The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction.

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.

Interaction of Synthetic Cannabinoid Receptor Agonists with Cannabinoid Receptor I: Insights into Activation Molecular Mechanism.

Synthetic cannabinoid receptor agonists (SCRAs) have become a wide group of new psychoactive substances since the 2010s. For the last few years, the X-ray structures of the complexes of cannabinoid receptor I (CB1) with SCRAs as well as the complexes of CB1 with its antagonist have been published. Based on those data, SCRA-CB1 interactions are analyzed in detail, using molecular modeling and molecular dynamics simulations. The molecular mechanism of the conformational transformation of the transmembrane domain of CB1 caused by its interaction with SCRA is studied. These conformational changes allosterically modulate the CB1-Gi complex, providing activation of the Gi protein. Based on the X-ray-determined structures of the CB1-ligand complexes, a stable apo conformation of inactive CB1 with a relatively low potential barrier of receptor activation was modeled. For that model, molecular dynamic simulations of SCRA binding to CB1 led to the active state of CB1, which allowed us to explore the key features of this activation and the molecular mechanism of the receptor's structural transformation. The simulated CB1 activation is in accordance with the previously published experimental data for the activation at protein mutations or structural changes of ligands. The key feature of the suggested activation mechanism is the determination of the stiff core of the CB1 transmembrane domain and the statement that the entire conformational transformation of the receptor to the active state is caused by a shift of alpha helix TM7 relative to this core. The shift itself is caused by protein-ligand interactions. It was verified via steered molecular dynamics simulations of the X-ray-determined structures of the inactive receptor, which resulted in the active conformation of CB1 irrespective of the placement of agonist ligand in the receptor's active site.

Quantum Mechanical Study of Oxygen Ligands Protonation for the Stable States of the Laccase Active Site.

Laccases are enzymes catalyzing the oxidation of a wide range of organic and inorganic substrates accompanied by molecular oxygen reduction to water. Recently, oxygen reduction by laccases has been studied by single-crystal serial X-ray crystallography with increasing absorption doses at subatomic resolution. There were two determined structures corresponding to the reduced and oxidized stable states of the laccase active site. However, the protonation of the oxygen ligands involved cannot be determined even at subatomic resolution. In the present work, the protonation of oxygen ligands in the active site of laccase for the two stable states determined in the X-ray study was explored using quantum mechanical and continuum-electrostatics calculations. This is important for understanding the reaction of the oxygen reduction mechanism in laccases. The high precision of X-ray data at subatomic resolutions allowed us to optimize the quantum mechanical calculations.

PCSK9 as a Target for Development of a New Generation of Hypolipidemic Drugs.

PCSK9 has now become an important target to create new classes of lipid-lowering drugs. The prevention of its interaction with LDL receptors allows an increase in the number of these receptors on the surface of the cell membrane of hepatocytes, which leads to an increase in the uptake of cholesterol-rich atherogenic LDL from the bloodstream. The PCSK9 antagonists described in this review belong to different classes of compounds, may have a low molecular weight or belong to macromolecular structures, and also demonstrate different mechanisms of action. The mechanisms of action include preventing the effective binding of PCSK9 to LDLR, stimulating the degradation of PCSK9, and even blocking its transcription or transport to the plasma membrane/cell surface. Although several types of antihyperlipidemic drugs have been introduced on the market and are actively used in clinical practice, they are not without disadvantages, such as well-known side effects (statins) or high costs (monoclonal antibodies). Thus, there is still a need for effective cholesterol-lowering drugs with minimal side effects, preferably orally bioavailable. Low-molecular-weight PCSK9 inhibitors could be a worthy alternative for this purpose.

Preliminary Assessment of the Anti-inflammatory Activity of New Structural Honokiol Analogs with a 4'-O-(2-Fluoroethyl) Moiety and the Potential of Their 18F-Labeled Derivatives for Neuroinflammation Imaging.

Neolignans honokiol and 4'-O-methylhonokiol (MH) and their derivatives have pronounced anti-inflammatory activity, as evidenced by numerous pharmacological studies. Literature data suggested that cyclooxygenase type 2 (COX-2) may be a target for these compounds in vitro and in vivo. Recent studies of [11C]MPbP (4'-[11C]methoxy-5-propyl-1,1'-biphenyl-2-ol) biodistribution in LPS (lipopolysaccharide)-treated rats have confirmed the high potential of MH derivatives for imaging neuroinflammation. Here, we report the synthesis of four structural analogs of honokiol, of which 4'-(2-fluoroethoxy)-2-hydroxy-5-propyl-1, 1'-biphenyl (F-IV) was selected for labeling with fluorine-18 (T1/2 = 109.8 min) due to its high anti-inflammatory activity confirmed by enzyme immunoassays (EIA) and neuromorphological studies. The high inhibitory potency of F-IV to COX-2 and its moderate lipophilicity and chemical stability are favorable factors for the preliminary evaluation of the radioligand [18F]F-IV in a rodent model of neuroinflammation. [18F]F-IV was prepared with good radiochemical yield and high molar activity and radiochemical purity by 18F-fluoroethylation of the precursor with Boc-protecting group (15) with [18F]2-fluoro-1-bromoethane ([18F]FEB). Ex vivo biodistribution studies revealed a small to moderate increase in radioligand uptake in the brain and peripheral organs of LPS-induced rats compared to control animals. Pretreatment with celecoxib resulted in significant blocking of radioactivity uptake in the brain (pons and medulla), heart, lungs, and kidneys, indicating that [18F]F-IV is likely to specifically bind to COX-2 in a rat model of neuroinflammation. However, in comparison with [11C]MPbP, the new radioligand showed decreased brain uptake in LPS rats and high retention in the blood pool, which apparently could be explained by its high plasma protein binding. We believe that the structure of [18F]F-IV can be optimized by replacing the substituents in the biphenyl core to eliminate these disadvantages and develop new radioligands for imaging activated microglia.

TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis.

Toll-Like Receptor 4 (TLR4) signal pathway plays an important role in initiating the innate immune response and its activation by bacterial endotoxin is responsible for chronic and acute inflammatory disorders that are becoming more and more frequent in developed countries. Modulation of the TLR4 pathway is a potential strategy to specifically target these pathologies. Among the diseases caused by TLR4 abnormal activation by bacterial endotoxin, sepsis is the most dangerous one because it is a life-threatening acute system inflammatory condition that still lacks specific pharmacological treatment. Here, we review molecules at a preclinical or clinical phase of development, that are active in inhibiting the TLR4-MyD88 and TLR4-TRIF pathways in animal models. These are low-molecular weight compounds of natural and synthetic origin that can be considered leads for drug development. The results of in vivo studies in the sepsis model and the mechanisms of action of drug leads are presented and critically discussed, evidencing the differences in treatment results from rodents to humans.