein during exchange. ACS Paragon Plus Environment

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Biochemistry

Streptavidin-conjugated AP is the preferred choice for the degradation as it can be easily separated by sizeexclusion chromatography because of its much larger size than Ras. A typical exchange reaction includes the recombinant protein KRas:GDP with 2- to 5-fold molar excess of nucleotide analogue in the exchange buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM TCEP, 2-5 mM EDTA), supplemented with streptavidin-conjugated AP (0.1 U/mg of protein). The incubation was done either at room temperature for 1-2 hours or at 4 °C overnight. Degradation of KRas-bound GDP and the presence of non-hydrolysable nucleotide was confirmed by HPLC using a C-18 column (Prontosil, 5 µm), equilibrated with 50 mM potassium phosphate buffer pH 6.6, 10 mM tetrabutylammonium bromide, 16% acetonitril). The protein was further purified by size exclusion chromatography (Superdex S75; GE Healthcare) using the assay buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP). Stopped-flow experiments: All stopped-flow experiments were performed in a SX-20 stopped-flow apparatus (Applied Photophysics) at 25 °C in the assay buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP), unless otherwise stated. The measurements were performed using excitation with a 360 nm LED and emission detection with a 420 nm cut-off filter. All stopped flow results that were analyzed are averages of 3 to 6 individual traces. Single exponential functions were fit using the Applied Photophysics software or Origin (OriginLab). Fluorescence spectrometry: Fluorescence intensity measurements were performed using a Fluoromax-4 spectrofluorometer at 25 °C in the assay buffer. Experimental settings were typically: excitation 360 nm, emission 440 nm, excitation band width 1 nm and emission band width 4 nm, integration time 0.25 s when low concentrations of mant-labeled nucleotides (e.g. 1 µM) were used. For experiments requiring high concentrations of mant-labeled nucleotides, excitation and emission band width were set to 1 and 2 nm, respectively. For experiments with a long time base, such as those involving dissociation of strongly bound species, intermittent opening of the excitation shutter was used to minimize photobleaching. Typically, these fluorescence intensities were recorded for an 0.25 s (integration time) every 1 min (time interval) with the shutter closed between recordings. Data analysis: KinTek Explorer11 was used to simulate and analyze data using numerical integration. In most cases, this involved the inclusion of 3 reactions (KRas + GDPβMe (or mantdGDPβMe), KRas + 3’-


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mantdGDP and KRas + inhibitor). Equilibrium titrations were also analyzed using KinTek Explorer. Screen shots and explanations of examples of KinTek Explorer files are shown in the supplementary information.

Results We describe here experiments using inhibitors of GDP binding to Ras in a competitive manner, beginning with an initial assessment including equilibrium titrations and then going on to the kinetics of reversible and irreversible inhibition. In general, 3 different types of ligands were used: GDP/GTP and (fluorescent) derivatives thereof (mantdGDP, 2’-amino GTP) as examples of ligands with high affinity, guanosine-5’[β,γ-methylene]-triphosphate (GppCH2p) as an example of a ligand with intermediate affinity and the βmethyl ester of GDP (GDPβMe) and the fluorescent derivative (mantdGDPβMe) as well as the covalently binding SML-8-73-1 as examples of low affinity binders (Figure 1). The resulting sections constitute a guide to a fairly comprehensive characterization of inhibitor characteristics, which probably only needs to be followed thoroughly for compounds showing interesting inhibition properties in the initial tests. The strategy that can be adopted is outline in the flow chart shown in Scheme 1.


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Biochemistry

Scheme 1: Suggested workflow for the characterization of potential nucleotide competitive inhibitors of Ras proteins

Many of the procedures described here make use of the weakly bound GDPβMe (or mantdGDPβMe). As already shown, these GDP analogs have similar association kinetics to those of GDP with Ras, but dissociation rate constants that are ca. 4 orders of magnitude higher than for GDP4. Despite the dramatically reduced affinity, with a Kd value of 0.14 µM, complexes of these derivatives with KRas are still stable enough to allow isolation of a 1:1 complex with Ras proteins, and this complex is more thermally stable than nucleotide-free KRas (HPLC traces of KRas:GDPβMe and as a comparison KRas:GDP are shown in the supplement in Figure S1). This then raises the possibility of using such a complex to estimate the interactions of substances being investigated as inhibitors of GTP/GDP binding, even if their affinities are not nearly as high as those of the natural nucleotides, and avoiding the use of unstable nucleotide-free proteins.

Initial characterization of potential inhibitors without using rapid mixing methods

“One shot” assessment of approximate affinity and association rates The procedure described here can be performed without rapid reaction equipment and is quite revealing concerning the inhibitory potential of newly obtained candidate compounds. It relies on the effect of such a compound on the fluorescence change seen when a sample of the weakly bound KRas:GDPβMe is added to mantdGDP. Using a concentration of mantdGDP that is sufficient to saturate KRas (e.g. 2 µM mantdGDP with 1µM KRas:GDPβMe), this will lead to a large increase in fluorescence intensity, and this will happen on a rapid time scale because of the high dissociation rate of the initial complex (koff of GDPβMe = 0.8 s-1). If the experiment is then repeated, but this time using a 1:1 mixture of mantdGDP and an inhibitor (Figure 2), the following results are to be expected depending on the affinity and kinetics of the inhibitor binding: (1)

For a weakly bound reversible inhibitor (affinity similar to or lower than GDPβMe), the end point will be essentially the same as without inhibitor, and will be reached rapidly. ACS Paragon Plus Environment

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(2)

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For a strongly bound inhibitor (GDP-like affinity), the amplitude of the fluorescence increase in an initial phase will be smaller than in its absence, and the amplitude decrease is determined by the relative association rate constants of the inhibitor and mantdGDP. In fact, the ratio of the amplitudes corresponding to the 2 species (i.e. the change in fluorescence divided by the “missing” fluorescence change in comparison with the situation without inhibitor) is a good measure of the relative rate constants for the association reaction, as long as the ratio is not much greater or much smaller than 1 (see supplementary text for the deviation from this generalization at higher ratios of rate constants; even in this case, a calibration curve can be constructed with the known kinetics of the mantdGDP/KRas interaction; Figure S2). If the inhibitor has GDP-like affinity, the signal will be stable after the initial manual mixing phase, but waiting for long periods (unrealistically long for GDP-like kinetics) will lead to reequilibration after the initial rapid association phase if the affinity is not exactly equal to that of mantdGDP (see 3 below).

(3)

If the inhibitor has an affinity intermediate between that of GDPβMe and mantdGDP, it is still possible (indeed probable for guanosine nucleotide derivatives) that its association rate constant is similar to that of mantdGDP, leading to substantial inhibition of the initial binding of the fluorescent derivative, followed by a relatively slow re-equilibration in which the inhibitor is (partially) displaced by mantdGDP. In principle, this can be analyzed using appropriate software (e.g. Kintek Explorer; see Supplement for examples of its use) to give the dissociation rate constant of the inhibitor, but this is not initially the aim of the experiment. If the extent of displacement is not complete after equilibration, there is information in the amplitude relative to that seen without inhibitor that can be used to determine the affinity without a detailed kinetic analysis. In this situation, the ratio of affinities is given fairly exactly by the ratio of the relative amplitudes of the inhibitor bound species to the mantdGDP bound species multiplied by the ratio of unbound mantdGDP to unbound inhibitor at equilibrium, which can be calculated directly from the amplitudes (see Supplementary text). This allows calculation of the inhibitor affinity, and together with the association rate constant from the


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Biochemistry

initial amplitude leads to the value of koff of the inhibitor, which again should be consistent with the time course of the relatively slow equilibration step. (4)

In general, covalent adduct formation will result in displacement of initially bound mantdGDP with a time course determined by all the kinetic constants that define the system. However, this will necessarily be slow due to the slow dissociation of mantdGDP, meaning that this is not a practical approach.

Figure 2: Initial assessment of inhibitors without rapid mixing. A) KRas:GDPβMe (1 µM) was added to 2 µM mantdGDP in a fluorescence cell and the change in intensity increase (amplitude) in the absence of an inhibitor was recorded (on the left side of the diagram; symbol designated as “without inhibitor”). For each inhibitor, KRas:GDPβMe (1 µM) was added to a preformed 1:1 mixture of mantdGDP and inhibitor (2 µM each). The smaller initial fluorescence amplitude in the presence of inhibitor is caused by competition in the association step and allows estimation of the relative on-rates of the inhibitor and mantdGDP. GppCH2p, a moderately strong binder (similar kon, but larger koff, compared to mantdGDP; 2.5 x 10-3 s-1 from Figure 5A), is slowly displaced by mantdGDP soon after initial mixing. 2’-aminoGTP is very slowly displaced, in keeping with the koff from Figure 5C (4.8 x 10-4 s-1). Note that an inhibitor that has a smaller koff than mantdGDP would lead to a decrease of fluorescence from the initially attained level, but this would be extremely slow because of the very small value of koff for mantdGDP. For a relatively weakly bound inhibitor (acyclovir triphosphate; Kd = 78 nM, koff = 0.1 s-14), there is no clearly separated rapid phase in this ACS Paragon Plus Environment

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manual mixing experiment and essentially no inhibition of mantdGDP binding at the end of the slower phase. Inhibitors binding even more weakly such as SML-83-7-1 reach this endpoint even more rapidly and without a detectable rapid binding phase. B) The values of kon obtained for nucleotides by multiplying the amplitude ratio (inhibitor bound fraction/mantdGDP bound fraction) by kon of mantdGDP (= 3.2 µM-1s-1) for the first phase. The kon values are: GppCH2p = 3.25 ± 0.17, 2´-aminoGTP = 2.5 ± 0.05, GDP = 3.74 ± 0.27 and GTP = 2.77 ± 0.064 µM-1s-1. The results show the mean of 3 experiments and the standard error bars except in the case of mantdGDP, which reflects the value obtained in previous work4. The value for 2´aminoGTP agrees quite well with that obtained by a completely different stopped flow approach described in Figure 4 (2.32 µM-1s-1).

