Elevated circulating cystatin C is associated with cognitive impairment in non-Hispanic Whites, but its role in racial disparities in dementia is understudied. In a nationally representative sample of older non-Hispanic White, non-Hispanic Black, and Hispanic adults in the United States, we use mediation-interaction analysis to understand how racial disparities in the cystatin C physiological pathway may contribute to racial disparities in prevalent dementia.n = 9,923), we employed Poisson regression to estimate prevalence ratios and to test the relationship between elevated cystatin C (>1.24 vs. ≤1.24 mg/L) and impaired cognition, adjusted for demographics, behavioral risk factors, other biomarkers, and chronic conditions. Self-reported racialized social categories were a proxy measure for exposure to racism. We calculated additive interaction measures and conducted four-way mediation-interaction decomposition analysis to test the moderating effect of race/ethnicity and mediating effect of cystatin C on the racial disparity.In a pooled cross-sectional sample of the Health and Retirement Study . Among non-Hispanic Black relative to non-Hispanic White participants, the relative excess risk due to interaction was 0.7 , the attributable proportion was 0.1 , and the synergy index was 1.1  in a fully adjusted model. Elevated cystatin C was estimated to account for 2%  for the racial disparity in prevalent dementia, and the interaction accounted for 8% . Analyses for Hispanic relative to non-white participants suggested moderation by race/ethnicity, but not mediation.Elevated cystatin C was associated with dementia prevalence. Our mediation-interaction decomposition analysis suggested that the effect of elevated cystatin C on the racial disparity might be moderated by race/ethnicity, which indicates that the racialization process affects not only the distribution of circulating cystatin C across minoritized racial groups, but also the strength of association between the biomarker and dementia prevalence. These results provide evidence that cystatin C is associated with adverse brain health and this effect is larger than expected for individuals racialized as minorities had they been racialized and treated as non-Hispanic White. Racial disparities in cognitive aging are well-documented , and thoCystatin C is an inhibitor of cysteine protease activity and cathepsins  and an iIn a nationally representative racially/ethnically diverse sample of middle-aged and older adults in the United States, we tested the associations between circulating cystatin C levels and cognitive status. Using four-way mediation-interaction decomposition analysis , we aime1 and genetic data are available through dbGaP2.The United States Health and Retirement Study is an ongoing longitudinal study consisting of a representative sample of adults in the United States over age 50 . To ensuIn the HRS, race is documented by self-reports across different waves of data. Study participants are asked to select their primary race from three large categories White/Caucasian, Black/African Americans, or Other. In an additional question, participants indicate whether they also identified as Hispanic or Latino. From these responses, we created a three mutually exclusive categorial variable . We used participants’ self-reported racialized category as a proxy indicator of exposure to racism and we compared cognitive status on each minoritized racial and ethnic category (non-Hispanic Black and Hispanic) to non-Hispanic White participants.Dried blood spots were collected on a randomly selected half of the HRS cohort in the 2006 wave and the other half of the cohort in 2008. Levels of cystatin C were measured in blood spots using an optimized ELISA protocol . Assay pTo represent the additive interactions between elevated cystatin C levels and race/ethnicity, we operationalized cystatin C and each of race and ethnicity together . We refer to the second and third levels as the “single-exposed” groups, because they have only one of the two exposures (minoritized race/ethnicity status or high cystatin C). We created a similar four-level variable for Hispanic participants, using non-Hispanic White participants with low cystatin C as the reference group. This operationalization allowed us to estimate the joint effect of race/ethnicity and cystatin C on cognitive function and directly estimate measures of interaction in the additive scale .Cognitive function was assessed by the Telephone Interview Cognitive Status, which provides a total score of 0−27 points based on a battery of cognitive tests assessing immediate and delayed recall of 10 words, serial 7s, counting backward, object naming, and recalling the date, president, and vice-president to assess orientation . We used2, continuous, based on self-reported height and weight), exercise was operationalized as a dichotomous variable  to capture whether a participant engaged in vigorous, moderate, or light physical activity at least >1 time per week. Sleep disturbance  captures whether a participant had restless sleep , and number of chronic health conditions included high blood pressure (yes or no), diabetes (yes or no), cancer (yes or no), lung disease (yes or no), heart disease (yes or no), stroke (yes or no), psychiatric problems (yes or no), and arthritis (yes or no), and was operationalized in our models as continuous (0−8 conditions).Relevant covariates and known confounding factors between circulating cystatin C and cognitive status were identified from previous studies . CovariaOther biomarkers measured in the dried blood spots were included as covariates . Levels 4 allele (APOE-ε4) are at increased risk of cognitive impairment  and race/ethnicity categories. Because cystatin C is a marker of renal function  and kidnWe used multivariable-adjusted Poisson regression models with a logarithmic link function and robust error variance  to estimAPOE-ε4 was not available for the full study sample. Thus, we tested APOE-ε4 carrier status in a sensitivity analysis where the genetic marker was added to our previously specified demographic models (Model 2). These models were stratified by race/ethnicity. Because the Langa-Weir algorithm may have differential sensitivity across racial/ethnic groups, we also assessed dementia using the Power’s dementia algorithm , a recently developed algorithm with comparable sensitivity of dementia classification across HRS racial groups  and Stata (v14). For reproducibility purposes, we compared the code we developed to the Stata macro med4way . A seconThere were 9,923 participants included in our analytic sample . The samAmong those with cystatin C >1.24 mg/L, the prevalence of CIND was 25%, and among those with levels <0.86 mg/L was 11% . Among tIn our overall sample, model 4 showed that those with high cystatin C had 1.2  times higher prevalence of dementia and 1.1  times higher prevalence of CIND than those with low cystatin C levels . The assDementia was more prevalent among those who were non-Hispanic Black with high cystatin C (25% in this double exposed group) than those who were non-Hispanic Black with low cystatin C (8.2% in this single exposed group), non-Hispanic White with high cystatin C (5.5% in this single exposed group), or non-Hispanic White with low cystatin C  had higher prevalence of dementia than the unexposed group (non-Hispanic White participants with low cystatin C) . Model 4Among non-Hispanic Black participants, model 4 yielded a relative excess risk due to interaction of 0.7 , indicating that the joint effect of having a double exposure exceeded the simple addition of being either a non-Hispanic Black participant or having high cystatin C . AdditioPoint measures of additive interaction for the Hispanic group were equivalent to those observed for non-Hispanic Black participants . EstimatThe decomposition analysis comparing non-Hispanic Black to White participants, adjusting for demographic variables, showed that the mediating effect of cystatin C accounted for 2%  of the observed racial disparity in dementia , while tFor the Hispanic group, the demographic-adjusted decomposition analysis showed similar interaction results to that of the non-Hispanic Black group. Among Hispanic participants, the mediating effect of cystatin C in the racial disparity was virtually zero, while the portion attributable to the interaction accounted for 9%  of the racial disparity and the direct effect for 91%  . We did APOE-ε4 on dementia biased our mediation decomposition results. After adjusting for APOE-ε4, prevalence ratios of dementia for the double and single exposed groups , the prevalence of dementia was 2.6  times higher than for non-Hispanic White participants with low cystatin C (unexposed group) . For HisIn this large and diverse cross-sectional analysis of older adults in the United States, high levels of serum cystatin C (>1.24 mg/L) were associated with elevated prevalence of cognitive impairment non-dementia and dementia relative to normal cognition. The association between high cystatin C and dementia was suggestive of super-additivity for minoritized racial groups, but the confidence intervals for the additive measures of interaction were imprecise. We found that 8% of the disparity in prevalent dementia between non-Hispanic Black and non-Hispanic White participants was due to the interaction between race and high cystatin C levels, while 2% was mediated through cystatin C. Altogether, these results suggest that, among older adults who are racialized as non-Hispanic Black or Hispanic, the effect of high cystatin C levels on dementia prevalence is greater than expected had these individuals been racialized and treated as non-Hispanic White . HoweverThree observational studies previously documented an association between elevated cystatin C levels and adverse cognitive health outcomes . The AgiOf the previous studies, only the ABC study explored an interaction effect between race and cystatin C levels . InteracWe found that the effect of cystatin C on dementia may be modified by minoritized racial status, as evidenced by two of our measures: the excess risk due to interaction (a measure of additive interaction) and the proportion due to the interaction . We observed that for minoritized participants with high levels of cystatin C (double exposed) the prevalence of dementia was higher than that of non-Hispanic White participants with low cystatin C (unexposed) or participants with either one of the two factors alone (single exposed). These findings held independently of demographic, behavioral, and other biomarkers and chronic conditions. Mediation-interaction analysis results indicated that the interaction effect was responsible for explaining at least 9% percent of the racial disparity in prevalent dementia across both minoritized groups. The mediating effect of cystatin C only explained a small portion of the racial disparity for non-Hispanic Black participants and virtually none for Hispanic participants. Our interaction results suggest that when comparing Hispanic to non-Hispanic White participants, the association between elevated cystatin C on prevalent dementia was of lesser magnitude than when comparing the non-Hispanic Black participants to their non-Hispanic White counterparts. We attribute these findings to Hispanic participants being of younger age, having less chronic health conditions, and comparable average blood levels of cystatin C than their non-Hispanic White counterparts in our study sample. The lack of mediation effect between Hispanic participants relative to non-Hispanic White participants may be because disparities in high levels of cystatin C across both groups are accounted for by covariates such as behavioral risk factors and other biomarkers, and because it is unlikely that cystatin C is the only pathway implicated in the production of racial disparities in prevalent dementia. Future research should focus on incorporating multiple mediators to better understand which deteriorated physiological systems are more likely to mediate racial disparities in cognitive aging.CST3 gene .The current study is a secondary data analysis of existing data that are already available in a publicly accessible data repository. The data presented in this study were accessed from the The studies was involving human participants were reviewed and approved by the University of Michigan Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.CH, KB, and EW contributed to the conception and design of the study. CH organized the database and performed the statistical analysis. MF performed the code and manuscript revision. LK, MH, MZ, and BM contributed to the manuscript revision, read, and approved the submitted version. All authors contributed to the article and approved the submitted version.


Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using  The disease is characterized by the progressive degeneration of specific muscles leading to ptosis (eyelid drooping), dysphagia  and proximal limb weakness –3. OPMD PABPN1 has multiple functions in RNA metabolism. PABPN1 was discovered through its role in pre-mRNA cleavage and polyadenylation . In thisDrosophila and mouse models of OPMD [Drosophila OPMD model that increased proteasome activity arises during OPMD progression, potentially in part through proteasome plugging with mutant PABPN1 oligomers, leading to muscle atrophy by degradation of myofibrillar proteins [Defects in any of these molecular functions could participate in OPMD. Indeed, PABPN1 aggregates were reported to sequester the normal protein, leading to reduced levels of soluble PABPN1 that would contribute to OPMD pathogenesis through a loss-of-function mechanism ,26,27. I of OPMD –32. In lproteins . Thus, pDrosophila), activating transcription factor 6 (ATF6) and inositol-requiring protein 1α (Ire1) [Drosophila) binds PERK, Ire1 and ATF6 in normal conditions. Upon ER stress, BiP recognizes and binds misfolded proteins and dissociates from the three sensors (a). PERK activation depends on its oligomerization and autophosphorylation. Following its activation, PERK phosphorylates eukaryotic translation initiation factor 2α (eIF2α). This leads to global inhibition of cap-dependent translation and translational activation of mRNAs encoding stress response proteins, such as activating transcription factor 4 (ATF4) and growth arrest and DNA damage-inducible protein ). Translation activation depends on regulatory upstream ORFs (uORFs) present in the 5'-UTR of these mRNAs [α and recovery of translation, and thus ends the UPR [It has been established that reduced mitochondrial activity is one of the earliest defects in OPMD ,34. Thisα (Ire1) –37. The  sensors a. PERK ase mRNAs ,39. ATF4 the UPR –42. In t the UPR , while I the UPR ,45. Ire1 the UPR –48. Clea the UPR .Figure α2-adrenergic receptors. GA was shown to reduce muscle degeneration and PABPN1 aggregation upon oral treatment in a Drosophila model of OPMD [Drosophila model by preventing the PFAR activity of the ribosome [a). This leads to prolonged eIF2α phosphorylation and translation inhibition, and extended protection of the ER by reducing the accumulation of misfolded proteins [Drosophila and mouse models of OPMD suggest that the beneficial effect of GA on OPMD might involve both its effects in blocking the PFAR activity of the ribosome and extending the UPR.Among the small molecules identified using animal models to be active in OPMD, Guanabenz acetate (GA) is an FDA-approved antihypertensive drug that acts as an agonist of  of OPMD . GA was  of OPMD –51. Syneribosome . More reribosome ), improvribosome . GA is aproteins ,54. The proteins . Togethea), but devoid of its α2-adrenergic activity. As such, it has lower adverse effects and might be a better option for pharmacological treatments of proteinopathies. Indeed, no hypotensive effect of ICE was recorded during a phase 1 clinical trial on healthy volunteers (NCT03610334). Here, using an OPMD Drosophila model, we show that the UPR is activated in OPMD muscles. Using mutants of the PERK branch of the UPR, we functionally validate that this branch of the UPR participates in OPMD. We further show that ICE has a positive effect when provided orally to OPMD flies, in decreasing muscle weakness and reducing the PABPN1 aggregation load. ICE acts synergistically with reduced GADD34 gene dosage, suggesting that its effect in OPMD depends on its known role in preventing eIF2α dephosphorylation by the GADD34–PP1c phosphatase complex. Together these data reveal the functional implication of the ER stress response in OPMD and identify ICE as a potential candidate for future pharmacological treatments of OPMD and other proteinopathies involving the ER stress response.Icerguastat (ICE), previously called IFB-088 and Sephin1, is a GA derivative with a similar effect on the UPR  figure a, but de. 2. 2.1Drosophila models of OPMD by expressing mammalian alanine-expanded PABPN1  in muscles. PABPN1-17ala is specifically expressed in muscles either from larval stages using the UAS/Gal4 system and the Myosin heavy chain-Gal4  driver or in adult thoracic muscles using the Act88F-PABPN1-17ala transgene [Drosophila model, we monitored ER changes in thoracic muscles of Act88F-PABPN1-17ala/+ flies using the GFP:KDEL reporter protein that marks the ER lumen thanks to ER targeting and retention signals [b) indicating a high level of ER reorganization consistent with ER stress.We previously developed ransgene ,34,55. Bransgene ,34,55. T signals ,57. ImmuAct88F-PABPN1-17ala model, at days 2 and 6 of adulthood. In the PERK branch, ATF4 is known to be regulated at transcriptional level, in addition to its translational regulation by eIF2α [Drosophila ATF4 is encoded by the cryptocephal (crc) gene [crc mRNA levels were higher in OPMD muscles compared to wild-type at both days 2 and 6 (c). By contrast, GADD34 that is only regulated at the translation level in Drosophila [c). We quantified Hsc70-3 (BiP) and Ire1 mRNAs to assess activation of the ATF6 and Ire1 branches of the UPR, respectively. Hsc70-3 is known to be transcriptionally activated by ATF6 (a) and Drosophila Ire1 is also activated at transcriptional level [Hsc70-3 mRNA levels were not higher than wild-type in OPMD muscles, suggesting a lack of activation of the ATF6 pathway, whereas Ire1 mRNA levels were higher at day 2, possibly consistent with its reported early activation under mild ER stress [α phosphorylation, a key marker of the PERK branch of the UPR, using western blots . eIF2α phosphorylation was higher in OPMD muscles compared to wild-type, at both day 2 and day 6 of adulthood.We then used RT-qPCR to quantify mRNA expression of UPR pathway components in the by eIF2α . Drosophrc) gene . We foun 2 and 6 c. By conosophila  was not  by ATF6 a and Droal level . Hsc70-3R stress ,63. We nDrosophila model.These results reveal ER stress and activation of the PERK branch of the UPR in the OPMD . 2.2Drosophila PERK pathway, namely of GADD34, crc and PEK genes  to analyse the functional role of this pathway in OPMD pathogenesis. We analysed the effect of heterozygous GADD34, crc or PEK mutants on the percentage of Act88F-PABPN1-17ala flies showing wing position defects. The 1crc mutant is a known point mutant allele [GADD34 and PEK mutants were uncharacterized P-element insertion alleles . We, therefore, validated that the corresponding mRNA levels were reduced in these two mutants . The number of flies expressing Act88F-PABPN1-17ala  with abnormal wing posture increased from 26% to 65% between day 2 and day 6 of adulthood . The presence of all three heterozygous GADD34, crc or PEK mutants led to a significant decrease in the number of flies with abnormal wing posture (b). The positive effects obtained with GADD34 and PEK heterozygous mutants were confirmed using UAS-RNAi transgenes specifically expressed in muscles with the Mhc-Gal4 driver . PEK and GADD34 have antagonist activities on eIF2α phosphorylation and thus translational regulation, therefore, the decrease of wing posture defects in the presence of both mutants was surprising. We analysed whether this positive phenotypic effect was linked to eIF2α phosphorylation by quantifying the levels of phosphorylated eIF2α in thoracic muscles, in the presence or absence of the three heterozygous mutants using western blots. A decrease of GADD34 function is expected to result in increased levels of phosphorylated eIF2α that might reduce the ER stress through enhanced UPR response. By contrast, the levels of phosphorylated eIF2α are expected to decrease in the PEK heterozygous mutant, which might contribute to restart cap-dependent translation and increase the ER stress. It should be noted though, that variations in eIF2α phosphorylation levels might be difficult to record in vivo as quantifications were not performed under acute ER stress, but rather in flies several days old, which might blur the response. Quantification of eiF2α phosphorylation in thoracic muscles showed that indeed the presence of the −/+GADD34 mutant in Act88F-PABPN1-17ala/+ flies led to higher levels of phosphorylated eIF2α at day 2, whereas −/+PEK mutant reduced these levels with a significant decrease at day 6, and −/+crc mutant had no significant effect on eIF2α phosphorylation . These results show that the level of eIF2α phosphorylation alone cannot explain the phenotypic improvement of OPMD flies by mutants of the PERK pathway, since reduced eIF2α phosphoryation in the −/+PEK mutant is not consistent with the positive effect of this mutant through the UPR. Therefore, the decrease of muscle defects by PERK pathway mutants involves different mechanisms. This is consistent with data in mouse models of neuropathy showing that reduced function of PERK or GADD34 have similar effects in improving neuronal function [Activation of the PERK branch of the UPR has a protective role under acute ER stress through the repression of general translation. However, PERK activation is detrimental under chronic stress through its role in activating apoptosis ,65. We ut allele , howeverdulthood a,b. The  posture b. The pofunction ,68.FiguHsc70-3 and Ire1 mRNAs were quantified in thoracic muscles using RT-qPCR. Hsc70-3 mRNA highly accumulated in Act88F-PABPN1-17ala/+ thoracic muscles in the presence of the −/+crc mutant at day 2 (d). This increased levels of Hsc70-3 mRNA might contribute to reduce muscle defects in −/+Act88F-PABPN1-17ala/+; crc flies through higher synthesis of the chaperone Hsc70-3/BiP. Ire1 mRNA levels were not significantly affected in any of the three PERK pathway mutants (d).Because of the crosstalk between the three branches of the UPR, we investigated whether the ATF6 and Ire1 branches might be activated in response to reduced activity of the PERK branch in heterozygous mutants. The expression levels of at day 2 d. This i mutants d.Drosophila OPMD model. This positive effect involves the UPR and increased eIF2α phosphorylation for −/+GADD34 mutant. In addition, the PEK kinase acts in OPMD through a distinct mechanism.These results reveal that targeting the PERK branch of the UPR strongly reduces muscle defects in the . 2.3Act88F-PABPN1-17ala/+ thoracic muscles at days 2, 4 and 6 of adulthood. As previously reported [a–d). Strikingly, in the presence of all three heterozygous GADD34, crc and PEK mutants, PABPN1-17ala aggregation was reduced at days 4 and 6. Although the percentage of nuclei with aggregates was not significantly reduced, the size of aggregates was reduced at days 4 and 6, with all three mutants (a–d). Because muscle defects and PABPN1-17ala aggregate formation are highly dependent on the levels of mutant PABPN1 [−/+GADD34 and −/+crc mutants did not affect PABPN1-17ala levels, however, the −/+PEK mutant reduced its levels, with a significant decrease at day 6 . This low level of PABPN1-17ala in the presence of the PEK heterozygous mutant is likely the molecular basis of the reduced muscle defects in the presence of this mutant (b).Aggregation of alanine-expanded PABPN1 is a hallmark of OPMD and genetic or pharmacological improvement of muscle function often correlates with reduced aggregation load ,31,32,69reported ,55, the  mutants a–d. Becas mutant b.Figurec) appears proportional to the positive effect of these mutants on the wing position phenotype (b), suggesting that these mutants might act primarily through PABPN1-17ala aggregation.Intriguingly, the decrease in the PABPN1-17ala aggregation load in the presence of the three heterozygous mutants c appearshenotype b, sugges. 2.4a) that, as GA, was reported to prolong the UPR response by binding GADD34 and preventing eIF2α dephosphorylation [α2-adrenergic activity present in GA and has therefore less side effects when used in proteinopathies. We tested the effect of ICE on two different OPMD Drosophila models.ICE is a GA derivative a that, arylation . ICE is UAS/Gal4 system to express PABPN1-17ala in muscles with the Mhc-Gal4 driver. Flies expressing PABPN1-17ala were fed with ICE at 2 to 4 mM in the food from day 1 of adulthood and the number of flies with wing position defects were quantified at days 3 to 6. GA that has a positive effect on OPMD in Drosophila and mouse models [b). ICE efficiency was stronger at day 3 when the wing position defects was lower, and was comparable between the three concentrations and comparable to GA efficiency at 2 mM. This indicated that the positive effect of the drug had already reached a plateau at the 2 mM concentration. We therefore analysed the effect of ICE at the lower concentrations of 1 and 0.5 mM in the food (c). The drug conserved a positive effect on the wing posture phenotype at these concentrations, but its efficiency decreased with a significant reduction of wing position defects at days 4 and 5 only (c).First, we used the e models ,52 was uthe food c. The drd 5 only c.Act88F-PABPN1-17ala model. Similarly, Act88F-PABPN1-17ala/+ flies were fed with 2 mM ICE in the food and wing position defects were recorded at days 3 to 6. ICE showed a positive effect in this model also, with reduced numbers of flies showing wing position defects at all time points, compared to DMSO alone (d). As with the UAS-PABPN1-17ala model, the drug efficiency was higher at day 3 and was comparable to that of GA. We then asked whether the positive effect of ICE involves its known activity in inhibiting eIF2α dephosphorylation. We used western blots to quantify the levels of phosphorylated eIF2α in thoracic muscles of Act88F-PABPN1-17ala/+ flies fed with 2 mM ICE or GA in the food. eIF2α phosphorylation was higher following treatment with both ICE and GA at day 3 . At day 6, eIF2α phosphorylation tended to be higher following ICE or GA treatment, but the increase was not significant , possibly consistent with the lower efficiency of the compounds with time (figure 4d). These data suggest that ICE efficiency on muscle defects depends on its activity in the UPR in favouring translational repression. In addition, the same activity contributes to the beneficial effect of GA in OPMD.Second, to confirm the positive effect of ICE on OPMD, we analysed its efficiency on the SO alone d. As witDrosophila, with an efficiency similar to that of GA. These data also suggest that the positive effect of ICE involves its activity in targeting the PERK pathway, lengthening the UPR response.We conclude that ICE is active in reducing OPMD muscle defects in . 2.5Act88F-PABPN1-17alaR strain contains the same transgene as the Act88F-PABPN1-17ala strain, but its genetic background was changed by consecutive backcrosses. The resulting Act88F-PABPN1-17alaR/+ individuals showed a stronger OPMD phenotype, namely more individuals had wing position defects than Act88F-PABPN1-17ala/+ (compare figure 4d and figure 5a), and importantly, they were not sensitive to oral treatment with 2 mM ICE (figure 5a). We took advantage of this condition where 2 mM ICE concentration was suboptimal for Act88F-PABPN1-17alaR/+ flies to address a potential synergy with the GADD34 heterozygous mutant. We found that the −/+GADD34 mutant was able to reduce the percentage of flies with abnormal wing posture of Act88F-PABPN1-17alaR/+ flies. When ICE at 2 mM was provided to −/+; Act88F-PABPN1-17alaR/+GADD34 flies, the small molecule acted synergistically with −/+GADD34 in decreasing the percentage of flies with abnormal wing posture to a higher level than −/+GADD34 alone (figure 5a). A similar synergistic effect with −/+GADD34 but more pronounced was observed when ICE was provided at 3 mM (figure 5b). To confirm that the observed synergy depended on a direct link between ICE and −/+GADD34 mutant, we used another small molecule, metixene that is active in the OPMD Drosophila model but acts by reducing protein aggregation through the PFAR activity of the ribosome, not through the UPR [Act88F-PABPN1-17alaR/+ flies (figure 5a), whereas it shows positive effect on Act88F-PABPN1-17ala/+ flies [−/+GADD34 as the number of −/+; Act88F-PABPN1-17alaR/+GADD34 flies with abnormal wing posture did not decrease in the presence of metixene (figure 5a).We used the genetic approach to confirm that ICE positive phenotypic effect depended on its ability to prevent GADD34-PP1c phosphatase complex activity. The  the UPR . As ICE,/+ flies . HoweverGFP:KDEL reporter. We confirmed the enlargement of the ER lumen in OPMD muscles compared to wild-type (c). Strikingly, GFP:KDEL staining was significantly reduced when OPMD flies were fed with food containing 2 mM ICE, compared to DMSO alone, revealing a reduction of the ER, and thus decreased ER stress in the presence of the small molecule (c).We, next analysed a direct effect of ICE on the ER by recording changes in the ER structure using immunostaining of muscles expressing the ild-type c. Strikimolecule c.α dephosphorylation by the GADD34-PP1c phosphatase complex and maintaining translation inhibition to reduce ER stress.Together, these results are consistent with ICE acting in OPMD through its known function in inhibiting eIF2. 2.6a–d), the number of nuclei containing aggregates was not affected following oral treatment with 2 mM ICE (a), but the size of aggregates was significantly reduced over time (b–d). This smaller size of PABPN1-17ala aggregates was not due to lower levels of PABPN1-17ala protein in thoracic muscles following ICE treatment .The decrease of muscle defects with PERK pathway heterozygous mutants correlates with reduced PABPN1-17ala aggregation in muscle nuclei. We, therefore, analysed whether ICE oral treatment also led to reduced PABPN1-17ala aggregate formation. Similarly to GA treatment  figure a–d, the 2 mM ICE a, but thver time b–d. ThisDrosophila OPMD muscles, similarly to GADD34 heterozygous mutant. They reinforce the notion that ICE would act positively on OPMD muscles through GADD34-PP1c complex activity.These results show that oral treatment with ICE decreases PABPN1-17ala aggregation in . 3Drosophila model. ER stress and activation of the UPR have been recently reported in the A17 mouse model of OPMD [Several molecular pathways have previously been described to participate in OPMD pathogenesis. Here, we show that the ER stress and the downstream UPR pathway play a functional role in OPMD defects using the  of OPMD . Here weDrosophila model of OPMD and maintained with time. In particular, eIF2α phosphorylation that prevents cap-dependent translation is increased in Drosophila OPMD muscles. Analysis of mRNA levels indicates that the Ire1 branch also appears to be activated, however, only at early stages. The Ire1 branch has been reported to be activated upon mild ER stress and beneficial [α phosphorylation. However, during chronic stress the PERK branch can activate an apoptotic cascade followed by cell death [α phosphorylation shows that it increases in OPMD Drosophila muscles in the presence of GADD34 heterozygous mutant, at early stages. This is consistent with a beneficial role of the PERK branch for OPMD at this early stage, through translation inhibition that would decrease the load of misfolded proteins in the ER. This beneficial effect would be enhanced by reducing the level of active GADD34-PP1c phosphatase in GADD34 heterozygous mutant. By contrast, the positive effect of PEK heterozygous mutant on OPMD muscle defects, associated with the decrease of eIF2α phosphorylation at later stages (day 6) is not consistent with a positive role of PEK through preventing translation. Thus, PEK would also act through an additional mechanism in OPMD pathogenesis. PEK function could involve apoptosis as it is the case during chronic stress, since apoptosis has been described to participate in OPMD defects [Drosophila muscles in the presence of PEK heterozygous mutant. Although the molecular mechanisms leading to this decrease remain unknown, a lower amount of PABPN1-17ala is expected to substantially contribute to the decrease of muscle defects since the strength of these defects correlates with PABPN1-17ala levels in the Drosophila model [We find that the PERK branch is the main pathway activated in the neficial –73. By cneficial . Upon mill death ,65. Both defects . In addila model . Despitela model ,75. Simila model ,68. Althα phosphorylation levels. Moreover, our genetics data indicate that GADD34 heterozygous mutant acts synergistically with ICE in lessening OPMD muscle defects. This provides functional evidence that ICE mechanism of action in OPMD involves GADD34-PP1c targeting. ICE was described to bind GADD34 and increase eIF2α phosphorylation levels from analyses in cell cultures [in vitro set up [Drosophila model reinforce the conclusion that ICE can target the PERK branch of the UPR in vivo, although it could do so through a different mechanism than a direct interaction with GADD34. Interestingly, ICE has lower adverse effects than GA since it is devoid of the α2-adrenergic activity and thus has no hypotensive effect. Therefore, this small molecule has a higher potential for future development of pharmacological approaches to treat OPMD.In this study, we identify a new molecule, ICE with a positive effect when provided orally to OPMD flies. This small molecule is a GA derivative and has the same property as GA in inactivating the GADD34–PP1c phosphatase complex, which leads to the maintenance of global translation repression ,54,76. Wo set up ,78. Our Intriguingly, both the genetic modulation of the PERK pathway and oral treatment of flies with ICE strongly reduces the size of PABPN1-17ala aggregates. Although reduction of muscle defects in OPMD animal models is not systematically associated with reduced PABPN1-17ala aggregation , this isThe positive effect of ICE in an animal model of OPMD along with its lower adverse effects make of this drug a promising therapeutic molecule for future treatments of OPMD. ICE was also shown to improve neuronal function in neurodegenerative diseases, such as Charcot–Marie–Tooth disease and amyotrophic lateral sclerosis ,80. Ther. 4. 