Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology.

Accelerating the Validation of Endogenous On-Target Engagement and In Cellulo Kinetic Assessment for Covalent Inhibitors of KRAS in Early Drug Discovery.

Covalent inhibition is a valuable modality in drug discovery because of its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in vitro inhibition assessment to the appropriate in cellulo target engagement parameter by covalent probes.

Structure-Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family.

The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca-independent production of -acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive -acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified α-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor.

Resurrecting the phoenix: When an assay fails.

Understanding protein-small-molecule interactions is a critical component of rational drug-design. Structure-activity relationship (SAR)-guided medicinal chemistry is informed by the biological outcome, as assessed by biochemical activity or cellular effect, of chemical modifications on small molecules. The effectiveness of SAR is reliant on the sturdiness and durability of assay design and the quality of information garnered from assays.

Identification of α,β-Hydrolase Domain Containing Protein 6 as a Diacylglycerol Lipase in Neuro-2a Cells.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid (RA)-induced neurite outgrowth of Neuro-2a cells. First, we confirmed that RA stimulation of Neuro-2a cells increases 2-AG production and neurite outgrowth.

Mechanistic and Quantitative Understanding of Pharmacokinetics in Zebrafish Larvae through Nanoscale Blood Sampling and Metabolite Modeling of Paracetamol.

Zebrafish larvae are increasingly used for pharmacological research, but internal drug exposure is often not measured. Understanding pharmacokinetics is necessary for reliable translation of pharmacological results to higher vertebrates, including humans. Quantification of drug clearance and distribution requires measurements of blood concentrations.

Impact of post-hatching maturation on the pharmacokinetics of paracetamol in zebrafish larvae.

Zebrafish larvae are increasingly used in pharmacological and toxicological studies, but it is often overlooked that internal exposure to exogenous compounds, rather than the incubation medium concentration, is driving observed effects. Moreover, as the zebrafish larva is a developing organism, continuous physiological changes impact pharmacokinetic or toxicokinetic processes like the absorption and elimination of exogenous compounds, influencing the interpretation of observations and conclusions drawn from experiments at different larval ages. Here, using paracetamol as paradigm compound, mathematical modelling is used to quantify absorption and elimination rates from internal exposure over time profiles after waterborne treatment, as well as changes in these parameters in post-hatching larvae of 3, 4, and 5 days post fertilisation (dpf).

High Fat Diet Increases Circulating Endocannabinoids Accompanied by Increased Synthesis Enzymes in Adipose Tissue.

The endocannabinoid system (ECS) controls energy balance by regulating both energy intake and energy expenditure. Endocannabinoid levels are elevated in obesity suggesting a potential causal relationship. This study aimed to elucidate the rate of dysregulation of the ECS, and the metabolic organs involved, in diet-induced obesity.

Activity-Based Protein Profiling Identifies α-Ketoamides as Inhibitors for Phospholipase A2 Group XVI.

Phospholipase A2, group XVI (PLA2G16) is a thiol hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of picornaviruses. Chemical tools are essential to visualize and control PLA2G16 activity, but they have not been reported to date. Here, we show that MB064, which is a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16.

Gene Expression of Endocannabinoid System Components in Skeletal Muscle and Adipose Tissue of South Asians and White Caucasians with Overweight.

Objective: The study aimed to investigate whether markers of endocannabinoid signaling differed between men with overweight of South Asian and white Caucasian descent.

Methods: We included South Asian (n = 10) and white Caucasian (n = 10) men with overweight and prediabetes aged 35 to 50 years. Plasma samples were analyzed for endocannabinoids, their congeners, and lipids.

Caloric restriction lowers endocannabinoid tonus and improves cardiac function in type 2 diabetes.

Background/objectives: Endocannabinoids (ECs) are associated with obesity and ectopic fat accumulation, both of which play a role in the development of cardiovascular disease (CVD) in type 2 diabetes (T2D). The effect of prolonged caloric restriction on ECs in relation to fat distribution and cardiac function is still unknown. Therefore, our aim was to investigate this relationship in obese T2D patients with coronary artery disease (CAD).

Endocannabinoid tone is higher in healthy lean South Asian than white Caucasian men.

South Asians have a higher risk to develop obesity and related disorders compared to white Caucasians. This is likely in part due to their lower resting energy expenditure (REE) as related with less energy-combusting brown adipose tissue (BAT). Since overactivation of the endocannabinoid system is associated with obesity and low BAT activity, we hypothesized that South Asians have a higher endocannabinoid tone.

Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474.

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues.

Integration of pharmacometabolomics with pharmacokinetics and pharmacodynamics: towards personalized drug therapy.

Personalized medicine, in modern drug therapy, aims at a tailored drug treatment accounting for inter-individual variations in drug pharmacology to treat individuals effectively and safely. The inter-individual variability in drug response upon drug administration is caused by the interplay between drug pharmacology and the patients' (patho)physiological status. Individual variations in (patho)physiological status may result from genetic polymorphisms, environmental factors (including current/past treatments), demographic characteristics, and disease related factors.

Quantitative profiling of endocannabinoids and related -acylethanolamines in human CSF using nano LC-MS/MS.

Endocannabinoids, a class of lipid messengers, have emerged as crucial regulators of synaptic communication in the CNS. Dysregulation of these compounds has been implicated in many brain disorders. Although some studies have identified and quantified a limited number of target compounds, a method that provides comprehensive quantitative information on endocannabinoids and related -acylethanolamines (NAEs) in cerebrospinal fluid (CSF) is currently lacking, as measurements are challenging due to low concentrations under normal physiological conditions.

Pharmacokinetic Modeling of Paracetamol Uptake and Clearance in Zebrafish Larvae: Expanding the Allometric Scale in Vertebrates with Five Orders of Magnitude.

Zebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling.

Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons.

Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes.