Amélie Bret
email: amelie.bret@univ-nantes.fr

Brice Beffara

Adrien Mierop

Martial Mermillod

Differentiated Evaluation of Counter-Conditioned Stimuli as a Function of Right-Wing Authoritarianism

Keywords: authoritarianism, attitude, change, evaluative counter-conditioning

Right Wing Authoritarianism (i.e., RWA) is associated with enhanced conservatism and social prejudice. Because research linking RWA to attitudes is largely correlational (i.e., it provides control for neither RWA nor attitude learning), it is not clear how RWA relates to attitude learning dynamics. We addressed this question in 11 evaluative conditioning experiments that ensured rigorous control of the affective learning setting. Results from two integrative data analyses suggest that (i) individuals scoring higher in RWA show a stronger acquisition of positive attitudes, and that (ii) the residuals of this stronger acquisition remain even after exposure to counterattitudinal information. Implications of these findings for research on RWA and its link to social prejudice are discussed.

Right Wing Authoritarianism (RWA) is characterized by a covariation of authoritarian aggression, authoritarian submission, and conventionalism [START_REF] Altemeyer | Right-wing authoritarianism[END_REF]. Higher lev els of RWA are typically associated with more negative attitudes concerning outgroups [START_REF] Altemeyer | The other "authoritarian personality[END_REF][START_REF] Duckitt | Right wing authoritarianism, social dominance orientation and the dimensions of generalized prejudice[END_REF][START_REF] Duriez | The march of modern fascism: A comparison of social dominance orientation and authoritarianism[END_REF][START_REF] Hodson | Bowing and kicking: Rediscovering the fundamental link between generalized authoritarianism and generalized prejudice[END_REF]. To date, however, research on RWA and attitudes has been largely observational: both RWA and social attitudes are often studied as observed variables (i.e., without consider ing causal determinism). This observational strategy has advanced our understanding of how RWA relates to prejudice and social attitudes. However, it does not allow optimal control when investigating attitude learning dynamics as a function of RWA. The aim of the present research was to contribute to the latter question by making use of a carefully controlled attitude learning paradigm. It should be noted, however, that RWA will not be studied as an experimentally manipulated variable here. In this series of experiments, we shall simply try to manipulate the acquisition and change of attitudes. We start by introducing RWA and its relation to attitudes and by considering evaluative conditioning as a controlled attitude learning procedure. We then report and present two integrative data analyses conducted on data from 11 experiments that shed light on our research question, namely how attitudes are acquired and how they resist counter-attitudinal information as a function of RWA.

RWA and Attitudes

RWA is a classic predictor of prejudice [START_REF] Altemeyer | Right-wing authoritarianism[END_REF]. It correlates with negative attitudes to a range of social groups [START_REF] Ekehammar | What matters most to prejudice: Big Five personality, Social Dominance Orientation, or Right-Wing Authoritarianism?[END_REF]: African Americans [START_REF] Altemeyer | The other "authoritarian personality[END_REF][START_REF] Lambert | Perceptions of disadvantage versus conventionality: Political values and attitudes toward the elderly versus Blacks[END_REF], homosexuals, [START_REF] Altemeyer | The other "authoritarian personality[END_REF][START_REF] Lippa | Gender, personality, and prejudice: The display of authoritarianism and social dominance in interviews with college men and women[END_REF], women [START_REF] Altemeyer | The other "authoritarian personality[END_REF], and immigrants [START_REF] Zakrisson | Construction of a short version of the Right-Wing Authoritarianism (RWA) scale[END_REF]. RWA is commonly described as social conservatism because it is positively correlated with con servative beliefs in the socio-political domain (e.g., [START_REF] Van Hiel | Explaining conservative beliefs and political preferences: A comparison of social dominance orientation and authoritarianism[END_REF].

Furthermore, beyond the correlation between RWA and negative attitudes towards outgroups, RWA is associated with more stable attitudes over a period of six months [START_REF] Asbrock | Right-wing authoritarianism and social dominance orientation and the dimensions of generalized prejudice: A longitudinal test[END_REF] and with a lower likelihood that these attitudes will change [START_REF] Huckfeldt | Political consequences of inconsistency: The accessibility and stability of abortion attitudes[END_REF]. This enhanced stability of attitudes in high-RWA individu als is consistent with studies suggesting that RWA is associated with lower cognitive flexibility [START_REF] Onraet | The association of cognitive ability with Right-wing ideological attitudes and prejudice: A metaanalytic review[END_REF][START_REF] Sidanius | Cognitive functioning and sociopolitical ideology revisited[END_REF][START_REF] Zmigrod | The role of cognitive rigidity in political ideologies: Theory, evidence, and future directions[END_REF]. Several studies have highlighted the importance of adaptation to change in understanding the emergence and maintenance of prejudice [START_REF] Dhont | Does lower cognitive ability predict greater prejudice?[END_REF][START_REF] Roets | Allport's prejudiced personality today: Need for closure as the motivated cognitive basis of prejudice[END_REF][START_REF] Stern | Ideological differences in race and gender stereotyping[END_REF]. In addition, RWA is typically linked with Need for Closure, defined as the motivation to have a definite answer or knowledge instead of uncertainty or doubts about the social environment, and can therefore be linked to a stronger 'primacy effect' (i.e., higher levels of RWA are associated with a better memory for the first items on a list as compared to the last items; [START_REF] Jost | Political conservatism as motivated social cognition[END_REF][START_REF] Kruglanski | Groups as epistemic providers: Need for closure and the unfolding of group centrism[END_REF].

It should be noted that the conceptualization of right-wing authoritarianism has important implications for its general understanding and even more so in this type of research [START_REF] Van Hiel | The impact of need for closure on conservative beliefs and racism: Differential mediation by authoritarian submission and authoritarian dominance[END_REF]. Some authors consider right-wing authoritarianism to be a personality trait [START_REF] Adorno | The authoritarian personality[END_REF], while others consider it to be a belief [START_REF] Duckitt | A dual-process cognitive-motivational theory of ideology and prejudice[END_REF]. If authoritarianism is viewed as a personality trait, the associated cognitive differences should be considered a consequence of authoritarian thinking [START_REF] Van Hiel | The impact of need for closure on conservative beliefs and racism: Differential mediation by authoritarian submission and authoritarian dominance[END_REF]. In this case, authoritarianism can be seen as a predis position to cognitive rigidity (as opposed to cognitive flexibility). By contrast, if we consider authoritarianism to be a belief, individuals with high cognitive rigidity would be predisposed to developing authoritarian thinking. Various studies corroborate this last hypothesis, showing an influence of cognitive style on the development of negative exo-group attitudes during adolescence. This suggests that authoritarianism is derived from political socialization (i.e., development) [START_REF] Alwin | Aging, cohorts, and the stability of sociopolitical orientations over the life span[END_REF][START_REF] Duckitt | A dual-process cognitive-motivational theory of ideology and prejudice[END_REF]. Whatever the case may be, this series of experiments is not intended to test these hypotheses or to establish any causal link.

The above literature suggests that high-RWA individuals may be less likely to revise their attitudes once they are formed. In other words, they may form evaluations (i.e., possibly acquired by evaluative conditioning, "EC" hereinafter) that are less sensitive to new and counter-attitudinal information (i.e., due to lower flexibility; for a meta-analyti cal review, see [START_REF] Jost | Political conservatism as motivated social cognition[END_REF]. This could partly explain the link between RWA and prejudice. The current literature links RWA to attitudinal stability over time, but does not allow us to understand this lack of change except in terms of a multitude of social factors (group membership). At present, we do not know precisely how attitudes are formed and even less how they evolve in relation to RWA. What we propose here is to study RWA in a context free of social information. Therefore, if the hypothesis of a lower sensitivity to counter-attitudinal information is confirmed, the net outcome of assessing exposure to attitudinal and then counter-attitudinal information should reflect higher levels of attitudinal information than counter-attitudinal information as RWA increases.

Evaluative Conditioning as a Controlled Attitude Learning Paradigm

To our knowledge, no prior work has examined how RWA modulates this attitude dy namic. The exclusive observational nature of research on RWA and attitudes has allowed for little control of attitude formation and change. These observational studies typically relate RWA levels to attitudes toward existing social groups. As such, they introduce a series of uncontrolled factors, all of which are capable of accounting for the observed relation between RWA and attitudes (i.e., the social nature of the attitudinal objects, previous knowledge and interactions regarding the attitudinal objects, the number of in stances of prior attitudinal and counter-attitudinal information regarding the attitudinal objects, the relevance of the context of the investigation for evaluations of the attitudinal objects, the social desirability of and the norms governing the expression of attitudes toward the attitudinal object, etc.). In the current research, we overcame this limitation (i) by employing a carefully controlled evaluative conditioning paradigm that permitted the causal control of attitude formation by means of an experimentally manipulated variable and (ii) by using a set of socially irrelevant and unfamiliar stimuli, for which parameters such as prior knowledge, time and frequency of exposure, social relevance, or group variability were controlled. Here, we consider the RWA variable as a general ideology.

Evaluative conditioning (EC) is a simple and reliable attitude learning paradigm, with an average Cohen's d = 0.52 [START_REF] Hofmann | Evaluative conditioning in humans: A meta-analysis[END_REF]. It consists of pairing a neutral stimu lus (conditioned stimulus, CS) with another positive or negative stimulus (unconditioned stimulus, US) [START_REF] De Houwer | Association learning of likes and dislikes: A review of 25 years of research on human evaluative conditioning[END_REF][START_REF] De Houwer | A conceptual and theoretical analysis of evaluative conditioning[END_REF]. Using this pairing procedure, the CS typically acquires the valence of the US it was paired with. For example, if a neutral stimulus such as headphones is presented at the same time as a cute baby, the headphones will take on the baby's valence and become positive. The process is the same for negative images.

