Investigating bisubstrate inhibitors of PRMT1 and CARM1

Protein arginine methyltransferases (PRMTs) are gaining traction as a novel drug target class for a wide range of diseases. The high sequence conservation within the active sites of the nine human PRMTs, particularly in the co-factor (S-adenosyl methionine [SAM]) binding site, makes the design of isozymeselective PRMT inhibitors challenging. Linking guanidine or substrate peptides to SAM mimics has shown potential as an approach to designing isozymeselective inhibitors, but the peptidic nature of such compounds limits their in vivo utility. Herein, the binding of a novel series of non-peptidic bisubstrate inhibitors to CARM1 and PRMT1 was evaluated. The bisubstrate nature of the inhibitors was confirmed by crystal structures of the inhibitors bound to CARM1. Isothermal titration calorimetry (ITC) and activity assays showed that varying the alkyl linker length between the guanidine and adenosine moieties of the inhibitors tested does not confer selectivity for PRMT1 or CARM1. These findings were supported by co-crystal structures of the inhibitors bound to CARM1, which showed that the guanidinium group is thermally mobile within the CARM1 active site. Conversely, isosteric replacement of the substrate-mimic with an N-alkyl-2- aminopyridine group was found to confer selectivity for CARM1 over PRMT1 (Kd values of 1.1 μM and 43.7 μM, respectively, for the most selective inhibitor). A CARM1 N265Y mutant displayed a significant reduction in affinity for the CARM1-selective aminopyridine inhibitor (judged by ITC), suggesting that this residue, located in the substrate binding channel, may contribute to the CARM1-selectivity of this inhibitor. The substitution of ribose for a morpholino group in PRMT bisubstrate inhibitors was found to abrogate inhibitor binding to PRMT1 and CARM1 in the majority of cases. Additionally, two putative FRET probes designed to bind to PRMT1 and CARM1 were designed and partially synthesised

Structural and biochemical evaluation of bisubstrate inhibitors of protein arginine N-methyltransferases PRMT1 and CARM1 (PRMT4)

Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the co-factor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts

Childhood adversity and self-poisoning:A hospital case control study in Sri Lanka

This is the final version. Available on open access from Public Library of Science via the DOI in this recordData Availability: The research data is available as follows: Data are available at the University of Bristol data repository, data.bris, at https://doi.org/10.5523/bris.37pg6mv6x35r12b98aoq4blcgs.” In a reference list: Knipe, D., Kidger, J. (2020): Data from ACE & Self harm Sri Lanka (10-2020). https://doi.org/10.5523/bris.37pg6mv6x35r12b98aoq4blcgs. The dataset appears in the repository catalogue, on Explore Bristol Research, and in the DataCite registry.Introduction Adverse childhood experiences (ACE) have been recognized as an important risk factor for suicidal behaviour among adults, but evidence from low and middle-income countries is lacking. This study explored associations between ACE and hospital admission due to non-fatal self-poisoning in Sri Lanka. Methods This was a case-control study. Adults admitted to a tertiary care hospital for medical management of self-poisoning were included as cases, and age and sex matched controls were recruited from the outpatient department. ACE were measured using the World Health Organization’s Childhood Adversity Scale. Logistic regression models adjusting for age, sex, ethnicity, and religion were used to quantify the association between ACE and self-poisoning. Results The study included 235 cases and 451 controls. Cases were 2.5 times (95% CI 1.8, 3.6) more likely to report an ACE than controls and had higher ACE scores. Childhood physical abuse (OR 4.7, 95% CI 1.2, 19.0) and emotional abuse or neglect (OR 3.7, 95% CI 1.3, 10.1, and 3.7, 95% CI 2.3, 6.0 respectively), increased the risk of self-poisoning in adulthood, as did witnessing household violence (OR 2.2, 95% CI 1.4, 3.4), growing up in a household with a mentally ill or suicidal household member (OR 2.1, 95% CI 1.2, 3.4), and experiencing parental death/separation/divorce (OR 3.1, 95% CI 2.0, 4.9) as a child. Conclusions Reducing exposures to ACEs should be a priority for prevention of suicide and self-harm in Sri Lanka. Innovative methods to increase support for children facing adversity should be explored.Medical Research Council (MRC)Elizabeth Blackwell Institute for Health Research, University of BristolWellcome Trus

Childhood adversity, pubertal timing and self-harm: a longitudinal cohort study.

BACKGROUND: The occurrence of early childhood adversity is strongly linked to later self-harm, but there is poor understanding of how this distal risk factor might influence later behaviours. One possible mechanism is through an earlier onset of puberty in children exposed to adversity, since early puberty is associated with an increased risk of adolescent self-harm. We investigated whether early pubertal timing mediates the association between childhood adversity and later self-harm. METHODS: Participants were 6698 young people from a UK population-based birth cohort (ALSPAC). We measured exposure to nine types of adversity from 0 to 9 years old, and self-harm when participants were aged 16 and 21 years. Pubertal timing measures were age at peak height velocity (aPHV - males and females) and age at menarche (AAM). We used generalised structural equation modelling for analyses. RESULTS: For every additional type of adversity; participants had an average 12-14% increased risk of self-harm by 16. Relative risk (RR) estimates were stronger for direct effects when outcomes were self-harm with suicidal intent. There was no evidence that earlier pubertal timing mediated the association between adversity and self-harm [indirect effect RR 1.00, 95% confidence interval (CI) 1.00-1.00 for aPHV and RR 1.00, 95% CI 1.00-1.01 for AAM]. CONCLUSIONS: A cumulative measure of exposure to multiple types of adversity does not confer an increased risk of self-harm via early pubertal timing, however both childhood adversity and early puberty are risk factors for later self-harm. Research identifying mechanisms underlying the link between childhood adversity and later self-harm is needed to inform interventions.Medical Research Foundation and Medical Research Council (MR/R004889/1)

An exploration of the genetic epidemiology of non-suicidal self-harm and suicide attempt.

BACKGROUND: Empirical evidence supporting the distinction between suicide attempt (SA) and non-suicidal self-harm (NSSH) is lacking. Although NSSH is a risk factor for SA, we do not currently know whether these behaviours lie on a continuum of severity, or whether they are discrete outcomes with different aetiologies. We conducted this exploratory genetic epidemiology study to investigate this issue further. METHODS: We explored the extent of genetic overlap between NSSH and SA in a large, richly-phenotyped cohort (the Avon Longitudinal Study of Parents and Children; N = 4959), utilising individual-level genetic and phenotypic data to conduct analyses of genome-wide complex traits and polygenic risk scores (PRS). RESULTS: The single nucleotide polymorphism heritability of NSSH was estimated to be 13% (SE 0.07) and that of SA to be 0% (SE 0.07). Of the traits investigated, NSSH was most strongly correlated with higher IQ (rG = 0.31, SE = 0.22), there was little evidence of high genetic correlation between NSSH and SA (rG = - 0.1, SE = 0.54), likely due to the low heritability estimate for SA. The PRS for depression differentiated between those with NSSH and SA in multinomial regression. The optimal PRS prediction model for SA (Nagelkerke R2 0.022, p < 0.001) included ADHD, depression, income, anorexia and neuroticism and explained more variance than the optimal prediction model for NSSH (Nagelkerke R2 0.010, p < 0.001) which included ADHD, alcohol consumption, autism spectrum conditions, depression, IQ, neuroticism and suicide attempt. CONCLUSIONS: Our findings suggest that SA does not have a large genetic component, and that although NSSH and SA are not discrete outcomes there appears to be little genetic overlap between the two. The relatively small sample size and resulting low heritability estimate for SA was a limitation of the study. Combined with low heritability estimates, this implies that family or population structures in SA GWASs may contribute to signals detected

An exploration of the genetic epidemiology of non-suicidal self-harm and suicide attempt

Abstract: Background: Empirical evidence supporting the distinction between suicide attempt (SA) and non-suicidal self-harm (NSSH) is lacking. Although NSSH is a risk factor for SA, we do not currently know whether these behaviours lie on a continuum of severity, or whether they are discrete outcomes with different aetiologies. We conducted this exploratory genetic epidemiology study to investigate this issue further. Methods: We explored the extent of genetic overlap between NSSH and SA in a large, richly-phenotyped cohort (the Avon Longitudinal Study of Parents and Children; N = 4959), utilising individual-level genetic and phenotypic data to conduct analyses of genome-wide complex traits and polygenic risk scores (PRS). Results: The single nucleotide polymorphism heritability of NSSH was estimated to be 13% (SE 0.07) and that of SA to be 0% (SE 0.07). Of the traits investigated, NSSH was most strongly correlated with higher IQ (rG = 0.31, SE = 0.22), there was little evidence of high genetic correlation between NSSH and SA (rG = − 0.1, SE = 0.54), likely due to the low heritability estimate for SA. The PRS for depression differentiated between those with NSSH and SA in multinomial regression. The optimal PRS prediction model for SA (Nagelkerke R2 0.022, p < 0.001) included ADHD, depression, income, anorexia and neuroticism and explained more variance than the optimal prediction model for NSSH (Nagelkerke R2 0.010, p < 0.001) which included ADHD, alcohol consumption, autism spectrum conditions, depression, IQ, neuroticism and suicide attempt. Conclusions: Our findings suggest that SA does not have a large genetic component, and that although NSSH and SA are not discrete outcomes there appears to be little genetic overlap between the two. The relatively small sample size and resulting low heritability estimate for SA was a limitation of the study. Combined with low heritability estimates, this implies that family or population structures in SA GWASs may contribute to signals detected

Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway