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Abstract Background Two types of methods are used to assess learning curves: outcome assessment and process assessment. Outcome measures are usually dichotomous rare events like complication rates and survival or require an extensive follow-up and are therefore often inadequate to monitor individual learning curves. Time-action analysis (TAA) is a tool to objectively determine the level of efficiency of individual steps of a surgical procedure. Methods/Design We are currently using TAA to determine the number of cases needed for surgeons to reach proficiency with a new innovative hip implant prior to initiating a multicentre RCT. By analysing the unedited video recordings of the first 20 procedures of each surgeon the number and duration of the actions needed for a surgeon to achieve his goal and the efficiency of these actions is measured. We constructed a taxonomy or list of actions which together describe the complete surgical procedure. In the taxonomy we categorised the procedure in 5 different Goal Oriented Phases (GOP): 1. the incision phase 2. the femoral phase 3. the acetabulum phase 4. the stem phase 5. the closure pase Each GOP was subdivided in Goal Oriented Actions (GOA) and each GOA is subdivided in Separate Actions (SA) thereby defining all the necessary actions to complete the procedure. We grouped the SAs into GOAs since it would not be feasible to measure each SA. Using the video recordings, the duration of each GOA was recorded as well as the amount of delay. Delay consists of repetitions, waiting and additional actions. The nett GOA time is the total GOA time – delay and is a representation of the level of difficulty of each procedure. Efficiency is the percentage of nett GOA time during each procedure. Discussion This allows the construction of individual learning curves, assessment of the final skill level for each surgeon and comparison of different surgeons prior to participation in an RCT. We believe an objective and comparable assessment of skill level by process assessment can improve the value of a surgical RCT in situations where a learning curve is expected.

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells., Competing Interests: Declaration of interests G.M.S., V.F.V., and L.R.W. are employees of Vividion Therapeutics, and B.F.C. is a founder and member of the Board of Directors of Vividion Therapeutics. G.W.Y. is a co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y.’s interests have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. A US provisional patent has been filed related to the work disclosed in this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hypertension and kidney disease have been repeatedly associated with genomic variants and alterations of lysine metabolism. Here, we combined stable isotope labeling with untargeted metabolomics to investigate lysine's metabolic fate in vivo. Dietary 13 C 6 labeled lysine was tracked to lysine metabolites across various organs. Globally, lysine reacts rapidly with molecules of the central carbon metabolism, but incorporates slowly into proteins and acylcarnitines. Lysine metabolism is accelerated in a rat model of hypertension and kidney damage, chiefly through N-alpha-mediated degradation. Lysine administration diminished development of hypertension and kidney injury. Protective mechanisms include diuresis, further acceleration of lysine conjugate formation, and inhibition of tubular albumin uptake. Lysine also conjugates with malonyl-CoA to form a novel metabolite Nε-malonyl-lysine to deplete malonyl-CoA from fatty acid synthesis. Through conjugate formation and excretion as fructoselysine, saccharopine, and Nε-acetyllysine, lysine lead to depletion of central carbon metabolites from the organism and kidney. Consistently, lysine administration to patients at risk for hypertension and kidney disease inhibited tubular albumin uptake, increased lysine conjugate formation, and reduced tricarboxylic acid (TCA) cycle metabolites, compared to kidney-healthy volunteers. In conclusion, lysine isotope tracing mapped an accelerated metabolism in hypertension, and lysine administration could protect kidneys in hypertensive kidney disease., (© 2022. The Author(s).)

