The life-saving benefit of dexamethasone in severe COVID-19 is linked to a reversal of monocyte dysregulation.

Dexamethasone is a life-saving treatment for severe COVID-19, yet its mechanism of action is unknown, and many patients deteriorate or die despite timely treatment initiation. Here, we identify dexamethasone treatment-induced cellular and molecular changes associated with improved survival in COVID-19 patients. We observed a reversal of transcriptional hallmark signatures in monocytes associated with severe COVID-19 and the induction of a monocyte substate characterized by the expression of glucocorticoid-response genes. These molecular responses to dexamethasone were detected in circulating and pulmonary monocytes, and they were directly linked to survival. Monocyte single-cell RNA sequencing (scRNA-seq)-derived signatures were enriched in whole blood transcriptomes of patients with fatal outcome in two independent cohorts, highlighting the potential for identifying non-responders refractory to dexamethasone. Our findings link the effects of dexamethasone to specific immunomodulation and reversal of monocyte dysregulation, and they highlight the potential of single-cell omics for monitoring in vivo target engagement of immunomodulatory drugs and for patient stratification for precision medicine approaches.

Validating Small Molecule Chemical Probes for Biological Discovery.

Small molecule chemical probes are valuable tools for interrogating protein biological functions and relevance as a therapeutic target. Rigorous validation of chemical probe parameters such as cellular potency and selectivity is critical to unequivocally linking biological and phenotypic data resulting from treatment with a chemical probe to the function of a specific target protein. A variety of modern technologies are available to evaluate cellular potency and selectivity, target engagement, and functional response biomarkers of chemical probe compounds. Here, we review these technologies and the rationales behind using them for the characterization and validation of chemical probes. In addition, large-scale phenotypic characterization of chemical probes through chemical genetic screening is increasingly leading to a wealth of information on the cellular pharmacology and disease involvement of potential therapeutic targets. Extensive compound validation approaches and integration of phenotypic information will lay foundations for further use of chemical probes in biological discovery.

In situ identification of cellular drug targets in mammalian tissue.

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.

Quantifying Target Occupancy of Small Molecules Within Living Cells.

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

Horizontal Cell Biology: Monitoring Global Changes of Protein Interaction States with the Proteome-Wide Cellular Thermal Shift Assay (CETSA).

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.

Enzyme inhibitor discovery by activity-based protein profiling.

Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.

Drug Target Identification in Tissues by Thermal Proteome Profiling.

Drug target deconvolution can accelerate the drug discovery process by identifying a drug's targets (facilitating medicinal chemistry efforts) and off-targets (anticipating toxicity effects or adverse drug reactions). Multiple mass spectrometry-based approaches have been developed for this purpose, but thermal proteome profiling (TPP) remains to date the only one that does not require compound modification and can be used to identify intracellular targets in living cells. TPP is based on the principle that the thermal stability of a protein can be affected by its interactions. Recent developments of this approach have expanded its applications beyond drugs and cell cultures to studying protein-drug interactions and biological phenomena in tissues. These developments open up the possibility of studying drug treatment or mechanisms of disease in a holistic fashion, which can result in the design of better drugs and lead to a better understanding of fundamental biology.

Improved drug target deconvolution with PISA-DIA using an extended, overlapping temperature gradient.

Thermal proteome profiling (TPP) is a powerful tool for drug target deconvolution. Recently, data-independent acquisition mass spectrometry (DIA-MS) approaches have demonstrated significant improvements to depth and missingness in proteome data, but traditional TPP (a.k.a. CEllular Thermal Shift Assay "CETSA") workflows typically employ multiplexing reagents reliant on data-dependent acquisition (DDA). Herein, we introduce a new experimental design for the Proteome Integral Solubility Alteration via label-free DIA approach (PISA-DIA). We highlight the proteome coverage and sensitivity achieved by using multiple overlapping thermal gradients alongside DIA-MS, which maximizes efficiencies in PISA sample concatenation and safeguards against missing protein targets that exist at high melting temperatures. We demonstrate our extended PISA-DIA design has superior proteome coverage as compared to using tandem-mass tags (TMT) necessitating DDA-MS analysis. Importantly, we demonstrate our PISA-DIA approach has the quantitative and statistical rigor using A-1331852, a specific inhibitor of BCL-xL. Due to the high melt temperature of this protein target, we utilized our extended multiple gradient PISA-DIA workflow to identify BCL-xL. We assert our novel overlapping gradient PISA-DIA-MS approach is ideal for unbiased drug target deconvolution, spanning a large temperature range whilst minimizing target dropout between gradients, increasing the likelihood of resolving the protein targets of novel compounds.

End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.

Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.

Stability-based approaches in chemoproteomics.

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.

Investigating Protein Binding with the Isothermal Ligand-induced Resolubilization Assay.

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.

Cellular thermal shift assay: an approach to identify and assess protein target engagement.

INTRODUCTION: A comprehensive and global knowledge of protein target engagement is of vital importance for mechanistic studies and in drug development. Since its initial introduction, the cellular thermal shift assay (CETSA) has proven to be a reliable and flexible technique that can be widely applied to multiple contexts and has profound applications in facilitating the identification and assessment of protein target engagement. AREAS COVERED: This review introduces the principle of CETSA, elaborates on western blot-based CETSA and MS-based thermal proteome profiling (TPP) as well as the major applications and prospects of these approaches. EXPERT OPINION: CETSA primarily evaluates a given ligand binding to a particular target protein in cells and tissues with the protein thermal stabilities analyzed by western blot. When coupling mass spectrometry with CETSA, thermal proteome profiling allows simultaneous proteome-wide experiment that greatly increased the efficiency of target engagement evaluation, and serves as a promising strategy to identify protein targets and off-targets as well as protein-protein interactions to uncover the biological effects. The CETSA approaches have broad applications and potentials in drug development and clinical research.

Population pharmacokinetic/target engagement modelling of tozorakimab in healthy volunteers and patients with chronic obstructive pulmonary disease.

AIMS: This study describes the pharmacokinetic (PK)/target engagement (TE) relationship of tozorakimab, an anti-interleukin (IL)-33 antibody, by building a mechanistic population PK/TE model using phase 1 biomarker data. METHODS: The analysis included tozorakimab PK and TE in serum assessed in 60 tozorakimab-treated participants, including healthy adults and patients with mild chronic obstructive pulmonary disease. Scenarios evaluated three dose frequencies (once every 2, 4 or 6 weeks) administered subcutaneously at seven doses of tozorakimab (30, 60, 90, 120, 150, 300 or 600 mg). For each dose, simulations were performed with 5000 virtual individuals to predict systemic TE. Inhibition of IL-33/soluble ST2 (sST2) complex levels at trough PK at steady state was assessed in each dosing scenario. The PK/TE modelling analyses were performed using a nonlinear mixed-effect modelling approach. RESULTS: The final two-compartment PK model with tozorakimab binding IL-33 in the central compartment adequately described the systemic PK and TE of tozorakimab at population and individual levels. The mean PK parameter estimates of absorption rate, central volume of distribution and clearance were 0.48 (90% confidence interval [CI]: 0.40-0.59, 1/day), 12.64 (90% CI: 8.60-18.62, L) and 0.87 (90% CI: 0.65-1.16, L/day), respectively. Consistent with the observed value, tozorakimab bioavailability was 45%. For all three dose frequencies, predicted inhibition of systemic IL-33/sST2 levels was more than 95% at doses greater than 90 mg. CONCLUSIONS: The PK/TE model reliably quantified the relationship between PK and systemic TE of tozorakimab, with potential utility for predicting clinical dose-response relationships and supporting clinical dose selection.

Chemo-proteomics in antimalarial target identification and engagement.

Humans have lived in tenuous battle with malaria over millennia. Today, while much of the world is free of the disease, areas of South America, Asia, and Africa still wage this war with substantial impacts on their social and economic development. The threat of widespread resistance to all currently available antimalarial therapies continues to raise concern. Therefore, it is imperative that novel antimalarial chemotypes be developed to populate the pipeline going forward. Phenotypic screening has been responsible for the majority of the new chemotypes emerging in the past few decades. However, this can result in limited information on the molecular target of these compounds which may serve as an unknown variable complicating their progression into clinical development. Target identification and validation is a process that incorporates techniques from a range of different disciplines. Chemical biology and more specifically chemo-proteomics have been heavily utilized for this purpose. This review provides an in-depth summary of the application of chemo-proteomics in antimalarial development. Here we focus particularly on the methodology, practicalities, merits, and limitations of designing these experiments. Together this provides learnings on the future use of chemo-proteomics in antimalarial development.

Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue.

BACKGROUND: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. METHODS: Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. RESULTS: Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622-2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127-2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0-18% CV across consecutive tissue sections and 5-20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). CONCLUSIONS: This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

Evidence that a novel transdiagnostic eating disorder treatment reduces reward region response to the thin beauty ideal and high-calorie binge foods.

BACKGROUND: Findings from brain imaging studies with small samples can show limited reproducibility. Thus, we tested whether the evidence that a transdiagnostic eating disorder treatment reduces responsivity of brain valuation regions to thin models and high-calorie binge foods, the intervention targets, from a smaller earlier trial emerged when we recruited additional participants. METHODS: Women with DSM-5 eating disorders (N = 138) were randomized to the dissonance-based body project treatment (BPT) or a waitlist control condition and completed functional magnetic resonance imaging (fMRI) scans assessing neural response to thin models and high-calorie foods at pretest and posttest. RESULTS: BPT v. control participants showed significantly greater reductions in responsivity of regions implicated in reward valuation (caudate) and attentional motivation (precuneus) to thin v. average-weight models, echoing findings from the smaller sample. Data from this larger sample also provided novel evidence that BPT v. control participants showed greater reductions in responsivity of regions implicated in reward valuation (ventrolateral prefrontal cortex) and food craving (hippocampus) to high-calorie binge foods v. low-calorie foods, as well as significantly greater reductions in eating disorder symptoms, abstinence from binge eating and purging behaviors, palatability ratings for high calorie foods, monetary value for high-calorie binge foods, and significantly greater increases in attractiveness ratings of average weight models. CONCLUSIONS: Results from this larger sample provide evidence that BPT reduces valuation of the thin ideal and high-calorie binge foods, the intervention targets, per objective brain imaging data, and produces clinically meaningful reductions in eating pathology.

The shortcoming of using glibenclamide in exploratory clinical headache provocation studies.

OBJECTIVE: Preclinical and clinical studies implicate the vascular ATP-sensitive potassium (KATP) channel in the signaling cascades underlying headache and migraine. However, attempts to demonstrate that the KATP channel inhibitor glibenclamide would attenuate triggered headache in healthy volunteers have proven unsuccessful. It is questionable, however, whether target engagement was achieved in these clinical studies. METHODS: Literature data for human glibenclamide pharmacokinetics, plasma protein binding and functional IC50 values were used to predict the KATP receptor occupancy (RO) levels obtained after glibenclamide dosing in the published exploratory clinical headache provocation studies. RO vs. time profiles of glibenclamide were simulated for the pancreatic KATP channel subtype Kir6.2/SUR1 and the vascular subtype Kir6.1/SUR2B. RESULTS: At the clinical dose of 10 mg of glibenclamide used in the headache provocation studies, predicted maximal occupancy levels of up to 90% and up to 26% were found for Kir6.2/SUR1 and Kir6.1/SUR2B, respectively. CONCLUSIONS: The findings of the present study indicate that effective Kir6.1/SUR2B target engagement was not achieved in the clinical headache provocation studies using glibenclamide. Therefore, development of novel selective Kir6.1/SUR2B inhibitors, with good bioavailability and low plasma protein binding, is required to reveal the potential of KATP channel inhibition in the treatment of migraine.

Phase 1 safety, tolerability, pharmacokinetics and pharmacodynamic results of KCL-286, a novel retinoic acid receptor-ß agonist for treatment of spinal cord injury, in male healthy participants.

AIMS: KCL-286 is an orally available agonist that activates the retinoic acid receptor (RAR) ß2, a transcription factor which stimulates axonal outgrowth. The investigational medicinal product is being developed for treatment of spinal cord injury (SCI). This adaptive dose escalation study evaluated the tolerability, safety and pharmacokinetics and pharmacodynamic activity of KCL-286 in male healthy volunteers to establish dosing to be used in the SCI patient population. METHODS: The design was a double blind, randomized, placebo-controlled dose escalation study in 2 parts: a single ascending dose adaptive design with a food interaction arm, and a multiple ascending dose design. RARß2 mRNA expression was evaluated in white blood cells. RESULTS: At the highest single and multiple ascending doses (100 mg), no trends or clinically important differences were noted in the incidence or intensity of adverse events (AEs), serious AEs or other safety assessments with none leading to withdrawal from the study. The AEs were dry skin, rash, skin exfoliation, raised liver enzymes and eye disorders. There was an increase in mean maximum observed concentration and area under the plasma concentration-time curve up to 24 h showing a trend to subproportionality with dose. RARß2 was upregulated by the investigational medicinal product in white blood cells. CONCLUSION: KCL-286 was well tolerated by healthy human participants following doses that exceeded potentially clinically relevant plasma exposures based on preclinical in vivo models. Target engagement shows the drug candidate activates its receptor. These findings support further development of KCL-286 as a novel oral treatment for SCI.

Multiple-ascending doses of ACT-1014-6470, an oral complement factor 5a receptor 1 (C5a1 receptor) antagonist: Tolerability, pharmacokinetics and target engagement.

AIMS: Targeting the complement factor 5a receptor 1 (C5a1 receptor) offers potential to treat various autoimmune diseases. The C5a1 receptor antagonist ACT-1014-6470 was well tolerated in a single-ascending dose study in healthy subjects. This double-blind, randomized, placebo-controlled study aimed to investigate the safety, tolerability, pharmacokinetics (PK) and target engagement of multiple-ascending doses of ACT-1014-6470. METHODS: Per dose level, 10 healthy male and female subjects of nonchildbearing potential (1:1 sex ratio) were enrolled to assess 30, 60 and 120 mg ACT-1014-6470 administered twice daily for 4.5 days under fed conditions. Adverse events, clinical laboratory data, vital signs, electrocardiogram and PK blood samples were collected up to 120 h post last dose and ex vivo stimulated matrix metalloproteinase 9 was quantified as target engagement biomarker. At the 60-mg dose level, PK samples were collected until 8 weeks post last dose. RESULTS: The total adverse event number was 57 and no treatment-related safety pattern was apparent. At steady state, ACT-1014-6470 reached maximum plasma concentrations after 2-3 h and the half-life estimated up to Day 10 was 115-146 h across dose levels. Exposure parameters increased dose-proportionally, steady state was attained between Day 3-5, and ACT-1014-6470 accumulated 2-fold. At the 60-mg dose level, ACT-1014-6470 was quantifiable until 8 weeks after the last dose. Matrix metalloproteinase 9 release was suppressed to endogenous background concentrations up to the last sampling time point, confirming sustained target engagement of ACT-1014-6470. CONCLUSION: The compound was generally safe and well tolerated at all dose levels, warranting further clinical investigations.

Determination of RNA-ligand interactions with the photoaffinity platform PEARL-seq.

In the development of therapeutics, it is important to establish engagement of a compound to its intended target and identify other targets it binds to. Methods for demonstrating target engagement in the growing field of RNA-targeted therapeutics are therefore needed. We present a detailed protocol for Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), a platform for determining interactions between small molecule ligands and their target RNA(s). PEARL-seq allows detection of binding and crosslinking events with single nucleotide resolution and allows measurement of enrichment of the target RNA relative to all other RNAs. PEARL-seq is a valuable tool in the effort to verify bona fide RNA-ligand interactions.