Virtual high throughput screening of a large compound library against the regulatory enzyme phospholipase C (PLC) led to the discovery of the thieno[2,3-b]pyridine-2-carboxamides as potential inhibitors. Subsequent biological testing verified the antiproliferative activity of this compound class. Morphology and motility assays, using a number of triple negative breast cancer cell lines, led to the conclusion that PLC is the most probable biomolecular target. Using a combination of computer-aided drug design and synthesis, further analogues have been prepared and tested for their antiproliferative activity, allowing a comprehensive SAR to be developed. Numerous analogues with low nano-molar growth inhibition against various cancers have been prepared. SAR studies suggest that the core structure can be fine-tuned to specific cancers, potentially due to enzyme/isoform specificity. Additionally, mouse xenograft assays showed significant reduction in tumour size after treatment, whilst showing no adverse effects to non-cancerous mice. Here, we report on our recent development of novel thienopyridines and derivatives, expanding the SAR against PLC, and our efforts to prepare potent, soluble, and bioavailable compounds. Full article

The Src, a protein kinase, is a family of protein tyrosine kinases (SFKs), and this protein catalyses the phosphorylation of tyrosine. The studies have revealed its key roles in regulating signal transduction from cell surface receptors. The Src kinases act as cytoplasmic signalling machinery through regulating various cellular processes, such as cell growth, differentiation, migration, and survival. The pleiotropic functions of the Src family emphasise the importance of family members which have also been accepted as cellular oncogenes. Indole phytoalexins, which have been identified in various plants, have a structure with indole nucleus with the side chain or a heterocycle containing nitrogen and sulphur atoms. The antiproliferative effects of some phytoalexins have been demonstrated in various cancers. Among the members of phytoalexins, brassinin is known with a dithiocarbamate moiety and S-alkyl piece linked to indole core, and camalexin has an indole structure substituted at position 3 by the 1,3-thiazol-2-yl group. The inhibitory effects of these compounds on cancer cell proliferation have been reported. The aim of this study is to evaluate the effects of compounds on Src kinase activity. Human MCF-7 breast carcinoma and SW480 colorectal carcinoma cells were treated with compounds, and the effects of compounds on Src kinase activity were evaluated by Src-tyrosine kinase assay. The data were also compared with the growth inhibitory potential of compounds. The results have shown that both brassinin and camalexin have significantly inhibited the activity of Src kinase at 10 mM and higher concentrations in MCF-7 and SW480 cell lines (p < 0.05). In conclusion, this study is the first to evaluate the role of indole phytoalexins on the Src kinase activity of cancer cells. The data obtained have proven that the indole phytoalexin structure can show anticancer activity as Src mediated. It is thought that existing data will shed light on novel anticancer drug development studies. Full article

Soil fungi are known to produce and secrete antibiotics with a strong antimicrobial effect towards eukaryotic organisms. In many occasions, these compounds belong to peptides that are products of non-ribosomal biosynthesis and are called peptaibols. Many peptaibols are cytotoxic and some of them suppress tumor cell lines much better than normal cells by inducing calcium-mediated apoptosis. The main antimicrobial lipoaminopeptaibol—emericellipsin A—isolated from the fungus Emericellopsis alkalina strain VKPM F-1428, which demonstrates promising antifungal activity against different fungal taxons,has been found to exhibit selective cytotoxic activity against HepG2 and Hela cell lines (EC₅₀ 2.8 and 0.5 μM, respectively) in MTT assays in vitro. This result corresponds to the standard antitumor antibiotic doxorubicin, which has an EC₅₀ value of 440 nM. In a fibroblast toxicity test, emericellipsin A exhibited less cytotoxic activity than doxorubicin (EC₅₀ 14 and 0.34 μM, respectively). Therefore, it is less toxic to normal cells than doxirubicin (~40 times), but it yields a more potent cytotoxic effect on tumor cell lines. That is why emericellipsin A can be considered for future more detailed investigations to be an effective antitumor substance. Full article

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Therefore, exploring effective anticancer agents and their modes of action is essential for the prevention and treatment of HCC. Glycosylation can significantly improve the physicochemical and biological properties of small molecules, such as high solubility, stability increase, and lower toxicity. In this study, for the first time, we evaluated the anticancer activities of mangostin-3-O-β-d-2-deoxyglucopyranoside (Man-3DG) and mangostin 6-O-β-d-2-deoxyglucopyranoside (Man-6DG), glycosides of mangostin, against human hepatoma Hep3B cells. Our results demonstrated that Man-3DG and Man-6DG significantly suppressed growth and migration of Hep3B cells. In addition, they induced apoptosis of Hep3B cells by regulating apoptosis-related proteins of mitochondria. Noticeably, Man-3DG and Man-6DG also caused autophage, while cotreatment of the mangostin glycosides with an autophage inhibitor 3MA enhanced the inhibitory effect on Hep3B cell growth, compared to single agent treatment. Moreover, Man-3DG and Man-6DG inhibited the c-Met signaling pathway, which plays a critical role in the pathogenesis of liver cancer. Furthermore, the mangostin glycosides decreased tumor cell-induced angiogenesis in vitro through downregulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). These findings suggest that Man-3DG and Man-6DG might be promising anticancer agents for HCC treatment with superior pharmacological properties than parent molecule mangostin. Full article