The type of experiment described here should give a clear indication of the affinity of a reversible complex, allowing the classification as GDP-like or higher (marked influence on the initial amplitude followed by no or very slow further change), low or no affinity (same end point reached as without inhibitor on a time scale of tens of seconds) or intermediate affinity (marked effect on the initial amplitude followed by a slow but observable further fluorescence increase). In addition, a first assessment of the association rate constant will be possible with high or intermediate affinity inhibitors. It should be noted that the stoichiometric amount of GDPβMe present in the 1:1 KRas:GDP complex will not have a significant effect on the results of these experiments, but must be considered in the other approaches described below. Note also that this is not meant to be an accurate determination of association rate constants, which can only be measured by transient kinetic experiments of the type reported previously1, 4 and those described below.

Assessing binding affinity using displacement titrations Equilibrium methods can generally be applied to the characterization of reversible inhibitors, but there is a fundamental problem if the starting point is a highly stable complex such as KRas:GDP because of the very low value of the dissociation rate constant for GDP. This also applies to many other GTPases, with the GDP off rate constants often in the range of 10-5-10-3 s-1 1, 12, 13. In early work on examining the relative affinities of various nucleotides to that of GDP, the rate of equilibration after addition of a nucleotide to Ras:GDP was increased dramatically by reducing the magnesium concentration to very low levels, increasing the GDP off 10 ACS Paragon Plus Environment

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Biochemistry

rate by a factor of more than 20014. After incubation, excess Mg2+ was added to “freeze” the nucleotide populations, and the data were used to calculate relative affinities15. This procedure actually leads to relative affinities in the absence of Mg2+, which might or might not be a good reflection of the value when the physiological Mg2+ ion is bound. Additionally, the affinity of nucleotides binding tightly to Ras cannot be measured by direct titration to nucleotide-free protein. Besides the fact that nucleotide-free Ras is difficult to generate and is thermally unstable, with affinities in the several picomolar range, the protein concentration used would have to be in the low picomolar range, and association rates would be prohibitively slow. Thus, at 3 pM GDP and 3 pM Ras, the half-life for association would be ca. 10,000 seconds, which is not commensurate with the low stability of nucleotide-free Ras. Some of the problems discussed can be avoided using the weakly bound β-methyl ester of GDP (GDPβMe). For such titrations, the previously described fluorescently labeled version of GDPβMe, mantdGDPβMe (a derivative harboring a methylanthraniloyl group on the 3’-oxygen of dGDP as well as the methyl group on the β-phosphate4) offers attractive possibilities. In the case of potential or proven covalent inhibitors that react with specific mutants, it is probably best to perform these experiments initially with the wild type protein so that only the reversible binding properties are assessed. Obviously, the reversible binding constants obtained are not necessarily identical to those that will pertain for a mutant that can also react covalently with the inhibitor. It is however unlikely that there will be a dramatic difference in most cases, and the constants derived will be a guide to the more complex analysis of the reaction between the mutant and a covalent inhibitor. Indeed, for a number of oncogenic mutants of KRas the largest difference reported is an approximately ten-fold increase in the dissociation rate constant of GDP from the G13D mutant16. For a particular mutant, the affinities and kinetics of GDPβMe and mantdGDPβMe will need to be determined using the methods described here and elsewhere4. Using the results reported by Müller et al.4, it can be calculated that at the initial concentration of the 1:1 mixture of KRas and mantdGDPβMe used, significant dissociation occurs before starting the titration. According to reported kinetic data (kon = 4.9 µM-1s-1, koff = 0.4 s-1, Kd = 82 nM for the complex KRas:mantdGDPβMe), 1 µM initial concentration would dissociate rapidly until 0.752 µM of the complex remains, while free KRas and free mantdGDPβMe are present at 0.248 µM (see Figure S3). Titration of the ACS Paragon Plus Environment

