Unveiling Colombia's medicinal Cannabis sativa treasure trove: Phenotypic and Chemotypic diversity in legal cultivation.

INTRODUCTION: Cannabis sativa is a highly versatile plant with a long history of cultivation and domestication. It produces multiple compounds that exert distinct and valuable therapeutic effects by modulating diverse biological systems, including the endocannabinoid system (ECS). Access to standardized, metabolically diverse, and reproducible C. sativa chemotypes and chemovars is essential for physicians to optimize individualized patient treatment and for industries to conduct drug-discovery campaigns. OBJECTIVE: This study aimed to characterize and assess the phytochemical diversity of C. sativa chemotypes in diverse ecological regions of Colombia, South America. METHODOLOGY: Ten cannabinoids and 23 terpenes were measured using liquid and gas chromatography, in addition to other phenotypic traits, in 156 C. sativa plants that were grown in diverse ecological regions in Colombia, a hotspot for global biodiversity. RESULTS: Our results reveal significant phytochemical diversity in Colombian-grown C. sativa plants, with four distinct chemotypes based on cannabinoid profile. The significant amount of usually uncommon terpenes suggests that Colombia's environments may have unique capabilities that allow the plant to express these compounds. Colombia's diverse climates offer enormous cultivation potential, making it a key player in both domestic and international medicinal and recreational C. sativa trade. CONCLUSION: These findings underscore Colombia's capacity to pioneer global C. sativa production diversification, particularly in South America with new emerging markets.

Structure-Based Design and Synthesis of Covalent Inhibitors for Deubiquitinase and Acetyltransferase ChlaDUB1 of Chlamydia trachomatis.

Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.

Fluoxetine exerts anti-inflammatory effects on human epidermal keratinocytes and suppresses their endothelin release.

Fluoxetine is a safe antidepressant with remarkable anti-inflammatory actions; therefore, we aimed to investigate its effects on immortalized (HaCaT) as well as primary human epidermal keratinocytes in a polyinosinic-polycytidylic acid (p(I:C))-induced inflammatory model. We found that a non-cytotoxic concentration (MTT-assay, CyQUANT-assay) of fluoxetine significantly suppressed p(I:C)-induced expression and release of several pro-inflammatory cytokines (Q-PCR, cytokine array, ELISA), and it decreased the release of the itch mediator endothelins (ELISA). These effects were not mediated by the inhibition of the NF-κB or p38 MAPK pathways (western blot), or by the suppression of the p(I:C)-induced elevation of mitochondrial ROS production (MitoSOX Red labeling). Instead, unbiased activity profiling revealed that they were most likely mediated via the inhibition of the phosphoinositide 3-kinase (PI3K) pathway. Importantly, the PI3K-inhibitor GDC0941 fully mimicked the effects of fluoxetine (Q-PCR, ELISA). Although fluoxetine was able to occupy the binding site of GDC0941 (in silico molecular docking), and exerted direct inhibitory effect on PI3K (cell-free PI3K activity assay), it exhibited much lower potency and efficacy as compared to GDC0941. Finally, RNA-Seq analysis revealed that fluoxetine deeply influenced the transcriptional alterations induced by p(I:C)-treatment, and exerted an overall anti-inflammatory activity. Collectively, our findings demonstrate that fluoxetine exerts potent anti-inflammatory effects, and suppresses the release of the endogenous itch mediator endothelins in human keratinocytes, most likely via interfering with the PI3K pathway. Thus, clinical studies are encouraged to explore whether the currently reported beneficial effects translate in vivo following its topical administration in inflammatory and pruritic dermatoses.

Development of an Indole-Amide-Based Photoswitchable Cannabinoid Receptor Subtype 1 (CB1R) "Cis-On" Agonist.

Activation of the human cannabinoid receptor type 1 (hCB1R) with high spatiotemporal control is useful to study processes involved in different pathologies related to nociception, metabolic alterations, and neurological disorders. To synthesize new agonist ligands for hCB1R, we have designed different classes of photoswitchable molecules based on an indole core. The modifications made to the central core have allowed us to understand the molecular characteristics necessary to design an agonist with optimal pharmacological properties. Compound 27a shows high affinity for CB1R (Ki (cis-form) = 0.18 µM), with a marked difference in affinity with respect to its inactive "trans-off" form (CB1R Ki trans/cis ratio = 5.4). The novel compounds were evaluated by radioligand binding studies, receptor internalization, sensor receptor activation (GRABeCB2.0), Western blots for analysis of ERK1/2 activation, NanoBiT ßarr2 recruitment, and calcium mobilization assays, respectively. The data show that the novel agonist 27a is a candidate for studying the optical modulation of cannabinoid receptors (CBRs), serving as a new molecular tool for investigating the involvement of hCB1R in disorders associated with the endocannabinoid system.

"Photo-Rimonabant": Synthesis and Biological Evaluation of Novel Photoswitchable Molecules Derived from Rimonabant Lead to a Highly Selective and Nanomolar "Cis-On" CB1R Antagonist.

Human cannabinoid receptor type 1 (hCB1R) plays important roles in the regulation of appetite and development of addictive behaviors. Herein, we describe the design, synthesis, photocharacterization, molecular docking, and in vitro characterization of "photo-rimonabant", i.e., azo-derivatives of the selective hCB1R antagonist SR1411716A (rimonabant). By applying azo-extension strategies, we yielded compound 16a, which shows marked affinity for CB1R (Ki (cis form) = 29 nM), whose potency increases by illumination with ultraviolet light (CB1R Kitrans/cis ratio = 15.3). Through radioligand binding, calcium mobilization, and cell luminescence assays, we established that 16a is highly selective for hCB1R over hCB2R. These selective antagonists can be valuable molecular tools for optical modulation of CBRs and better understanding of disorders associated with the endocannabinoid system.

Structural Basis of Substrate Recognition and Covalent Inhibition of Cdu1 from Chlamydia trachomatis.

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into the substrate recognition of Cdu1, active-site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong basis for future structure-guided medicinal chemistry optimization of this cyanopyrimidine scaffold into more potent and selective Cdu1 inhibitors.