This paradigm is efficient for almost any type of stimulus. As a result, this paradigm can be used to manipulate the acquisition and change of an attitude towards a stimulus never previously encountered by the participants. The advantage of this method is that it allows us to examine the dynamics of attitudes in a setting without prior knowledge of or social motivation regarding the stimuli. In other words, it permits a purer exami nation of how RWA influences attitude dynamics, without being interfered with by uncontrolled variables (e.g., pre-existing attitudes, knowledge and motivations regarding existing social groups), which may and, indeed, are very likely to be at work when forming and adapting attitudes about social groups. At the same time, increasing our knowledge of basic learning mechanisms makes it possible to infer how social knowledge and motivations may influence attitudinal processes with regard to real social groups. However, it seems important (independently of the possible generalization to real social groups) to undertake a causal study of the acquisition of attitudes towards completely new stimuli in RWA (by means of experimentally manipulated variables), particularly in the light of the literature on cognitive skills and RWA.

Method Integrative Data Analyses

Several EC studies were conducted by our research team between 2015 and 2018 in order to provide information relevant to the question of interest: Is RWA positively related to a resistance toward counter-attitudinal information? In each of the experiments, a set of Greebles (fictive characters, the CSs) were paired with valenced pictures (the USs), with half of the Greebles being paired with positive pictures and the other half being paired with negative pictures. This pairing procedure constitutes the "conditioning phase". In ten of these experiments, evaluative RWA and CS ratings were collected directly after a "counter-conditioning phase". To do this, the Greebles that had been paired with a given valence during the conditioning phase were then subsequently paired with USs of the opposite valence prior to the RWA and evaluative measures. In two experiments, RWA and evaluative ratings were collected immediately after the conditioning phase (meaning that there was no counter-conditioning phase in these experiments).

Here, we report and discuss two separate integrative data analyses on these two sets of experiments. These integrative data analyses have two main goals: to test whether RWA is related to more resistant attitudes in a counter-conditioning procedure (integra tive data analysis 1), and to test whether this effect is due to differences in RWA present in acquisition attitudes in a conditioning procedure (integrative data analysis 2). First of all, we hypothesized that RWA would be positively related to a resistance to novel counter-attitudinal information. This should result in a final evaluation that is more consistent with the conditioning than the counter-conditioning phase. Let us illustrate this hypothesis with an example. In the conditioning phase, participant 1 sees CS-A with a positive US and CS-B with a negative US. In the counter-conditioning phase, these pairings are then reversed (i.e., CS-A is now presented with a negative US and CS-B with a positive US). In such an example, we would expect CS-A to be evaluated more positively than CS-B due to resistance to the second -counter-attitudinal -item of information. Secondly, we did not hypothesize any difference in the acquisition of information with RWA, i.e., in (simple) conditioning effects. As a methodological precau tion, we chose only to evaluate the attitude after counter-conditioning in integrative data analysis 1. This approach makes it possible to reduce the demand effect bias that might result from multiple measures [START_REF] Charness | Experimental methods: Between-subject and within-subject design[END_REF]. However, this choice also reduces the amount of information available for understanding the dynamics of attitudes in RWA. We therefore chose to evaluate the attitude after the conditioning phase in integrative data analysis 2 in order to determine whether the link between RWA and the relative resistance to counter-attitudinal information is the reflection of a link between RWA and the acquisition of attitudes.

The experiments included in these two integrative data analyses were designed to test a variety of research questions, which involved running both control and experimental conditions. However, the main goal of this line of research was the same: to understand the roots of resistance to new information in RWA. Stimuli, numbers of trials, measures of attitudes, time of presentation and counter-balanced design were constant across these various control conditions which are the focus of the analyses presented here (as they are directly relevant to our questions). The additional experimental conditions are not relevant to our current questions and will not be discussed. In most cases, they provided mixed or non-replicated evidence (see Table 1 for an overview of the experimental settings of the different experiments).

Sample Size

We initially performed a priori power analyses to determine the required sample size for the individual studies taken separately. However, after data collection, we realized that these power analyses were derived from models which were not suitable for the type of data we had collected. In addition, the estimated expected sample size was inadequate because the initially specified parameters were inaccurate. What is more, the final sample sizes for the individual studies are highly heterogeneous as they depended on the availability of resources at the time we were running each study. Because the present article focuses on the integrative data analysis of several studies, we explain how we subsequently considered its a posteriori sensitivity. This sensitivity results from the analysis described in the data analysis section and makes it possible to determine our smallest detectable effect size [START_REF] Lakens | Sample size justification[END_REF]. More details can be found in the Supplementary Materials section.

The smallest effect size we were able to detect was determined based on the precision of the estimation of our parameter of interest [START_REF] Lakens | Equivalence tests: A practical primer for t tests, correlations, and meta-analyses[END_REF][START_REF] Lakens | Equivalence testing for psychological research: A tutorial[END_REF]. We considered that a null effect corresponded to a parameter value lower than |0.13| (see the Supplementary Materials for more information on how we determined this value). In other words, we would consider a 95% HDI (Highest Density Interval) lying between -0.13 and 0.13 as a null effect. Parameter values < |0.13| are considered practically equiva lent to 0 [START_REF] Kruschke | Rejecting or accepting parameter values in Bayesian estimation[END_REF].

Integrative Data Analysis 1: Net Outcome of a Conditioning-Then-Counter-Conditioning Procedure

Ten studies were run examining the association between RWA and the evaluation of stimuli using a conditioning-then-counter-conditioning procedure. We hypothesized that the net outcome of a conditioning-then-counter-conditioning procedure would indicate that more evaluative information is acquired in the conditioning than in the coun ter-conditioning phase at higher levels of RWA. In other words we expected negative evaluations of negatively conditioned (and positively counter-conditioned) stimuli in higher-RWA individuals. We expected positive evaluations of positively conditioned (and negatively counter-conditioned) stimuli in higher-RWA individuals.

Participants and Design

A total of 720 participants were recruited in exchange for credits (for laboratory stud ies) or money (for online studies). The participants in the laboratory studies were French-speaking (French and Belgian students) and those in the online studies were English-speaking (from the general population). Participants were randomly assigned to one of the two counter-balanced groups in which half of the CSs (i.e., group A) were paired either with positive or negative USs and the other half (i.e., group B) were paired either with negative or positive USs, respectively. This counter-balanced design did not moderate the effects and so will not be discussed further.

Stimuli and Measures

These studies were conducted in the laboratory or online. We used Testable and Prolific Academic for the online studies and E-prime software 2.0 for the laboratory studies [START_REF] Schneider | Psychology Software Tools[END_REF]. The US set consisted of eight unpleasant and eight pleasant pictures from the International Affective Picture System (IAPS; [START_REF] Lang | International affective picture system (IAPS): Affective ratings of pictures and instruction manual[END_REF]; see Supplementary Materials). Pleasant pictures were rated more positively than unpleasant pictures, t(14) = 21,14, p < .001, but it was not possible to conclude that there was a difference in arousal, t(14) = .76, p = .46. The CSs were 16 pictures from two different groups of neutral characters that were equalized on contrast and luminance (i.e., "Gree bles", [START_REF] Sheinberg | Objects of expertise[END_REF]. RWA was assessed using 10 items (Cronbach's α = .837) from the French translation of Altemeyer's 20-item (1988;[START_REF] Bougie | Validation francophone préliminaire de l'échelle écourtée du Right-Wing Authoritarianism d'Altemeyer[END_REF]) RWA Scale (from 1 = totally disagree to 7 = totally agree).

Procedure

For a visualization of the experimental design see Figure 1.

Categorization stage: In the first step, participants were asked to carefully examine, for an unlimited duration, one slide displaying the two groups of CSs (i.e., Greebles groups A and B). They were then randomly presented with individual CSs and had to sequentially categorize them into one of the two groups by pressing a corresponding key. After each trial, participants received feedback about the accuracy of the answer 1 . Conditioning and counter-conditioning stage: In the second step, participants took part in a conditioning procedure in which Greebles were positively or negatively conditioned (depending on their group and the counter-balancing condition). More specifically, par ticipants were presented with individual CSs (i.e., Greeble exemplars) that were paired either with individual positive or negative USs (i.e., a positive or negative picture). Each individual Greeble was paired with a given US four times, resulting in 64 trials. Any given group (A or B, composed of several Greebles) was therefore paired 32 times with either positive or negative USs (pairing of groups with positive or negative USs was counterbalanced among participants). A trial consisted in the simultaneous presentation of a CS and a US for 1000ms, directly followed by a black screen displayed for 1500ms (inter-trial interval, ITI). The conditioning phase was immediately followed by the coun ter-conditioning. CSs were paired with USs whose valence was opposite to that used in the conditioning phase for the same number of trials and using the same pairing procedure. Nothing indicated to the participants that a new phase had started.

Evaluative stage and scale completion: After the counter-conditioning phase, partici pants were asked to state their attitude towards each individual Greeble (from 1 = "very negative" to 9 = "very positive"). CSs were presented in a random order. Finally, the participants completed the RWA scale, were thanked, debriefed and dismissed.

Statistics and Results

We report posterior means and 95% Highest Density Intervals (HDIs) for each parameter (β) based on a multilevel Bayesian ordinal regression [START_REF] Bürkner | Ordinal regression models in psychological research: A tutorial[END_REF]Liddell & Kruschke, 2018) computed with the "brms" package (Rstudio; Bürkner, 2017a, 2017b[START_REF] Team | RStudio: Integrated Development Environment for R. RStudio[END_REF]. HDIs correspond to the probability density of "credible" values of β. We interpret these values in the light of the 95% highest probability mass ("confidence interval" in the Bayesian perspective). For an accessible introduction to Bayesian data analysis and interpretation, see [START_REF] Kruschke | The Bayesian New Statistics: Hypothesis testing, estimation, meta-analysis, and power analysis from a Bayesian perspective[END_REF]. More information about this model can be found in the Supplementary Materials.

We included the initial valence (i.e., valence corresponding to the US valence initially attached to a CS family in the evaluative conditioning stage; within-participant dichoto mous variable: negative = -0.5 or positive = +0.5) and RWA (observed variable initially bounded between 7 and 70 and with precision 1 and then transformed into a z-score), as well as their interaction as fixed factors and varying intercepts for participants and stimuli in the model. The dependent variable is the evaluation of Greebles on the ordinal scale from 1 (very negative) to 9 (very positive) (Liddell & Kruschke, 2018). Integrative data analysis was performed on the data of all experiments: Experiments 1 and 11 for integrative data analysis 1 and Experiments 1 through 10 for integrative data analysis 2 (see Table 1 for an overview). The same parameters were kept for the integrative data analyses and a varying intercept for experiments was added. Before running the models, and before centering and scaling variables, we checked for the presence of possible extreme RWA scores in our sample (possibly due to measurement errors including -for instance-misunderstanding the questions or disengaging from the task). This step was based on quartile statistics, i.e., first quartile (Q1 = 25% of the sample), third quartile (Q3 = 75% of the sample) and interquartile range (IQR = Q3 -Q1). RWA scores were considered extreme when > Q3 + 3*IQR or < Q1 -3*IQR. We excluded two participants over these 10 experiments (see Table 1 for an overview of all the experiments). The results are reported in accordance with the guidelines proposed by [START_REF] Makowski | Indices of effect existence and significance in the Bayesian framework[END_REF], as adapted for our ordinal models. For easier reading, we only report the results concerning our main parameters of interest in the manuscript. However, two tables detail the results concerning all parameters in the Supplementary Materials.

The interaction between the initial valence and RWA has a probability of 100% of be ing positive (Median = 0.21, 95% HDI [0.15, 0.27], see Figure 2 and Figure 3.1) and can be considered to be very small [START_REF] Sawilowsky | New effect size rules of thumb[END_REF] according to standard benchmarks 2 but significant (0.50% in the region of practical equivalence, R mz 2 = .003 ≈ d = 0.12 3 ). Higher RWA scores were associated with higher evaluations of the positively conditioned CSs (Median = 0.16, 95% HDI [0.11, 0.22], probability of 100% of being positive, see Figure 2 and Figure 3.2). It is uncertain whether this effect is practically significant (11.68% in the region of practical equivalence). However, for the negatively conditioned CSs, despite the fact that the association between RWA and evaluation has a 93.75% probability of being negative, (Median = -0.04, 95% HDI [-0.10, 0.01], see Figure 2 and Figure 3.3), the effect seems to be practically equivalent to 0 (99.72% in the region of practical equivalence).

2) The effect sizes reported here should be interpreted in the context of future similar studies rather than compared to benchmarks. Comparisons to benchmarks are simply given in order to permit familiarity. So far, it is complicated to determine the smallest practically significant effect in the context of this study. The reader is free to interpret the data. The most important part of the analysis is the ongoing estimation of parameters that can be used for meta-analyses or integrative analyses.

3) See the Supplementary Materials for more details about the computation of effect sizes. Note. The purple area corresponds to a region of practical equivalence where the values are considered equivalent to 0 [START_REF] Kruschke | Rejecting or accepting parameter values in Bayesian estimation[END_REF]. More information can be found in the Supplementary Materials.

Discussion of Integrative Data Analysis 1

The outcomes of integrative data analysis 1 are partially consistent with our predictions. As in the case of social attitudes, evaluations become more closely aligned with attitu dinal than counter-attitudinal information as the RWA level increases. However, this interaction between valence and RWA does not seem to rely significantly on negative conditioning and is probably mainly driven by positive conditioning, although more data are probably needed to (in)validate this point. We did not initially predict this possible asymmetry. As mentioned above, an interesting question that remains unanswered at this stage is whether the link between RWA and the relative resistance to counter-attitu dinal information reflects a link between RWA and the acquisition of attitudes. Indeed, we hypothesized that RWA was associated with a lack of flexibility, namely a difficulty in processing and assimilating new (counter-attitudinal) information. However, while this hypothesis is compatible with the results of this IDA, at least one other explanation remains possible. Indeed, these findings could instead result from a stronger effect of initial conditioning for higher RWA individuals. One possible way to address this issue is to compare the evaluations of Greebles in two experimental conditions (across the RWA continuum): one condition where the CSs are counter-conditioned and another condition where they are only conditioned.

Since we would still have (only) one CS evaluation per participant using this design, we would not increase the demand effect. Thus, if the link between RWA and the relative resistance to counter-attitudinal information found in integrative data analysis 1 simply reflects attitude acquisition differences, then the higher the RWA score, the higher the ratings should be when a positive attitude is acquired.

Integrative Data Analysis 2: Acquisition of Positive and Negative Attitudes

Two studies examined the acquisition of positive and negative attitudes as a function of RWA in an evaluative conditioning procedure. A recent meta-analysis on the impact of conservatism on cognitive functions did not report an effect of RWA in (non-evalua tive) learning [START_REF] Van Hiel | The relationship between social-cultural attitudes and behavioral measures of cognitive style: A meta-analytic integration of studies[END_REF]. In line with this meta-analysis, we hypothesized that attitude acquisition would not vary as a function of RWA.

Participants and Design

A total of 317 participants were recruited in exchange for credits (for laboratory stud ies) or money (for online studies). The participants in the laboratory studies were French-speaking (French and Belgian students) and those in the online studies were English-speaking (from the general population). Participants were randomly assigned to one of the two counter-balanced groups in which half of the CSs (i.e., group A) were paired with either positive or negative USs and the other half (i.e., group B) were paired with either negative or positive USs, respectively. This counter-balanced design did not moderate effects and so will not be discussed further.

Stimuli and Measures

These two experiments were conducted online using Testable and Prolific Academic. Materials were the same as for integrative data analysis 1.

Procedure

Categorization stage: This step was similar to the one presented in integrative data analysis 1.

Conditioning stage: The conditioning phase, similar to that reported for integrative data analysis 1, was immediately followed by the CS evaluation.

Evaluative stage and scale completion: Participants were asked to state their attitude towards each individual Greeble (from 1 = "very negative" to 9 = "very positive"). CSs were presented in a random order. Finally, the participants completed the RWA scale, were thanked, debriefed and dismissed.

Statistics and Results

The rationale underlying the data analysis was the same as for integrative data analysis 1. We also checked for the presence of possible extreme RWA scores in our sample. We excluded two participants over these two experiments (see Table 1 for an overview of all the experiments). The interaction between the initial valence and RWA has a probability of 100% of being positive (Median = 0.22, 95% HDI [0.12, 0.33], see Figure 4 and Figure 5.1). It is uncertain whether this effect is statistically significant. Even though only 4.05% of the parameter values lie in the region of practical equivalence, the 95% HDI slightly overlaps with it. This effect can be considered to be very small [START_REF] Sawilowsky | New effect size rules of thumb[END_REF] according to standard benchmarks 4 (pseudo-R 2 = .003 ≈ d = 0.12 5 ). Higher RWA scores were associated with higher evaluations of the positively conditioned CSs (Median = 0.25, 95% HDI [0.14, 0.34], probability of 100% of being positive, see Figure 4 and Figure 5.2). This effect can be considered to be significant and small (0.95% in the region of practical equivalence). However, for the negatively conditioned CSs, the association between RWA and evaluation has a 70.85% probability of being negative, (Median = -0.03, 95% HDI [-0.07, 0.13], see Figure 4 and Figure 5.3), but the effect seems to be practically equivalent to 0 (97.67% in the region of practical equivalence).

4) The effect sizes reported here should be interpreted in the context of future similar studies rather than compared to benchmarks. Comparisons to benchmarks are simply given in order to permit familiarity. So far, it is complicated to determine the smallest practically significant effect in the context of this study. The reader is free to interpret the data. The most important part of the analysis is the ongoing estimation of parameters that can be used for meta-analyses or integrative analyses. 5) See the Supplementary Materials for more details about the computation of effect sizes.

Forest Plot Showing Simple Slope Parameter Estimates for RWA in the Evaluation of Positively Conditioned Greebles

Note. The purple area corresponds to a region of practical equivalence where the values are considered equivalent to 0 [START_REF] Kruschke | Rejecting or accepting parameter values in Bayesian estimation[END_REF]. More information can be found in the Supplementary Materials.

Forest Plot Showing Simple Slope Parameter Estimates for RWA in the Evaluation of Negatively Conditioned Greebles

Note. The purple area corresponds to a region of practical equivalence where the values are considered equivalent to 0 [START_REF] Kruschke | Rejecting or accepting parameter values in Bayesian estimation[END_REF]. More information can be found in the Supplementary Materials.

Discussion of Integrative Data Analysis 2

Even though some uncertainty remains at the statistical level, it is important to note that the results of the second IDA tend to run counter to our initial hypothesis. Indeed, contrary to our hypotheses, higher RWA levels were associated with stronger attitude acquisition. In line with the results of the first IDA, this effect is reliably observed only in the acquisition of positive attitudes, but not in the acquisition of negative attitudes. It is noteworthy that this effect was replicated in a third conditioning experiment that could not be included in integrative data analysis 1 because it used difference scale anchors for the evaluative measure. Hence, the RWA effect in the acquisition of positive attitudes seems to be reliable and robust, at least for the materials and procedures considered here. However, a larger sample size would enhance the precision and sensitivity of the test of the interaction between RWA and valence. These findings suggest that the results obtained in the first IDA are likely to be driven by a positivity effect in attitude acquisition (but not attitude change) in higher-RWA individuals. We elaborate on this point in the general discussion.

General Discussion

The two integrative data analyses reported here permit a first examination of how RWA relates to attitude learning dynamics in a controlled evaluative learning setting. Because RWA is associated with more stable attitudes over time and is negatively associated with cognitive flexibility [START_REF] Sidanius | Cognitive functioning and sociopolitical ideology revisited[END_REF][START_REF] Zmigrod | The role of cognitive rigidity in political ideologies: Theory, evidence, and future directions[END_REF], we hypothesized that individuals with higher levels of RWA would exhibit lower sensitivity to information encountered in the second stage (counter-attitudinal information) than to the information encountered first (attitudinal information, resulting from conditioning).

Together, the data collected here suggest that high RWA scorers (here, RWA is under stood as a general ideology) exhibit a preferential acquisition of positive attitudes. We observe the residuals of this higher sensitivity to initial (positive) conditioning after the exposure to counter-attitudinal information. When we commenced the present research project, we initially predicted that RWA would be associated with more resistance to counter-attitudinal information but not with facilitated acquisition of evaluations. This prediction was not supported by the results. More specifically, and against expectations, we observed initial evidence suggesting a positivity bias in attitude acquisition as a function of RWA.

At first sight, this positivity effect is difficult to reconcile with the literature pointing to a negativity bias in high RWA scorers [START_REF] Castelli | Ideology is related to basic cognitive processes involved in attitude formation[END_REF][START_REF] Shook | Political ideology, exploration of novel stimuli, and attitude formation[END_REF], which is defined as a higher psychological sensitivity to negative information [START_REF] Baumeister | Bad is stronger than good[END_REF]. In addition, RWA has been linked to a greater allocation of resources to negative information [START_REF] Hibbing | Differences in negativity bias underlie variations in political ideology[END_REF]. More surprisingly, when partici pants are allowed to look at images (positive and negative) without any time limitation and without any particular task to perform, there is a positive correlation between the level of conservatism (which is an important dimension of RWA) and fixation time for negative images 6 [START_REF] Dodd | The political left rolls with the good and the political right confronts the bad: Connecting physiology and cognition to preferences[END_REF]. Such an attentional bias should have favored rather than lowered negative conditioning effects in high-RWA individuals.

One first possible explanation for this discrepancy would be that individuals who are more sensitive to negative information are also those who are more likely to try to stop negative experiences from arising when initially given a chance to form positive evaluations [START_REF] Hibbing | Differences in negativity bias underlie variations in political ideology[END_REF]. From a functional perspective, RWA is considered an ideological response designed to reduce a high level of anxiety and threat perception [START_REF] Katz | The functional approach to the study of attitudes[END_REF]. The direct consequence of this threat perception is the implementation of safety strategies to prevent negative events [START_REF] Doty | Threat and authoritarianism in the United States, 1978-1987[END_REF][START_REF] Lavine | Threat, authoritarianism, and selective exposure to information[END_REF]. Based on this literature and our current results, we can hypothesize (for future studies) that an increased sensitivity to positive information could serve this preventive purpose. In addi tion, the greater conditioning for positive stimuli that we observed in the conditioning phase may be consistent with an enhanced Need for Closure in high RWA scorers [START_REF] Napier | Why are conservatives happier than liberals?[END_REF]. Indeed, more conservative participants tend to prefer very simple things that quickly satisfy their needs with a minimum of ambiguity [START_REF] Kruglanski | Groups as epistemic providers: Need for closure and the unfolding of group centrism[END_REF].

A second important point to consider is the social context. Previous studies indicating a negativity bias in high-RWA individuals have focused on effects that occur after an initial learning phase (e.g., during memory consolidation, retrieval, decision making, or behavioral enactment) and this is not optimally taken into account in the deliberately simplified paradigm used here. Intergroup processes, prior knowledge, and social motiva tions can undoubtedly modulate attitudinal effects when dealing with real social groups that involve complex social knowledge and motivations. In the light of this observation, it should be noted that the effect sizes of the statistically significant effects described in our study are very small. This is not surprising given the absence of social context in our protocols, as we discussed above (see also our discussion about the ecological aspects of our study below). This should be considered in combination with the nature of our task, which is not a self-reported measure of cognitive style. Indeed, the meta-analysis carried-out by [START_REF] Van Hiel | The relationship between Right-wing attitudes and cognitive style: A comparison of self-report and behavioural measures of rigidity and intolerance of ambiguity[END_REF] indicates that behavioral measures of cognitive style usually yield smaller effect sizes than self-reported measures when addressing the question of right-wing attitudes (see also [START_REF] Zmigrod | The role of cognitive rigidity in political ideologies: Theory, evidence, and future directions[END_REF], for a more recent overview). Further studies comparing social vs. non-social tasks and laboratory vs. field experiments could inform us about the minimum practically significant effect size in this field of research. Our integrative data analysis shows the number of observations needed to detect effect sizes of d ≈ 0.1 (and null effects corresponding to d < 0.07) in this context [START_REF] Lakens | Equivalence tests: A practical primer for t tests, correlations, and meta-analyses[END_REF][START_REF] Lakens | Sample size justification[END_REF][START_REF] Lakens | Equivalence testing for psychological research: A tutorial[END_REF].

A third (complementary and not exclusive) possible way of accounting for asymmet rical valence effect in both the conditioning and counter-conditioning designs is to consider that some CSs were evaluated more positively even before the beginning of the 6) The same database we used in the experiments presented here.

experiment. Thus, we could have added pre-conditioning ratings in order to address this issue. However, even if we can hypothesize that ratings are more positive with increasing RWA (as suggested by the results presented in the two integrative data analyses), we also hypothesize that all the stimuli (CS-A and CS-B) should be rated the same. In addition, CSs were counterbalanced between participants, that is to say some participants got to see CS-A with positive conditioning while others saw CS-B with positive conditioning. Thus, the valence asymmetry cannot be explained by a more positive evaluation of one specific group of CSs before conditioning.

Even if the evidence clearly suggests that RWA is associated with a certain approach to negative stimuli and is consequently not associated with particularly positive evalua tions [START_REF] Dodd | The political left rolls with the good and the political right confronts the bad: Connecting physiology and cognition to preferences[END_REF], we still do not know with certainty whether RWA is associated with a more positive evaluation of neutral or mildly positive stimuli. It would be useful to evaluate the attitude towards neutral stimuli in the light of RWA scores without any acquisition of attitude phase. This would allow us to identify the net impact of conditioning or counter-conditioning on the CS.

Related to the previous point, the evaluation of the resistance to change in these studies can be criticized. Indeed, there are no before-and-after measurements that permit a real assessment of resistance. Conditioning has certainly taken place (whatever the level of RWA and the associated valence). Indeed, in the second IDA, we observed that all participants (regardless of RWA level) were receptive to the evaluative conditioning (but with different magnitudes associated with RWA). Based on these experiments, we can assume that the evaluation of attitudes (after counter-conditioning) reflects a combination of the conditioning and counter-conditioning effects. There are probably no substantial differences between lower and higher RWA levels in the assimilation of counter-attitudinal information. Instead, RWA is likely to be associated with stronger initial conditioning (at least in the positive valence). The observations when attitudes were evaluated after counter-conditioning therefore probably reflect a residual effect of this stronger conditioning. Seen in this light, we provide (still incomplete) evidence that RWA is not specifically associated with cognitive rigidity but rather with higher sensitivity to initial (positive) information.

We accept that in order to permit a clear-cut test of attitude change in these exper imental designs, it would be useful to conduct new studies which include two new measures of attitudes: one before the first conditioning phase and one between the con ditioning and counter-conditioning phases (while also continuing to measure attitudes after counter-conditioning). This might allow us to observe the detailed evolution of attitudes across the different conditioning phases (and thus clearly measure the potential resistance to change in relation to RWA). However, it is important to be aware of other biases that such a procedure introduces. Measuring attitudes toward the same stimuli three times could lead to 1) a considerable demand bias, 2) an artificial focus on the change of valence between the two conditioning phases, and 3) a certain weariness in performing the experiment.

We want to emphasize that this research examined a restricted kind of stimuli, thus limiting the generalizability to different types of stimuli. Unpublished data from our team 7 show that sensitivity to initial positive information compared to counter-attitudi nal information is also observed with even less social stimuli (other types of Greebles were used in this experiment). Nevertheless, we believe that this effect should be tested with other types of stimuli, for example, in the form of more ecologically valid stimuli, or with additional measures involving, for example, the control or manipulation of the so cial distance between the stimuli and the participants. Indeed, in many studies, RWA has been found to be correlated with social distance (see for example: [START_REF] Koc | Social distance toward Syrian refugees: The role of intergroup anxiety in facilitating positive relations[END_REF][START_REF] Sayılan | Keeping my distance: Prejudice towards transwomen and its socio-political and moral predictors[END_REF]. However, this is a more complex question because its investigation requires us to reduce the level of control of attitude acquisition. These limitations reveal the need for much further research on attitudinal and counter-attitudinal acquisition. Another dimension relating to the ecological nature of the research concerns the context in which the participants respond. Future research could address the issue of attitudinal change while taking participants' political context into account. Indeed, recent studies have shown a link between national identity and RWA [START_REF] Vargas-Salfate | Right-wing authoritarianism and national identification: The role of democratic context[END_REF]. More specifically, RWA positively predicts national identification. It would therefore be very relevant to look at the interaction between RWA and the political context of the home country in the evolution of attitudes. More specifically, it would be very interesting to investigate this effect in authoritarian countries in order to (1) obtain a larger panel of high-RWA participants and (2) investigate the cross-cultural components of this effect.

The ecological problem leads to a critical question: from an attitude-learning perspec tive, why are higher RWA levels related to greater prejudice? One intriguing possibility relates to the positivity bias found here. More specifically, it might be assumed that peo ple's environments are such that individuals are mostly exposed to positive information, on the one hand, and in-group information, on the other [START_REF] Unkelbach | Good things come easy: Subjective exposure frequency and the faster processing of positive information[END_REF]. Hence, high-RWA individuals may form positive in-group attitudes which emerge from the initial information (normally) available in the environment. This, in turn, may contribute to out-group derogation effects and out-group avoidance in these individuals. More generally, more positive in-group views may fuel the resistance of high-RWA individuals to social changes and motivate various confirmatory biases in these individuals. Because the current study revealed residuals of positively acquired attitudes after exposure to counter-attitudinal information, one way to proceed would be to promote less biased exposure to social information when social attitudes form in these individuals. 7) A second condition of Experiment 2. This condition was not included in the integrative data analysis because the stimuli used in this condition were not equivalent to the stimuli used in the other condition and other experiments.

