Exploring novel dilazep derivatives as hENT1 inhibitors and potentially covalent molecular tools.

The human equilibrative nucleoside transporter 1 (SLC29A1, hENT1) is a solute carrier that modulates the passive transport of nucleosides and nucleobases, such as adenosine. This nucleoside regulates various physiological processes, such as vasodilation and -constriction, neurotransmission and immune defense. Marketed drugs such as dilazep and dipyridamole have proven useful in cardiovascular afflictions, but the application of hENT1 inhibitors can be beneficial in a number of other diseases. In this study, 39 derivatives of dilazep's close analogue ST7092 were designed, synthesized and subsequently assessed using [3H]NBTI displacement assays and molecular docking. Different substitution patterns of the trimethoxy benzoates of ST7092 reduced interactions within the binding pocket, resulting in diminished hENT1 affinity. Conversely, [3H]NBTI displacement by potentially covalent compounds 14b, 14c, and 14d resulted in high affinities (Ki values between 1.1 and 17.5 nM) for the transporter, primarily by the ability of accommodating the inhibitors in various ways in the binding pocket. However, any indication of covalent binding with amino acid residue C439 remained absent, conceivably as a result of decreased nucleophilic residue reactivity. In conclusion, this research introduces novel dilazep derivatives that are active as hENT1 inhibitors, along with the first high affinity dilazep derivatives equipped with an electrophilic warhead. These findings will aid the rational and structure-based development of novel hENT1 inhibitors and pharmacological tools to study hENT1's function, binding mechanisms, and its relevance in (patho)physiological conditions.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study.

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.

Computational Characterization of Membrane Proteins as Anticancer Targets: Current Challenges and Opportunities.

Cancer remains a leading cause of mortality worldwide and calls for novel therapeutic targets. Membrane proteins are key players in various cancer types but present unique challenges compared to soluble proteins. The advent of computational drug discovery tools offers a promising approach to address these challenges, allowing for the prioritization of "wet-lab" experiments. In this review, we explore the applications of computational approaches in membrane protein oncological characterization, particularly focusing on three prominent membrane protein families: receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and solute carrier proteins (SLCs). We chose these families due to their varying levels of understanding and research data availability, which leads to distinct challenges and opportunities for computational analysis. We discuss the utilization of multi-omics data, machine learning, and structure-based methods to investigate aberrant protein functionalities associated with cancer progression within each family. Moreover, we highlight the importance of considering the broader cellular context and, in particular, cross-talk between proteins. Despite existing challenges, computational tools hold promise in dissecting membrane protein dysregulation in cancer. With advancing computational capabilities and data resources, these tools are poised to play a pivotal role in identifying and prioritizing membrane proteins as personalized anticancer targets.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays.

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches.

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

Collaborative SAR Modeling and Prospective In Vitro Validation of Oxidative Stress Activation in Human HepG2 Cells.

Oxidative stress is the consequence of an abnormal increase of reactive oxygen species (ROS). ROS are generated mainly during the metabolism in both normal and pathological conditions as well as from exposure to xenobiotics. Xenobiotics can, on the one hand, disrupt molecular machinery involved in redox processes and, on the other hand, reduce the effectiveness of the antioxidant activity. Such dysregulation may lead to oxidative damage when combined with oxidative stress overpassing the cell capacity to detoxify ROS. In this work, a green fluorescent protein (GFP)-tagged nuclear factor erythroid 2-related factor 2 (NRF2)-regulated sulfiredoxin reporter (Srxn1-GFP) was used to measure the antioxidant response of HepG2 cells to a large series of drug and drug-like compounds (2230 compounds). These compounds were then classified as positive or negative depending on cellular response and distributed among different modeling groups to establish structure-activity relationship (SAR) models. A selection of models was used to prospectively predict oxidative stress induced by a new set of compounds subsequently experimentally tested to validate the model predictions. Altogether, this exercise exemplifies the different challenges of developing SAR models of a phenotypic cellular readout, model combination, chemical space selection, and results interpretation.

Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer.

PURPOSE: Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer. METHODS: EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed. RESULTS: Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers. CONCLUSION: Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

3DDPDs: describing protein dynamics for proteochemometric bioactivity prediction. A case for (mutant) G protein-coupled receptors.

Proteochemometric (PCM) modelling is a powerful computational drug discovery tool used in bioactivity prediction of potential drug candidates relying on both chemical and protein information. In PCM features are computed to describe small molecules and proteins, which directly impact the quality of the predictive models. State-of-the-art protein descriptors, however, are calculated from the protein sequence and neglect the dynamic nature of proteins. This dynamic nature can be computationally simulated with molecular dynamics (MD). Here, novel 3D dynamic protein descriptors (3DDPDs) were designed to be applied in bioactivity prediction tasks with PCM models. As a test case, publicly available G protein-coupled receptor (GPCR) MD data from GPCRmd was used. GPCRs are membrane-bound proteins, which are activated by hormones and neurotransmitters, and constitute an important target family for drug discovery. GPCRs exist in different conformational states that allow the transmission of diverse signals and that can be modified by ligand interactions, among other factors. To translate the MD-encoded protein dynamics two types of 3DDPDs were considered: one-hot encoded residue-specific (rs) and embedding-like protein-specific (ps) 3DDPDs. The descriptors were developed by calculating distributions of trajectory coordinates and partial charges, applying dimensionality reduction, and subsequently condensing them into vectors per residue or protein, respectively. 3DDPDs were benchmarked on several PCM tasks against state-of-the-art non-dynamic protein descriptors. Our rs- and ps3DDPDs outperformed non-dynamic descriptors in regression tasks using a temporal split and showed comparable performance with a random split and in all classification tasks. Combinations of non-dynamic descriptors with 3DDPDs did not result in increased performance. Finally, the power of 3DDPDs to capture dynamic fluctuations in mutant GPCRs was explored. The results presented here show the potential of including protein dynamic information on machine learning tasks, specifically bioactivity prediction, and open opportunities for applications in drug discovery, including oncology.

Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A3 Receptor Expression.

The adenosine A3 receptor (A3AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A3AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A3AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A3AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A3AR in pathologies.

DrugEx: Deep Learning Models and Tools for Exploration of Drug-Like Chemical Space.

The discovery of novel molecules with desirable properties is a classic challenge in medicinal chemistry. With the recent advancements of machine learning, there has been a surge of de novo drug design tools. However, few resources exist that are user-friendly as well as easily customizable. In this application note, we present the new versatile open-source software package DrugEx for multiobjective reinforcement learning. This package contains the consolidated and redesigned scripts from the prior DrugEx papers including multiple generator architectures, a variety of scoring tools, and multiobjective optimization methods. It has a flexible application programming interface and can readily be used via the command line interface or the graphical user interface GenUI. The DrugEx package is publicly available at https://github.com/CDDLeiden/DrugEx.

Large-Scale Modeling of Sparse Protein Kinase Activity Data.

Protein kinases are a protein family that plays an important role in several complex diseases such as cancer and cardiovascular and immunological diseases. Protein kinases have conserved ATP binding sites, which when targeted can lead to similar activities of inhibitors against different kinases. This can be exploited to create multitarget drugs. On the other hand, selectivity (lack of similar activities) is desirable in order to avoid toxicity issues. There is a vast amount of protein kinase activity data in the public domain, which can be used in many different ways. Multitask machine learning models are expected to excel for these kinds of data sets because they can learn from implicit correlations between tasks (in this case activities against a variety of kinases). However, multitask modeling of sparse data poses two major challenges: (i) creating a balanced train-test split without data leakage and (ii) handling missing data. In this work, we construct a protein kinase benchmark set composed of two balanced splits without data leakage, using random and dissimilarity-driven cluster-based mechanisms, respectively. This data set can be used for benchmarking and developing protein kinase activity prediction models. Overall, the performance on the dissimilarity-driven cluster-based split is lower than on random split-based sets for all models, indicating poor generalizability of models. Nevertheless, we show that multitask deep learning models, on this very sparse data set, outperform single-task deep learning and tree-based models. Finally, we demonstrate that data imputation does not improve the performance of (multitask) models on this benchmark set.

CalcAMP: A New Machine Learning Model for the Accurate Prediction of Antimicrobial Activity of Peptides.

To combat infection by microorganisms host organisms possess a primary arsenal via the innate immune system. Among them are defense peptides with the ability to target a wide range of pathogenic organisms, including bacteria, viruses, parasites, and fungi. Here, we present the development of a novel machine learning model capable of predicting the activity of antimicrobial peptides (AMPs), CalcAMP. AMPs, in particular short ones (<35 amino acids), can become an effective solution to face the multi-drug resistance issue arising worldwide. Whereas finding potent AMPs through classical wet-lab techniques is still a long and expensive process, a machine learning model can be useful to help researchers to rapidly identify whether peptides present potential or not. Our prediction model is based on a new data set constructed from the available public data on AMPs and experimental antimicrobial activities. CalcAMP can predict activity against both Gram-positive and Gram-negative bacteria. Different features either concerning general physicochemical properties or sequence composition have been assessed to retrieve higher prediction accuracy. CalcAMP can be used as an promising prediction asset to identify short AMPs among given peptide sequences.

Proteochemometric Modeling Identifies Chemically Diverse Norepinephrine Transporter Inhibitors.

Solute carriers (SLCs) are relatively underexplored compared to other prominent protein families such as kinases and G protein-coupled receptors. However, proteins from the SLC family play an essential role in various diseases. One such SLC is the high-affinity norepinephrine transporter (NET/SLC6A2). In contrast to most other SLCs, the NET has been relatively well studied. However, the chemical space of known ligands has a low chemical diversity, making it challenging to identify chemically novel ligands. Here, a computational screening pipeline was developed to find new NET inhibitors. The approach increases the chemical space to model for NETs using the chemical space of related proteins that were selected utilizing similarity networks. Prior proteochemometric models added data from related proteins, but here we use a data-driven approach to select the optimal proteins to add to the modeled data set. After optimizing the data set, the proteochemometric model was optimized using stepwise feature selection. The final model was created using a two-step approach combining several proteochemometric machine learning models through stacking. This model was applied to the extensive virtual compound database of Enamine, from which the top predicted 22,000 of the 600 million virtual compounds were clustered to end up with 46 chemically diverse candidates. A subselection of 32 candidates was synthesized and subsequently tested using an impedance-based assay. There were five hit compounds identified (hit rate 16%) with sub-micromolar inhibitory potencies toward NET, which are promising for follow-up experimental research. This study demonstrates a data-driven approach to diversify known chemical space to identify novel ligands and is to our knowledge the first to select this set based on the sequence similarity of related targets.

UnCorrupt SMILES: a novel approach to de novo design.

Generative deep learning models have emerged as a powerful approach for de novo drug design as they aid researchers in finding new molecules with desired properties. Despite continuous improvements in the field, a subset of the outputs that sequence-based de novo generators produce cannot be progressed due to errors. Here, we propose to fix these invalid outputs post hoc. In similar tasks, transformer models from the field of natural language processing have been shown to be very effective. Therefore, here this type of model was trained to translate invalid Simplified Molecular-Input Line-Entry System (SMILES) into valid representations. The performance of this SMILES corrector was evaluated on four representative methods of de novo generation: a recurrent neural network (RNN), a target-directed RNN, a generative adversarial network (GAN), and a variational autoencoder (VAE). This study has found that the percentage of invalid outputs from these specific generative models ranges between 4 and 89%, with different models having different error-type distributions. Post hoc correction of SMILES was shown to increase model validity. The SMILES corrector trained with one error per input alters 60-90% of invalid generator outputs and fixes 35-80% of them. However, a higher error detection and performance was obtained for transformer models trained with multiple errors per input. In this case, the best model was able to correct 60-95% of invalid generator outputs. Further analysis showed that these fixed molecules are comparable to the correct molecules from the de novo generators based on novelty and similarity. Additionally, the SMILES corrector can be used to expand the amount of interesting new molecules within the targeted chemical space. Introducing different errors into existing molecules yields novel analogs with a uniqueness of 39% and a novelty of approximately 20%. The results of this research demonstrate that SMILES correction is a viable post hoc extension and can enhance the search for better drug candidates.

DrugEx v3: scaffold-constrained drug design with graph transformer-based reinforcement learning.

Rational drug design often starts from specific scaffolds to which side chains/substituents are added or modified due to the large drug-like chemical space available to search for novel drug-like molecules. With the rapid growth of deep learning in drug discovery, a variety of effective approaches have been developed for de novo drug design. In previous work we proposed a method named DrugEx, which can be applied in polypharmacology based on multi-objective deep reinforcement learning. However, the previous version is trained under fixed objectives and does not allow users to input any prior information (i.e. a desired scaffold). In order to improve the general applicability, we updated DrugEx to design drug molecules based on scaffolds which consist of multiple fragments provided by users. Here, a  Transformer model was employed to generate molecular structures. The Transformer is a multi-head self-attention deep learning model containing an encoder to receive scaffolds as input and a decoder to generate molecules as output. In order to deal with the graph representation of molecules a novel positional encoding for each atom and bond based on an adjacency matrix was proposed, extending the architecture of the Transformer. The graph Transformer model contains growing and connecting procedures for molecule generation starting from  a given scaffold based on fragments. Moreover, the generator was trained under a reinforcement learning framework to increase the number of desired ligands. As a proof of concept, the method was applied to design ligands for the adenosine A2A receptor (A2AAR) and compared with SMILES-based methods. The results show that 100% of the generated molecules are valid and most of them had a high predicted affinity value towards A2AAR with given scaffolds.

Artificial intelligence in multi-objective drug design.

The factors determining a drug's success are manifold, making de novo drug design an inherently multi-objective optimisation (MOO) problem. With the advent of machine learning and optimisation methods, the field of multi-objective compound design has seen a rapid increase in developments and applications. Population-based metaheuris-tics and deep reinforcement learning are the most commonly used artificial intelligence methods in the field, but recently conditional learning methods are gaining popularity. The former approaches are coupled with a MOO strat-egy which is most commonly an aggregation function, but Pareto-based strategies are widespread too. Besides these and conditional learning, various innovative approaches to tackle MOO in drug design have been proposed. Here we provide a brief overview of the field and the latest innovations.

Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

BACKGROUND AND AIMS: The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. APPROACH AND RESULTS: To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. CONCLUSIONS: We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

Cancer-Associated Mutations of the Adenosine A2A Receptor Have Diverse Influences on Ligand Binding and Receptor Functions.

The adenosine A2A receptor (A2AAR) is a class A G-protein-coupled receptor (GPCR). It is an immune checkpoint in the tumor micro-environment and has become an emerging target for cancer treatment. In this study, we aimed to explore the effects of cancer-patient-derived A2AAR mutations on ligand binding and receptor functions. The wild-type A2AAR and 15 mutants identified by Genomic Data Commons (GDC) in human cancers were expressed in HEK293T cells. Firstly, we found that the binding affinity for agonist NECA was decreased in six mutants but increased for the V275A mutant. Mutations A165V and A265V decreased the binding affinity for antagonist ZM241385. Secondly, we found that the potency of NECA (EC50) in an impedance-based cell-morphology assay was mostly correlated with the binding affinity for the different mutants. Moreover, S132L and H278N were found to shift the A2AAR towards the inactive state. Importantly, we found that ZM241385 could not inhibit the activation of V275A and P285L stimulated by NECA. Taken together, the cancer-associated mutations of A2AAR modulated ligand binding and receptor functions. This study provides fundamental insights into the structure-activity relationship of the A2AAR and provides insights for A2AAR-related personalized treatment in cancer.

Dose escalations in phase I studies: Feasibility of interpreting blinded pharmacodynamic data.

AIMS: During phase I study conduct, blinded data are reviewed to predict the safety of increasing the dose level. The aim of the present study was to describe the probability that effects are observed in blinded evaluations of data in a simulated phase I study design. METHODS: An application was created to simulate blinded pharmacological response curves over time for 6 common safety/efficacy measurements in phase I studies for 1 or 2 cohorts (6 active, 2 placebo per cohort). Effect sizes between 0 and 3 between-measurement standard deviations (SDs) were simulated. Each set of simulated graphs contained the individual response and mean ± SD over time. Reviewers (n = 34) reviewed a median of 100 simulated datasets and indicated whether an effect was present. RESULTS: Increasing effect sizes resulted in a higher chance of the effect being identified by the blinded reviewer. On average, 6% of effect sizes of 0.5 between-measurement SD were correctly identified, increasing to 72% in 3.0 between-measurement SD effect sizes. In contrast, on average 92-95% of simulations with no effect were correctly identified, with little effect of between-measurement variability in single cohort simulations. Adding a dataset of a second cohort at half the simulated dose did not appear to improve the interpretation. CONCLUSION: Our analysis showed that effect sizes <2× the between-measurement SD of the investigated outcome frequently go unnoticed by blinded reviewers, indicating that the weight given to these blinded analyses in current phase I practice is inappropriate and should be re-evaluated.

Cancer-Related Somatic Mutations in Transmembrane Helices Alter Adenosine A1 Receptor Pharmacology.

Overexpression of the adenosine A1 receptor (A1AR) has been detected in various cancer cell lines. However, the role of A1AR in tumor development is still unclear. Thirteen A1AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. We have investigated the pharmacology of the mutations located at the 7-transmembrane domain using a yeast system. Concentration-growth curves were obtained with the full agonist CPA and compared to the wild type hA1AR. H78L3.23 and S246T6.47 showed increased constitutive activity, while only the constitutive activity of S246T6.47 could be reduced to wild type levels by the inverse agonist DPCPX. Decreased constitutive activity was observed on five mutant receptors, among which A52V2.47 and W188C5.46 showed a diminished potency for CPA. Lastly, a complete loss of activation was observed in five mutant receptors. A selection of mutations was also investigated in a mammalian system, showing comparable effects on receptor activation as in the yeast system, except for residues pointing toward the membrane. Taken together, this study will enrich the view of the receptor structure and function of A1AR, enlightening the consequences of these mutations in cancer. Ultimately, this may provide an opportunity for precision medicine for cancer patients with pathological phenotypes involving these mutations.