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weakly bound SML 8-73-1 (for the simulation, the kinetic data from Müller et al4, kon = 7.4 µM-1s-1 and koff = 1 s-1 were used) would then lead to the binding curve shown in Figure 3A (orange line), with complete displacement of the fluorescent nucleotide occurring at high excess of SML 8-73-1. In contrast, titration with the tightly bound GDP (the kinetic values were taken from Müller et al4 and John et al5, kon = 8.2 µM1 -1

s and koff = 1.6 × 10-5 s-1) leads to a completely different curve shape, with the slightly sigmoidal shape at

the beginning of the titration arising from the fact that GDP will initially bind to free Ras (Figure 3A; red line). This is followed by an approximately linear phase which saturates abruptly at a 1:1 stoichiometry. When the displacing agent has intermediate affinity, this end point is reached less abruptly, as shown in the simulated curve for GppCH2p assuming an affinity that is ca. 100 fold lower than that of GDP (Figure 3A; green line). The following protocol describes this type of titration: 1. Prepare the fluorescence spectrometer with desired settings (the settings used here are noted in the materials and methods section). 2. Dilute KRas:mantdGDPβMe to a 1 µM final concentration in a cuvette (500 µL volume, 1 cm path-length) and place into the cuvette holder of a fluorescence spectrophotometer. 3. Add the nucleotide analogues in a stepwise manner. With a half-life of ca. 1 s for its dissociation, mantdGDPβMe will rapidly exchange with titrated ligands, resulting in a decrease of fluorescence intensity (but see the point discussed below about equilibration times near saturation for stronglybound inhibitors). Prepare the stocks of nucleotides at concentrations that avoid addition of large volumes, which will generally imply changing the concentration of the stock solution as the titration proceeds. 4. Continue the measurements until there is no further decrease of fluorescence intensity (this corresponds to the fluorescence signal of free mantdGDPβMe).


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Biochemistry

Figure 3: Titration experiments to assess inhibitory potential of Ras binders. A) Simulations showing the titration of a strong Ras binder (GDP), an intermediate binder (GppCH2p) and a weak binder (SML 8-73-1) against KRas:mantdGDPβMe. Note that a steep, non hyperbolic curve is expected in the case of strong binder, whereas compounds with low affinity will show less steep (approximately) hyperbolic behavior. Note that the starting concentration of KRas:mantdGDPβMe is not the nominal concentration of 1 µM expected from the dilution from a concentrated stock but is ca. 0.75 µM. The simulations were calculated using KinTek Explorer with the kinetic values as follows: for mantdGDPβMe, kon = 4.9 µM-1s-1 and koff = 0.4 s-1 (from Müller et al4); for GDP, kon = 8.2 µM-1s-1 and koff = 1.6 × 10-5 s-1 (from Müller et al4 and John et al5); for GppCH2p, kon = 2.52 µM-1s-1 and koff = 0.0025 s-1 (from this study, see below) and for SML 873-1, kon = 7.3 µM-1s-1 and koff = 1 s-1 (from Müller et al4); equilibration time = 2000 s. B) Titration of SML 8-73-1 against 1 µM KRas:mantdGDPβMe. Fitting using KinTek Explorer software resulted in a Kd value of 135 nM (reported Kd value for SML 8-73-1 to KRas is 140 nM4). C) Titration of GDP and GppCH2p against 1 µM KRas:mantdGDPβMe. Note that the fluorescence intensity drops steeply to the background value already at a 1:1 concentration ratio. No fits are shown since the data do not contain enough information to allow Kd values to be determined for these tightly binding ligands. The point at which saturation is reached in the GDP titration (1:1 stoichiometry) shows that all of the KRas in the KRas:GDPβMe is active and able to bind GDP. It should be noted that around the equivalence point with GDP, there is slight deviation from the expected behavior. Simulations show that this is probably due to insufficient waiting time between titration steps, since there is actually a relatively long equilibration time under these concentration conditions (i.e. near equivalence), which arises from the now high concentration of free mantdGDPβMe competing in the association reaction with the very low concentration of free GDP in terms of the association rate. This problem would be solved by increasing the length of time 13 ACS Paragon Plus Environment

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between titration steps, or in a less time-consuming manner using automatic titration in microtiter plates with one well for each concentration of titrant and a constant incubation time of at least 5 minutes. However, even with this approach, the affinity of ligands binding with GDP-like affinity will not be measurable, and kinetic methods described in the next section will be required to estimate the affinity. The results of Figure 2 show that equilibrium titrations reveal a distinction between weakly and strongly binding inhibitors, but that affinities can only be determined for those that bind weakly. However, it is likely that the approach can be used in high-throughput format in 96 or 384 well plates, allowing a quick assessment of larger libraries of compounds (Scheme 1).

