The pyrazole derivative of usnic acid inhibits the proliferation of pancreatic cancer cells in vitro and in vivo.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. METHODS: The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. RESULTS: Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. CONCLUSIONS: UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5.

Divergent reactivity of usnic acid and evaluation of its derivatives for antiproliferative activity against cancer cells.

Natural products continue to be an inspiration for new drugs to treat debilitating diseases such as cancer. Usnic acid is a secondary metabolite isolated predominately from lichen species and has been shown to exhibit antiproliferative properties, however its application is limited by poor drug-like properties and low specificity. We report our work on investigating the reactivity of usnic acid for incorporating heterocyclic rings and the divergent reactivity that can be obtained by simply altering the reaction solvent and temperature. The synthesised derivatives were then tested against HeLa cancer cells for their antiproliferative properties. A number of promising compounds were obtained including 4, 5 and 9 that showed an IC50 of 878, 311 and 116 nM, respectively, against HeLa cancer cells after 48 h of treatment.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

The Current Status of Drug Discovery for the Oxytocin Receptor.

The oxytocin receptor plays a significant role in peripheral regulation of parturition and lactation. Given this important role, multiple drug discovery programs have been conducted to develop agonists and antagonists for peripheral activity. The role of the oxytocin receptor in the central nervous system is also significant, promoting social interaction, trust, and empathy in humans. As such, molecules that can access the central nervous system and target the oxytocin receptor are of significant interest. Due to the role of the oxytocin receptor in regulating social function and psychological well-being, agonists of this receptor have considerable promise for the treatment of numerous neuropsychiatric conditions. The poor pharmacokinetic properties and blood-brain barrier penetration of peptide-based molecules means nonpeptide compounds have more commonly been the focus for central nervous system activity. This chapter aims to summarize the current standing of peptide and nonpeptide drug discovery for antagonists and agonists of the oxytocin receptor and focusses on centrally active nonpeptidic agonists.

Tobramycin and Colistin display anti-inflammatory properties in CuFi-1 cystic fibrosis cell line.

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and 1H NMR. HRMS showed pairing of Ibuprofen and Tobramycin, further confirmed by 1H NMR whilst no pairing was observed for either Ibuprofen:Colistin or Ibuprofen:Tadim combinations. The anti-bacterial activity of the combinations against Pseudomonas aeruginosa showed that neither paired nor non-paired Ibuprofen:antibiotic therapies altered the anti-bacterial activity. The anti-inflammatory efficacy of the combination therapies was next determined at two different concentrations (Low and High) using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines. Differential response in the anti-inflammatory efficacy of Ibuprofen:Tobramycin combination was observed between the two concentrations due to changes in the structural conformation of the paired Ibuprofen:Tobramycin complex at High concentration, confirmed by 1H NMR. In contrast, the non-pairing of the Ibuprofen:Colistin and Ibuprofen:Tadim combinations showed a significant decrease in IL-8 secretion at both the concentrations. Importantly, all antibiotics alone showed anti-inflammatory properties, highlighting the inherent anti-inflammatory properties of these antibiotics.

Tricyclic heterocycles display diverse sensitivity to the A147T TSPO polymorphism.

The 18 kDa translocator protein (TSPO) is a target for the development of imaging agents to detect neuroinflammation. The clinical utility of second-generation TSPO ligands has been hindered by the presence of a polymorphism, rs6971, which causes a non-conservative substitution of alanine for threonine at amino acid residue 147 (TSPO A147T). Given the complex nature of TSPO binding, and the lack of non-discriminating high-affinity ligands at both wild type and A147T forms of TSPO, a series of novel TSPO ligands containing various heterocyclic scaffolds was developed to explore the pharmacophoric drivers of affinity loss at TSPO A147T. In general, N-benzyl-N-methyl-substituted amide ligands showed increased affinity at TSPO A147T, and a pyrazolopyrimidine acetamide containing this motif displayed low nanomolar binding affinities to both TSPO forms.

Rapid Antibacterial Activity of Cannabichromenic Acid against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has proven to be an imminent threat to public health, intensifying the need for novel therapeutics. Previous evidence suggests that cannabinoids harbour potent antibacterial activity. In this study, a group of previously inaccessible phytocannabinoids and synthetic analogues were examined for potential antibacterial activity. The minimum inhibitory concentrations and dynamics of bacterial inhibition, determined through resazurin reduction and time-kill assays, revealed the potent antibacterial activity of the phytocannabinoids against gram-positive antibiotic-resistant bacterial species, including MRSA. One phytocannabinoid, cannabichromenic acid (CBCA), demonstrated faster and more potent bactericidal activity than vancomycin, the currently recommended antibiotic for the treatment of MRSA infections. Such bactericidal activity was sustained against low-and high-dose inoculums as well as exponential- and stationary-phase MRSA cells. Further, mammalian cell viability was maintained in the presence of CBCA. Finally, microscopic evaluation suggests that CBCA may function through the degradation of the bacterial lipid membrane and alteration of the bacterial nucleoid. The results of the current study provide encouraging evidence that cannabinoids may serve as a previously unrecognised resource for the generation of novel antibiotics active against MRSA.

Reversing binding sensitivity to A147T translocator protein.

The translocator protein (TSPO) is a target for the development of neuroinflammation imaging agents. Clinical translation of TSPO PET ligands, such as [11C]DPA-713, has been hampered by the presence of a common polymorphism (A147T TSPO), at which all second-generation TSPO ligands lose affinity. Little is known about what drives binding at A147T compared to WT TSPO. This study aimed to identify moieties in DPA-713, and related derivatives, that influence binding at A147T compared to WT TSPO. We found changes to the nitrogen position and number in the heterocyclic core influences affinity to WT and A147T to a similar degree. Hydrogen bonding groups in molecules with an indole core improve binding at A147T compared to WT, a strategy that generated compounds that possess up to ten-times greater affinity for A147T. These results should inform the future design of compounds that bind both A147T and WT TSPO for use in neuroinflammation imaging.

First Nondiscriminating Translocator Protein Ligands Produced from a Carbazole Scaffold.

Development of neuroinflammation agents targeting the translocator protein (TSPO) has been hindered by a common single nucleotide polymorphism (A147T) at which TSPO ligands commonly lose affinity. To this end, carbazole acetamide scaffolds were synthesized and structure activity relationships elaborated to explore the requirements for high-affinity binding to both TSPO wild type (WT) and the polymorphic TSPO A147T. This study reports high binding affinity and nondiscriminating TSPO ligands.

An overview of late-stage functionalization in today's drug discovery.

Introduction: Late-stage functionalization (LSF) can introduce important chemical groups in the very last steps of the synthesis. LSF has the potential to speed up the preparation of novel chemical entities and diverse chemical libraries and have a major impact on drug discovery. Functional group tolerance and mild conditions allows access to new molecules not easily accessible by conventional approaches without the need for laborious de novo chemical synthesis. Areas Covered: A historical overview of late-stage functionalization and its applicability to drug discovery is provided. Pioneering methodologies that laid the foundations for the field are briefly covered and archetypal examples of their application to drug discovery are discussed. Novel methodologies reported in the past few years mainly stemming from the recent renaissances of photoredox catalysis and radical chemistry are reviewed and their application to drug discovery considered. Expert opinion: It is envisioned that late-stage functionalization will improve the efficiency and efficacy of drug discovery. There is evidence of the widespread uptake of LSF by the medicinal chemistry community and it is expected that the recent and continuing endeavors of many academic laboratories and pharmaceutical companies will soon have an impact on drug development.

Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells.

Usnic acid is a secondary metabolite abundantly found in lichens, for which promising cytotoxic and antitumor potential has been shown. However, knowledge concerning activities of its derivatives is limited. Herein, a series of usnic acid derivatives were synthesized and their antiproliferative potency against cancer cells of different origin was assessed. Some of the synthesized compounds were more active than usnic acid. Compounds 2a and 2b inhibited survival of all tested cancer cell lines in a dose- and time-dependent manner. Their IC50 values after 48 h of treatment were ca. 3 µM for MCF-7 and PC-3 cells and 1 µM for HeLa cells, while 3a and 3b revealed antiproliferative activity only against HeLa cells. All active usnic acid derivatives induced G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7 cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of derivatives with more favorable properties against cancer cells.

Cubanes in Medicinal Chemistry.

Cubane is a highly strained saturated hydrocarbon system that has historically been of interest in theoretical organic chemistry. More recently it has become a molecule of interest for biological applications due to its inherent stability and limited toxicity. Of greater significance is the ability to potentially functionalize cubane at each of its carbon atoms, providing complex biologically active molecules with unique spatial arrangements for probing active sites. These characteristics have led to an increased use of cubane in pharmaceutically relevant molecules. In this Perspective we describe synthetic methodology for accessing a range of functionalized cubanes and their applications in pharmaceuticals. We also provide some perspectives on challenges and future directions in the advancement of this field.

Targeting the Oxytocin System: New Pharmacotherapeutic Approaches.

Deficits in social behavioral domains, such as interpersonal communication, emotion recognition, and empathy, are a characteristic symptom in several neuropsychiatric disorders, including schizophrenia and autism spectrum disorder (ASD). The neuropeptide oxytocin (OT) has emerged as a key regulator of diverse social behaviors in vertebrates and, thus, has been identified as a potential therapeutic target for improving social dysfunction. In recent years, the field of OT research has seen an explosion of scientific inquiry, producing a more comprehensive picture of oxytocinergic signaling and the pathways that regulate its release and degradation in the brain. In this review, we provide an analysis of how this information is being exploited to accelerate the discovery of novel oxytocinergic therapeutics.

Imaging glial activation in patients with post-treatment Lyme disease symptoms: a pilot study using [11C]DPA-713 PET.

The pathophysiology of post-treatment Lyme disease syndrome (PTLDS) may be linked to overactive immunity including aberrant activity of the brain's resident immune cells, microglia. Here we used [11C]DPA-713 and positron emission tomography to quantify the 18 kDa translocator protein, a marker of activated microglia or reactive astrocytes, in the brains of patients with post-treatment Lyme disease symptoms of any duration compared to healthy controls. Genotyping for the TSPO rs6971 polymorphism was completed, and individuals with the rare, low affinity binding genotype were excluded. Data from eight brain regions demonstrated higher [11C]DPA-713 binding in 12 patients relative to 19 controls. [11C]DPA-713 PET is a promising tool to study cerebral glial activation in PTLDS and its link to cognitive symptoms.

In vivo assessment of neuroinflammation in progressive multiple sclerosis: a proof of concept study with [18F]DPA714 PET.

BACKGROUND: Over the past decades, positron emission tomography (PET) imaging has become an increasingly useful research modality in the field of multiple sclerosis (MS) research, as PET can visualise molecular processes, such as neuroinflammation, in vivo. The second generation PET radioligand [18F]DPA714 binds with high affinity to the 18-kDa translocator-protein (TSPO), which is mainly expressed on activated microglia. The aim of this proof of concept study was to evaluate this in vivo marker of neuroinflammation in primary and secondary progressive MS. METHODS: All subjects were genotyped for the rs6971 polymorphism within the TSPO gene, and low-affinity binders were excluded from participation in this study. Eight patients with progressive MS and seven age and genetic binding status matched healthy controls underwent a 60 min dynamic PET scan using [18F]DPA714, including both continuous on-line and manual arterial blood sampling to obtain metabolite-corrected arterial plasma input functions. RESULTS: The optimal model for quantification of [18F]DPA714 kinetics was a reversible two-tissue compartment model with additional blood volume parameter. For genetic high-affinity binders, a clear increase in binding potential was observed in patients with MS compared with age-matched controls. For both high and medium affinity binders, a further increase in binding potential was observed in T2 white matter lesions compared with non-lesional white matter. Volume of distribution, however, did not differentiate patients from healthy controls, as the large non-displaceable compartment of [18F]DPA714 masks its relatively small specific signal. CONCLUSION: The TSPO radioligand [18F]DPA714 can reliably identify increased focal and diffuse neuroinflammation in progressive MS when using plasma input-derived binding potential, but observed differences were predominantly visible in high-affinity binders.

Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A.

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.

Peptides, Peptidomimetics, and Carbohydrate-Peptide Conjugates as Amyloidogenic Aggregation Inhibitors for Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder accounting for 60-80% of dementia cases. For many years, AD causality was attributed to amyloid-ß (Aß) aggregated species. Recently, multiple therapies that target Aß aggregation have failed in clinical trials, since Aß aggregation is found in AD and healthy patients. Attention has therefore shifted toward the aggregation of the tau protein as a major driver of AD. Numerous inhibitors of tau-based pathology have recently been developed. Diagnosis of AD has shifted from measuring late stage senile plaques to early stage biomarkers, amyloid-ß and tau monomers and oligomeric assemblies. Synthetic peptides and some derivative structures are being explored for use as theranostic tools as they possess the capacity both to bind the biomarkers and to inhibit their pathological self-assembly. Several studies have demonstrated that O-linked glycoside addition can significantly alter amyloid aggregation kinetics. Furthermore, natural O-glycosylation of amyloid-forming proteins, including amyloid precursor protein (APP), tau, and α-synuclein, promotes alternative nonamyloidogenic processing pathways. As such, glycopeptides and related peptidomimetics are being investigated within the AD field. Here we review advancements made in the last 5 years, as well as the arrival of sugar-based derivatives.

Conformationally rigid derivatives of WAY-267,464: Synthesis and pharmacology at the human oxytocin and vasopressin-1a receptors.

WAY-267,464 (1) and twelve conformationally rigid analogues (3a-f-4a-f) were synthesised, characterised and evaluated in cellular assays with the aim of systematically exploring interactions with the oxytocin receptor (OTR). Each analogue was evaluated in radioligand binding displacement assays at both human OTR and arginine vasopressin 1a receptors (V1aR). Physiological characterisation was determined by whole cell IP1 accumulation assays on stably transfected human embryonic kidney (HEK) cells. Incorporation of the rigid, optionally substituted benzene ring abolished OTR activity and diminished V1aR pharmacology when compared to 1. A general trend was observed in V1aR affinity for the propyl analogues (3d-3f) which identified the ortho-substituted analogue as the best in series (Ki = 251 nM) followed by a decrease in affinity through the meta and para-derivatives (3e; Ki = 874 nM and 3f; Ki = 1756 nM respectively). This study confirms the importance of the central pharmacophoric motifs of WAY-267,464 and illuminates the differences in the binding pocket of the highly conserved OTR and V1aR.

Investigation of pyrazolo-sulfonamides as putative small molecule oxytocin receptor agonists.

The neuropeptide oxytocin has been implicated in multiple central nervous system functions in mammalian species. Increased levels have been reported to improve trust, alleviate symptoms related to autism and social phobias, and reduce social anxiety. Hoffman-La Roche published a patent claiming to have found potent small molecule oxytocin receptor agonists, smaller than the first non-peptide oxytocin agonist reported, WAY 267,464. We selected two of the more potent compounds from the patent and, in addition, created WAY 267,464 hybrid structures and determined their oxytocin and vasopressin receptor activity. Human embryonic kidney and Chinese hamster ovary cells were used for the expression of oxytocin or vasopressin 1a receptors and activity assessed via IP1 accumulation assays and calcium FLIPR assays. The results concluded that the reported compounds in the patent and the hybrid structures have no activity at the oxytocin or vasopressin 1a receptors.