Over the last decades, pyridazine derivatives are considered “privileged structures” in medicinal chemistry, with special attention being given to pyridazinones derivatives, which were found to have a large range of biological activities, including anticancer. On the other hand, because of the huge difficulties in cancer treatment, there is an urgent need from the pharmaceutical industry for new anticancer drug candidates. As part of our ongoing efforts in searching for new biologically active entities with anticancer potential, we report here the design, synthesis, structure and in vitro anticancer activity of a new class of pyridazinones derivatives, namely bis-pyridazinones. The structures of the compounds were proven by elemental and spectral analysis: IR, LC-MS, 1H-NMR, 13C-NMR, two-dimensional experiments 2D-COSY, HMQC, and HMBC. A few of the compounds were accepted by the National Cancer Institute (USA) for anticancer screening and were evaluated for their in vitro cytotoxic activity against a panel of 60 human tumor cell lines, representing cancers of the brain, breast, colon, kidney, lung, ovary, prostate, as well as leukemia and melanoma. Three of the tested compounds have proven to be active against non-small cell lung cancer HOP 92 and NCI-H226, CNC cancer SNB-75, renal cancer A498 and UO-31, with a growth inhibition between 50–80 mM. Interestingly, one compound (unsubstituted bis-pyridazinones I) has a selective anticancer activity, being active only on non-small cell lung cancer HOP 92, with a growth inhibition of 51.45 mM. SAR correlation has been performed. Full article

Malaria remains a devastating disease with nearly half a million deaths per year. The WHO reports a stagnating number of new infections every year without a significant decline, as a result of insufficient access to antimalarials in endemic regions as well as complex resistance mechanisms of the parasites against current treatments. Due to its critical role during the parasite’s life cycle, the invasion motor myosin A is a promising target, which has not yet been considered in drug discovery. Myosins appeared to be undruggable since they are ubiquitously expressed and involved in a wide range of cellular processes. In total, the protein superfamily of myosins comprises 35 known subclasses. However, recent studies highlighted the possibility to modulate the myosin motor activity of specific myosin isoforms and classes using small allosteric effector molecules. Exploiting the concept of reversible covalent binding, we show the development of highly potent and specific inhibitors of the key motor myosin A of the glideosome—a sophisticated motor machinery involved in parasite motility and host cell invasion. Combining chemical synthesis with biophysical in vitro analysis confirmed the preferential inhibition of the target protein in the submicromolar range. The developed compounds show significant antiparasitic activities and block efficiently glideosome-associated processes, parasite proliferation, and parasitemia of the malaria parasites. Our findings demonstrate the high potential of our approach using reversible covalent binding to develop new allosteric inhibitors, targeting specifically the key invasion motor as a novel drug target to treat infections caused by malaria parasites. Full article

Cancer continues to be one of the leading causes of death, according to the World Health Organization, and chemotherapy is its principal treatment. Organometallic complexes with copper (II) and zinc (II) with Schiff bases as ligand, capable of interacting with cancer cells’ DNA under physiological conditions, may work as good chemotherapy agents because they are less toxic to healthy cells than to cancerous ones. In view of the above, this work focuses on obtaining new Schiff base ligands and their copper and zinc complexes and characterizing them by nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, and other spectroscopic and spectrometric techniques. The objective is to evaluate the toxic activity of the new molecules using the fast proliferating cells of the zebrafish model during development. The toxicity test was performed in zebrafish embryos at 8, 24, 48 and 72 h post fertilization, and a survival and malformation index were registered. Preliminary results show a dose-related effect of the designed Schiff ligands and complexes on the toxicity of the zebrafish embryo and larvae. The survival and malformation index are more severe when exposure occurs during early developmental stages, when cell division is higher due to rapid organization and growth of the new organism. This is a promising result, as the molecules might be cytotoxic to highly proliferating cells, as it occurs in cancer cells. This work represents one of the very few examples that use the zebrafish model to evaluate the cytotoxic activity of Schiff base complexes. Developing the animal model to test the effect of Schiff ligands and their complexes is an important first step to assess the effectiveness of new molecules as antineoplastic agents. Full article

Chemotherapy is an essential treatment that still plays a vital role in cancer treatment worldwide. The ferrocene derivatives of the general formula [Fe{(η⁵‑C₅H₄CH₂(p‑C₆H₄)CH₂(N‑het)}₂] bearing modified six and five membered N-heterocycles were tested in vitro for their cytotoxic properties against ovarian cancer cell lines A2780 and SK-OV-3. These ferrocene complexes displayed cytotoxicity in low micromolar concentrations against both cell lines. To study cellular uptake of particular ferrocenes into tumor cells, we used differential pulse voltammetry and ICP-MS. We confirmed the crucial role of transferrin receptors in the process of intracellular accumulation of these ferrocenes. Interestingly, the rate of intracellular accumulation of particular ferrocenes clearly mirrored the cytotoxicity of these organometallic compounds. Deeper investigation of the mechanism by which ferrocenes kill tumor cells revealed induction of apoptosis associated with significant increase of reactive oxygen species. In conclusion, our screening identified several ferrocene derivatives exerting promising cytostatic activity in vitro. Further investigation led to the identification of the mechanism of action of these potential anticancer agents, which represents an important milestone in preclinical anticancer drug discovery programs. This work was supported by the project MEYS-NPS I-LO1413, MH CZ-DRO (MMCI, 00209805) and Czech Science Foundation project 17-05838S. Full article