4.11118w was used as a control. OPMD was induced either by using the transgenic line Act88F-PABPN1-17ala to express PABPN1-17ala in adult indirect flight muscles [UAS-PABPN1-17ala crossed with the Gal4 driver line Mhc-Gal4 to express PABPN1-17ala in all muscles from larval stages [EY0957PEK (#17582 at Bloomington Drosophila Stock Center (BDSC)), 1/SM5crc [HP20979/CyOPPP1R15 (#22330 at BDSC). Stocks containing UAS-RNAi constructs were TRiP.GL00030-attP2  and TRiP.HMS00811-attP2 ; the attP2 stock (#36303 at BDSC) was used as a negative control. The UAS-KDEL:GFP stock [UAS-PABPN1-17ala and Mhc-Gal4 that were maintained at 18°C; flies from these crosses were maintained at 18°C during the whole experiments. muscles ,34 or thl stages . Mutant crc1/SM5   reconstituted with 2 ml of 1% yeast in water and supplemented with various concentrations of either Guanabenz acetate salt , metixene (a gift from Dr Cécile Voisset) or ICE (InFlectis BioScience) diluted in DMSO . DMSO alone was used as control. Note that the concentration of 2 mM ICE in the food corresponds to an exposure of 0.23 mg kg−1/12 h, considering that a Drosophila ingests 20 nanoL h−1 [−1 day−1) [Drosophila medium. Wing posture phenotype was scored every day from day 2 to day 6.Drug-supplemented medium was prepared with Instant anoL h−1 . This qu1 day−1) . Adult m. 4.3Drosophila sop mRNA that encodes a ribosomal protein. Primers used are listed in Total RNA was prepared from five thoraxes per genotype using Trizol (Invitrogen) following recommendations of the manufacturer. DNA was digested using RQ1 RNase-Free DNase (Promega) for 30 min at 37°. Total RNA concentration was determined with nanodrop ND-1000 spectrophotometer. 1 µg of RNA was reverse transcribed for RT-qPCR using SuperScript III (Invitrogen) and random hexamers (Invitrogen). Quantitative PCR was performed on LightCycler LC480 (Roche) using LightCycler 480 SYBR Green I Master (Roche), and normalized with the . 4.4−1 DAPI (Sigma-Aldrich) in 1× PBS, 0.1% Triton X-100. Fluorescent images were acquired with a Leica SP8 confocal microscope. Quantification of KDEL:GFP staining was performed using ImageJ and calculated with the CTCF  as follows: CTCF = integrated density – (selection area* mean fluorescence of the background), where integrated density = selection area × mean fluorescence of the selection. The area of nuclei was subtracted from the area of the selection. Surface of aggregates was measured and quantified using the ImageJ software. For western blots, protein extracts were obtained from five thoraxes per genotype crushed in 50 µl of 2× Laemmli buffer supplemented with 10% β-mercaptoethanol and boiled for 10 min at 95°. Samples were loaded onto 4–15% or 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Each membrane was blocked for 2 h in 5% milk diluted in 1× PBS, 0.1% Tween 20. Primary antibody incubation was performed overnight at 4°C in 1× PBS, 0.1% Triton X-100, 1% BSA, 0.02% azide. Antibodies used were: rabbit anti-eIF2α at 1:1000 , rabbit anti-phosphorylated eIF2α at 1:1000 , rabbit anti-PABPN1 at 1:1000 [Dissection and immunostaining of adult indirect flight muscles (IFMs) were performed as described previously  with somt 1:1000 , mouse a


Mutants of Ras are oncogenic drivers of a large numberof humantumors. Despite being recognized as an attractive target for the treatmentof cancer, the high affinity for its substrate tagged the proteinas undruggable for a few years. The identification of cryptic pocketson the protein surface gave the opportunity to identify moleculescapable of acting as allosteric modulators. Several molecules weredisclosed in recent years, with sotorasib and adagrasib already approvedfor clinical use. The present study makes use of computational methodsto characterize eight prospective allosteric pockets (P1–P8)in K-Ras, four of which (P1–P4) were previously characterizedin the literature. The present study also describes the results ofa virtual screening study focused on the discovery of hit compounds,binders of the P4 site that can be considered as peptidomimetics ofa fragment of the SOS αI helix, a guanine exchange factor ofRas. After a detailed description of the computational procedure followed,we disclose five hit compounds, prospective binders of the P4 allostericsite that exhibit an inhibitory capability higher than 30% in a cellproliferation assay at 50 μM. The two forms exhibit differentconformations, granting the protein the ability to recognize diversesubstrates. Two types of proteins facilitate the exchange betweenthe active and inactive forms. On the one hand, guanine exchange factors(GEFs) catalyze nucleotide exchange from GDP to GTP, and on the otherhand, GTPase activating proteins (GAPs) facilitate the hydrolysisof the γ-phosphate of the bound GTP.4 Specifically,the Ras–Raf–MEK–ERK pathway controls cell proliferationby modulating the levels of diverse cell cycle regulators. Alternately,Ras can also promote proliferation and cell survival through the Ras–PI3K–Aktsignaling pathway. In addition, Ras can activate other proteins likethe Ral guanine nucleotide dissociation stimulator  or theT-cell lymphoma invasion and metastasis 1 (TIAM1), involved in theregulation of vesicle trafficking and cytoskeletal organization, respectively.6 Constitutive activation of Ras is the cause of severe pathologiesincluding cancer.7 Actually, about 30%of human cancers are due to Ras constitutive activation, with thehighest incidence in lung, colon, and pancreatic cancers.1 Dysregulation of Ras is mainly due to the occurrenceof point mutations, most frequently in residues G12, G13, and Q61that cover ca. 98% of all mutations associated with Ras,1 with K-Ras being the predominantly mutated isoform(85%).8 These mutations leave the proteinin a constitutively active state due to a decreased intrinsic GTPaseactivity and/or to an increased resistance to GAP-mediated hydrolysis9 resulting in aberrant signaling.This family of proteins engages and activatesdiverse downstreamsignaling pathways regulating different cellular responses, includingproliferation, survival, and differentiation.10 Moreover, the lack of obviousalternative pockets in its crystal structure made the protein to beconsidered for a long time as undruggable.11 However, analysis of the protein dynamical behavior carried outby means of computational studies revealed the existence of transientpockets susceptible to be exploited for the design of allosteric inhibitors.13 As a proof of concept, diverse low-affinity inhibitors were subsequentlyidentified and reported in the literature.18 Today, sotorasib19 andadagrasib,20 allosteric inhibitors specificallytargeting the G12C mutant, are already approved for clinical use.Recognized as an attractivetarget for the treatment of cancer,the high cytosolic concentrations of GTP and GDP, together with theirhigh affinity for Ras, rendered the design of competitive inhibitorselusive.21 and the C-terminalor allosteric lobe (residues 87–166) engaged in dimerization.22 Diverse allosteric sites were identified fromcomputational studies,13 with four of them (P1–P4)subsequently validated experimentally.24 The P1 siteor switch I/II pocket is located in the effector lobe between theβ1−β3 strands and the α2 helix. Most of theallosteric inhibitors disclosed to date bind to this site includingcompounds described by Fesik’s group,14 Genetech,15 Kobe0065,16 0375-0604,25 Gorfe’sgroup,26 BI-2852,27 or Rabbitts’s group.29 The P2 site or switchII pocket is also located on the effector lobe between helices α2and α3 and the P-loop. This is the binding site of irreversibleinhibitors targeting the G12C K-Ras mutant like sotorasib,19 adagrasib,20 or ARS-853.30 Moreover, recent reports suggest that inhibitionof K-Ras through this site can be achieved beyond the G12C mutants,opening the door for designing more general therapeutic agents.31 In this direction, a reversible ligand (TH-Z835)targeting the G12D mutant32 and a reversiblepan K-Ras compound (BI-2865) were recently disclosed.33 P3 is located in the allosteric lobe between the C-terminusof helix α3, the L7 loop, and the C-terminus of helix α5.Compound KAL-21404358 is one of the few compounds disclosed shownto bind to this site.34 Interestingly,the compound Zn2+-cyclen binds with low affinity to a P3subpocket termed P3b.35 Both compoundsfavor the state 1 versus the state 2 of the active, GTP bound K-Rasstructure, characterized by the loss of hydrogen bonding interactionsbetween the γ-phosphate and residues Thr35 and Gly60 and associated with a weaker affinity to effector molecules.36 Finally, the P4 site is located between the effector and the allostericlobe close to the nucleotide binding site. It involves residues ofthe switch I, the α1 helix, and β2 and emerges when theswitch I adopts an open conformation bound to GDP. Located at theinteraction interface of some downstream effectors such as Raf, PI3K,or RalGDS,37 there is no definitively recognizedsmall molecule binder yet. Andrographolide and its derivatives SRJ09and SRJ23 were proposed to bind the site,38 although subsequent studies point to the switch I/II