Kinetics of reversible inhibition

Determining association rate constants by generating nucleotide-free KRas by dilution of KRas:GDPβMe The direct measurement of nucleotide association kinetics requires the use of a stopped flow apparatus, but note that a good indication of the magnitude of the association rate constant can be obtained in a standard fluorescence spectrophotometer, as described in an earlier section. Association rate constants have previously been measured using nucleotide-free protein preparations with all the inherent problems this implies in the case of Ras superfamily proteins1. The use of the relatively low affinity KRas:GDPβMe complex allows this to be done without isolating nucleotide-free Ras. The basis of the method is that given the affinity of the KRas:GDPβMe interaction (Kd = ca. 0.2 µM), diluting a 1:1 mixture of a concentrated stock solution to sub-micromolar concentrations will result in significant dissociation of the complex. Thus, in a stopped flow experiment, a known (i.e. calculable) concentration of free KRas can be generated. The following procedure can be used: (1)

Dilute 1 µM KRas:GDPβMe in the drive syringe of the stopped flow apparatus. At 1 µM of the mixture, using the association and dissociation rate constants reported, a concentration of 0.308 µM free KRas will be generated (in the drive syringe of the stopped flow apparatus; note that in the next step, the starting concentration of free KRas will be 50% of this value if a 1:1 mixing ratio is used).


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Biochemistry

(2)

This solution is mixed with e.g. 2 µM (final concentration) mantdGDP and the association kinetics of the fluorescent nucleotide with KRas can be monitored and interpreted in terms of the known kinetic constants.

(3)

In the next step, a candidate inhibitor is included with mantdGDP in the second syringe of the stopped flow machine, and the influence of this on the kinetics of association of the fluorescent nucleotide will be modified according to the kinetic properties of both competing species. The different types of kinetic traces to be expected with varying ligand affinities are shown in Figure 4A-B.

In Figure 4A, the curve without inhibitor presence shows a rapid initial phase, corresponding to mantdGDP binding to free Ras, followed by a slower phase arising from the release of GDPβMe. In principle, this experiment has information on all 4 rate constants describing this system and can be analyzed by numerical simulation and fitting to extract these parameters in case they are not known under the particular conditions in the experiments performed. As also shown in Figure 4A, inclusion of 2 µM of the weakly-binding SML 83-7-1 with mantdGDP has a marked effect on the time course of the reaction, and this can be analyzed to yield both the association and dissociation rate constants, as shown in Figure 4B. The dissociation rate constant should agree with that obtained from experiments such as those described in Figure 5. For the more slowly released acyclovir triphosphate as a competitor of mantdGDP, there is a more dramatic effect with a slower second phase, while the strongly bound GTP has a marked effect on the overall amplitude due to its high affinity. An example of fitting such data is shown in Figure 4B for SML 83-7-1, which results in kinetic constants that are in quite good agreement with values derived earlier using a different approach (here kon = 5.44 µM-1 s-1 cf. 7.4 µM-1 s-14, koff = 0.61 s-1 cf. 1.0 s-1 4) .

Determining association rate constants by in situ degradation of GDPβMe from the KRas:GDPβMe complex The procedure described in the previous section has the disadvantage that a relatively sophisticated analysis of the results is required, and that this analysis might not be robust unless experiments at varying concentrations of the competing nucleotides combined with a global analysis of the data is performed, increasing the level of expertise required still further. A solution can be to use the following approach,


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which is again based on the relatively rapid rate of dissociation of GDPβMe from its complex with KRas. Because of this, treatment of a dilute solution of a 1:1 mixture of KRas and GDPβMe with phosphodiesterase, which can degrade the derivative to GMP and methyl phosphate, will generate nucleotide-free KRas. Despite its thermal instability, this can be used in association experiments in the stopped flow apparatus according to the following protocol. (1)

Dilute 1:1 KRas:GDPβMe complex to 0.5 µM in the solution for the stopped flow syringe.

(2)

Add a small amount of phosphodiesterase (ca. 0.0001 units for 1 ml of solution). In the time needed to fill the solution into the stopped flow drive syringe, most or all of the nucleotide is degraded.

(3)

Drive this solution against mantdGDP (at e.g. 1 µM final concentration) with and without the inhibitor (increasing concentrations) under investigation.

If this is done using an excess of mantdGDP over KRas in the presence of varying amounts of inhibitor, a plot of the observed first order rate constant for the association curve against the concentration of inhibitor leads to the value of the second order rate constant for association of the inhibitor (slope of the plot) (Figure 4C). The amount of PDE and the time required for it to digest Ras- bound GDPβMe can be checked by mixing Ras:mantdGDPβMe rapidly with PDE. The ensuing time-dependent loss of fluorescence intensity indicates the time required for degradation. The amount of PDE used must be chosen so that it does not degrade the nucleotides before their association with KRas, but this is an easy requirement to fulfill due to the rapid association reaction under these conditions. Note that longer periods of treatment with PDE should be avoided, and will result in loss of amplitude of the observed signal. However, if the reaction is performed under pseudo-first order conditions (in practical terms with at least a 3-fold excess of mantdGDP over KRas) the rate constant will not be changed by decreasing amplitude (i.e. by loss of active KRas).