Cancer is one of the most serious and merciless health problems of humankind, and the number of new cases is expected to increase in the next decades. Despite extensive cancer research in order to find more effective drugs and treatments, cancer chemotherapy is complex and complicated, because of the limited efficacy of drugs, significant levels of toxicity, and lack of selectivity, and the emergence of drug resistance and multidrug resistance make the situation even worse. We report here the design, synthesis, structure, and in vitro anticancer activity of two series of compounds derived from pyridazine and phthalazine. The in vitro anticancer activity was tested on a panel of 60 human tumor cell lines representing cancers of the brain, breast, colon, kidney, lung, ovary, prostate, as well as leukemia and melanoma, to the National Cancer Institute (USA). The test was conducted on a single dose and five dose assay. Notably, from the tested compounds, five of them show very good anticancer activity (superior to Doxorubicin, the NCI standard drug for this type of analysis), with a growth inhibition in the area of nanomolar, between 20–100 nM, on several cancer cell lines: breast cancer MCF7, colon cancer HCT-15, KM12 and SW-620, leukemia K562 and SR, melanoma MDA-MB-435, SK-MEL-5, and UACC-62, and renal cancer A498. SAR correlation in the two series and in between the two series has been performed. One compound has an excellent anticancer activity against breast cancer MCF7 cell, leukemia SR cell, and melanoma MDA-MB-435 cell, in the area of 20 nM. Full article

The orphan G protein coupled receptor 6 (GPR6) is a cannabinoid-related Class A GPCR. It is highly expressed in the central nervous system and exhibits high constitutive activation of adenylyl cyclase. Several research groups have demonstrated that GPR6 represents a possible target for the treatment of neurodegenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. Several patents claim the use of a wide range of pyrazine derivatives as GPR6 inverse agonists for the treatment of Parkinson’s disease and other dyskinesia syndromes. Using cyclic AMP accumulation assays in hGPR6-CHO cells as a readout, the most potent GPR6 pyridopyrazine inverse agonist compounds identified thus far have been found to display IC₅₀ values in the low nanomolar range. A subset of these compounds was used here as starting points for the design of novel potent GPR6 inverse agonists using a core hopping approach. In parallel with the core hopping studies, we employed a recently constructed homology model of the GPR6 inactive state. The X-ray crystal structure of the Sphingosine-1-phosphate receptor 1 (S1P1) receptor structure was used as the template and the conformational memories technique was used to explore the conformational consequences of sequence differences between S1P1 and GPR6. The most potent GPR6 pyridopyrazine inverse agonists were docked in the resultant GPR6 inactive state model, first as tests of the model. Once we had identified the binding site of each inverse agonist, we used this site to identify amino acid sites in proximity that we can use to build additional interactions for ligands output from core hopping studies above. ADME properties and the absence of PAINS will also be considered for the hit selection process. These potential GPR6 chemotypes may serve as research tools for further understanding the biological role of this orphan receptor. Full article

Epilepsy is recognized as one of the most common neurological disorders with a high risk of drug resistance. Notably, about one-third of the patients with epilepsy are not responsive to pharmacological treatment. Thus, the search for new, more effective anticonvulsants with a novel mechanism of action is undoubtedly necessary. The most recent neurobiological studies implicate central TRPV1 receptors in the induction of epileptic seizures. Moreover, it is suggested that TRPV1 desensitization is one of the crucial mechanisms of action responsible for the anticonvulsant activity of cannabidiol (CBD), which was proven to be effective against drug-resistant epilepsy. Bearing in mind the aforementioned facts, we developed in our recent studies a series of chemically original TRPV1 antagonists. Their structures were designed as integrated hybrids that join on the common chemical template the structural fragments of anticonvulsants identified by our team in the previous studies and known TRPV1 antagonists (described in the literature). As a result, these compounds revealed potent anticonvulsant activity in the preclinical studies using the most widely employed animal seizure models, namely, the maximal electroshock (MES) test, and the psychomotor 6 Hz (32 mA and 44 mA) seizure model in mice. In addition, selected substances demonstrated potent effectiveness by decreasing pain responses in formalin-induced tonic pain, in capsaicin-induced neurogenic pain, as well as in oxaliplatin-induced neuropathic pain in mice. Full article

17β-Estradiol (E2), the main human estrogen, has been known to exert multiple actions throughout the body, including in the central nervous system (CNS). In particular, it has been shown that E2 is gender-independently needed for brain and eye health. Lack of E2 due to normal aging and/or pathological processes leads to neurological and psychiatric diseases as well as accelerated neurodegeneration. Current estrogen replacement therapies, however, cannot be used as therapeutic interventions to treat these maladies due to a profound, unwanted hormonal exposure to the rest of the body. In this presentation, we show that the small-molecule bioprecursor prodrug 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) produces E2 only in the CNS but remains inert in the rest of the body, both upon chronic systemic and topical administrations, thereby avoiding the detrimental side-effects of the hormone, such as stimulation of the uterus and tumor growth. The highly localized production of E2 in the CNS will be shown through a series of bioanalytical assays and efficacy studies using animal models of estrogen-responsive maladies pertaining to the brain and the retina. Owing to DHED’s significantly more favorable physicochemical properties than the highly lipophilic parent E2 for transport through biological membranes such as the blood-brain barrier or the cornea, a highly effective E2 therapy can be achieved in rodents upon prodrug administration, which further enhances therapeutic safety. Altogether, our patented DHED approach shows unprecedented selectivity to deliver E2 into the CNS and, thus, promises a high translation value in terms of efficacious and safe treatment against neurodegeneration as well as neurological and psychiatric symptoms arising from estrogen deficiency. Full article

