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BACKGROUND: The importance of Chlamydia pneumoniae respiratory tract infection in childhood is under discussion. PATIENTS AND METHODS: 798 hospitalized children with respiratory tract diseases were prospectively studied during a 2-year period by polymerase chain reaction and enzyme immunoassay (PCR-EIA) detection from throat swabs. Paired serum samples were used to screen for Chlamydia antibodies. RESULTS: C. pneumoniae was detected by PCR-EIA in 74 children. Prevalence was 11% in lower and 4% in upper respiratory tract disease (p = 0.049) without age dependency. From November to February prevalence was elevated (42/277 vs. 32/521; p < 0.001). Using serology, prevalence of acute Chlamydia infection increased with age (p < 0.001) and the number of coinfections (p < 0.001), without seasonal variation. CONCLUSION: Characteristics of C. pneumoniae carriage in the respiratory tract in childhood differ from those in systemic infection. [ABSTRACT FROM AUTHOR]

Adenosine A(2A) antagonists can exert antiparkinsonian effects in animal models. Recent experiments studied the ability of MSX-3 (an adenosine A(2A) antagonist) to reverse the locomotor suppression and tremor produced by dopamine antagonists in rats. MSX-3 reversed haloperidol-induced suppression of locomotion, and reduced the tremulous jaw movements induced by haloperidol, pimozide, and reserpine. Infusions of MSX-3 into the nucleus accumbens core increased locomotion in haloperidol-treated rats, but there were no effects of infusions into the accumbens shell or ventrolateral neostriatum. In contrast, MSX-3 injected into the ventrolateral neostriatum reduced pimozide-induced tremulous jaw movements. Dopamine/adenosine interactions in different striatal subregions are involved in distinct aspects of motor function. [ABSTRACT FROM AUTHOR]

The G protein-coupled receptor 17 (GPR17) is an orphan receptor involved in inflammatory diseases. GPR17 antagonists have been proposed for the treatment of multiple sclerosis due to their potential to induce remyelination. Potent, selective antagonists are required to enable target validation. In the present study, we describe the discovery of a novel class of GPR17 antagonists based on an anthranilic acid scaffold. The compounds' potencies were evaluated in calcium mobilization and radioligand binding assays, and structure-activity relationships were analyzed. Selected antagonists were additionally studied in cAMP and G protein activation assays. The most potent antagonists were 5-methoxy-2-(5-(3'-methoxy-[1,1'-biphenyl]-2-yl)furan-2-carboxamido)benzoic acid ( 52 , PSB-22269, K i 8.91 nM) and its 3'-trifluoromethyl analog ( 54 , PSB-24040, K i 83.2 nM). Receptor-ligand docking studies revealed that the compounds' binding site is characterized by positively charged arginine residues and a lipophilic pocket. These findings yield valuable insights into this poorly characterized receptor providing a basis for future drug development.

Inflammation is a protective response to pathogens and injury. To be effective it needs to be resolved by endogenous mechanisms in order to avoid prolonged and excessive inflammation, which can become chronic. Specialized pro-resolving mediators (SPMs) are a group of lipids derived from omega-3 fatty acids, which can induce the resolution of inflammation. How SPMs exert their anti-inflammatory and pro-resolving effects is, however, not clear. Here, we show that SPMs such as protectins, maresins, and D-series resolvins function as biased positive allosteric modulators (PAM) of the prostaglandin E 2 (PGE 2 ) receptor EP4 through an intracellular binding site. They increase PGE 2 -induced G s -mediated formation of cAMP and thereby promote anti-inflammatory signaling of EP4. In addition, SPMs endow the endogenous EP4 receptor on macrophages with the ability to couple to G i -type G-proteins, which converts the EP4 receptor on macrophages from an anti-phagocytotic receptor to one increasing phagocytosis, a central mechanism of the pro-resolving activity of synthetic SPMs. In the absence of the EP4 receptor, SPMs lose their anti-inflammatory and pro-resolving activity in vitro and in vivo. Our findings reveal an unusual mechanism of allosteric receptor modulation by lipids and provide a mechanism by which synthetic SPMs exert pro-resolving and anti-inflammatory effects, which may facilitate approaches to treat inflammation., Competing Interests: Competing interests statement:The authors declare no competing interest.

The COVID-19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96- well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (M pro ) of SARS-CoV-2. Subsequent direct biological screening identifies novel 1,2,3-triazole derivatives as robust M pro inhibitors with high anti-SARS-CoV-2 activity. Notably, C5N17B demonstrates sub-micromolar M pro inhibitory potency (IC 50 = 0.12 µM) and excellent antiviral activity in Calu-3 cells determined in an immunofluorescence-based antiviral assay (EC 50 = 0.078 µM, no cytotoxicity: CC 50 > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC 50 = 1.95 µM) and similar efficacy to ensitrelvir (EC 50 = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS-CoV-2 variants (Gamma, Delta, and Omicron, EC 50 = 0.13 - 0.26 µM) and HCoV-OC43, indicating its broad-spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir-resistant strains with T21I/E166V and L50F/E166V mutations in M pro (EC 50 = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non-covalent multi-site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up-and-coming drug candidate targeting SARS-CoV-2 M pro for clinical therapy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (M pro ). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including M pro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC 50 values in the low nanomolar range, k inact / K i values of >100,000 M -1 s -1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho -fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p -ethoxy- o -fluorobenzoyl chloropyridyl ester (PSB-21110, 29b , MW 296 g/mol; EC 50 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.

Competing Interests: Disclosure M Kleemiss and FG Bauernfeind have received lecture fees from the healthcare business of Merck KGaA, Darmstadt, Germany. R Strotman, K Orlowski and M Yanik are employees of the healthcare business of Merck KGaA, Darmstadt, Germany. K Goteti is an employee of EMD Serono.

The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough. Aiming to develop novel, selective P2X3R antagonists, tetrazolopyrimidine-based hit compound 9 was optimized through structure-activity relationship studies by modifying the tetrazole core as well as side chain substituents. The optimized antagonist 26a , featuring a cyclopropane-substituted triazolopyrimidine core, displayed potent P2X3R-antagonistic activity (IC 50 = 54.9 nM), 20-fold selectivity versus the heteromeric P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive P2X3R blockade was experimentally confirmed by calcium influx assays. Cryo-electron microscopy revealed that 26a stabilizes the P2X3R in its desensitized state, acting as a molecular barrier to prevent ions from accessing the central pore. In vivo studies in a rat neuropathic pain model (spinal nerve ligation) showed dose-dependent antiallodynic effects of 26a , thus presenting a novel, promising lead structure.

Brain aging is associated with cognitive decline, reduced synaptic plasticity, and altered metabolism. The activity of mechanistic target of rapamycin (mTOR) has a major impact on aging by regulating cellular metabolism. Although reduced mTOR signaling has a general antiaging effect, it can negatively affect the aging brain by reducing synaptogenesis and thus cognitive functions. Increased mTOR activity facilitates aging and is responsible for the amnestic effect of the cannabinoid receptor 1 agonist Δ 9 -tetrahydrocannabinol (THC) in higher doses. Long-term low-dose Δ 9 -THC had an antiaging effect on the brain by restoring cognitive abilities and synapse densities in old mice. Whether changes in mTOR signaling and metabolome are associated with its positive effects on the aging brain is an open question. Here, we show that Δ 9 -THC treatment has a tissue-dependent and dual effect on mTOR signaling and the metabolome. In the brain, Δ 9 -THC treatment induced a transient increase in mTOR activity and in the levels of amino acids and metabolites involved in energy production, followed by an increased synthesis of synaptic proteins. Unexpectedly, we found a similar reduction in the mTOR activity in adipose tissue and in the level of amino acids and carbohydrate metabolites in blood plasma as in animals on a low-calorie diet. Thus, long-term Δ 9 -THC treatment first increases the level of energy and synaptic protein production in the brain, followed by a reduction in mTOR activity and metabolic processes in the periphery. Our study suggests that a dual effect on mTOR activity and the metabolome could be the basis for an effective antiaging and pro-cognitive medication., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A study comprising 74 colic and 74 control horses admitted to an animal hospital was performed. Faecal samples were collected and analysed for dry matter concentration, particle size distribution using wet-sieving, and sand presence through a sand sedimentation test. Data on horse breed, age, gender and basic feeding variables was collected and analysed using χ 2 -tests. Faecal dry matter concentration, particle size distribution and sand score was compared between colic and non-colic horses, and between horses with different colic types, using one-way ANOVA. Results showed that colic and non-colic horse groups were similar in breed, age, gender and basic feeding variables. Faecal dry matter concentration, particle size distribution and sand score were similar among colic and non-colic horses. Horses diagnosed with "unknown colic cause" had higher proportion of particles >0.5 <1.0 mm size compared to horses with colic due to impactions in caecum or colon, torsion or gas accumulation (P<0.05), but this difference was very small and most likely not of biological importance. Faecal dry matter concentration and sand score were similar among horses with different types of colic. Increased knowledge of the composition of particles of different size in equine faeces may enhance our understanding of digesta passage rate in colic and non-colic horses, which is needed to develop preventative measures of certain types of colic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of G q proteins by blocking GDP/GTP exchange. The inhibitor binding site of G q has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The human orphan G protein-coupled receptor GPR18, activated by Δ 9 -tetrahydrocannabinol (THC), constitutes a promising drug target in immunology and cancer. However, studies on GPR18 are hampered by the lack of suitable tool compounds. In the present study, potent and selective GPR18 agonists were developed showing low nanomolar potency at human and mouse GPR18, determined in β-arrestin recruitment assays. Structure-activity relationships were analyzed, and selectivity versus cannabinoid (CB) and CB-like receptors was assessed. Compound 51 (PSB-KK1415, EC 50 19.1 nM) was the most potent GPR18 agonist showing at least 25-fold selectivity versus CB receptors. The most selective GPR18 agonist 50 (PSB-KK1445, EC 50 45.4 nM) displayed >200-fold selectivity versus both CB receptor subtypes, GPR55, and GPR183. The new GPR18 agonists showed minimal species differences, while THC acted as a weak partial agonist at the mouse receptor. The newly discovered compounds represent the most potent and selective GPR18 agonists reported to date.

Background: Advanced Trauma Life Support (ATLS) is the gold standard of initial assessment of trauma patients and therefore a widely used training program for medical professionals. Practical application of the knowledge taught can be challenging for medical students and inexperienced clinicians. Simulation-based training, including virtual reality (VR), has proven to be a valuable adjunct to real-world experiences in trauma education. Previous studies have demonstrated the effectiveness of VR simulations for surgical and technical skills training. However, there is limited evidence on VR simulation training specifically for trauma education, particularly within the ATLS curriculum. The purpose of this pilot study is to evaluate the feasibility, effectiveness, and acceptance of using a fully immersive VR trauma simulation to prepare medical students for the ATLS course., Methods: This was a prospective randomised controlled pilot study on a convenience sample of advanced medical students (n = 56; intervention group with adjunct training using a commercially available semi-automated trauma VR simulation, n = 28, vs control group, n = 28) taking part in the ATLS course of the Military Physician Officer School. Feasibility was assessed by evaluating factors related to technical factors of the VR training (e.g. rate of interruptions and premature termination). Objective and subjective effectiveness was assessed using confidence ratings at four pre-specified points in the curriculum, validated surveys, clinical scenario scores, multiple choice knowledge tests, and ATLS final clinical scenario and course pass rates. Acceptance was measured using validated instruments to assess variables of media use (Technology acceptance, usability, presence and immersion, workload, and user satisfaction)., Results: The feasibility assessment demonstrated that only one premature termination occurred and that all remaining participants in the intervention group correctly stabilised the patient. No significant differences between the two groups in terms of objective effectiveness were observed (p = 0.832 and p = 0.237 for the pretest and final knowledge test, respectively; p = 0.485 for the pass rates for the final clinical scenario on the first attempt; all participants passed the ATLS course). In terms of subjective effectiveness, the authors found significantly improved confidence post-VR intervention (p < .001) in providing emergency care using the ATLS principles. Perceived usefulness in the TEI was stated with a mean of 4 (SD 0.8; range 0-5). Overall acceptance and usability of the VR simulation was rated as positive (System Usability Scale total score mean 79.4 (SD 11.3, range 0-100)., Conclusions: The findings of this prospective pilot study indicate the potential of using VR trauma simulations as a feasible and acceptable supplementary tool for the ATLS training course. Where objective effectiveness regarding test and scenario scores remained unchanged, subjective effectiveness demonstrated improvement. Future research should focus on identifying specific scenarios and domains where VR can outperform or enhance traditional learning methods in trauma simulation., (© 2024. The Author(s).)

Given the crucial role of the main protease (M pro ) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of M pro . Our systematic approach combined an M pro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent M pro inhibitor 84 with an IC 50 value of 3.23 nM and a second-order rate constant of inactivation, k inac / K i , of 448,000 M -1 s -1 . The open-chain M pro inhibitor 58 , along with the macrocyclic compounds 83 and 84 , a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC 50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic M pro inhibitors as anti-SARS-CoV-2 agents.

P2X4 receptors are ATP-gated cation channels that were proposed as novel drug targets due to their role in inflammation and neuropathic pain. Only few potent and selective P2X4 receptor antagonists have been described to date. Labeled tool compounds suitable for P2X4 receptor binding studies are lacking. Here, we present a novel allosteric P2X4 receptor antagonist possessing high potency in the low nanomolar range. We describe its tritium-labeling resulting in the P2X4-selective radiotracer [ 3 H]PSB-OR-2020 with high specific activity (45 Ci/mmol; 1.67 TBq/mmol). A radioligand binding assay was developed using human embryonic kidney (HEK293) cell membranes recombinantly expressing the human P2X4 receptor. Competition binding studies with structurally diverse P2X4 receptor antagonists revealed different allosteric binding sites indicating that the new class of P2X4 receptor antagonists, to which PSB-OR-2020 belongs, interacts with an unprecedented allosteric site. [ 3 H]PSB-OR-2020 may become a useful tool for research on P2X4 receptors and for promoting drug development., (© 2024. The Author(s).)

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2 ) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3 ) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3 , suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

A titrant for the SARS-CoV-2 main protease (M pro ) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active M pro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme-titrant complex. Four fluorogenic substrates of M pro , including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the M pro -catalyzed cleavage of these substrates were determined., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors.)

The adenosine A 2A receptor (A 2A AR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A 2A AR agonist 2-amino-6-[(1 H -imidazol-2-ylmethyl)sulfanyl]-4- p -hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A 2A AR construct A 2A -PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p -hydroxy group of LUF5834 with T88 3.36 was observed, an amino acid that was mutated to alanine in the most frequently used A 2A AR crystallization constructs thus preventing the discovery of its interactions in most of the previous A 2A AR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N -ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S91 3.39 K mutation, although with an affinity lower than that at the native receptor. This could explain the compound's partial agonistic activity: while full A 2A AR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)