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Biochemistry

Figure 4: Kinetic measurements to obtain association rate constants. A) 1 µM KRas:GDPβMe was mixed with a pre-mixed solution of 4 µM mantdGDP and 4 µM ligands (final concentration in the cell is 0.5 µM for protein and 2 µM for ligands) in a stopped-flow instrument. B) A fit using KinTek Explorer (data as in orange curve in 4A) for SML 83-7-1revealed a kon value of (5.44 ± 0.081) µM-1s-1 and koff value of (0.61 ± 0.0014) s-1 for SML 8-73-1 (the following kinetics parameters were fixed: for GDPβMe, kon = 5.8 µM-1s-1 and koff = 0.8 s-1, and for mantdGDP, koff = 1.6 × 10-5 s-1). The dashed gray lines denote the approximate end of the first (association) and the second (dissociation) phases, respectively. C) 1 µM KRas:GDPβMe was diluted in the syringe of the stopped flow machine and a small amount of PDE was added just prior to the measurements. This was then rapidly mixed with mantdGDP and increasing concentrations of 2’-amino GTP. The slope of the observed first order rate constant against the concentration of 2’-amino GTP is (2.3 ± 0.19) µM-1s-1.

Determination of dissociation rate constants Kinetic studies of nucleotide dissociation are readily performed using displacement reactions. This is most easily done if the ligand to be characterized bears a fluorescent label, but this will not be the case in general. The dissociation kinetics of a non-fluorescent nucleotide can be examined using displacement by a fluorescent nucleotide, and this is easily achieved for relatively weakly bound ligands that can be displaced by, for example, mantdGDP. Starting with the simplest case, i.e. with a ligand which is bound strongly enough to allow generation of a stoichiometric complex at a concentration of e.g. 1 µM but which is still bound much less strongly than mantdGDP, the ligand can be displaced by a small excess of mantdGDP. This is illustrated for the ligand GppCH2p, which is displaced in a single exponential reaction by 10-fold 17 ACS Paragon Plus Environment

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molar excess of mantdGDP, with an observed rate constant of 0.0025 s-1 (Figure 5A). To check whether this really reflects the limiting rate constant of GppCH2p release, this can be repeated using an increased mantdGDP concentration, which should lead to the same result for the rate constant. Ideally, testing for saturation of the rate constant would require further doubling of the concentration of displacing agent, but the observable signal becomes very small in comparison to the background, making meaningful estimates difficult. The steps in this experiment are: (1)

Set the fluorescence spectrophotometer to the desired settings for high mantdGDP concentrations.

(2)

Dilute mantdGDP from a concentrated stock solution to a 10-fold higher concentration than the chosen concentration of the 1:1 mixture of KRas:GppCH2p to be used in the next step into a 1 ml quartz fluorescence cuvette at 25°C and insert this into the cuvette holder of a fluorescence spectrophotometer.

(3)

Add a 1:1 mixture of KRas:GppCH2p to a concentration of, e.g. 1 µM and stir well before recording the fluorescence intensity as a function of time.


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Biochemistry

Figure 5: The dissociation rate constants of ligands bound to Ras can be obtained by monitoring competition with mantdGDP. A) Displacement of GppCH2p from its 1:1 complex with KRas (1 µM) by 10 µM mantdGDP. A single exponential fit for the increase of intensity over time yielded a koff value of 0.0025 (± 0.00014) s-1. B) Principle of the experiment to determine dissociation rate constants for tightly bound inhibitors (simulated curves). 1 µM KRas:mantdGDP is mixed with 0.25 (solid curve) or 0.5 µM (dashed curve) of an inhibitor with kon = 3.2 µM-1s-1 and koff = 1.6 × 10-5 s-1. Note that equilibrium is reached more rapidly with the lower inhibitor concentration added, but the amplitude is smaller. C) 1 µM KRas:mantdGDP was mixed with 0.5 µM 2´-amino GTP. After monitoring for 7 hours, the final amplitude corresponding to complete mantdGDP displacement was determined by adding 5 mM EDTA and 100 µM GDP (final concentrations). This provides a fixed input value for the simulation/fit procedure for the fluorescence intensity arising from unbound mantdGDP. A fit with KinTek Explorer resulted in a koff value for 2´-aminoGTP of 4.8 x 10-4 s-1, using fixed values of kon for mantdGDP = 3.2 µM-1s-1, kon for 2´aminoGTP = 2.3 µM-1s-1 and koff for mantdGDP was fitted to 2.3 × 10-5 s-1. D) KRas:GDPβMe (0.5 µM) was mixed with a 10-fold excess mantdGDP in a stopped flow apparatus. Note that 1 µM Ras:GDPβMe upon dilution in the syringe generates 0.308 µM free Ras, using the known values of kon = 5.8 µM-1s-1 and koff = 0.8 s-1. This leads to initial rapid binding of mantdGDP to free Ras (phase ending approximately as designated by the gray dashed line), followed by dissociation of bound ligand (GDPβMe) that is reflected by the second phase. Fitting with KinTek Explorer using initial concentrations of KRas:GDPβMe = 0.346 µM, free Ras and GDPβMe = 0.154 µM led to a koff value of 0.62 (± 0.0009) s-1for GDPβMe. If the affinity of GDPβMe were not known, it could also be calculated approximately as follows: the amplitude corresponding to free Ras (initial mantdGDP binding phase) is 0.23 and the amplitude of second phase is 0.44 (total amplitude = 0.67). This means that about 30 % of the complex dissociates upon dilution to 1 µM i.e. 0.3 µM free Ras (and free GDPβMe) and 0.7 µM Ras:GDPβMe. This corresponds to a Kd value of 0.13 µM, in good agreement with the previously reported value of 0.14 µM4.

If the inhibitor being tested has high affinity (i.e. approaching that of GDP or potentially higher, which should already have been detected at least qualitatively in equilibrium experiments), measurement of offACS Paragon Plus Environment

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rates is more difficult because of the long time course of classical displacement experiments, and because of the difficulty of using sufficiently high concentrations of displacing nucleotide. In this case, a different approach can be adopted to shorten the measurement time, and this is illustrated in Figure 5B. A general procedure begins with the 1:1 KRas:mantdGDP complex (e.g. at 1 µM), and a low concentration of inhibitor (e.g. 0.5 µM) is added to this and the time-course of the loss of fluorescence intensity is recorded (Figure 5C). Because mantdGDP displacement at equilibrium is not complete, equilibrium is reached at relatively short times, and the time course of the transient is determined by all 4 rate constants defining the system. If the association rate constant for inhibitor has been determined (see above), fitting by numerical simulation allows extraction of the dissociation rate constant of the inhibitor (Figure 5C) For an unknown ligand that has already been shown to be capable of displacing mantdGDPβMe in titration experiments of the type described in the previous section, if the dissociation reaction is too fast to measure using mixing by hand, suggesting weak binding, the experiment must be performed under similar conditions in a stopped-flow apparatus (Figure 5D). In this case, there will be a fraction of free Ras upon dilution, resulting in two binding phases, namely rapid mantdGDP binding to free Ras, followed by a second phase dictated by the dissociation of the weak binder (Figure 5D). There is information on both affinity and rate constants from this result. The ratio of the amplitude of the 2 phases defines the affinity of the unknown ligand, while a full fit using numerical simulation with KinTek Explorer will lead to all unknown constants (i.e. on and off rate constants for the ligand).

Kinetics of Irreversible inhibition Irreversible inhibitors that first form a complex by interacting with the binding site of the natural ligand and then react covalently will, in general, have two significant aspects that define their effectivity. The first is characterized by the reversible association and dissociation rate constants of the initial binding step and usually happens on a relatively rapid time scale (assuming the binding site is not occupied by a slowly dissociating ligand, such as GDP in the case of GTPases). The second step will generally be slower, and is dependent on the (first order) rate constant for the covalent reaction occurring in the reversible Ras:inhibitor complex. This basic mechanism is shown in scheme 1. Ki

kinact


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Biochemistry

Ras + I → Ras:I → Ras.I ←

Scheme 1. Ki (= koff/kon) is the dissociation constant of the inhibitor for the initial binding step, kinact is the rate constant for the covalent reaction. Ras:I is the reversibly bound complex, Ras.I the covalent adduct.

In the absence of an already bound ligand, the effective overall rate constant for formation of the covalent complex is given by equation 1.

=

1 +

Equation 1. Dependence of the observed first order rate constant for formation of the covalent complex for an irreversible inhibitor if the inhibitor is in excess over the protein.

In the case of Ras, for reasons discussed above, it is inconvenient to perform experiments starting with the nucleotide-free protein. A general approach can be most easily to start using the weakly bound KRas:GDPβMe complex. If covalent reaction is suspected, mass spectrometry experiments will normally lead to easy and rapid detection of the resulting adduct. In earlier work starting with the Ras:GDP complex, the rate of covalent reaction was limited not only by kinact but by all constants in the system, including the association and dissociation rate constants of GDP and the inhibitor4. A partial solution to this problem is to use EDTA to remove free Mg2+, but the rate constant for GDP release is still quite small (ca. 0.004 s-1, corresponding to a half-life for the dissociation reaction of ca. 3 min14), but we are then looking at the covalent reaction in the absence of Mg2+, which is a non-physiological situation. Starting with KRas:GDPβMe, the dissociation rate constant of 0.8 s-1 means that the half-life for dissociation is ca. 1 sec, even in the presence of Mg2+, making the use of EDTA unnecessary. This means that for an inhibitor that binds reversibly considerably more strongly than GDPβMe (which is a general requirement for a potentially useful inhibitor), the rate constant for the covalent reaction could be measured directly using mass spectrometry as long as the reaction is not too rapid and a way can be found to stop the reaction at specified


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times before mass-spec analysis. Addition of a high concentration of a thiol reagent or a mixture of 30% acetonitrile + 0.1% trifluoroacetic acid might serve this purpose. It should be noted that other methods have also been used successfully to assess the time course of covalent reaction of GDP derivatives with KRas G12C (AktivAlpha method)8. For inhibitors that initially bind weakly, experiments of the type shown in Figure 6 can lead to determination of the rate constant for inactivation. Mixing a 1:1 solution of KRas:mantdGDPβMe with a small excess of a similarly weak-binding inhibitor will lead to rapid establishment of an equilibrium on the time scale of a few seconds (gray curve in Figure 6A). If the inhibitor reacts covalently, this will be followed by a phase in which the covalent reaction occurs (if this is indeed slower than the initial equilibration), proceeding to complete displacement (which is defined here by the GTP displacement curve) (orange curve in Figure 6A) and the rate constant for inactivation can be obtained from the data (Figure 6B). Note that an inhibitor that binds reversibly with GDP/GTP like affinity will lead to complete displacement in the initial phase, so that no phase corresponding to the covalent reaction will be observed.

Figure 6: Kinetic experiments to determine the rate of covalent adduct formation. A) 0.5 µM KRas G12C:mantdGDPβMe was rapidly mixed with 1 µM GDPβMe (gray curve), 1 µM SML 8-73-1 (light orange curve), 2 µM SML 8-73-1 (orange curve) or 1 µM GTP (red curve). B) Fitting the orange curve (SML 8-73-1) using KinTek Explorer. Keeping the constants for mantdGDPβMe fixed to their known values (kon = 4.9 µM-1s-1, koff = 0.4 s-1) led to a value of 0.053 s-1 for kinact for SML-8-73-1). The rapid phase is due to reversible binding of the inhibitor while the second phase arises from the covalent reaction. ACS Paragon Plus Environment

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Biochemistry

Conclusion The procedures reported here make use of weakly bound KRas-nucleotide complexes to provide easy access to information on the affinities and kinetics of compounds that compete with binding of GDP. The methods presented avoid the relatively inconvenient process of generation of thermally unstable GDP-free KRas. As shown in Scheme 1, we suggest a simple rapid procedure to estimate the approximate inhibitory potential of a compound (or compounds) under investigation, followed by more detailed analyses in the case of initially positive results. In the case of covalently reacting inhibitors, additional methods to those presented here will be needed for a full characterization, except in the case of inhibitors that initially bind with low affinity, as shown in Figure 6B, where rate constants can be measured using the assay described here. However, in general the more interesting covalently binding inhibitors will have a high affinity of the reversible binding step to a Ras mutant (i.e. GDP-like affinity), as we have previously argued4. In this case, a full kinetic characterization is difficult, especially if the rate constant for the covalent reaction is much faster than the koff value, which will in fact be the case for the most potent compounds. In this case, a probably very good assessment of the binding/dissociation kinetics can be obtained from experiments with wild type Ras, and these can be combined with a measurement of the rate constant of inactivation starting from the nucleotide-free Ras mutant or its complex with GDPβMe, which will be rapidly and effectively displaced by such a compound. Mass spectrometry or other methods can be used for this purpose4, 8, but for rapidly reacting compounds this will have to be combined with rapid mixing and stopping the reaction using the classical quenched flow technique.

AUTHOR INFORMATION Corresponding Author Email: [email protected]


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Funding This work was supported by Max-Planck-Society and the Deutsche Forschungsgemeinschaft ((DFG/ANR grant GO 284/8-1 and SFB 642).

Notes The authors declare no competing financial interest Supporting Information available A brief text for the derivation of the amplitude ratio, related to Figure 2, and Figures S1-S4 with an outline of the use of Kintek Explorer for data analysis are supplied as Supporting Information.

References

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[4] 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, Scientific reports 7, 3687.

[5] John, J., Rensland, H., Schlichting, I., Vetter, I., Borasio, G. D., Goody, R. S., and Wittinghofer, A. (1993) Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras, J Biol Chem 268, 923-929.

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[11] 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.

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

[13] 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.

[14] John, J., Rensland, H., Schlichting, I., Vetter, I., Borasio, G. D., Goody, R. S., and Wittinghofer, A. (1993) Kinetic and Structural-Analysis of the Mg2+-Binding Site of the Guanine Nucleotide-Binding Protein P21(H-Ras), J Biol Chem 268, 923-929.

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Assays for Nucleotide Competitive Reversible and Irreversible

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