Despite decades of research, only few drugs have been approved that interact with purine receptors. Recently, new hypes and hopes have been created in the field, mainly due to the gold rush fever in immuno-oncology. Adenosine is one of the strongest immunosuppressant agents of the innate immune system. Cancer cells and tissues can release large amounts of ATP, which is immediately hydrolyzed by ectonucleotidases. These ecto-enzymes, including ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1, CD203a), ectonucleoside diphospho­hydrolase 1 (NTPDase1, CD39), and ecto-5′-nucleotidase (CD73), are upregulated on many cancer cells, leading to the production of adenosine. The cloud of adenosine formed around cancer tissues contributes to immune escape by interacting with adenosine A_2A and A_2B receptor subtypes (A_2AAR, A_2BAR) on immune cells. In addition, activation of A_2BARs by adenosine enhances cancer cell proliferation, metastasis, and angiogenesis. Blockade of A_2A and A_2B adenosine receptors and/or inhibition of adenosine formation by blocking ectonucleotidases are being pursued as novel principles that activate the immune system to defeat cancer. Recent progress in the development of adenosine receptor antagonists and ectonucleotidase inhibitors will be presented and discussed. Full article

Our previous research showed that a novel series of phenylsulfonyl hydrazide derivatives reduced LPS-induced PGE₂ levels in RAW 264.7 macrophage cells via an inhibition of mPGES-1 enzyme. As a continuous work, new phenylsulfonamide derivatives (5a-5k) as methylene analogues of phenylsulfonyl hydrazide derivatives, including MPO-0063, were synthesized and biologically evaluated in vitro. Among synthetic compounds, 5a (MPO-0112) showed decreased inhibitory activity against PGE₂ production (IC₅₀: 0.34 µM) compared to MPO-0063 (IC₅₀: 0.04 µM) but inhibited the mPGES-1 enzyme (IC₅₀: 7.37 µM) similar to MPO-0063 (IC₅₀: 0.10 µM) together with excellent selectivity over COX-enzymes (COX-1 and 2). According to recent studies on the close correlation between up-regulation of mPGES-1 and Alzheimer's disease, we investigated whether 5a could ameliorate scopolamine-induced memory impairment using the passive avoidance test. The memory impairment-ameliorating effect of 5a (1.0 mg/kg, p.o.) was found to be effective, comparable to that of donepezil (5 mg/kg, p.o.) as a positive control. On the other hand, 5a exhibited little or weak AChE and BuChE inhibitory activity, which implies that 5a could ameliorate scopolamine-induced memory impairment by inhibiting mPGES-1 enzyme instead of cholinesterase enzymes. In addition, MPO-0112 exhibited a favorable in vitro CYP profile, which is suggestive of no potential drug–drug interactions. Therefore, these overall results suggest that 5a as a selective mPGES-1 inhibitor may be a novel therapeutic agent for diseases associated with cognitive deficits, such as Alzheimer’s disease. Full article

Microsomal prostaglandin E synthase-1 (mPGES-1) is responsible for the massive prostaglandin E₂ (PGE₂) formation during inflammation. Increasing evidence reveals mPGES-1 inhibitors as a safe alternative to nonsteroidal anti-inflammatory drugs. Recently, we reported that a novel series of phenylsulfonyl hydrazide derivatives could reduce LPS-induced PGE₂ levels in RAW 264.7 macrophage cells via an inhibition of the mPGES-1 enzyme. However, a few of the phenylsulfonyl hydrazide derivatives showed poor metabolic stability in liver microsomes. In order to identify new mPGES-1 inhibitors with improved metabolic stability, therefore, a series of arylsulfonamide derivatives has been synthesized and biologically evaluated against PGE₂ production and the mPGES-1 enzyme. Among them, MPO-0186 inhibits the production of PGE₂ (IC₅₀ = 0.20 μM) in A549 cells via inhibition of mPGES-1 (IC₅₀ = 0.49 μM in a cell-free assay) together with high selectivity over both COX-1 and COX-2. A molecular docking study theoretically suggests that MPO-0186 could inhibit PGE₂ production by blocking the PGH₂ binding site of the mPGES-1 enzyme. Furthermore, MPO-0186 demonstrated good metabolic stability in human liver microsomes and no significant inhibition observed in clinically relevant CYP isoforms. Full article

Transition metal complexes attract continuous research interest as potential antitumor agents. The most popular compounds are ruthenium, gold, titanium, osmium, iridium, zinc, and palladium complexes, which have already displayed cytotoxic features that are not typical for classical platinum-containing chemotherapeutic agents. Substantially lower attention is drawn to organometallic compounds of rhenium. However, the known examples of cytotoxic organometallic rhenium derivatives with bidentate heterocyclic, organophosphorus, labile alkoxide, and hydroxide ligands render further studies in this field very promising. As for their analogs with multidentate ligands, a literature survey has revealed only a few examples of cytotoxic rhenium complexes, whereas the antitumor activity of cyclometallated derivatives has not been studied at all. At the same time, it is known that the use of pincer-type ligands having specific tridentate monoanionic frameworks, which offer multiple options for directed structural modifications, allows one to finely tune the thermodynamic and kinetic stability of the resulting metal complexes. Therefore, we synthesized and studied the cytotoxic properties of a series of rhenium(I) complexes with tridentate pincer-type ligands based on functionalized carboxamides bearing ancillary donor groups both in the acid and amine components. It was shown that the target complexes can be obtained not only by the conventional solution-based method, but also under solvent-free conditions according to the solid-phase methodology recently developed in our group. The results obtained were used to define the main structure–activity relationships for a principally new class of potential antitumor agents and to choose the most promising compounds for further studies in order to create new pharmaceuticals. Full article

Abstract: Physicochemical and pharmacokinetic properties of compounds marked with acronyms PQA-AZ-4 and PQA-AZ-6 were studied using experimental methods. Selected compounds (dual-active effective inhibitors of phosphodiesterase (PDE) 10A and serotonin 5-HT_1A and 5-HT₇ receptor ligands) in prescreening pharmacological studies revealed antipsychotic-like, antidepressant-like, and anxiolytic activities. The liquid chromatographic–tandem mass spectrometric method with electrospray ionization (LC/ESI-MS/MS) system was calibrated and validated for lipophilicity studies. Lipophilicity was assessed from the y axis intercept (y₀) of the linear regression line of ln((t − t₀)/t₀) against % concentration of organic eluent. Finally, lipophilicity was calculated using a linear regression equation of the logP value against y₀ values obtained for the reference compounds in the same experimental conditions. Next, the thermodynamic aqueous solubility of selected compounds was detected with UPLC detection. The LC/ESI-MS/MS system was used for the simultaneous determination of PQA-AZ-4 and PQA-AZ-6 in mouse plasma, hippocampus, striatum, and frontal cortex, developed and validated according to GLP procedures. Finally, drug-likeness properties of selected compounds were evaluated using a predictive bioavailability radar model from the SwissADME web tool. The descriptors of physicochemical properties (lipophilicity, size, polarity, solubility, flexibility, and saturation) for selected compounds were projected next on the optimal range for each property to be considered drug-like. Full article

The Forkhead boX M1 (FOXM1) is an essential transcription factor for normal activation of the cell cycle and cell replication. However, increasing evidence suggests that overexpression of this protein correlates with cancer development and poor patient prognosis, which makes FOXM1 a promising drug target in medicinal chemistry. Based on a computer-based molecular modeling protocol reported by our group, we hypothesized that FOXM1 inhibitors bind to the FOXM1 DNA binding domain (DBD) by (i) a pi-sulfur interaction with His287, and (ii) a halogen bonding with Arg297 within the FOXM1 DNA binding domain. To test this hypothesis, we modified the chemical structure of a known “forkhead domain inhibitor” (FDI) to synthesize and screen a series of FDI-derivatives. In this regard, we removed or replaced two essential groups in FDI-6, namely (i) the 4-fluorophenyl position and (ii) the heterocyclic sulfur atoms. We determined the inhibitory effects of test molecules on the protein expression of FOXM1 using a triple negative breast cancer cell line (MDA-MB-231), and then we measured their binding affinity to DNA by electrophoretic mobility shift assay (EMSA). Next, using a site-directed mutagenesis technique, we confirmed specific binding interactions exerted by these molecules. These results validate the role of essential binding interactions (pi-sulfur binding) predicted by computer simulations and provide preliminary evidence to postulate a mechanism of action exerted by “direct” FOXM1 inhibitors. Full article

Skin cancer has evolved as the most common malignant disease, accounting for 4.5% of all new cancer cases. Melanoma, the most aggressive skin cancer type, develops in melanocytes and has high mortality rates due to its biological features and frequent failures of therapeutic alternatives. On the other hand, non-melanoma skin cancers (NMSC), the most common malignant tumors in humans, are developed in keratinocytes of the basal or spinous layer, and increased exposure to ultraviolet (UV) light remains the most important modifiable risk factor. To date, treatment alternatives for melanoma are limited to surgery, in cases of early diagnosis, and a few pharmacological options in inoperable tumors. These limitations for skin cancer management evidence the need to develop therapeutic options for prevention and treatment. The antiproliferative effects of Amaryllidaceae alkaloids have been tested for different types of cancer. However, their activity on skin models is not well-established. Pure alkaloids and alkaloidal fractions characterized by GC-MS of several Amaryllidaceae species from Crinum, Zephyranthes, Hippeastrum, and Eucharis genera were assayed by their effects on skin cancer. Photoprotective effects of the alkaloids and fractions were determined through cell viability assay, and the quantification of intracellular reactive oxygen species (ROS) and inflammation biomarker IL-6 in UVB-stimulated keratinocytes (HaCaT). Cytotoxicity were assessed in human metastatic melanoma cells (CRL-3229) to evaluate therapeutic potential, and chemometric techniques were used to analyze data. Most substances enhanced HaCaT proliferation at 5.0 µg/mL. E. caucana and Z. carinata bulbs alkaloidal fractions significantly reduced intracellular ROS and IL-6 production in UVB-stimulated HaCaT, respectively. Tazettine and lycoramine showed photoprotective effects. Additionally, E. caucana bulbs alkaloidal fraction was selectively cytotoxic in melanoma cells at 20.0 µg/mL. Collectively, these results demonstrate that Amaryllidaceae alkaloids could represent a new option in skin cancer management, acting as photoprotective agents in healthy UVB-exposed keratinocytes and therapeutic agents in melanoma. Full article

In continuation of synthetic pursuit of metallo-nucleosides, in particular dicobalt hexacarbonyl 5-alkynyl-2′-deoxyuridines, novel compounds with alkynyl groups were synthesized, starting from 5-iodo-2′-deoxyuridine. Reactions of dicobalt octacarbonyl [Co₂(CO)₈] with 2′-deoxy-5-oxopropynyluridines and related compounds gave dicobalt hexacarbonyl nucleoside complexes (83–31%). The growth inhibition of HeLa and K562 cancer cell lines by organometallic nucleosides was examined and compared to that by alkynyl nucleoside precursors. Coordination of the dicobalt carbonyl moiety to the 2′-deoxy-5-alkynyluridines led to a significant increase in its cytotoxic potency. The cobalt compounds antiproliferative activities against the HeLa cell line and the K562 cell line will be described. Coordination of an acetyl-substituted cobalt nucleoside was expanded using the 1,1-bis(diphenylphosphino)methane (dppm) ligand, resulting in cytotoxicity at comparable levels. The formation of reactive oxygen species in the presence of cobalt compounds was determined in K562 cells. The results indicate that the mechanism of action for most antiproliferative cobalt compounds may be related to the induction of oxidative stress. Full article

Epigenetic regulation of gene expression without changing DNA sequences is a promising strategy in developing therapeutic agents for human diseases, especially cancer. Histone deacetylases (HDACs) are a family of enzymes involved in epigenetic modulation of gene expression by chromatin remodeling. Deacetylation of the lysine side chains of histones by HDAC proteins renders DNA transcriptionally inactive, resulting in the inhibition of expression of tumor suppressor genes, leading to tumorigenesis and tumor progression. Therefore, inhibiting HDAC enzymes has become an attractive strategy to modulate gene expression as a strategy for developing anticancer drugs. There are currently four FDA-approved cancer drugs in clinical use, and many more are in different stages of clinical trials. However, their clinical utility is limited due to undesirable side effects, mainly attributed to their lack of selectivity and the presence of a hydroxamic acid moiety as the metal-binding group. There are eighteen different isoforms of HDACs belonging to four classes that have been identified in humans. A major challenge in HDAC inhibitor development is how to make them selective for these HDAC isoforms and classes. We report the design and synthesis of new classes of HDAC inhibitors and the evaluation of their anticancer activity and selectivity. Full article

The subfamily Amaryllidoideae within of the Amaryllidaceae family has an exclusive group of compounds called Amaryllidaceae alkaloids. Galanthamine, the most known Amaryllidaceae alkaloid, is approved by the FDA as an inhibitor of the enzyme acetylcholinesterase (AChE) for the palliative treatment of Alzheimer Disease (AD). However, butyrylcholinesterase (BChE) contributes critically to cholinergic dysfunction associated with AD. Thus, the development of novel therapeutics may involve the inhibition of both cholinesterase enzymes. Zephyranthes carinata, a species of the Amaryllidaceae family, has been reported to have inhibitory activity against cholinesterases. In order to determine the enzymatic inhibition potential, the major alkaloids of bulbs and leaves of Z. carinata were evaluated in both AChE and BChE. A purification and characterization process was made using different chromatographic and spectrometric techniques, and the inhibitory activity was evaluated with the Ellman method. Alkaloidal extracts of bulbs and leaves exhibited an inhibitory activity with IC₅₀ values of 5.8 ± 0.2 and 8.7 ± 0.3 μg/mL, respectively, against AChE. Further, bulb extract showed IC₅₀ values of 77.9 ± 3.4 μg/mL against BChE. Amaryllidaceae alkaloids hamayne, pseudolycorine, galanthine, criasbetaine, tazettine, lycoramine, hippeastidine, galanthamine, trisphaeridine, 3-epimacronine, haemanthamine, lycorine, and vittatine were purified and evaluated for their AChE and BChE inhibitory activities. Lycoramine (galanthamine type) presented the lowest IC₅₀ value in AChE (17 ± 0.7 μg/mL), and trisphaeridine (narciclasine type) showed the lowest IC₅₀ value in BChE (33.1 ± 3.6 μg/mL). Combined major alkaloids (>10%) were analyzed to observe synergistic behavior. The mixture alkaloids lycoramine and galanthine presented IC₅₀ values of 14.55 ± 1.0 μg/mL against AChE, and the lycoramine, trisphaeridine, and vittatine mix presented IC₅₀ values of 38.42 ± 3.4 μg/mL in BChE. These results showed prominent inhibitory activity against AChE and BChE enzymes, indicating their potential as agents for treating AD through a combined strategy. Full article

The entry of HIV-1 into permissible cells remains an extremely attractive and underexploited therapeutic intervention point. We have previously demonstrated the ability to extend the chemotypes available for optimization in the entry inhibitor class using computational means. Here, we continue this effort, designing and testing three novel compounds with the ability to inhibit HIV-1 entry. We demonstrate that alteration of the core moiety of these entry inhibitors directly influences the potency of the compounds, despite common proximal and distal groups. Moreover, by establishing for the first time a surface plasmon resonance (SPR)-based interaction assay with soluble recombinant SOSIP Env trimers, we demonstrate that the off-rate (kd) parameter shows the strongest correlation with potency in an antiviral assay. Finally, we establish an underappreciated relationship between the potency of a ligand and its degree of electrostatic complementarity (EC) with its target, the Env complex. These findings not only broaden the chemical space in this inhibitor class, but also establish a rapid and simple assay to evaluate future HIV-1 entry inhibitors. Full article

Fibroblast activation protein (FAP) is a type II transmembrane serine protease that belongs to the dipeptidyl peptidase (DPP) family. Although FAP expression is practically non-existent in the majority of healthy adult tissues, it is clearly upregulated in tissue remodeling processes associated with several diseases. These include cancer, atherosclerosis, arthritis, hepatic, and pulmonary fibrosis. This finding has recently sparked intensive research aiming at the clinical implementation of FAP as a diagnostic and/or prognostic biomarker for the aforementioned diseases. Several immunochemical approaches have been reported that can quantify FAP expression. The main drawback of these approaches, however, lies in the fact that some of the commercially available FAP antibodies have been reported to lack specificity. On the other hand, an orthogonal line of research focuses on the quantification of the enzymatically active fraction of FAP, generally relying on peptidic activity-based probes. Developing a selective activity-based assay for FAP has proven to be challenging, owing to the frequently encountered lack of probe selectivity towards prolyl oligopeptidase (PREP, PO). In response, we report a novel series of activity-based FAP probes, based on our potent and selective inhibitor UAMC-1110. Full article

Alzheimer’s disease (AD) is a complex and progressive neurodegenerative disorder. The available therapy is limited to symptomatic treatment, and its efficacy remains unsatisfactory. In view of the prevalence and expected increase in the incidence of AD, the development of an effective therapy is crucial for public health. Since the therapeutic paradigm “one compound–one-target” has shown its limits in the treatment of AD, new strategies are emerging to overcome the lack of efficiency of the current pharmacotherapy in the past decade. The most promising is the multitarget-directed ligands (MTDLs) strategy. This project consists of the development of new multifunctional agents, which will act simultaneously on the different players in AD pathology by combining an AChE inhibitory activity based on the structures of a well-known AChE inhibitor (Rivastigmine) with an α-7 nAChR activation. Indeed, nAChRs were recently put forward as potential targets for the treatment of central nervous system (CNS) diseases, such as AD. Because of their distribution and abundance in the CNS, the α-7 subtypes are potential therapeutic targets for this disorder. Full article

The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the Nedd4-1 E3 Ubiquitin ligase is an enzyme that may be targeted either covalently, or non-covalently, there are few studies that demonstrate effective inhibitors of the enzyme. In this work, we aimed to identify covalent inhibitors of Nedd4-1. This task however, proved to be challenging due to the limited available electrophilic moieties in virtual libraries. We therefore opted to divide an existing covalent Nedd4-1 inhibitor in two parts: A non-covalent binding part and a pre-selected α, β-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on the active site binding investigations followed by validating the covalent conjugation. Thirty compounds were selected and covalently docked into the catalytic site of the Nedd4-1. Multiple filtrations were effected before selecting 5 hits that were later analysed by molecular dynamic simulations to check their stability and explore their binding landscape in complex with the protein. All in all, two inhibitors with optimum overall stability and more stabilising interactions were kept for eventual biological evaluation. Our improved pharmacophore model approach serves as a robust method that will illuminate the screening for novel covalent inhibitor in drug discovery. Full article

Fragment-based drug discovery (FBDD) has become a major strategy to derive novel lead candidates for both new and established therapeutic targets, as it promises efficient exploration of chemical space by employing fragment-sized (MW 300) compounds. One of the first challenges in implementing a FBDD approach is the design of a fragment library, and more specifically, the choice of its size and individual members. In order to construct a library that maximises the chances of discovering novel chemical matter, a large number of fragments with sufficient structural diversity are often sought. However, the exact diversity of a certain collection of fragments remains elusive, which hinders direct comparisons among different selections of fragments. Building upon structural fingerprints that are commonly utilised in cheminformatics, we herein introduced quantitative measures for the structural diversity of fragment libraries. Structures of commercially available fragments were retrieved from the ZINC database and filtered by physicochemical properties, after which they were subject to selections with library sizes ranging from 100 to 100,000 compounds. The selected libraries were evaluated and compared quantitatively, resulting in interesting size-diversity relationships. Our results suggested the existence of an optimal size for structural diversity and demonstrated that such quantitative measures can guide the design of diverse fragment libraries under various circumstances Full article

Phospholipid-hydrolyzing enzymes, such as phospholipases A₂ (PLA₂s) and autotaxin (ATX), have attracted medicinal interest because they are involved in the generation of various inflammatory mediators. PLA₂s hydrolyze membrane phospholipids initiating the arachidonic acid cascade, and in particular calcium-independent PLA₂ (iPLA₂), have been recognized as a participant in biological processes underlying diabetes development and autoimmune-based disorders. ATX hydrolyzes lysophosphatidylcholine, generating lysophosphatidic acid; both ATX and lysophosphatidic acid are involved in pathological conditions, such as fibrosis and cancer. We have developed several classes of potent PLA₂ inhibitors. In this presentation, we will present new β-lactones as highly potent inhibitors of iPLA₂ and a novel class of ATX inhibitors containing the zinc binding functionality of hydroxamic acid. Various novel hydroxamic acids based on either 4-aminophenylacetic acid or non-natural δ-amino acids were synthesized and evaluated (J. Med Chem. 2018, 61, 3697–3711). Hydroxamic acids that incorporate a δ-amino acid residue exhibit high in vitro inhibitory potency over ATX. Inhibitor GK442 (IC₅₀ 60 nM), based on δ-norleucine, was tested for its efficacy in a mouse model of pulmonary inflammation and fibrosis induced by bleomycin and exhibited promising efficacy. New β-lactones have been synthesized and evaluated for inhibitory potency over iPLA₂ (J. Med Chem. 2019, 62, 2916–2927). A β-lactone substituted at position-3 by a four-carbon chain carrying a phenyl group, and at position-4 by a n-propyl group (GK563), was identified as being the most potent iPLA₂ inhibitor ever reported (X_I(50) 0.0000021). GK563 was found to reduce β-cell apoptosis induced by pro-inflammatory cytokines, raising the possibility that it can be beneficial in countering autoimmune diseases, such as type-1 diabetes. Full article

Today, drug abuse has developed into a social problem and begun to demand specific measures from different social sectors and government agencies all over the world. Despite significant efforts made toward relevant mechanistic targets, such as the dopamine transporter (DAT), the development of pharmacotherapeutic treatments of psychostimulant abuse has remained a challenge so far. Using a set of 49 2-[(diphenylmethyl)sulfanyl]ethanamines described as DAT inhibitors, 2D-QSAR/PLS studies were performed using two different approaches of variable selection: Ordered predictors selection (OPS) and genetic algorithm (GA). All structures were optimized at the B3LYP/6-311G++(d,p) level of theory. The molecular descriptors were obtained in the Dragon 6 program (topological, geometric, molecular, and constitutional) and GaussView 03 (electronic). Both models were formed by two latent variables. Model 1 (OPS) was constructed with four molecular descriptors (GATS3m, Mor15p, SpMin3_Bh(s), and HOMO-1), while six (Mor13m, CATS2D_09_LL, RDF110u, RDF085m, Mor24s, and RDF010s) were required to obtain model 2 (GA). The models can be considered reasonably different: In model 1, electronic features predominate, whereas in model 2, steric and geometric effects do. The overall test indicated that models 1 and 2 have equivalent predictive ability (Average r²_m Overall = 0.730 versus 0.710 and Delta r²_m Overall = 0.122 versus 0.151). However, model 1 is simpler (it has only four descriptors, which facilitates its interpretation), presents more relevant information used in the construction of its two latent variables (75.99% versus 64.07%), and its calibration is more significant than that of model 2 (F_n,n−p−1 = 115.814 versus 80.888, for the same tabled F value, where n = 36, and n − p − 1 = 3.256, with alfa = 0.05). Considering these results, although model 2 may also be considered a good result, model 1, obtained using the OPS approach for variable selection, may be considered more reliable for prediction purposes. This result is in agreement with good results previously obtained using the OPS methodology. Full article

Bivalent degrader molecules (also termed PROTACs) target proteins for degradation through recruitment to E3 ligases. PROTACs are a revolutionary new modality class with therapeutic potential. Formation of a ternary complex between the degrader, the ligase, and the target leads to tagging by ubiquitination and proteasomal degradation of the target protein. In 2015, we disclosed MZ1, a potent degrader made of a ligand we had previously discovered for the E3 ligase von Hippel–Lindau (VHL), and a pan-selective ligand for the BET proteins Brd2, Brd3, and Brd4. We made the unexpected but fascinating observation that MZ1 induces preferential degradation of Brd4 over Brd2 and Brd3—despite engaging BET proteins with the same binary affinity. This demonstrated a now well-established feature of PROTACs: They can achieve a narrower degradation profile in spite of broad target engagement. Our co-crystal structure of a PROTAC ternary complex (VHL:MZ1:Brd4) illuminated the role of cooperative molecular recognition inducing de novo contacts to form a stable ternary. Our work is revealing the structural basis and guiding principles of PROTAC degradation selectivity and mode of action. Full article

Nowadays, the incidence of neurodegenerative diseases is increasing, and these disorders will become one of the main challenges for medicine and public health in future years. Particularly, memory loss characterizes many neurodegenerative pathologies, and it is often related to low levels of cyclic adenosine monophosphate (cAMP). [...] Full article

The phosphatidyl–choline-specific phospholipase C (PC-PLC) enzyme has been shown to be an important enzyme involved in various cell-signaling processes. Furthermore, PC-PLC has been shown to be upregulated in various cancer cell lines, thereby presenting itself as a potential anti-cancer therapeutic target. Current PC-PLC inhibitors, including the literature standard inhibitor D609 possess characteristics making them unsuitable for clinical use. We have discovered a new class of potent PC-PLC inhibitors with much improved activity and drug-like properties than D609. The synthesis and SAR study of this class of active compounds is presented. Full article

Muramyl dipeptide (MDP, N-acetylmuramyl-l-alanyl-d-isoglutamine) is known as the smallest synthetic adjuvant molecule capable of replacing whole Mycobacteria in Freund’s adjuvant. Numerous MDP derivatives were synthesized with the aim to avoid MDP unwanted side-effects. Many of them have therapeutic potential, including clinical use. A very important parameter in the improvement of pharmacological properties of MDP is lipophilicity, e.g., it eliminates drawbacks caused by poor macrophage penetration and rapid elimination. On the other side, mannose receptors (MR), present on immunocompetent cells (such as macrophages and dendritic cells), are considered to be pattern-recognition receptors and responsible for the binding, among others, of mannosylated antigens or relevant biologically active molecules containing mannose, thus affecting the immune reactions. Up to now, our research was directed towards desmuramyl peptides which contain adamantylglycine and mannosylated adamantylglycine moieties bound to the essential part of MDP, l-Ala-d-isoGln. Here, we present the design and synthesis of novel mannosylated muropeptide analogs containing 2-aminoadamantane-2-carboxylic acid. Prepared desmuramyl peptides have lipophilic 2-aminoadamantane-2-carboxylic acid attached at the N-terminus of desmuramy dipeptide core and mannose connected to the tripeptide over a glycolyl linker. Immunostimulating activities of prepared compounds will be evaluated in the mice model using ovalbumin as an antigen and compared with previously prepared derivatives. Full article

Taking into account that the identification of novel molecules for the effective treatment of inflammatory and immune diseases is one of the main present medical needs and one of the major goals of the pharmaceutical industry, the aim of our work is intended to provide new therapeutic strategies and a deeper understanding of the mechanism of action of new drugs related to such disorders. [...] Full article

Cysteine proteases belonging to the papain superfamily have been recognized as interesting therapeutic targets for the search for new drugs against infectious tropical diseases such as malaria (falcipain), Chagas’ disease (curtain), leishmaniasis, and Sleeping sickness (rhodesian), and a number of human pathologies, including cancer, Alzheimer’s disease, and osteoporosis (cathepsins). [...] Full article

Docking studies of identified GPR55 ligands using a GPR55 inactive state model allow rationalizing key structural features involved in ligand–receptor binding. On this molecular basis, we have designed novel quinolone sulfonamide derivatives with optimized potency and efficacy. These novel molecules compounds are being synthesized and evaluated using a β-arrestin recruitment assay in CHO cells overexpressing human GPR55 and βarr2-GFP. Full article