Conclusion

Here, we presented data showing that RWA is linked to a higher sensitivity to initial positive information in a controlled attitude acquisition paradigm. This finding contra dicted our hypothesis about cognitive rigidity in RWA. As mentioned above, from a motivated social cognition perspective, specific information processing in RWA would develop in such a way as to meet the need for closure and the need for structure [START_REF] Jost | Political conservatism as motivated social cognition[END_REF][START_REF] Napier | Why are conservatives happier than liberals?[END_REF] and would be found particularly in right-wing ideologies [START_REF] Jost | Ideological asymmetries and the essence of political psychology[END_REF]. However, other authors defend the idea that these motives are not specific to right-wing ideologies but derive rather from a general ideological rigidity due to in-group favoritism [START_REF] Greenberg | Psychological motives and political orientation -The left, the right, and the rigid: Comment on Jost et al[END_REF][START_REF] Van Prooijen | Psychological features of extreme political ideologies[END_REF]. Seen from this this perspective, the entire continuum of ideologies, from right wing to left wing, should be affected by the impact of ideology on information processing, at least when group identities are engaged. Further research should examine the association between ideology and cognitive style within such a paradigm in order to understand their underlying mechanisms and their causal relationship.

Figure 1

 1 Figure 1Description of the Experimental Procedure: From the First Phase (Left) "Categorization Stage" to the Last One "RWA Scale Completion"(Right)

Figure 2

 2 Figure 2Evaluation of Stimuli After Counter-Conditioning as a Function of RWA and Conditioning Valence

Figure 3. 1 Forest

 1 Figure 3.1 Forest Plot Showing Interaction Parameter Estimates for the Interaction Effect Between the Initial Valence and RWA in the Evaluation of the Greebles

Figure 3. 2 Forest

 2 Figure 3.2 Forest Plot Showing Simple Slope Parameter Estimates for RWA in the Evaluation of Positively Conditioned Greebles (Following Negative Counter-Conditioning)

Figure 3. 3 Forest

 3 Figure 3.3 Forest Plot Showing Simple Slope Parameter Estimates for RWA in the Evaluation of Negatively Conditioned Greebles (Following Positive Counter-Conditioning)

Figure 4

 4 Figure 4Evaluation of Stimuli After Conditioning as a Function of RWA and Conditioning Valence

Figure 5. 1 Forest

 1 Figure 5.1 Forest Plot Showing Parameter Estimates for the Interaction Effect Between RWA and the Initial Valence in the Evaluation of Conditioned Greebles

Figure

  Figure 5.2

Figure

  Figure 5.3

Table 1

 1 Overview of the Eleven Experiments Included in the Two Integrative Data Analyses (IDAs)

	Experiment	IDA	N	Data collection	Outliers
	1	IDA 1	31	Laboratory	N = 0
	2	IDA 1	57	Laboratory	N = 0
	3	IDA 1 and 2	202 and 197	Online	N = 2
	4	IDA 1	37	Laboratory	N = 0
	5	IDA 1	54	Online	N = 0
	6	IDA 1	58	Online	N = 0
	7	IDA 1	75	Online	N = 0
	8	IDA 1	61	Online	N = 0
	9	IDA 1	59	Laboratory	N = 0
	10	IDA 1	91	Online	N = 0
	11	IDA 2	115	Online	N = 0

Social Psychological Bulletin | 2569-653Xhttps://doi.org/10.32872/spb.6593

1) Across all the experiments, accuracy was high (above 98%), thus indicating participants' high capacity to distin guish between the two groups.RWA and Evaluative Counter-Conditioning

Bret, Beffara, Mierop, & Mermillod

Acknowledgments:

We thank the reviewers for insightful comments and suggestions during the peer-review process.

Funding: This work benefited from the Alpes Grenoble Innovation Recherche Grant from the University Grenoble Alpes and a "CNRS-Attentats" grant provided by the CNRS. We thank the Université de Nantes, the Université Grenoble Alpes, and the Université Catholique de Louvain for supporting our research. The authors received no specific financial support for the research, authorship, and/or publication of this article.

Competing Interests:

The authors declare no potential conflicts of interest with respect to research, authorship, and/or publication of this article.

Ethics Statement: This research project was approved by the IPSY ethics committee in Louvain la Neuve (Belgium).

Data Availability: For this article, a dataset is freely available (Bret, Beffara, Mierop, & Mermillod, 2021).

Supplementary Materials

Materials (basic scripts and information about/links to the stimuli), data, and analysis scripts are available on OSF (for access see Index of Supplementary Materials below).

Index of Supplementary Materials


Saonli Roy

Loganathan Rangasamy

Assia Nouar

Christiane Koenig

Vanessa Pierroz

Simon Kaeppeli

Stefano Ferrari
email: sferrari@imcr.uzh.ch

Malay Patra
email: malay.patra@tifr.res.in

Gilles Gasser
email: gilles.gasser@chimeparistech.psl.eu

Synthesis and Biological Evaluation of Metallocene-Tethered Peptidyl Inhibitors of CDC25

Keywords: Bioorganometallic Chemistry, CDC25, Enzyme Inhibition, Metallocene, Peptide Conjugates

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The Cell Division Cycle 25 (CDC25) family of enzymes encompasses three highly conserved members of dual specificity phosphatases that selectively target Cyclin-Dependent Kinases (CDKs). The latter are key controllers of cell cycle transitions. By removing inhibitory phosphorylation from residues located in the catalytic domain of CDKs, CDC25 cause CDK activation and, in turn, promote cell cycle progression. [START_REF] Russell | cdc25+ functions as an inducer in the mitotic control of fission yeast[END_REF][START_REF] Dunphy | The cdc25 protein contains an intrinsic phosphatase activity[END_REF] Specifically, CDC25A acts at the G1/S transition and in S-phase, controlling Cyclin E-and Cyclin A-dependent kinases, though it also cooperates with CDC25B at the onset of mitosis. [START_REF] Hoffmann | Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition[END_REF][START_REF] Lindqvist | Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome[END_REF] CDC25B initiates CDK1 activation at centrosomes during the G2/M transition, whereas CDC25C causes full activation of nuclear CDK1 at mitotic entry. [START_REF] Gabrielli | Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells[END_REF][START_REF] Gabrielli | Hyperphosphorylation of the N-terminal Domain of Cdc25 Regulates Activity toward Cyclin B1/Cdc2 But Not Cyclin A/Cdk2*[END_REF] Experimental evidence suggested roles for CDC25s that are additional to the control of the cell cycle. Explicitly, the observation that CDC25B loss-offunction led to reduced expression of neuronal differentiation markers in Xenopus laevis and the evidence that CDC25B controls neuronal production during embryogenesis, in a manner that is independent of interaction with CDKs, points to a role for CDC25B in neurogenesis. [START_REF] Ueno | FoxM1-driven cell division is required for neuronal differentiation in early Xenopus embryos[END_REF][START_REF] Bonnet | Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase[END_REF] Regarding pathology, overexpression of CDC25A and CDC25B has been reported in a variety of human cancers and was found to correlate with poor clinical prognosis. [START_REF] Boutros | CDC25 phosphatases in cancer cells: key players? Good targets?[END_REF] Of note, CDC25 was reported to be the therapeutic target of choice in triple-negative breast cancers that are nonresponsive to standard therapy. [START_REF] Liu | Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer[END_REF] Furthermore, the established role of CDC25A in the cell death that is characteristic of Alzheimer disease, led to the suggestion that inhibition of CDC25A could allow blocking pathological neuron degeneration and death. [START_REF] Chatterjee | Role and regulation of Cdc25A phosphatase in neuron death induced by NGF deprivation or β-amyloid[END_REF] Given the physiological role played by CDC25 phosphatases in the cell cycle and their involvement in human diseases, a number of laboratories have engaged in drug discovery programs aimed at identifying specific CDC25 inhibitors. [START_REF] Pestell | Small molecule inhibitors of dual specificity protein phosphatases[END_REF][START_REF] Kar | Binding and Inhibition of Cdc25 Phosphatases by Vitamin K Analogues[END_REF][START_REF] Lavergne | Synthesis and biological evaluation of novel heterocyclic quinones as inhibitors of the dual specificity protein phosphatase CDC25C[END_REF][START_REF] Brezak | A Novel Synthetic Inhibitor of CDC25 Phosphatases[END_REF][START_REF] Brun | synthesis, and biological evaluation of novel naphthoquinone derivatives with CDC25 phosphatase inhibitory activity[END_REF][START_REF] Chen | Dual-Specific Cdc25B Phosphatase: In Search of the Catalytic Acid[END_REF][START_REF] Lavecchia | Discovery of New Inhibitors of Cdc25B Dual Specificity Phosphatases by Structure-Based Virtual Screening[END_REF][START_REF] Kabakci | Pharmacophoreguided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression[END_REF] Tao et al. summarized the approaches so far adopted to discover CDC25 inhibitors in a recent review. [START_REF] Tao | Medicinal chemistry insights into novel CDC25 inhibitors[END_REF] Most compounds so far identified failed to keep up with the expectations, either due to rapid metabolism in tumor-bearing SCID mice, [START_REF] Guo | Pharmacology and Antitumor Activity of a Quinolinedione Cdc25 Phosphatase Inhibitor DA3003-1 (NSC 663284)[END_REF] or for not completing clinical trials. [START_REF] Lavecchia | Inhibitors of Cdc25 phosphatases as anticancer agents: a patent review[END_REF] Of note, a recent pharmacophore-guided drug discovery program identified naphthoquinone compounds displaying excellent inhibition of CDC25 in enzymatic and cellular assays. These compounds not only induced growth arrest and differentiation of mouse Apc/K-Ras mutant duodenal organoids, but they also displayed efficacy in tumor regression and inhibition of metastasis formation in an in vivo xenograft model, [START_REF] Kabakci | Pharmacophoreguided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression[END_REF] making them promising candidates for the development of clinically effective drugs. The Rudolph research group in 2000s patented several short peptides which could potentially inhibit the function of CDC25s. [START_REF] Lavecchia | Inhibitors of Cdc25 phosphatases as anticancer agents: a patent review[END_REF][START_REF] Taylor | Method of identifying inhibitors of[END_REF] One of their candidates, the pentapeptide 1 (Scheme 1) could be crystallized with CDC25B and it was shown that the sulfonic group of 1 mimics the phosphate and forms hydrogen bonding interaction with amino acid residues at the catalytic site. [START_REF] Taylor | Method of identifying inhibitors of[END_REF][START_REF] Bäurle | Design and synthesis of macrocyclic inhibitors of phosphatase Cdc25B[END_REF] Organometallic derivatization or mimic of clinically used organic drugs was demonstrated to improve therapeutic properties of parent drugs. [START_REF] Patra | The medicinal chemistry of ferrocene and its derivatives[END_REF][START_REF] Jaouen | Ferrocifen type anti cancer drugs[END_REF][START_REF] Dive | Ferrocene Conjugates of Chloroquine and other Antimalarials: the Development of Ferroquine, a New Antimalarial[END_REF][START_REF] Dubar | The Antimalarial Ferroquine: Role of the Metal and Intramolecular Hydrogen Bond in Activity and Resistance[END_REF][START_REF] Patra | Small Organometallic Compounds as Antibacterial Agents[END_REF][START_REF] Dubar | The ferroquine antimalarial conundrum : redox activation and reinvasion inhibition[END_REF][START_REF] Keiser | In vitro and in vivo Antischistosomal Activity of Ferroquine Derivatives[END_REF] One of the leading examples is ferroquine, a ferrocene derivative of antimalarial chloroquine (Scheme 1a). [START_REF] Dive | Ferrocene Conjugates of Chloroquine and other Antimalarials: the Development of Ferroquine, a New Antimalarial[END_REF][START_REF] Dubar | The Antimalarial Ferroquine: Role of the Metal and Intramolecular Hydrogen Bond in Activity and Resistance[END_REF][START_REF] Biot | Investigation of the redox behavior of ferroquine, a new antimalarial[END_REF] While chloroquine and ferroquine exert comparable activity against chloroquine-sensitive Plasmodium falciparum, comparison of activity against chloroquine resistant P. falciparum strain revealed that ferroquine is ten times more active compared to the parent organic drug. [START_REF] Patra | The medicinal chemistry of ferrocene and its derivatives[END_REF][START_REF] Dive | Ferrocene Conjugates of Chloroquine and other Antimalarials: the Development of Ferroquine, a New Antimalarial[END_REF][START_REF] Biot | Investigation of the redox behavior of ferroquine, a new antimalarial[END_REF][START_REF] Biot | Insights into the Mechanism of Action of Ferroquine. Relationship between Physicochemical Properties and Antiplasmodial Activity[END_REF] Jaouen et al. demonstrated a very successful bio-isosteric replacement of aromatic phenyl ring in tamoxifens and hydroxytamoxifens with ferrocene (Scheme 1b). [START_REF] Jaouen | Ferrocifen type anti cancer drugs[END_REF] The resulting bioorganometallic compounds, termed ferrocifens, were shown to be active against hormone independent breast cancer, where the parent organic drugs are inactive. [START_REF] Patra | The medicinal chemistry of ferrocene and its derivatives[END_REF][START_REF] Jaouen | Ferrocifen type anti cancer drugs[END_REF] Meggers and co-workers introduced the fascinating concept of mimicking structurally complicated organic scaffolds with relatively simple organometallic moieties to obtain potent and selective kinase inhibitors (Scheme 1c). [START_REF] Dörr | Metal complexes as structural templates for targeting proteins[END_REF][START_REF] Feng | Structurally Sophisticated Octahedral Metal Complexes as Highly Selective Protein Kinase Inhibitors[END_REF] Encouraged by these results, we embarked on an effort to design organometallic peptide inhibitor of CDC25 enzymes inspired by the structure of organic inhibitor 1 (Scheme 1d).

Analysis of crystal structure of 1 bound to CDC25B revealed that the naphthyl group (color coded red) is not involved in any hydrogen bonding interaction with active side residues. In addition, based on results of a previous report showing that negatively-charged COOH groups of small molecule CDC25 inhibitors interact with Arginine residues of the enzyme pocket, we inferred that interaction of the COOH groups of 1, 3 and 5 with Arginine residues that line up the catalytic site of CDC25s may stabilize the peptide-enzyme interaction. [START_REF] Kabakci | Pharmacophoreguided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression[END_REF] Therefore, as shown in Scheme 1d, we hypothesized that the naphthyl group could be substituted using triazole-bearing metallocene moieties such as ferrocene, ruthenocene, CpMn(CO)3 and CpRe(CO)3 moieties. Herein, we report synthesis, aqueous stability and biological evaluation of peptides 1-5.

RESULTS AND DISCUSSION

Design and synthesis of peptide inhibitors

The design of metallocene containing peptides 2-5 was based on co-crystallization data obtained from peptide 1 bound to the active site of CDC25B. [START_REF] Taylor | Method of identifying inhibitors of[END_REF][START_REF] Bäurle | Design and synthesis of macrocyclic inhibitors of phosphatase Cdc25B[END_REF] Peptides 1-5 were synthesized employing solid phase Fmoc strategy following one step solution phase deprotection of the side chain. For peptide 1, Fmoc protected amino acids were sequentially coupled using HBTU as coupling agent (Scheme S2). The peptide was then cleaved from the Tentagel resin using a solution consisting of TFA:H2O:TIPS 95:2.5:2.5. Next, the crude peptide was subjected to Pd/C mediated hydrogenolysis in solution to remove trichloro-ethyl protecting group on the sulphonic acid residue. Finally, pure peptide was isolated from HPLC purification and characterized using ESI mass spectroscopy, which showed a peak at m/z = 1025.3 that can be assigned as the [M-H] -adduct of 1. Similarly, as shown in Scheme 2, the metallocene containing peptides 2-5 was synthesized using solid phase followed by a solution phase synthesis. First, metallocenyl-azides of ferrocene (Fc-N3), [START_REF] Gasser | Synthesis of organometallic PNA oligomers by click chemistry[END_REF] cymantrene (cym-N3) [START_REF] Day | Cymantrene-Triazole "Click" Products: Structural Characterization and Electrochemical Properties[END_REF] and cyrhetrene (cyr-N3) [START_REF] Day | Synthesis and characterization of redox active cyrhetrene-triazole click products[END_REF] were synthesized following previously reported procedures. Ruthenocenyl azide (Rc-N3) was synthesized from ruthenocene following an analogous route as used for ferrocene (Scheme S1). Then, solid phase peptide synthesis was used to generate the peptide precursor with an alkyne side chain (Scheme 2). Cu-catalyzed click reaction was performed between the intermediate peptide and azide containing metallocenes as previously performed by Metzler-Nolte et al. [START_REF] Gasser | Synthesis of organometallic PNA oligomers by click chemistry[END_REF][START_REF] Köster | Spectroscopic and Electrochemical Studies of Ferrocenyl Triazole Amino Acid and Peptide Bioconjugates synthesized by Click Chemistry[END_REF] Next, a similar reaction sequence, as described for 1, was used to cleave the peptide as well as remove all protecting groups from the side chains. As presented in Table 1, the observed m/z peak corresponding to [M-H] -adducts unambiguously confirmed the formation of the targeted peptides 2-5. The purities were confirmed to be 92-98% using analytical UHPLC (See ESI for HPLC chromatograms and ESI-MS spectra).

Fe = 2 Ru 3 Mn OC CO CO Re OC CO CO 4 5 Organometallic CDC25 inhibitors ( 2 -5 ) NHFmoc N H H N N H H N N H H N O O O O O O S O O O O H 3 C TentaGel S RAM Resin O O(tBu) O(tBu) O O(tBu) O Cl Cl Cl M N 3 CuI, Sodium ascorbate, DIPEA, 2,6-Leutidine N H H N N H H N N H H N O O O O O O S O O O O H 3 C O O(tBu) O(tBu) O O(tBu) O Cl Cl Cl N N N M i) TFA/Phenol/TIPS (85/10/5) ii) NH 4 HCO 3 , Pd/C, H 2 N H H N N H H N N H NH 2 O O O O O O S O O OH O H 3 C O OH OH O OH O N N N M S1 S2 S3 S4 S5

Stability of 1-5 in aqueous media

Prior to assessment of biological activity, we examined the stability of the newly synthesized (organometallic-containing) peptides in aqueous media. To this end, peptides were incubated in water at room temperature and UHPLC was recorded up to 7 days. As shown in Figure S6, no significant changes were observed in the chromatograms of peptide 1, 3 and 5, confirming their stability in aqueous media. By contrast, 2 and 4 were found to be unstable under the same experimental conditions. A 26% and 57 % of 2 and 4, respectively, remained intact after 7 days (Figure S6). In order to obtain detail insight to the decomposition mechanism of 2 and 4, the mixtures were analyzed using UHPLC-MS. As shown in Figures S7 andS8, the major decomposition product of 2 as well as 4 showed a similar mass spectrum patterns with a major peak at m/z = 1030.3 (ESI negative mode), which can be assigned to the peptide 6 (calculated m/z for [6-H] -= 1030.29, structure of 6 is shown in Scheme S3 in the ESI). Based on the mass spectrometry data, it is reasonable to hypothesize that molecular oxygen oxidized the ferrocene moiety of 2, which then slowly decomposed, leading to formation of 6 as the end product (Scheme S3, ESI). Similarly, we observed that 4 released the Mn(CO)3 moiety in water to form 6 (Scheme S3, ESI). Based on the stability results, we chose 1, 3 and 5 for biological evaluation.

Enzymatic characterization of CDC25 inhibitors

To conduct an initial assessment of 1, 3 and 5, the compounds were tested at a fixed concentration (1 mM) on CDC25B, using the established naphthoquinone inhibitor NSC-663284 as positive control. The data showed that all peptides effectively inhibited CDC25B phosphatase activity, with the order of potency being 3>5>1 (91%>85>79% inhibition relative to untreated CDC25B) (Figure 1).

As next step, we performed a dose-response experiment with the three peptides. The data confirmed the potency of inhibition observed in Figure 1 and indicated that 0.25 mM organic peptide 1 was sufficient to yield ~50% inhibition of CDC25B phosphatase activity, with the metallocene containing peptides 3 and 5 reaching ~60% inhibition of the phosphatase at this concentration (Figure 1). Next, the most potent of the three peptides, namely the ruthenocene containing peptide 3, was tested in a dose-response assay on the three CDC25 phosphatases. The data showed that 3 was most effective on CDC25A and CDC25B, with a less potent inhibition of CDC25C (Figure 2). Finally, we examined the ability of 3 to inhibit CDC25 in an ex vivo assay. To this end, we monitored CDC25-mediated CDK1 dephosphorylation in cellular extracts by means of an antibody detecting phosphorylated Tyrosine 15 in CDK1, an inhibitory site of phosphorylation that is used as read-out for CDK1 activity. Upon preparing cellular extracts of human embryo kidney cells (HEK-293), we supplemented them with purified CDC25B and low doses of 3, in order to prevent peptide precipitation in the assay. We expected that the presence of low amounts of 3 would at least partially rescue dephosphorylation of endogenous CDK1 by the CDC25B that was added to the reaction. Densitometric analysis of the protein band corresponding to CDK1 phosphorylated at Tyrosine 15 revealed that this was indeed the case, as shown in Figure 3 (bottom panel, lanes 4 and 5 vs. 2).

To support these findings, we examined the effect of 3 on extracts obtained from HeLa cells that were synchronized in the cell cycle. As expected, we observed robust phosphorylation of Tyrosine 15 in CDK1 till the end of the G2-phase (Fig. S9, lane 1), a time when the action of CDK1 is not required and the kinase is kept inactive, followed by maximum activation of CDK1 in mitosis through dephosphorylation of Tyrosine 15 (Fig. S9, lane 2). Addition of exogenous CDC25 did not substantially alter the pattern of CDK1 dephosphorylation caused by endogenous CDC25 (Suppl. The promising enzyme inhibition results shown above prompted us to study the effects of these peptidyl inhibitors on cell cycle progression. Namely, we decided to monitor the two cell cycle transitions that depend on CDC25 activity, G1/S or G2/M. To this end, HeLa cells were treated with 3 (0.5 mM) for 17 h, stained with DAPI and DNA content was analyzed using flow cytometry. To facilitate shuttling of the peptidyl inhibitor 3 through the plasma membrane, we used the cell penetrating peptide Pep-1 at different molar ratios to 3. Pep-1 was demonstrated to shuttle small peptides or large protein cargos into cells in a matter of minutes. [START_REF] Morris | A peptide carrier for the delivery of biologically active proteins into mammalian cells[END_REF] Results presented in Figure 4 show that cells did not accumulate at cell cycle transitions controlled by CDC25, indicating that 3 failed to inhibit cellular CDC25 activity. At this point, we decided to address the issue of cellular uptake to verify whether the observed inactivity of compounds in cellular assays was related to poor compound penetration in the cells. To this end, we studied 3 as representative of this class of inhibitors and we used U2OS cells that display a wider surface compared to HeLa. U2OS were incubated with 100 µM 3 and Pep-1 at different molar ratios (from 5:1 to 40:1) for 48 h and Ru content in the cell was measured using ICP-MS. The data showed that the amount of ruthenium present in the cells was below the quantification limit of ICP-MS, evidencing extremely poor cellular uptake of 3 (data not shown). The inefficient cellular uptake of the peptidyl inhibitor is most likely due to the presence of several carboxylic and sulphonic acid side chains that are negatively charged at physiological pH.

CONCLUSION

Today's trend in the therapy of disease is toward tailored treatment of patients, a strategy that is better known as personalized medicine. The strength of this approach relies on the ability to predict drug responses and overall treatment outcome based on the genetic and biochemical profiling of individual patients. [START_REF] Chan | Personalized Medicine: Progress and Promise[END_REF] Despite the great progress made in this direction, the onset of resistance poses a constant threat also in precision medicine. [START_REF] Vasan | A view on drug resistance in cancer[END_REF] In order to satisfy the continuous demand for therapeutic anti-cancer agents, we set out to develop novel CDC25 inhibitors. In this study, we designed and successfully synthesized the first organometallic-containing CDC25 inhibitors based on lead structure of an organic pentapeptide inhibitor. [START_REF] Taylor | Method of identifying inhibitors of[END_REF][START_REF] Bäurle | Design and synthesis of macrocyclic inhibitors of phosphatase Cdc25B[END_REF] While the ferrocene and CpMn(CO)3 containing peptides were unstable, the ruthenocene and CpRe(CO)3 containing peptides displayed robust stability in aqueous media. Biochemical studies conducted on purified CDC25 phosphatases evidenced that all peptidyl compounds were able to effectively inhibit enzymatic activity in a dosedependent manner, with the ruthenocene derivative being the most potent of the series. Ex vivo studies conducted on endogenous CDC25 present in cellular extracts as well as exogenously supplemented CDC25, demonstrated that low doses of 3 could partially rescue CDC25mediated dephosphorylation of its physiological target CDK1. On the other hand, cellular studies aimed at assessing overall cytotoxicity of the peptidyl inhibitors as well as specific effects on cell cycle perturbance, such as arrest at checkpoints that depend on CDC25 activity, were unsuccessful, even when compound shuttling to the cell interior was facilitated by a cellpenetrating carrier peptide. Dedicated mass spectrometry studies revealed that, under all conditions tested, peptidyl compounds could not reach the cell interior, a fact that likely depends on the high extent of negative charges present on the compounds at physiological pH.

In conclusion, the compounds described here represent an innovative concept in drug design to target key components of oncogenic pathways. Biochemical data provided here confirm the validity of this approach, but further chemical modification or caging of the compounds to generate pro-drugs will be necessary to improve activity as well as cell membrane permeability.

EXPERMENTAL SECTION

Materials and methods

Chemicals and solvents were of reagent grade or better and were purchased from commercial suppliers. They were used without further purification unless otherwise specified. Ferroceneazode (Fc-N3) [START_REF] Gasser | Synthesis of organometallic PNA oligomers by click chemistry[END_REF] , cymantrene-azide (cym-N3) [START_REF] Day | Cymantrene-Triazole "Click" Products: Structural Characterization and Electrochemical Properties[END_REF] and cyrhetrene-azide (cyr-N3) [START_REF] Day | Synthesis and characterization of redox active cyrhetrene-triazole click products[END_REF] were synthesized by following previously published procedures. The analytical data matched with those previously reported. [START_REF] Gasser | Synthesis of organometallic PNA oligomers by click chemistry[END_REF][START_REF] Day | Cymantrene-Triazole "Click" Products: Structural Characterization and Electrochemical Properties[END_REF][START_REF] Day | Synthesis and characterization of redox active cyrhetrene-triazole click products[END_REF] Caution! Care should be taken during the preparation of such potentially explosive organometallic azides, especially on scaled-up reactions. Synthesis of ruthenocene-azide (Rc-N3) is presented in the SI.

NSC-663284 (Sigma-Aldrich, Buchs, Switzerland) was dissolved in DMSO at 10 mM stock concentration and stored in aliquots at -20 °C. The dye 4',6-Diamidino-2-Phenylindole (DAPI) was from Sigma-Aldrich. The rabbit monoclonal antibody to CDK1-pTyr15 (#9111) was purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-conjugated anti-rabbit secondary antibody was obtained from GE-Healthcare (Otelfingen, Switzerland). [START_REF] Russell | cdc25+ functions as an inducer in the mitotic control of fission yeast[END_REF] H and [START_REF] Kar | Binding and Inhibition of Cdc25 Phosphatases by Vitamin K Analogues[END_REF] C NMR spectra were recorded using a Bruker 400 spectrometer. The chemical shifts, δ, are reported in ppm (parts per million). The residual solvent peaks have been used as an internal reference. The abbreviations for the peak multiplicities are as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet) and br (broad). Infrared spectra were recorded on an ATR unit using a Perkin Elmer FTIR spectrophotometer at 4 cm - 1 resolution. Signal intensity is abbreviated br (broad). Elemental microanalyses were obtained on a LecoCHNS-932 elemental analyser. LC-MS spectra were obtained using a Bruker Daltonics HCT 6000 mass spectrometers. LC-MS spectra were measured using an Acquity™ from Water's system equipped with a PDA detector and an auto sampler using an Agilent Zorbax 300SB-C18 analytical column (3.5 μm particle size, 300 Å pore size, 150 × 4.6 mm).

The LC run (flow rate: 0.5 mL min -1 ) was performed with a linear gradient of A (double distilled water containing 0.1% v/v TFA) and B (acetonitrile containing 0.1% v/v TFA); t = 0 min, 5% B; t = 3 min, 5% B; t = 17 min, 100% B; t = 20 min, 100% B; t = 25 min, 5% B. HPLC purification was performed using a Varian ProStar system and an Agilent Zorbax 300 SB-C18 prep column (5 μm particle size, 300 Å pore size, 150 × 21.1 mm; flow rate: 20 mL min -1 ). The runs were carried out with a linear gradient of A (double distilled water containing 0.1% v/v TFA) and B (acetonitrile containing 0.1% v/v TFA, Sigma-Aldrich HPLC-grade).

Preparative runs: t = 0 min, 5% B; t = 25 min, 100% B; t = 30 min, 100% B; t = 32 min, 5% B.

General Procedure for synthesis of peptide and metallocene-peptide conjugates

The peptide synthesis was performed similarly to a procedure reported by Metzler-Nolte et al. [START_REF] Gasser | Synthesis of organometallic PNA oligomers by click chemistry[END_REF][START_REF] Raszeja | A Novel Organometallic ReI Complex with Favourable Properties for Bioimaging and Applicability in Solid-Phase Peptide Synthesis[END_REF] More specifically, the peptides with C-terminal amide were prepared manually in oneway polypropylene syringes (10 mL) equipped with a frit using a standard solid-phase peptide synthesis procedure. All reactions were carried out using a mechanical shaker. Polystyrene resin beads of TentaGel S RAM resin (capacity 0.23 mmol g -1 or 0.26 mmol g -1 ) were swollen in DMF for 2 h before use. The resin was deprotected with 2-methylpiperidine (20% v/v in DMF) for 2 × 10 min. The resin was then washed with DMF (5x), DCM (5x), and DMF (5x).

A Fmoc-protected amino acid was first pre-activated (4.5 equiv. amino acid, 4 equiv. TBTU) in 1 mL of base solution (11.63 mL of DMF, 0.437 mL of DIPEA and 0.437 mL of 2-lutidine) for 2 min and then reacted with the resin for 1.5 h for natural amino acid and 3 h for unnatural amino acids. Afterwards, the resin beads were washed with DMF (5x) and DCM (5x), and a Kaiser test was performed to check if the coupling was complete. The fritted syringe containing the peptide was swollen during 2 h in DMF. Fe-N3, Rc-N3, cym-N3, cyr-N3 (4.65 equivalents) and CuI (3 equivalents) were added into the syringe from the top. A mixture of DIPEA (150 equivalents), DMF (100 µL per µm of resin) were sucked up into the syringe, which was placed into a Shlenk tube and shaken for 48 h at room temperature under N2 atmosphere and in the absence of light. The resin was washed with DMF (5x), DCM (5x), and DMF (5x) successively and dried under vacuo for 2 h. Prior to cleavage, the resin was shrunk in methanol and dried.

Peptides were cleaved using a mixture of trifluoroacetic acid-water-triisopropylsilane, 95:2.5:2.5 v/v/v (3 × 1 mL, for 2.5 h each). Metal containing peptides were cleaved using a mixture of TFA-phenol-triisopropylsilane, 85:10:5 v/v/v (3 × 1 mL, for 2.5 h each). The collected solutions were combined and dried and the crude peptides were precipitated with cold ether. The t-butyl ester is a standard acid-labile carboxy protecting group which is removed during the cleavage step. The Tce group is cleaved by hydrogenolysis (H2/Pd in the presence of ammonium formate). Finally, they were purified by preparative HPLC.

  Scheme 1. a-c) Previously reported organometallic derivatization or mimic of organic drugs and kinase inhibitors provided bioorganometallic with improved properties. d) Structures of organic CDC25 inhibitor 1 and its organometallic derivatives 2-5 designed through organometallic mimic of the naphthyl group (coded in red color) with metallocenes.

S6 MScheme 2 . 5 .

 S625 Scheme 2. Synthesis of peptides 2-5. Various protected amino acids used in the coupling steps S1-S6 on solid phase are as follows: S1: Fmoc-Glu(tBu)-OH, S2: Fmoc-Pra-OH, S3: Fmoc-Glu(tBu)-OH, S4: Fmoc-Glu(tBu)-OH, S5: Fmoc-4-sulfomethyl-Phe(TCE)-OH, S6: 2methoxy-napthoic acid.

Figure 1 .

 1 Figure 1. CDC25B phosphatase activity was assayed in duplicate on 0.1 mM OMFP for 60 min in the presence or the absence of different concentrations of the peptides 1, 3 and 5. The activity of CDC25B in presence of positive control NSC-6632845 (5 µM) was -2.49 ±0.4 %. Significant differences: *p < 0.05 (n = 2). n.s. = no statistical significance, p > 0.05 (n = 2).

Figure 2 .

 2 Figure 2. CDC25A/B/C phosphatase activity was assayed in duplicate on 0.1 mM OMFP for 60 min in the absence or the presence of different concentrations of peptide 3. Significant differences: *p < 0.05 (n = 2). n.s. = no statistical significance, p > 0.05 (n = 2).

  Fig. S9, lane 3). Under these conditions, supplementation of the reaction with 3 resulted in a partial rescue of CDK1-Tyrosine 15 dephosphorylation, indicative of inhibition of endogenous and exogenous CDC25 by 3 (Fig. S9, lanes 4 and 5).

Figure 3 .

 3 Figure 3. Western blot analysis of CDK1 phosphorylation at Tyrosine 15 (pY15) in HEK293 cell extracts treated as indicated. The asterisk indicates an unspecific band that was used as loading control for the normalization of Western blot data. Quantifications obtained by densitometric analysis of protein bands are shown at the bottom of the panel.

Figure 4 .

 4 Figure 4. Flow cytometric analysis of DNA content (DAPI staining) for HeLa cells incubated with 0.5 mM 3 at three different molar ratios of Pep-1. The percentage of cells in S-and G2/M phase are boxed.

  element standard and verified by two controls (Agilent5188-6524 PA Tuning 2 and a freshly prepared mix of Ru, Ni and Nb 1 ppb standards). Ruthenium content of the samples was determined by means of a 7-step serial dilution in the range between 0 and 300 ppb of Ru (R>0.99) with a background equivalent concentration of BEC: 8.5 ppt and a detection limit of DL: 28.4 ppt. The isotope 99 Ru (12.76% abundance) 101 Ru (17.06% abundance) was evaluated in "no-gas" mode and He-gas mode. Spiking the samples with untreated negative controls (to account for eventual carbon content from the biological samples) resulted in equivalent values within error ranges. A solution of Indium (100 ppb) and Tungsten (100 ppb) was used as internal standard. The Ru content in the samples were below the quantification limit of the ICP-MS.Expression and purification of CDC25 phosphatasesCDC25A (kind gift of I. Hoffmann, Heidelberg, Germany), CDC25B and CDC25C (kind gift of B. Gabrielli, Queensland, Australia) in pGEX were expressed in E. coli BL21 cells and purified on 1 ml Glutathione Sepharose 4 Fast Flow columns as described.[START_REF] Kabakci | Pharmacophoreguided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression[END_REF] Enzymatic assays CDC25A, CDC25B and CDC25C were appropriately diluted in 10 mM Tris-HCl pH 8.0, 50 mM NaCl and assayed in 20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EGTA, 1 mM DTT, 0.1 mg/ml BSA and defined concentrations of peptides. Reactions were started by addition of 0.1 mM substrate 3-O-methyl fluorescein phosphate (OMFP). Assays were carried out in duplicate at room temperature using transparent-bottom, V-shaped, 96 well plates and read at defined time intervals (15-30-45-60 min) on a Molecular Devices SpectraMax microplate reader (ex: 490 nm / em: 525 nm). Data were plotted using Origin software.

Table 1 .

 1 Characterization data of newly synthesized peptides 1-5.ICP-MS measurements were performed on an Agilent QQQ 8800 Triple quad ICP-MS spectrometer (Agilent Technologies) with a ASX200 autosampler (Agilent Technologies), equipped with standard nickel cones and a "micro-mist" quartz nebulizer fed with 0.3 ml/min analytic flow (as a 2% HNO3 aqueous solution). Ruthenium was measured against a Ru single

	Peptide	HPLC retention time (tR,	ESI-MS, m/z found	Calculated m/z for [M-H] -
		Min)
	1	1.440	1025.3	1025.32
	2	1.445	1150.2	1150.29
	3	1.452	1196.2	1196.27

RNAse and DNA was stained with 1 µg/ml DAPI. Samples were measured on an Attune™ NxT Cytometer (ThermoFisher Scientific, Carlsbad, CA, USA) and data were analyzed with FlowJo® software v10 (FlowJo, LLC, Ashland, OR, USA).

Acknowledgements

This work was financially supported by an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679), the Swiss National Science Foundation (Professorships N° PP00P2_133568 and PP00P2_157545 to G.G.), the University of Zurich (G.G), and has received support under the program Investissements d'Avenir launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). M.P. gratefully acknowledges the financial supports under project no. RTI4003 from Department of Atomic Energy (DAE) and Tata Institute of Fundamental Research (TIFR), India. S.F. received support from the Stiftung für wissenschaftliche Forschung of the University of Zurich. R.L received support from the Swiss Government Excellence Scholarship for Postdoctoral Researcher 2014.0942/India/OP.

Cell culture and preparation of cell extracts

HeLa (human cervical cancer) and HEK-293 (human embryo kidney) cells were grown using DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured in an incubator using 5% CO2 with humidified atmosphere at 37 °C.

Cell synchronization was obtained through the double-thymidine procedure. [START_REF] Gentili | Chromosome Missegregation Associated with RUVBL1 Deficiency[END_REF] Upon release from the second thymidine treatment, cells were grown in complete medium and harvested at 8h (G2-phase) or treated with 200 ng/ml nocodazole at 8h from the release and then harvested at 10h (M-phase).

Ex-vivo enzymatic assay and Western blot analysis

Purified CDC25B was incubated at room temperature for 5 min in the presence or the absence of 3 (50-100 µM) and then added to HEK-293 or HeLa cell extracts (50 µg). Incubation was protracted for 30 min at room temperature and the reactions were terminated by addition of Laemmli sample buffer (60 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue). Proteins were resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and immunoblot analysis was performed as previously described. [START_REF] El-Shemerly | ATR-dependent pathways control hEXO1 stability in response to stalled forks[END_REF] Proteins were revealed using the Western blotting detection kit WesternBright™ ECL (Advansta, Menlo Park, CA, USA) and signal was imaged using the Fusion Solo system (Vilber Lourmat).

Quantification of signal in protein bands was performed using Fiji-ImageJ 2.1.0/1.53c.

Flow cytometric analysis

To quantify DNA content, cells were harvested, fixed with 70% ethanol (-20 o C) and stored overnight at +4 o C. Cells were washed with PBS, resuspended in PBS containing 0.1 mg/ml

Conflict of interest

The authors declare no competing financial interests

ASSOCIATED CONTENT

Supporting Information

Additional experimental details, HPLC traces, 1H NMR spectra for compounds are available as electronic supplementary information.