pocket as themost likely spot.39The structure of K-Ras consists of a globular GTPase domain (residues1–166) and a hypervariable region (167–188). Furthermore,the globular domain is composed of two lobes including the N-terminalor effector lobe (residues 1–86), where all the downstreameffectors and other major regulators such as GEF and GAP proteinsmake major contacts,40 This raises thequestion of whether the site is affordable only to macromolecule ligands.Aimed at exploring the viability of the site for designing novel smallmolecule K-Ras inhibitors, we report in the present work the resultsof a virtual screening study on the site and disclose novel hit moleculesas prospective allosteric modulators. For this purpose, we first carriedout a study of the dynamic behavior of the protein using moleculardynamics to better characterize the structural features of the P4site. These results permitted defining a query that was used for screeningthe ZINC chemical library.41 As a result,we conclude this report by disclosing the structures of five molecularhits, prospective binders of the P4 site.Druggabilityof K-Ras by inhibition through the P4 site is puzzlingbecause there is no direct evidence of a small molecule binder, althoughthe antibody mimetic DARPin K27 binds to the site and inhibits K-Rasdownstream signaling.242 Specifically, protein wassubjected to a 500 ns accelerated molecular dynamics (aMD) calculationwithin the classical (NVT) ensemble using the protocol implementedin Amber12.43 The solvent was treated explicitlyusing the TIP3P water model, and periodic boundary conditions wereapplied. Energy was computed using the Amber ff99SB force field,44 although parameters for GDP were taken froman alternative source.45 A cutoff distanceof 10 Å was set for short-range noncovalent interactions, whereaselectrostatic interactions were treated using the PME method.46 The temperature was kept constant at 300 K bymeans of a Langevin thermostat.47The computationalprocedure followed to identify transient sites on the protein is explainedelsewhere.4EPX),14 whose atomic coordinates were retrieved from the RCSB PDBwebsite.48 Protonation states of the diverseresidues were assigned using the Protonate3D function of the MOE program.49 The structure was soaked in a rectangular boxof 12,876 water molecules, to which six Na+ ions were addedto maintain the neutrality of the system. The system was energy minimizedto relax the initial structure in several steps using the steepestdescent method. In the first step, only side chains were free to move,applying to the protein backbone atoms and GDP-Mg a positional constrainingharmonic potential with a force constant of 1 kcal/mol Å2. In the following steps, restrictions were gradually lifted,first on the protein backbone and then on the GDP-Mg system. Next,the temperature of the system was gradually increased using 100 psof NVT molecular dynamics at a rate of 30 K every 10 ps. Subsequently,the system was subjected to an equilibration process consisting of1 ns of molecular dynamics in the NPT ensemble and 1 ns of moleculardynamics in the NVT ensemble. The SHAKE algorithm was used to restrictthe elongation motion of the bonds involving hydrogen atoms,50 which allowed using an integration time of 2fs.The simulation was carried out on the structure of the human K-RasG12V bound to GDP solved at 1.76 Å resolution  and theaverage dihedral energy  from a previous 50 nsclassical molecular dynamics trajectory and the use of system sizedependent tuning parameters αP = 8275 and αD = 120.43 To identify the diverse conformationsthe protein attains during the sampling process, we carried out ahierarchical cluster analysis by means of the average link algorithm52 using the backbone Cα root-mean-squaredeviation as a measure for the distance between two conformations.Subsequently, mapping of transient binding sites on the protein surfacewas carried out on each of the cluster representative structures usingthe FTMap procedure.53 It consists of samplingbillions of positions of small organic molecules used as probes andscoring their poses using a detailed energy expression. Regions thatbind several probe types serve to identify binding hot spots.Conformational analysiswas carried out using 50,000 structuresof the trajectory taken at regular intervals of 10 ps. Structureswere aligned using the Cα coordinates of the residues includedin the invariant nucleus  to the firststructure of the trajectory using the cpptraj module of the Amber12program.54 The procedure computesa druggability score for the target site. The computation includesdiverse terms that promote ligand binding including adequate size,isolation from the solvent, and penalizes increasing hydrophilicity.The druggability ofeach of the prospective binding sites was assessed by means of theSiteMap program.49 In addition, an exclusion volumewas included as restriction to disregard structures that, despitecomplying with the pharmacophore, may exhibit steric effects withthe protein.A pharmacophore was developed forany binding site of interest by means of the Site Finder program ofMOE.41 filtered to containlead-like molecules only.55 Prior to undertakingthe virtual screening process, up to 1000 conformations of each moleculewere generated using the conformational import function of MOE.49 Molecules were first screened to determine thepharmacophore of the site of interest. Then, the compounds selectedwere subjected to a molecular docking process using the Glide program.57 Next, poses resulting from the docking process were filtered againfor pharmacophore fulfillment using a lower degree of compliance.Finally, a diversity analysis of the set of compounds was performedby means of the canvas program59 using a molecular fingerprintthat encodes the three-point pharmacophores that meet the 3D structuresof the generated protein–ligand poses. Visual inspection ofthe complex structures permitted selecting those compounds to be procuredfrom commercial vendors.Virtual screening was carried out using the ZINCchemical library3 cells/cm2 and incubated for 24 h at 37 °C in a humidified atmospherecontaining 5% CO2. Test compounds were first dissolvedin DMSO 100% and subsequently diluted to 50 μM, embedding amaximum 0.5% DMSO concentration, and incubated in the cell culturefor another 72 h. After incubation, proliferation was quantified usingthe CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay-MTS(Promega 10 #G5421) following manufacturer's instructions. Absorbanceat 490 nm that is directly proportional to the number of living cellswas recorded with a BMG Fluostar Optima Microplate Reader and normalizedrelative to control cells treated with the vehicle.Cell proliferationinhibition assays were conducted using the NIH/3T3 fibroblast cellline transformed with K-RAS oncogene DNA with the G12 V mutation bytransfection. Cells were cultured in DMEM supplemented with 5% fetalbovine serum (FBS). The cultured cells were seeded in tissue culturemicroplates at a density of 2 × 10353 was used to identify hot spots on each of the diverse clusters characterized.Transient binding pockets on K-Ras were identified from the analysisof 50,000 structures taken from the aMD trajectory at regular intervalsof 10 ps. The first step consisted of classifying the diverse conformationsinto groups by means of cluster analysis. This required the computationof a distance matrix for all pairs of structures using the root-mean-squaredeviation (rmsd) of the backbone Cα atoms once the structureswere previously aligned using the less flexible elements of the protein,as explained below. In the second step, FTMapFigure S1 shows the root-mean-square fluctuationsof each residue. Inspection of both figures suggests that switch Iis the most flexible structural motif, together with the N-terminusof the β2-strand (red in 52 that permitted to group the structures into six clusters. Clusterswere represented by the structure with the closest root-mean-squaredeviation (rmsd) to the average value of the group. Figure S2 shows the representative structures of each clustersuperimposed with the crystal structure used as starting point forthe MD simulations in this work (PDB ID: 4EPX).14 Inspectionof Figure S2 suggests that the differencesbetween structures are located on the switch I and switch II regions,in addition to specific secondary elements linked to them like theβ2 strand and the α3 helix, as well as the loops L4 andL7.53 For comparison purposes, we also mapped thecrystallographic structure used in the present work. The aggregatedresults of the mapping on each of the six representatives as wellas that of the crystal structure are shown in 24 The rest of the sites identified(P5–P8), despite a few of them being described previously incomputational studies,60 are not yet experimentally validated. Interestingly, only four ofthese sites  appear in both the crystal and atleast one of the representative structures, stressing that bindinghot spot identification requires inclusion of the dynamical behaviorof the protein.Mapping hot spots on the protein surface for eachof the six clusterrepresentative structures was performed using the FTMap program.54 This procedure permitsclassifying sites into druggable, difficult, and undruggable by means of a druggability score,defined as a composite index comprising diverse properties like size,exposure, enclosure, hydrophobicity, and hydrophilicity.54 However, the threshold of the druggability indexdifferentiating the diverse categories is fuzzy, and although it providesan overall view, values of other properties can be used to fine-tunethe classification of a specific site.54A druggability profile of the eight sites identifiedwas investigatedusing SiteMap.54 Inspection of the first two columns of druggable, with P3, P4, P1, P6, and P2 being difficult andthe rest undruggable. The present results agree withthe results of a previous analysis performed on sites P1–P4.23Two druggability indexes were computed in the present workandare shown in the first two columns of 61 Concerningsites already validated, site P1 corresponds to the switch I/II pocketdescribed above,18 the binding site of diverse ligands.29 There are also macromolecules described to bind to the site, actingas inhibitors, like the affimer K662 orthe monobody JAM20.63 In the present study,the site is identified in five of the cluster representative structures as well as in the crystal