LPCAT3 is an integral membrane acyltransferase in the Lands cycle responsible for generating C20:4 phospholipids and has been implicated in key biological processes such as intestinal lipid absorption, lipoprotein assembly, and ferroptosis. Small-molecule inhibitors of LPCAT3 have not yet been described and would offer complementary tools to genetic models of LPCAT3 loss, which causes neonatal lethality in mice. Here, we report the discovery by high-throughput screening of a class of potent, selective, and cell-active inhibitors of LPCAT3. We provide evidence that these compounds inhibit LPCAT3 in a biphasic manner, possibly reflecting differential activity at each subunit of the LPCAT3 homodimer. LPCAT3 inhibitors cause rapid rewiring of polyunsaturated phospholipids in human cells that mirrors the changes observed in LPCAT3 -null cells. Notably, these changes include not only the suppression of C20:4 phospholipids but also corresponding increases in C22:4 phospholipids, providing a potential mechanistic explanation for the partial but incomplete protection from ferroptosis observed in cells with pharmacological or genetic disruption of LPCAT3.

The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue., Competing Interests: Declaration of interests The design, steps, and applications of CATCH are covered in pending patent application material from The Scripps Research Institute. B.F.C. is a member of the Cell advisory board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Monoacylglycerol lipase (MAGL) is a 33 kDa serine protease primarily responsible for hydrolyzing 2-arachidonoylglycerol into the proinflammatory eicosanoid precursor arachidonic acid in the central nervous system. Inhibition of MAGL constitutes an attractive therapeutic concept for treating psychiatric disorders and neurodegenerative diseases. Herein, we present the design and synthesis of multiple reversible MAGL inhibitor candidates based on a piperazinyl azetidine scaffold. Compounds 10 and 15 were identified as the best-performing reversible MAGL inhibitors by pharmacological evaluations, thus channeling their radiolabeling with fluorine-18 in high radiochemical yields and favorable molar activity. Furthermore, evaluation of [ 18 F] 10 and [ 18 F] 15 ([ 18 F]MAGL-2102) by autoradiography and positron emission tomography (PET) imaging in rodents and nonhuman primates demonstrated favorable brain uptakes, heterogeneous radioactivity distribution, good specific binding, and adequate brain kinetics, and [ 18 F] 15 demonstrated a better performance. In conclusion, [ 18 F] 15 was found to be a suitable PET radioligand for the visualization of MAGL, harboring potential for the successful translation into humans.

The cis -stereoisomers of Δ 9 -THC [(-)- 3 and (+)- 3 ] were identified and quantified in a series of low-THC-containing varieties of Cannabis sativa registered in Europe as fiber hemp and in research accessions of cannabis. While Δ 9 - cis -THC ( 3 ) occurs in cannabis fiber hemp in the concentration range of (-)-Δ 9 - trans -THC [(-)- 1 ], it was undetectable in a sample of high-THC-containing medicinal cannabis. Natural Δ 9 - cis -THC ( 3 ) is scalemic (ca. 80-90% enantiomeric purity), and the absolute configuration of the major enantiomer was established as 6a S ,10a R [(-)- 3 ] by chiral chromatographic comparison with a sample available by asymmetric synthesis. The major enantiomer, (-)-Δ 9 - cis -THC [(-)- 3 ], was characterized as a partial cannabinoid agonist in vitro and elicited a full tetrad response in mice at 50 mg/kg doses. The current legal discrimination between narcotic and non-narcotic cannabis varieties centers on the contents of "Δ 9 -THC and isomers" and needs therefore revision, or at least a more specific wording, to account for the presence of Δ 9 - cis -THCs [(+)- 3 and (-)- 3 ] in cannabis fiber hemp varieties.

As a serine hydrolase, monoacylglycerol lipase (MAGL) is principally responsible for the metabolism of 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS), leading to the formation of arachidonic acid (AA). Dysfunction of MAGL has been associated with multiple CNS disorders and symptoms, including neuroinflammation, cognitive impairment, epileptogenesis, nociception and neurodegenerative diseases. Inhibition of MAGL provides a promising therapeutic direction for the treatment of these conditions, and a MAGL positron emission tomography (PET) probe would greatly facilitate preclinical and clinical development of MAGL inhibitors. Herein, we design and synthesize a small library of fluoropyridyl-containing MAGL inhibitor candidates. Pharmacological evaluation of these candidates by activity-based protein profiling identified 14 as a lead compound, which was then radiolabeled with fluorine-18 via a facile S N Ar reaction to form 2-[ 18 F]fluoropyridine scaffold. Good blood-brain barrier permeability and high in vivo specific binding was demonstrated for radioligand [ 18 F] 14 (also named as [ 18 F]MAGL-1902). This work may serve as a roadmap for clinical translation and further design of potent 18 F-labeled MAGL PET tracers., Competing Interests: The authors have no conflicts of interest to declare., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

The endocannabinoid system (ECS) is involved in a wide range of biological functions and comprises cannabinoid receptors and enzymes responsible for endocannabinoid synthesis and degradation. Over the past 2 decades, significant advances toward developing drugs and positron emission tomography (PET) tracers targeting different components of the ECS have been made. Herein, we summarized the recent development of PET tracers for imaging cannabinoid receptors 1 (CB1R) and 2 (CB2R) as well as the key enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), particularly focusing on PET neuroimaging applications. State-of-the-art PET tracers for the ECS will be reviewed including their chemical design, pharmacological properties, radiolabeling, as well as preclinical and human PET imaging. In addition, this review addresses the current challenges for ECS PET biomarker development and highlights the important role of PET ligands to study disease pathophysiology as well as to facilitate drug discovery.

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders., Competing Interests: Declaration of Interests B.F.C. is a founder and scientific advisor to Vividion Therapeutics. B.F.C., V.M.C., B.M., D.R., M.A.S., E.V.V., X.Z., and M.Y. are co-inventors on a patent application related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dysfunction of monoacylglycerol lipase (MAGL) is associated with several psychopathological disorders, including drug addiction and neurodegenerative diseases. Herein we design, synthesize, and evaluate several irreversible fluorine-containing MAGL inhibitors for positron emission tomography (PET) ligand development. Compound 6 (identified from a therapeutic agent) was advanced for 18 F-labeling via a novel spirocyclic iodonium ylide (SCIDY) strategy, which demonstrated high brain permeability and excellent specific binding. This work supports further development of novel 18 F-labeled MAGL PET probes.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the endocannabinoid system (eCB). Selective inhibition of MAGL has emerged as a potential therapeutic approach for the treatment of diverse pathological conditions, including chronic pain, inflammation, cancer, and neurodegeneration. Herein, we disclose a novel array of reversible and irreversible MAGL inhibitors by means of "tail switching" on a piperazinyl azetidine scaffold. We developed a lead irreversible-binding MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with 11 C or 18 F. [ 11 C]8 ([ 11 C]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible radioligands [ 11 C]17 ([ 11 C]PAD) and [ 18 F]37 ([ 18 F]MAGL-4-11) also demonstrated excellent in vivo binding specificity toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission tomography tracers with tunability in reversible and irreversible binding mechanisms.

Phytochemical studies on the liverwort Radula genus have previously identified the bibenzyl (-)- cis -perrottetinene ( cis -PET), which structurally resembles (-)-Δ 9 - trans -tetrahydrocannabinol (Δ 9 - trans -THC) from Cannabis sativa L. Radula preparations are sold as cannabinoid-like legal high on the internet, even though pharmacological data are lacking. Herein, we describe a versatile total synthesis of (-)- cis -PET and its (-)- trans diastereoisomer and demonstrate that both molecules readily penetrate the brain and induce hypothermia, catalepsy, hypolocomotion, and analgesia in a CB1 receptor-dependent manner in mice. The natural product (-)- cis -PET was profiled on major brain receptors, showing a selective cannabinoid pharmacology. This study also uncovers pharmacological differences between Δ 9 -THC and PET diastereoisomers. Most notably, (-)- cis -PET and (-)- trans -PET significantly reduced basal brain prostaglandin levels associated with Δ 9 - trans -THC side effects in a CB1 receptor-dependent manner, thus mimicking the action of the endocannabinoid 2-arachidonoyl glycerol. Therefore, the natural product (-)- cis -PET is a psychoactive cannabinoid from bryophytes, illustrating the existence of convergent evolution of bioactive cannabinoids in the plant kingdom. Our findings may have implications for bioprospecting and drug discovery and provide a molecular rationale for the reported effects upon consumption of certain Radula preparations as moderately active legal highs.

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.

The cannabinoid receptor 1 (CB1) is an inhibitory G protein-coupled receptor abundantly expressed in the central nervous system. It has rich pharmacology and largely accounts for the recreational use of cannabis. We describe efficient asymmetric syntheses of four photoswitchable Δ 9 -tetrahydrocannabinol derivatives (azo-THCs) from a central building block 3-Br-THC. Using electrophysiology and a FRET-based cAMP assay, two compounds are identified as potent CB1 agonists that change their effect upon illumination. As such, azo-THCs enable CB1-mediated optical control of inwardly rectifying potassium channels, as well as adenylyl cyclase.

A method for the enantioselective synthesis of carbo- and heterocyclic ring systems enabled through the combination of Lewis acid activation and iridium-catalyzed allylic substitution is described. The reaction proceeds with branched, allylic alcohols and carbon nucleophiles as well as heteronucleophiles to give a diverse set of ring systems in good yields and with high enantioselectivities. The utility of the method is highlighted by the asymmetric syntheses of erythrococcamides A and B.

The changing legal landscape including medicinal and recreational consumption of Cannabis sativa has led to renewed interest to study the chemistry and biology of cannabinoids. The chemistry in this chapter highlights approaches to cannabinoid total synthesis with an emphasis on the implementation of modern methods and tactics, which provide access to modified structures and enable investigations of the biology of the cannabinoid product family.

All four stereoisomers of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) were synthesized in concise fashion using stereodivergent dual catalysis. Thus, following identical synthetic sequences and applying identical reaction conditions to the same set of starting materials, selective access to the four stereoisomers of THC was achieved in five steps., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel bioconjugation strategy is presented that relies on the coupling of diazonium terephthalates with amines in proteins. The diazonium captures the amine while the vicinal ester locks it through cyclization, ensuring no reversibility. The reaction is highly efficient and proceeds under mild conditions and short reaction times. Densely functionalized, complex natural products were directly coupled to proteins using low concentrations of coupling partners.

We describe the fully stereodivergent, dual catalytic α-allylation of linear aldehydes. The reaction proceeds via direct iridium-catalyzed substitution of racemic allylic alcohols with enamines generated in situ. The use of an Ir(P,olefin) complex and a diarylsilyl prolinol ether as catalysts in the presence of dimethylhydrogen phosphate as the promoter proved to be crucial for achieving high enantio- and diastereoselectivity (>99% ee, up to >20:1 dr). The utility of the method is demonstrated in a concise enantioselective synthesis of the antidepressant (-)-paroxetine.

An important challenge in asymmetric synthesis is the development of fully stereodivergent strategies to access the full complement of stereoisomers of products bearing multiple stereocenters. In the ideal case, where four products are possible, applying distinct catalysts to the same set of starting materials under identical conditions would in a single step afford any given stereoisomer. Herein, we describe the realization of this concept in a fully stereodivergent dual-catalytic synthesis of γ,δ-unsaturated aldehydes bearing vicinal quaternary/tertiary stereogenic centers. The reaction is enabled by chiral iridium and amine catalysts, which activate the allylic alcohol and aldehyde substrates, respectively. Each catalyst exerts high local stereocontrol irrespective of the other's inherent preference.

A highly enantioselective polycyclization method has been developed using the combination of Lewis acid activation with iridium-catalyzed allylic substitution. This strategy relies on direct use of branched, racemic allylic alcohols and furnishes a diverse and unique set of carbo- and heteropolycyclic ring systems in good yields and ≥99% ee.