Investigations around RBM39 degrading arylsulfonamides

The cells in our body are a dynamic system that is constantly adapting to changing conditions. Their function is highly regulated by mechanisms, which go beyond the genome. The genes give the recipe to build the components in the system, but it is the molecular diversity that is introduced on the protein level which is crucial for the function. As such, proteins are the most diversified molecules in our body and have evolved complex networks that are involved in key cellular processes. Within these networks, they can either directly impact processes on a molecular level or indirectly do so by being part of a multi-protein complex. It is hence not surprising that protein misregulation or malfunction is at the bottom of a lot of diseases and is targeted in drug discovery. Recently, the ubiquitin proteasomal pathway (UPP) has been manipulated to mediate targeted protein degradation (TPD) using small molecules. The development of such molecules has emerged from different independent angles, but it was the discovery of the mode-of-action of the clinically approved immunomodulatory drugs (IMiDs) that paved the way. They engage a UPP component (E3 ligase) and induce the ubiquitination and degradation of key proteins. The IMiDs and similar E3 binding molecules were used to develop chimeric molecules (PROTACs) which could degrade other proteins. In this work, we analyzed such a series of arysulfonamides (ArSulfs) that engage the E3 ligase CRL4DCAF15 reminiscent of the IMiDs and degrade the splicing factor RBM39. In the first chapter, we look at PROTACs which were made to degrade the transcription elongation factor BRD4. The molecules entail the ArSulf Indisulam or derivatives thereof and the BRD4 inhibitor (+)JQ1. We show that, although the molecules can degrade BRD4 in a proteasomal-dependent manner, it is independent of the engagement with CRL4DCAF15. Our results show that degradation is CRL-dependent and suggest that it could be a general phenomenon when targeting BRD4 with (+)JQ1. The following chapter summarizes our structural investigations around the RBM39-ArSulf-DCAF15 interaction. We conduct immunoprecipitation and pull-down experiments with the complex. A structure-activity relationship on the ArSulfs with a focus on Indisulam and E7820 helps us identify core elements of the scaffold and a site for modification. We use the site to make more PROTACs. The overall results indicate a very tight and specific interaction between RBM39-ArSulf-DCAF15 that offers little scope for a binary interaction with the ArSulfs in the absence of either protein. Hence, we made Indisulam derivatives with electrophilic warheads, which would engage in the complex but stay attached once RBM39 dissociates. The derivates were made using recently published structural data of the complex. We show the effects of the molecules in cells as well as purified proteins. In the final chapter, we take a closer look at the effects of the ArSulfs in the cells. We look at what other proteins the ArSulfs can degrade but also investigate the other changes seen. We start by conducting tandem-mass tag (TMT) labelled proteomics experiments that help us identify other proteins affected by the treatment. By overlaying our proteomics data with RBM39 eCLIP data we identify splicing targets of RBM39 that show changes in protein levels upon RBM39 degradation. We validate these targets independently and hypothesize that the changes in protein levels come from aberrant splicing. Indeed, we demonstrate using transcript-specific qPCR that the relative level of transcript types can be correlated to the protein level change of RSRP1, as well as the kinesins KIF20A and KIF20B. A STRING network analysis with the significantly regulated proteins correlates with cell cycle and mitosis. Hence, we analyze the effect on the cell cycle with the ArSulfs to see if it can be attributed to the deregulation of the kinesins

Tailored meta-analysis:an investigation of the correlation between the test positive rate and prevalence

Background and Objective: Meta-analysis may produce estimates that are unrepresentative of a test’s performance in practice. Tailored meta-analysis (TMA) circumvents this by deriving an applicable region for the practice and selecting the studies compatible with the region. It requires the test positive rate, r and prevalence, p being estimated for the setting but previous studies have assumed their independence. The aim is to investigate the effects a correlation between r and p has on estimating the applicable region and how this affects TMA. Methods: Six methods for estimating 99% confidence intervals (CI) for r and p were investigated: Wilson’s 6 Bonferroni correction, Clopper-Pearson’s 6 Bonferroni correction, and Hotelling’s T2 statistic 6 continuity correction. These were analyzed in terms of the coverage probability using simulation trials over different correlations, sample sizes, and values for r and p. The methods were then applied to two published meta-analyses with associated practice data, and the effects on the applicable region, studies selected, and summary estimates were evaluated. Results: Hotelling’s T2 statistic with a continuity correction had the highest median coverage (0.9971). This and the Clopper-Pearson method with a Bonferroni correction both had coverage consistently above 0.99. The coverage of Hotelling’s CI’s varied the least across different correlations. For both meta-analyses, the number of studies selected was largest when Hotelling’s T2 statistic was used to derive the applicable region. In one instance, this increased the sensitivity by over 4% compared with TMA estimates using other methods. Conclusion: TMA returns estimates that are tailored to practice providing the applicable region is accurately defined. This is most likely when the CI for r and p are estimated using Hotelling’s T2 statistic with a continuity correction. Potentially, the applicable region may be obtained using routine electronic health data

Divergent Synthesis of Bioactive Dithiodiketopiperazine Natural Products Based on a Double C(sp3)−H Activation Strategy

This article provides a detailed report of our efforts to synthesize the dithiodiketopiperazine (DTP) natural products (−)‐epicoccin G and (−)‐rostratin A using a double C(sp3)−H activation strategy. The strategy's viability was first established on a model system lacking the C8/C8’ alcohols. Then, an efficient stereoselective route including an organocatalytic epoxidation was secured to access a key bis‐triflate substrate. This bis‐triflate served as the functional handles for the key transformation of the synthesis: a double C(sp3)−H activation. The successful double activation opened access to a common intermediate for both natural products in high overall yield and on a multigram scale. After several unsuccessful attempts, this intermediate was efficiently converted to (−)‐epicoccin G and to the more challenging (−)‐rostratin A via suitable oxidation/reduction and protecting group sequences, and via a final sulfuration that occurred in good yield and high diastereoselectivity. These efforts culminated in the synthesis of (−)‐epicoccin G and (−)‐rostratin A in high overall yields (19.6 % over 14 steps and 12.7 % over 17 steps, respectively), with the latter being obtained on a 500 mg scale. Toxicity assessments of these natural products and several analogues (including the newly synthesized epicoccin K) in the leukemia cell line K562 confirmed the importance of the disulfide bridge for activity and identified dianhydrorostratin A as a 20x more potent analogue

Dynamic kinetic energy potential for orbital-free density functional theory

A dynamic kinetic energy potential (DKEP) is developed for time-dependent orbital-free (TDOF) density function theory applications. This potential is constructed to affect only the dynamical (? = 0) response of an orbital-free electronic system. It aims at making the orbital-free simulation respond in the same way as that of a noninteracting homogenous electron gas (HEG), as required by a correct kinetic energy, therefore enabling extension of the success of orbital-free density functional theory in the static case (e.g., for embedding and description of processes in bulk materials) to dynamic processes. The potential is constructed by expansions of terms, each of which necessitates only simple time evolution (concurrent with the TDOF evolution) and a spatial convolution at each time-step. With 14 such terms a good fit is obtained to the response of the HEG at a large range of frequencies, wavevectors, and densities. The method is demonstrated for simple jellium spheres, approximating Na9 + and Na65+ clusters. It is applicable both to small and large (even ultralarge) excitations and the results converge (i.e., do not blow up) as a function of time. An extension to iterative frequency-resolved extraction is briefly outlined, as well as possibly numerically simpler expansions. The approach could also be extended to fit, instead of the HEG susceptibility, either an experimental susceptibility or a theoretically derived one for a non-HEG system. The DKEP potential should be a powerful tool for embedding a dynamical system described by a more accurate method (such as time-dependent density functional theory, TDDFT) in a large background described by TDOF with a DKEP potential. The type of expansions used and envisioned should be useful for other approaches, such as memory functionals in TDDFT. Finally, an appendix details the formal connection between TDOF and TDDFT.The Journal of Chemical Physics 134(14), 144101. (2011)0021-960

The optimal second-line therapy for older adults with type 2 diabetes mellitus: protocol for a systematic review and network meta-analysis using individual participant data (IPD)

Background: Due to increasing life expectancy, almost half of people with type 2 diabetes are aged 65 years or over worldwide. When metformin alone does not control blood sugar, the choice of which second-line therapy to prescribe next is not clear from currently available evidence. The existence of frailty and comorbidities in older adults further increases the complexity of medical decision-making. As only a relatively small proportion of trials report results separately for older adults, the relative efficacy and safety of second-line therapies in older adults with type 2 diabetes mellitus are unknown and require further investigation. This individual participant data (IPD) network meta-analysis evaluates the relative efficacy and safety of second-line therapies on their own or in combination in older adults with type 2 diabetes mellitus. Methods: All relevant published and unpublished trials will be identified. Studies published prior to 2015 will be identified from two previous comprehensive aggregate data network meta-analyses. Searches will be conducted in CENTRAL, MEDLINE, and EMBASE from 1st January 2015 onwards, and in clinicaltrials.gov from inception. Randomised controlled trials with at least 100 estimated older adults (≥ 65 years) receiving at least 24 weeks of intervention that assess the effects of glucose-lowering drugs on mortality, glycemia, vascular and other comorbidities outcomes, and quality of life will be eligible. The screening and data extraction process will be conducted independently by two researchers. The quality of studies will be assessed using the Cochrane risk of bias tool 2. Anonymised IPD of all eligible trials will be requested via clinical trial portals or by contacting the principal investigators or sponsors. Received data will be reanalysed where necessary to standardise outcome metrics. Network meta-analyses will be performed to determine the relative effectiveness of therapies. Discussion: With the increasing number of older adults with type 2 diabetes worldwide, an IPD network meta-analysis using data from all eligible trials will provide new insights into the optimal choices of second-line antidiabetic drugs to improve patient management and reduce unnecessary adverse events and the subsequent risk of comorbidities in older adults. Systematic review registration: PROSPERO CRD42021272686

Diet and physical activity in pregnancy to prevent gestational diabetes:A protocol for an individual participant data (IPD) meta-analysis on the differential effects of interventions with economic evaluation

IntroductionMothers with gestational diabetes (GDM) are at increased risk of pregnancy-related complications and developing type-2 diabetes after delivery. Diet and physical-activity based interventions may prevent GDM, but variations in populations, interventions and outcomes in primary trials have limited the translation of available evidence into practice. We plan to undertake an individual participant data (IPD) meta-analysis of randomised trials to assess the differential effects and cost-effectiveness of diet and physical-activity based interventions in preventing GDM and its complications.MethodsThe International Weight Management in Pregnancy Collaborative Network database is a living repository of IPD from randomised trials on diet and physical-activity in pregnancy identified through a systematic literature search. We shall update our existing search on MEDLINE, EMBASE, BIOSIS, LILACS, Pascal, Science Citation Index, CDSR, CENTRAL, DARE and HTA Database without language restriction, to identify relevant trials until March 2021. Primary researchers will be invited to join the Network and share their IPD. Trials including women with GDM at baseline will be excluded. We shall perform a one and two stage random effects meta-analysis for each intervention type (all interventions, diet-based, physical-activity based, and mixed approach), to obtain summary intervention effects on GDM with 95% confidence intervals, and summary treatment-covariate interactions. Heterogeneity will be summarised using I2 and tau2 statistics with 95% prediction intervals. Publication and availability bias will be assessed by examining small-study effects. Study quality of included trials will be assessed by the Cochrane Risk of Bias tool, and the GRADE approach will be used to grade the evidence in the results. A model-based economic analysis will be carried out to assess the cost-effectiveness of interventions to prevent GDM and its complications compared to usual care. Dissemination Ethics approval is not required. The study is registered on PROSPERO (CRD42020212884). Results will be submitted for publication in peer-reviewed journals. Strengths and Limitations• The IPD meta-analysis builds on an established global group’s (i-WIP) living database (24,766 women, 58 trials) with the potential to expand further with additional published data• Increased power in IPD compared to aggregate meta-analysis to determine the differential effects of lifestyle interventions on gestational diabetes• Detailed mapping of the of the intervention components will enable translation of the results to clinical practice. • Limitations include potential lack of access to new data and missingness in available data. <br/

A multicomponent reaction-based platform opens new avenues in Aryl Hydrocarbon Receptor modulation

A multidisciplinary platform is presented to address aryl hydrocarbon receptor (AhR) modulation. A rewired Yonemitsu multicomponent reaction with indole 2-carboxaldehydes and nucleophilic species was designed to access a family of 6-substituted indolocarbazoles. The conformational behavior of these compounds was examined to rationalize their axial chirality. In silico docking and molecular simulations highlighted key features implicated in their binding to AhR. Furthermore, the synthesis of linkable derivatives allowed the direct development of conjugated entities. Reporter gene and target gene expression analyses identified these novel structures as potent noncytotoxic activating AhR ligands, that can be extended to bifunctional molecules. The anti-inflammatory properties of these AhR agonists were assessed in interleukin-13 treated keratinocytes. Altogether, the synergistic research in synthetic and computational chemistry integrated with biological studies opens novel avenues toward understanding the biological roles of AhR and the development of targeted therapeutics