structure. Moreover,it appears number three in 23Ligands targeting these sites have a potential roleas allostericregulators or protein–protein modulators depending on theirspecific binding location. However, it should be borne in mind thatidentification of a binding transient site does not guarantee thatthe site is necessarily functional.30 ] including sotorasib and adagrasib.20 There are also macromolecules binding to the site acting as inhibitors,like the affimer K3.62 FTMap identifiesthe pocket in three of the cluster representative structures , in addition to the crystal structure. The site appears listedas difficult in 34 Compounds binding to the site are thought to stabilizea non-SOS-recognizable switch II conformation and, consequently, inhibitGDP/GTP exchange.17SiteP2 corresponds to the switch II pocket, the binding site ofdiverse irreversible inhibitors targeting the G12C mutant34 as wellas the Zn2+-cyclen in a P3b subpocket,35 as described above. The DARPins K13 and K19also bind to the site.64 In the presentstudy, the site is identified by FTMap in five cluster representativestructures , as well as in the crystal structure.However, the values listed in 23 point to the site as undruggable due to the lowhydrobophicity indicator. The site P3b should also be considered as undruggable due to its small size. Binding to this siteoccurs preferentially in state 1 of the active Ras-GTP, providingthe opportunity to stabilize the protein in this state that preventsthe activation of the downstream cascade.37P3 is the bindingsite of compound KAL-21404358,39 This uncertainty is probablydue to the fact that P4 is located in the vicinity of P1. Moreover,the P4 site is the target of the antibody mimetic DARPin K27 thatshows inhibition of downstream signaling.40 In the present work, the P4 site is identified in the cluster representative4; however, cluster representatives 2 and 5 identify a subsite. Incontrast, the site cannot be identified in the crystal structure.The difference between P4 in the structure of cluster 4 and in thestructures of clusters 2 and 5 is a consequence of the conformationadopted by switch I. Whereas, in the former, it is widely open, inthe latter, it adopts a partially closed form, making accessible onlya subsite of P4. Interestingly, the structure of the switch I adoptedin the representative of cluster 4 is very close to the conformationfound in the structure of the K-Ras/SOS complex (PDB entry: 1nvv), and it is associatedwith an increased GDP off rate.65 The results of difficult, a classificationthat agrees with previous findings.23Finally, P4 was previously proposed to be one of the prospectivebinding sites for andrographolide and its derivatives SRJ09 and SRJ23,together with P1.66 Subsequent directed mutagenesis experiments on residues Asn26 and Val45 confirmed that the pocket is part ofthe recognition interface of the cysteine-rich domain of the Raf protein,67 suggesting that binders to the pocket can actas protein–protein inhibitors of Raf. Site P6, not describedpreviously, is located between the C-terminus of helix α5, theβ1 strand, and the L3 loop. This site is identified in two clusterrepresentative structures (4 and 6) and appears as the result of themovement of the L3 loop and the β-sheet coordinated with themovement of switch I. According to the results of difficult. Sites P7 and P8 were described in previous computational studies.60 Site P7 is located between the α4 helix, the N-terminal partof the α5 helix, and the β6 strand. The site was recentlyidentified as the binding pocket of the monobody NS168 as well as the affimer K69.62 These proteins disrupt RAS dimerization, leading to the blockingof CRAF-BRAF heterodimerization and activation. P7 is analogous toa site previously described and associated with the calmodulin bindingsite.69 In the present study, P7 is identifiedin four cluster representative structures , beinglisted in undruggable due to its small size and hydrophobicity index.Finally, the P8 site is a small pocket located between helices α3and α4 and was identified only in the cluster representativestructure 4. Although the site may be considered as difficult according to the values of the Sscore and Dscore listed in undruggable due to the low hydrophobicity index.Concerning the rest of the sites identified, P5 and P6 are locatedbetween the effector and the allosteric lobes, whereas P7 and P8 arelocated on the allosteric lobe. P5 is located between the helix α5N-term, the helix α1 C-term, and the β2 strand. It appearsin six cluster representatives but not in the crystal structure. Thepocket was identified in previous computational studies.49 The features that binders shouldexhibit include  in the vicinity of the NH group of the Asp33 backboneand the hydroxyl group of Tyr40, respectively; a hydrophobicmoiety (Ph3) next to Ile21 and Thr20; and twohydrophobic or aromatic moieties (Ph5 and Ph6) adjacent to residuesLeu56 and Ile21, respectively. In addition,three constraints were added, two (R2 and R4) to define the directionof the hydrogen acceptor points (Ph2 and Ph4) and another to specifythe orientation of the aromatic ring (R1) of point Ph1, followingthe PHCD pharmacophore definition of MOE.49 In addition, an exclusion volume that defines the binding cavitywas included to eliminate structures that, despite complying withthe pharmacophore, overlap their structure with that of the protein.A six-point pharmacophorewas defined based on the K-Ras structure using the Site Finder moduleof MOE. include 3 an arom57 Next, to prevent the flexible docking process from taking ligandsaway from pharmacophore fulfillment, the diverse poses were filteredfor their degree of compliance of at least six pharmacophoric points,reducing the size of the library to 336 compounds. Finally, to facilitatethe selection of the final candidates, a diversity analysis was carriedout using the canvas program of the Schrödinger software.57 For this purpose, we used a molecular fingerprint as a descriptor,encoding all three-point pharmacophores that meet the 3D structuresof the generated protein–ligand poses. This permitted us toclassify the hits into 30 clusters. Finally, a subset of 16 clusterrepresentatives was selected according to their docking score andstereochemical complementarity with the binding site assessed by visualinspection. Of these, 13 compounds were purchased from commercialsuppliers (A55001 to A55013) and submitted for in vitro tests.Screening of the ZINC compound library for pharmacophorefulfillmentyielded a total of 1364 molecules and a total of 2372 conformations,with 11 of them fulfilling all restrictions. The resulting compoundswere subjected to a molecular docking process using the Glide program.71 We then searched the corresponding diversityclusters of positive hits for additional compounds and selected sixadditional compounds for screening according to their docking scoreand stereochemical complementarity with the binding site, assessedby visual inspection. Four of these compounds were purchased (compoundsA55014 to A55017) and submitted for in vitro tests,with one of them showing a cell proliferation inhibitory behaviorat least 30% at 50 μM. in vitro screening of all the compounds tested at 50 μM. CompoundsA55003 and A55016 show inhibitions close to 50%, whereas compoundsA55004, A55007 and A33013 inhibit cell proliferation at a 30–40%yield. The success rate of the screening process is about 30%, similarto the one observed in similar studies.76 The results shown in Screening showed four compounds with a cell proliferationinhibitionhigher than 30% at 50 μM. This is an arbitrary cutoff to retaincompounds for further investigation, loose enough not to miss anyinteresting hit and covering the maximal chemical diversity of theset.40 and close to Ile36. The hydrophobic/aromaticpoint Ph6 is also fulfilled in all five compounds with moieties asdiverse as a methyl group or a five-membered heterocyclic ring thatinteract with Pro34. Hydrophobic point Ph3 is fulfilledby means of a methyl or methylene group at a distance suitable fora van der Waals interaction with Ile21. All the compounds,except for A55003, fulfill the hydrophobic or aromatic pharmacophoricpoint Ph5. Specifically, compound A55004 fulfills the point by meansof a benzene ring, compound A55007 fulfills the point by means ofa methyl group, and compounds A55013 and A55016 fulfill the pointby means of a piperidine ring. These moieties establish a van derWaals interaction with Leu56. Regarding hydrogen bond interactions,except for A55016, compounds fulfill the pharmacophoric hydrogen acceptorpoints Ph2 and Ph4. Specifically, compound A55003 establishes a doublehydrogen bond interaction through the oxygen of an aliphatic hydroxylgroup with the amide hydrogens of the Tyr32 and Asp33 backbone. The same interaction is established with the oxygenof an amide group in compound A55004, with the sulfur atom of a thiophenering in compound A55007, and with the nitrogen in meta of the pyrazolering of compound A55013, although the interaction is established onlywith the nitrogen of the Asp33 backbone in the latter.In contrast, the amide carbonyl of compound A55016 is far from bothdonor groups of the receptor to establish a hydrogen bond, despiteit establishing a hydrogen bond with the hydroxyl group of Tyr40. All compounds, with the exception of A55013, have a centralamide group that forms a hydrogen bond by means of the oxygen atomof the carbonyl group with the hydroxyl group of Tyr40.In contrast, compound A55013 stabilizes the interaction by means ofan aliphatic-type hydroxyl group.After analysis of the prospective bound conformationsof the diversehits, we hypothesize that binding of compounds to the P4 site mayexert a stabilizing action on switch I that facilitates its open conformation.This conformation is similar to the one adopted by K-Ras when boundto SOS, more widely open than that shown in the structures of Ras-GTPin state 1 and associated with an increased GDP dissociation rate.The fact that this type of open conformation was sampled during thesimulation process suggests that the recognition between Ras and SOScould respond to a conformational selection mechanism. However, thetwo conformations are not identical showing some differences in thearrangement of diverse residues. Accordingly, a second step of inducedfit is required between the interfaces of both proteins in the recognitionprocess.78 interfering with the recognition between K-Ras and SOS and consequentlyarresting the GDP dissociation process. Inspection of the bound conformationof the active compounds showed that these compounds share a few interactionswith the K-Ras/SOS complex. Specifically, all the compounds occupyin part the area of the SOS αI helix C-terminus in the K-Ras/SOScomplex (40 in K-Ras mimics the interaction between Tyr40 and SOS His911 found in the K-Ras/SOS complex.Another interaction common to all compounds is the presence of anacceptor group forming a hydrogen bond with the backbone NH amideof Tyr32 and mimicking the interaction between Tyr32 and SOS Asn944 in the K-Ras/SOS complex.The compounds identified in the present work can beconsideredas peptidomimetics of the SOS helix αIcomplex 9.MoreovThe present work reports the results ofa modeling study aimedat identifying transient pockets of K-Ras as prospective allostericsites to modulate protein activity. For this purpose, the dynamicalbehavior of the protein was investigated by means of a 500 ns moleculardynamics trajectory at 300 K using accelerated molecular dynamicsas the sampling technique. Cluster analysis of the trajectory permittedus to identify six different conformations adopted by the protein.Each of the conformations was mapped for hot spots using diverse molecularprobes by means of the FTMap program. The results permitted identificationof eight cryptic pockets. Seven of them were previously described,with four of them already validated. We also analyzed the characteristicsof the pockets.in vitro testing.Four compounds exhibited cell proliferation inhibitory activity higherthan 30% at 50 μM. In the second step, we selected an additionalsix compounds from the clusters of the positive hits for screening.Four of these compounds were purchased and submitted to invitro screening. Only one of them showed cell proliferationinhibitory activity. Accordingly, the present work discloses fivemolecular hits with cell proliferation inhibitory activity higherthan 30% at 50 μM, targeting the P4 site of K-Ras. Further studiesneed to be carried out to validate binding to the site and to assesstheir role as allosteric modulators of the K-Ras activity.Moreover, a virtual screening process was conductedon site P4using the ZINC database. After a hierarchical process, we ended upwith a short list of 16 compounds, 13 of which were purchased fromcommercial suppliers and submitted for


Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1 Parthenogenesis has been used extensively as a model to study the biochemical and morphological events that occur during the early stages of embryonic development . During Oocyte activation protocols consist of the use of physical  or chemiDuring the meiotic cycle of mammalian oocytes, the activity of MPF follows a well-defined pattern. It is low during the prophase of the first meiotic division (MI), and gradually increases as oocytes resume meiosis until reaching its maximum level at MI metaphase. At the transition from MI metaphase to anaphase, MPF is inactivated. Then, as oocytes progress to the metaphase of the second meiotic division (MII), MPF activity increases again and remains high during MII arrest. After oocyte activation, MPF activity decreases sharply, promoting the resumption of the cell cycle .The MPF activity in female gametes cultivated in vitro is affected by various factors, such as the stage of the meiotic cycle, the species, the age and quality of the oocytes, and the oocyte activation protocol used. The molecular mechanisms behind these fluctuations are not yet fully understood.It has been recently reported that both sperm-induced activation (after IVF) and the chemically induced activation of bovine oocytes inactivate MPF by CDK1-specific phosphorylation without degrading CCNB1 . This isTherefore, the inhibition of CDK1 seems to be the mechanism by which in vitro matured bovine oocytes can be successfully activated. This is consistent with what was observed by ,23, who The activity of MPF is known to depend on the phosphorylation status of CDK1. Previous studies have shown that phosphorylation of both Thr14 and Tyr15 on CDK1 inhibits MPF activity ,25. On tThe meiotic arrest is also supported by the cytostatic factor (CSF), conformed by the proteins of the mitogen-activated protein kinases (MAPKs) signaling pathway . EvidencPreviously, we have reported a differential modulation in the activity of MPF between physiologic activation of oocytes through in vitro fertilization (IVF) and chemical activation using protein synthesis inhibitors such as ANY and CHX. Specifically, we determined that the activity of MPF after in vitro fertilization (IVF) is dependent on the activity of ERK1/2 signaling pathways. In contrast, in chemically activated oocytes, the inactivation of MPF occurs independently of ERK1/2 signaling .Current protocols for inducing the resumption of meiosis of bovine oocytes involve a sequential treatment, using calcium ionophore, specifically ionomycin, followed by treatment with protein kinase inhibitors such as 6-dimethylamino purine (6-DMAP), which inhibits protein phosphorylation, or alternatively with protein synthesis inhibitors such as cycloheximide (CHX) ,40,41,42The present study aimed to analyze the activation efficiency of bovine oocytes using combinations of the aforementioned inhibitors of protein synthesis and phosphorylation. We examined the developmental potential in vitro, the effects on the dynamics of MPF and the ERK1/2 MAPKs pathway, and the quality of bovine parthenogenetic embryos generated by these combinations.First, we investigated the effects of the different treatments on main cell-cycle regulators MPF and MAPKs.Thr161) or the inhibitory (p-CDK1Thr14-Tyr15) residues among the groups, as represented in The results indicated that chemical activation methods involving protein synthesis inhibition (ANY and ANY + CHX) or dual inhibition of protein synthesis and phosphorylation  did not affect the levels of CCNB1 protein, indicating that the chemical resumption of meiosis II of bovine oocytes does not rely on the degradation or loss of CCNB1. The semi-quantitative analysis is shown in p < 0.05); the semi-quantitative analysis is represented in Expression and profile of ERK1/2 phosphorylation followed. We continued to analyze the total expression of ERK1/2. The data indicated that the use of protein synthesis inhibitors (ANY or ANY + CHX) reduced the expression of ERK1/2 compared to dual inhibition  (p < 0.05); the semi-quantitative analysis is represented in For the analysis of phosphorylated ERK1/2 (p-ERK1/2), GAPDH was used as a loading control, since protein synthesis inhibitors affect ERK1/2 expression levels. The analysis showed that the dual inhibition decreased the levels of p-ERK1/2 compared to the treatments based only on protein synthesis inhibition (ANY and ANY + CHX) , which had no effect on CDK1 phosphorylation but had a marked impact on ERK1/2 phosphorylation.Once the effects of the different treatments on main cell-cycle regulators MPF and MAPKs were determined, we evaluated whether the different combinations of inhibitors would impact further embryonic development. The results of 582 oocytes (n = 5) activated using inhibition of protein synthesis or dual inhibition showed no differences in terms of the cleavage  and blastocysts rates , as can be seen in Similarly, the embryo morphology and quality in terms of the total number of cells, allocation to the TE and ICM, and the percentage of cells with fragmented DNA did not show differences among groups; see CDX2 transcript was similar among treatments; see POU5F1 were higher under activation by ANY + CHX + DMAP, compared to the activation with protein synthesis inhibitors (ANY and ANY + CHX) (p < 0.05); see The relative abundance of the BCL2A1 were high in embryos obtained by inhibition using ANY + DMAP compared to those of the other treatments, but it was not statistically significant; see BAX showed lower levels in embryos obtained under activation with ANY and dual inhibition with CHX + DMAP compared to the embryos obtained under activation with ANY + DMAP and ANY + CHX + DMAP treatments (p < 0.05), but the levels were similar to those of the ANY + CHX treatment. The treatment ANY + CHX + DMAP showed the highest level of BAX transcript, which was only similar to those of the ANY + DMAP treatment; see BCL2A1 to BAX (BCL2A1/BAX) showed a significative low level in embryos activated by ANY + CHX + DMAP only when compared to the ANY group (p < 0.05); see BCL2A1/BAX ratio and were not significatively different from the ANY or ANY + CHX + DMAP treatments.On the other hand, the levels of the anti-apoptotic gene Xenopus oocytes induced by phosphorylation inhibition (using DMAP) was characterized by the constant presence of CCNB1 protein. Likewise, the expression of the catalytic subunit of MPF, CDK1 kinase, is not affected by any of the inhibitors tested in this study. Conversely, the analysis of the phosphorylation on the activating (CDK1Thr161) and inhibitory (CDK1Thr14-Tyr15) residues demonstrated that the patterns of phosphorylation induced by all treatments evaluated in this study were consistent with the findings of our previously reported study [In the current study, we utilized combinations of protein synthesis and phosphorylation inhibitors to induce parthenogenetic activation of bovine oocytes. Our data demonstrated that neither inhibition of protein synthesis nor dual inhibition of synthesis and phosphorylation of proteins affected the developmental potential of the oocytes in vitro, up to the blastocyst stage. We focused specifically on the proportions of cleaved embryos and total blastocysts, which are critical indicators of successful embryonic development. Cleaved embryos are those that have undergone the first cell division, while total blastocysts include early, expanded, and hatched stages of development. By comparing the proportions of these different stages of development, we aimed to gain insight into the overall quality of the embryonic development process. However, our analysis revealed no significant differences in the proportions of cleaved embryos and total blastocysts, indicating that there were no variations in embryonic development between the groups being compared. Previous studies in other species have reported that combined inhibition of protein synthesis and phosphorylation improved in vitro developmental potential ,53,54,55ed study . Thus, ted study . It is nThr161 or pCDK1Thr14-Tyr15, generated by the ANY treatment at 4 h post-activation, align with the phosphorylation pattern previously reported by [Thr161 or CDK1Thr14-Tyr15. In this context, owing to the broad-spectrum nature of DMAP as a kinase inhibitor, it is likely that it also has the potential to deactivate specific kinases with intricate inactivation mechanisms, such as CDKs. For instance, Ref. [Furthermore, it is worth noting that the phosphorylation levels at residues pCDK1orted by  at the sce, Ref.  reportedThe dynamics of the inactivation of major MAPKs revealed that the inhibition of the protein synthesis decreased ERK1/2 expression, consistent with the previous literature . InteresFinally, the analysis of embryo quality carried out on the blastocyst produced parthenogenetically showed, in terms of the total number of cells, their cell allocation (MCI or TE), and the presence of DNA damage (number cell TUNEL+). The single or dual inhibition to activate the bovine oocyte did not impact the quality of the parthenogenetic embryos obtained in vitro. These results can be largely explained by the theoretical condition of diploidy of the parthenotes analyzed here, given the use of cytochalasin B in all the parthenogenetic activation treatments, which differs from what was observed by , who repCDX2, a known marker of trophectoderm (TE) differentiation [CDX2 expression have been associated with enhanced embryo developmental competence and quality [POU5F1  is also associated with embryo quality and developmental competence [CDX2 expression indicated similarities among groups. However, when oocytes were activated through the inhibition of both protein synthesis and phosphorylation using ANY + CHX + DMAP, the transcript levels of POU5F1 increased. This could indicate a higher level of embryonic competence. It is worth noting that [POU5F1 gene expression with respect to IVF zygotes. These results indicate that progression to the blastocyst stage of diploid parthenogenetic bovine embryos is not affected by the oocyte activation protocol, regardless of the selected chemical cocktail.In addition, we evaluated the relative abundance of the transcription factor caudal-related homeobox 2 ntiation ,62,63. I quality ,65, and  quality . Similarmpetence ,68,69. Ting that  demonstrBCL2A1 has been documented in oocytes and embryos of good quality, unlike the BAX transcript. [BCL2A1/BAX ratio is a better marker of cell survival or death [BCL2A1/BAX ratio observed for the ANY + CHX + DMAP would suggest a possible pro-apoptotic state in parthenotes derived from oocytes activated with the combination of the three inhibitors. However, this condition was inconsistent with the enhanced apoptotic phenotype in the parthenogenetic embryos. These findings call for further investigation.On the other hand, a higher expression of nscript. ,71. Addior death . TherefoUnless otherwise indicated, all of the chemical agents were acquired from Sigma .v/v) bovine fetal serum , and incubated at 38.5 °C in 5% (v/v) CO2 and saturation humidity.The ovaries were collected in the local slaughterhouse . The cumulus–oocyte complexes (COCs) were aspirated from follicles between 2 and 7 mm. Groups of 50 oocytes were matured in vitro for 24 h in maturation medium TCM-199 containing 6 μg/mL LH, 6 μg/mL FSH, and 1 μg/mL estradiol, supplemented with 10%  supplemented with (i) 1 μg/mL anisomycin (ANY treatment), (ii) 1 μg/mL anisomycin, plus 10 μg/mL cycloheximide (ANY + CHX treatment), (iii) 1 μg/mL anisomycin, plus 1.9 mM 6-dimethylaminopurine (ANY + DMAP treatment), (iv) 1 μg/mL of anisomycin, plus 10 μg/mL cycloheximide, plus 1.9 mM of 6-dimethylaminopurine (ANY + CHX + DMAP treatment) or (v) 10 μg/mL cycloheximide, plus 1.9 mM 6-dimethylaminopurine (CHX + DMAP treatment) for 5 h. All groups included cytochalasin B (5 µg/mL), a blocker of the formation of contractile microfilaments, to avoid extrusion of the second polar body and, by this, to derivate diploid zygotes which behave similarly to IVF embryos in terms of developmental potential [v/v) BME essential amino acids, 1% (v/v) MEM non-essential amino acids, and 4 mg/mL of fatty acid-free bovine serum albumin (BSA). The culture was carried out at 38.5 °C in a gas mixture of 5% CO2, 5% O2, 90% N2, and saturation humidity [After 24 h of maturation, the oocytes were denuded by vortexing in 1 mg/mL hyaluronidase and selected by the presence of the first polar body. The metaphase II (MII) oocytes were assigned randomly to the different activation treatments, consisting of incubation with 5 µM of ionomycin  for 5 min followed by incubation in potassium simplex optimization medium , the point at which the embryos were supplemented with 5% FBS. The embryos were cultured until day seven to record the rate of blastocyst formation.m/v) polyvinyl alcohol (PVA). Then, the oocytes were lysed by boiling for 5 min in sample buffer SDS 2× containing 5% 2-mercaptoethanol and stored at −80 °C until electrophoresis. Electrophoresis was performed in polyacrylamide gel and 12% sodium dodecyl sulfate (m/v) (SDS-PAGE), and then the proteins were transferred to polyvinylidene difluoride (PVDF) membranes . All the antibodies used were diluted in a buffer blot composed of TBS 1×, 0.1% (v/v) Tween-20, and 1% (m/v) BSA free of proteases, fatty acids, and globulins.Analysis of western blotting was performed as previously reported . BrieflyThe membranes were divided into two parts and subsequently incubated with the respective antibodies according to the molecular weight of the target proteins, with the primary antibodies for CCNB1 (55 Kda) , p-CDK1 (34 kDa) phospho Thr161 , p-CDK1 (34 kDa) phospho Thr14, Tyr15 , or p-ERK1/2 (42/44kDa) , followed by incubation with their respective conjugated secondary antibody with horseradish peroxidase (HRP) (1:2000) . In the same membranes after stripping and re-testing with the respective antibodies were detected; CDK1 (34 kDa) , ERK1/2 (42/44 kDa)  and GAPDH (37 kDa) . The immunoreaction was detected using the Westar Supernova chemiluminescence kit  according to the manufacturer’s instructions, and viewed using autoradiography development . The semi-quantification of the relative intensity of the CCNB1:GAPDH, CDK1:GAPDH, p-CDK1:total CDK1, p-ERK1/2:GAPDH, and ERK1/2:GAPDH ratios were quantified by means of optical densitometry of the bands using a free plug-in available for ImageJ 1.53t software  The experiment was performed in 3 different biological replicates corresponding to independent ovary collections on different days.Differential staining was used to assess the total number of cells with DAPI staining and their allocation to the inner cell mass cells (ICM) by immunodetection of SOX2, and indirectly to trophectoderm (TE), by counting SOX2-non-expressing cells in expanded blastocysts generated by parthenogenesis , using a free plug-in available for ImageJ 1.53t software  Immunostaining was performed as has been described previously , with a g for 2 min, and then stored at −80 °C until used. Total RNA was extracted from each pool of embryos  using the PicoPure RNA Isolation Kit  according to the manufacturer’s instructions. Residual genomic DNA was removed by DNase I digestion using the RNase-Free DNase Set . Reverse transcription was carried out using the RevertAid H Minus First Strand Kit .To analyze gene expression, groups of five Day-7 blastocysts from each treatment were lysed in 20 mL of extraction buffer  by incubation at 42 °C for 30 min, followed by centrifugation at 3000× To quantify the expression of six developmentally important genes , real-tiThe ANY blastocysts were used as calibrators and the values are presented as n times relative difference with respect to the calibrator. The MXPro-MX300P Version 4.10 program (Stratagene Agilent Technologies) was used to quantify the relative mRNA expression levels based on the ΔΔCt method and using the amplification efficiency of each gene as a correction factor ,77. The We performed descriptive statistics based on mean and standard deviation, calculated for each of the analyzed variables using Stat Graphics Plus 5.1 software . Each experiment was repeated at least three times with different biological replicates, in which each biological replicate corresponded to independent ovary collections on different days.Raw data were transformed to ratio from the baseline condition.CDX2, POU5F1, BCL2A1, BAX, BCL2A1/BAX ratio, and TUNEL assay, differences between treatments in each experiment were determined by one-way ANOVA. In cases where significant differences were observed, Schffe’s test was used to assess the magnitude of the differences.For calculations of relative quantification of CCNB1, CDK1, ERK1/2, p-ERK1/2, cell allocation analysis, the abundance of mRNA of In order to determine whether there were significant differences in the proportions of cleaved embryos and total blastocysts, we utilized a chi-squared test with Bonferroni’s correction. Our analysis was conducted using the Statgraphics Centurion XV software, version 15.1.02.The analysis of variation of the levels of phosphorylation of activating and inhibitory CDK1 residue was performed using GraphPad Prism 7.0 software , using two-way ANOVA prior to confirmation of the statistical assumptions of normality and homoscedasticity of variance, followed by Tukey’s multiple comparison test.In conclusion, the inactivation of MPF in oocytes activated parthenogenetically by dual inhibition of protein synthesis and phosphorylation is associated with phosphorylation in both activating and inhibitory CDK1 residues (pre-MPF) and constant levels of CCNB1. Moreover, the inactivation of MAPKs differs between inhibitors; the inhibition of protein synthesis reduces the expression of ERK1/2, whereas the dual inhibition inactivates MAPKs by dephosphorylation of ERK1/2. Importantly, the combined inhibition of protein synthesis and phosphorylation does not affect further in vitro development of parthenogenetic bovine embryos. In order to obtain a better understanding, future studies will be aimed at a more in-depth analysis involving global transcriptomic analysis in activated oocytes.To summarize, our findings indicate that utilizing these inhibitor combinations may enhance the effectiveness of reproductive techniques like intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT).