Ubiquitin-specific protease 7 (USP7): an emerging drug target for cancer treatment.

INTRODUCTION: Ubiquitin-specific protease 7 (USP7) also known as herpesvirus-associated ubiquitin-specific protease (HAUSP) is a well-characterized cysteine protease that belongs to the largest subfamily of deubiquitinating enzymes (DUBs). It is involved in multiple signaling pathways, some of them dysregulated in malignant tumors. USP7 inhibition can lead to cell growth arrest and apoptosis through inhibition of tumor promoters and stabilization of tumor suppressors, making it a promising druggable target for cancer therapy. AREAS COVERED: This review covers the structure of USP7, its function in multiple signaling pathways and relevance in cancer, as well as recent advances and future perspectives in the development of USP7 inhibitors for cancer therapy. EXPERT OPINION: Literature reports display the multiple antitumor activities of USP7 inhibitors, both in vitro and in vivo. Nonetheless, none have entered clinical trials so far, highlighting the need to delve into a deeper understanding of USP7 binding sites and the development of more accurate compound screening methods. Despite these challenges, further development of USP7 inhibitors is promising as a valuable new approach for cancer treatment, including the ability to address chemoresistance.

Probing the Interactions of Thiazole Abietane Inhibitors with the Human Serine Hydrolases ABHD16A and ABHD12.

12-Thiazole abietanes are highly selective reversible inhibitors of hABHD16A that could potentially alleviate neuroinflammation. In this study, we used synthetic chemistry, competitive activity-based protein profiling, and computational methodologies to try to establish relevant structural determinants of activity and selectivity of this class of compounds for inhibiting ABHD16A over ABHD12. Five compounds significantly inhibited hABHD16A but also very efficiently discriminated between inhibition of hABHD16A and hABHD12, with compound 35 being the most effective, at 100 µM (55.1 ± 8.7%; p < 0.0001). However, an outstanding switch in the selectivity toward ABHD12 was observed in the presence of a ring A ester, if the C2' position of the thiazole ring possessed a 1-hydroxyethyl group, as in compound 28. Although our data were inconclusive as to whether the observed enzyme inhibition is allosteric or not, we anticipate that the structure-activity relationships presented herein will inspire future drug discovery efforts in this field.

29th Annual GP2A Medicinal Chemistry Conference.

The 29th Annual GP2A (Group for the Promotion of Pharmaceutical chemistry in Academia) Conference was a virtual event this year due to the COVID-19 pandemic and spanned three days from Wednesday 25 to Friday 27 August 2021. The meeting brought together an international delegation of researchers with interests in medicinal chemistry and interfacing disciplines. Abstracts of keynote lectures given by the 10 invited speakers, along with those of the 8 young researcher talks and the 50 flash presentation posters, are included in this report. Like previous editions, the conference was a real success, with high-level scientific discussions on cutting-edge advances in the fields of pharmaceutical chemistry.

Liquorice for pain?

Liquorice has a long history of use in traditional Chinese, Ayurvedic and herbal medicine. The liquorice plant contains numerous bioactive compounds, including triterpenes, flavonoids and secondary metabolites, with glycyrrhizin being the main active compound. Liquorice constituents have been found to have anti-inflammatory, antioxidant, antiviral, anticancer, hepatoprotective and neuroprotective properties. In addition, they appear to have antidepressant actions and effects on morphine tolerance. Glycyrrhizin, its metabolite glycyrrhetic (glycyrrhetinic) acid and other liquorice-derived compounds such as isoflavonoids and trans-chalcones, exert potent anti-inflammatory effects via a wide range of mechanisms including high mobility group box 1 protein (HMGB1) inhibition, gap junction blockade and α2A-adrenoceptor antagonism. These properties, together with an increasing body of preclinical studies and a long history of use in herbal medicine, suggest that liquorice constituents may be useful for pain management. Glycyrrhizin is used widely in the confectionary, food and tobacco industries, but has documented adverse effects that may limit clinical use. Whether liquorice plant-derived compounds represent a novel class of analgesics is yet to be established. Having a host of bioactive compounds with a broad range of mechanisms of effect, liquorice is a plant that, in the future, may give rise to new therapies for pain.

Diagnosis of SARS-Cov-2 Infection by RT-PCR Using Specimens Other Than Naso- and Oropharyngeal Swabs: A Systematic Review and Meta-Analysis.

The rapid and accurate testing of SARS-CoV-2 infection is still crucial to mitigate, and eventually halt, the spread of this disease. Currently, nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the recommended standard sampling techniques, yet, these have some limitations such as the complexity of collection. Hence, several other types of specimens that are easier to obtain are being tested as alternatives to nasal/throat swabs in nucleic acid assays for SARS-CoV-2 detection. This study aims to critically appraise and compare the clinical performance of RT-PCR tests using oral saliva, deep-throat saliva/posterior oropharyngeal saliva (DTS/POS), sputum, urine, feces, and tears/conjunctival swab (CS) against standard specimens (NPS, OPS, or a combination of both). In this systematic review and meta-analysis, five databases (PubMed, Scopus, Web of Science, ClinicalTrial.gov and NIPH Clinical Trial) were searched up to the 30th of December, 2020. Case-control and cohort studies on the detection of SARS-CoV-2 were included. The methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS 2). We identified 1560 entries, 33 of which (1.1%) met all required criteria and were included for the quantitative data analysis. Saliva presented the higher accuracy, 92.1% (95% CI: 70.0-98.3), with an estimated sensitivity of 83.9% (95% CI: 77.4-88.8) and specificity of 96.4% (95% CI: 89.5-98.8). DTS/POS samples had an overall accuracy of 79.7% (95% CI: 43.3-95.3), with an estimated sensitivity of 90.1% (95% CI: 83.3-96.9) and specificity of 63.1% (95% CI: 36.8-89.3). The remaining index specimens could not be adequately assessed given the lack of studies available. Our meta-analysis shows that saliva samples from the oral region provide a high sensitivity and specificity; therefore, these appear to be the best candidates for alternative specimens to NPS/OPS in SARS-CoV-2 detection, with suitable protocols for swab-free sample collection to be determined and validated in the future. The distinction between oral and extra-oral salivary samples will be crucial, since DTS/POS samples may induce a higher rate of false positives. Urine, feces, tears/CS and sputum seem unreliable for diagnosis. Saliva testing may increase testing capacity, ultimately promoting the implementation of truly deployable COVID-19 tests, which could either work at the point-of-care (e.g. hospitals, clinics) or at outbreak control spots (e.g., schools, airports, and nursing homes).

Mitoxantrone, pixantrone and mitoxantrone (2-hydroxyethyl)piperazine are toll-like receptor 4 antagonists, inhibit NF-κB activation, and decrease TNF-alpha secretion in primary microglia.

Toll-like receptor 4 (TLR4) recognizes various endogenous and microbial ligands and is an essential part in the innate immune system. TLR4 signaling initiates transcription factor NF-κB and production of proinflammatory cytokines. TLR4 contributes to the development or progression of various diseases including stroke, neuropathic pain, multiple sclerosis, rheumatoid arthritis and cancer, and better therapeutics are currently sought for these conditions. In this study, a library of 140 000 compounds was virtually screened and a resulting hit-list of 1000 compounds was tested using a cellular reporter system. The topoisomerase II inhibitor mitoxantrone and its analogues pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were identified as inhibitors of TLR4 and NF-κB activation. Mitoxantrone was shown to bind directly to the TLR4, and pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were shown to inhibit the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) in primary microglia. The inhibitory effect on NF-κB activation or on TNFα production was not mediated through cytotoxity at ≤ 1 µM concentration for pixantrone and mitoxantrone (2-hydroxyethyl)piperazine treated cells, as assessed by ATP counts. This study thus identifies a new mechanism of action for mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine through the TLR4.

Non-leaching, Highly Biocompatible Nanocellulose Surfaces That Efficiently Resist Fouling by Bacteria in an Artificial Dermis Model.

Bacterial biofilm infections incur massive costs on healthcare systems worldwide. Particularly worrisome are the infections associated with pressure ulcers and prosthetic, plastic, and reconstructive surgeries, where staphylococci are the major biofilm-forming pathogens. Non-leaching antimicrobial surfaces offer great promise for the design of bioactive coatings to be used in medical devices. However, the vast majority are cationic, which brings about undesirable toxicity. To circumvent this issue, we have developed antimicrobial nanocellulose films by direct functionalization of the surface with dehydroabietic acid derivatives. Our conceptually unique design generates non-leaching anionic surfaces that reduce the number of viable staphylococci in suspension, including drug-resistant Staphylococcus aureus, by an impressive 4-5 log units, upon contact. Moreover, the films clearly prevent bacterial colonization of the surface in a model mimicking the physiological environment in chronic wounds. Their activity is not hampered by high protein content, and they nurture fibroblast growth at the surface without causing significant hemolysis. In this work, we have generated nanocellulose films with indisputable antimicrobial activity demonstrated using state-of-the-art models that best depict an "in vivo scenario". Our approach is to use fully renewable polymers and find suitable alternatives to silver and cationic antimicrobials.

Pyrazine-Fused Triterpenoids Block the TRPA1 Ion Channel in Vitro and Inhibit TRPA1-Mediated Acute Inflammation in Vivo.

TRPA1 is a nonselective cation channel, most famously expressed in nonmyelinated nociceptors. In addition to being an important chemical and mechanical pain sensor, TRPA1 has more recently appeared to have a role also in inflammation. Triterpenoids are natural products with anti-inflammatory and anticancer effects in experimental models. In this paper, 13 novel triterpenoids were created by synthetically modifying betulin, an abundant triterpenoid of the genus Betula L., and their TRPA1-modulating properties were examined. The Fluo 3-AM protocol was used in the initial screening, in which six of the 14 tested triterpenoids inhibited TRPA1 in a statistically significant manner. In subsequent whole-cell patch clamp recordings, the two most effective compounds (pyrazine-fused triterpenoids 8 and 9) displayed a reversible and dose- and voltage-dependent effect to block the TRPA1 ion channel at submicromolar concentrations. Interestingly, the TRPA1 blocking action was also evident in vivo, as compounds 8 and 9 both alleviated TRPA1 agonist-induced acute paw inflammation in mice. The results introduce betulin-derived pyrazine-fused triterpenoids as promising novel antagonists of TRPA1 that are potentially useful in treating diseases with a TRPA1-mediated adverse component.

Dehydroabietic oximes halt pancreatic cancer cell growth in the G1 phase through induction of p27 and downregulation of cyclin D1.

Low 5-year survival rates, increasing incidence, as well as the specific challenges of targeting pancreatic cancer, clearly support an urgent need for new multifunctional drugs for the prevention and treatment of this fatal disease. Natural products, such as abietane-type diterpenoids, are widely studied as promiscuous anticancer agents. In this study, dehydroabietic oximes were identified as potential compounds to target pancreatic cancer and cancer-related inflammation. The compounds inhibited the growth of human pancreatic cancer Aspc-1 cells with IC50 values in the low micromolar range and showed anti-inflammatory activity, measured as the inhibition of nitric oxide production, an important inflammatory mediator in the tumour microenvironment. Further studies revealed that the compounds were able to induce cancer cell differentiation and concomitantly downregulate cyclin D1 expression with upregulation of p27 levels, consistent with cell cycle arrest at the G1 phase. Moreover, a kinase profiling study showed that one of the compounds has isoform-selective, however modest, inhibitory activity on RSK2, an AGC kinase that has been implicated in cellular invasion and metastasis.

Synthesis of 7ß-hydroxy-8-ketone opioid derivatives with antagonist activity at mu- and delta-opioid receptors.

Despite extensive years of research, the direct oxidation of the 7,8-double bond of opioids has so far received little attention and knowledge about the effects of this modification on activity at the different opioid receptors is scarce. We herein report that potassium permanganate supported on iron(II) sulfate heptahydrate can be used as a convenient oxidant in the one-step, heterogeneous conversion of Δ7,8-opioids to the corresponding 7ß-hydroxy-8-ketones. Details of the reaction mechanism are given and the effects of the substituent at position 6 of several opioids on the reaction outcome is discussed. The opioid hydroxy ketones prepared are antagonists at the mu- and delta-opioid receptors. Docking simulations and detailed structure-activity analysis revealed that the presence of the 7ß-hydroxy-8-ketone functionality in the prepared compounds can be used to gain activity towards the delta opioid receptor. The 7ß-hydroxy-8-ketones prepared with this method can also be regarded as versatile intermediates for the synthesis of other opioids of interest.

Discovery of 12-Thiazole Abietanes as Selective Inhibitors of the Human Metabolic Serine Hydrolase hABHD16A.

Screening of an in-house library of compounds identified 12-thiazole abietanes as a new class of reversible inhibitors of the human metabolic serine hydrolase. Further optimization of the first hit compound lead to the 2-methylthiazole derivative 18, with an IC50 value of 3.4 ± 0.2 µM and promising selectivity. ABHD16A has been highlighted as a new target for inflammation-mediated pain, although selective inhibitors of hABHD16A (human ABHD16A) have not yet been reported. Our study presents abietane-type diterpenoids as an attractive starting point for the design of selective ABHD16A inhibitors, which will contribute toward understanding the significance of hABHD16A inhibition in vivo.

Catalytic, Tunable, One-Step Bismuth(III) Triflate Reaction with Alcohols: Dehydration Versus Dimerization.

Bi(OTf)3·xH2O is a powerful catalyst for the dehydration of tertiary alcohols into alkenes in apolar solvents. The reaction proceeds smoothly and selectively, with amounts as low as 0.01 mol % catalyst, in yields up to 93%. Moreover, in polar solvents, Bi(OTf)3·xH2O (0.1-1 mol %) selectively catalyzes the dimerization of the alcohols instead, forming new C-C bonds, in yields up to 96%. This mild, efficient, economic, and eco-friendly method is applicable across different chemical classes and amenable to several functional groups.

Trisubstituted barbiturates and thiobarbiturates: Synthesis and biological evaluation as xanthine oxidase inhibitors, antioxidants, antibacterial and anti-proliferative agents.

Barbituric and thiobarbituric acid derivatives have become progressively attractive to medicinal chemists due to their wide range of biological activities. Herein, different series of 1,3,5-trisubstituted barbiturates and thiobarbiturates were prepared in moderate to excellent yields and their activity as xanthine oxidase inhibitors, antioxidants, antibacterial agents and as anti-proliferative compounds was evaluated in vitro. Interesting bioactive barbiturates were found namely, 1,3-dimethyl-5-[1-(2-phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6c) and 1,3-dimethyl-5-[1-[2-(4-nitrophenyl)hydrazinyl]ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6e), which showed concomitant xanthine oxidase inhibitory effect (IC50 values of 24.3 and 27.9 µM, respectively), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (IC50 values of 18.8 and 23.8 µM, respectively). In addition, 5-[1-(2-phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6d) also revealed DPPH radical scavenger effect, with an IC50 value of 20.4 µM. Moreover, relevant cytotoxicity against MCF-7 cells (IC50 = 13.3 µM) was observed with 5-[[(2-chloro-4-nitrophenyl)amino]methylene]-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (7d). Finally, different 5-hydrazinylethylidenepyrimidines revealed antibacterial activity against Acinetobacter baumannii (MIC values between 12.5 and 25.0 µM) which paves the way for developing new treatments for infections caused by this Gram-negative coccobacillus bacterium, known to be an opportunistic pathogen in humans with high relevance in multidrug-resistant nosocomial infections. The most promising bioactive barbiturates were studied in silico with emphasis on compliance with the Lipinski's rule of five as well as several pharmacokinetics and toxicity parameters.

Antibacterial profiling of abietane-type diterpenoids.

Abietic and dehydroabietic acid are interesting diterpenes with a highly diverse repertoire of associated bioactivities. They have, among others, shown antibacterial and antifungal activity, potentially valuable in the struggle against the increasing antimicrobial resistance and imminent antibiotic shortage. In this paper, we describe the synthesis of a set of 9 abietic and dehydroabietic acid derivatives containing amino acid side chains and their in vitro antimicrobial profiling against a panel of human pathogenic microbial strains. Furthermore, their in vitro cytotoxicity against mammalian cells was evaluated. The experimental results showed that the most promising compound was 10 [methyl N-(abiet-8,11,13-trien-18-yl)-d-serinate], with an MIC90 of 60µg/mL against Staphylococcus aureus ATCC 25923, and 8µg/mL against methicillin-resistant S. aureus, Staphylococcus epidermidis and Streptococcus mitis. The IC50 value for compound 10 against Balb/c 3T3 cells was 45µg/mL.

Synthesis and Anti-Proliferative Activity of Sulfanyltriazolylnaphthalenols and Sulfanyltriazolylnaphthalene-1,4-diones.

A series of new sulfanyltriazolylnaphthalenols (10a-f and 13a-f) and sulfanyltriazolylnaphthalene-1,4-diones (14a-f) were synthesized and evaluated against a panel of cancer cell lines. Among the tested compounds, 10b and 10d showed the best anti-proliferative activity with GI50 values ranging from 2.72 to 10 and 3.13 to 13.1 µM, respectively, in several of the tumor cell lines tested. Compound 10d is highly selective toward leukemia cell lines and can be regarded as a good model for the development of new anti-leukemic agents.

Betulin Derivatives Effectively Suppress Inflammation in Vitro and in Vivo.

Betulin is a pharmacologically active triterpenoid found in the bark of the birch tree (Betula sp. L.). Betulin and betulinic acid are structurally related to anti-inflammatory steroids, but little is known about their potential anti-inflammatory properties. In the present study, the inflammatory gene expression and the anti-inflammatory properties of betulin, betulinic acid, and 16 semisynthetic betulin derivatives were investigated. Betulin derivatives 3, 4, and 5 selectively inhibited the expression of the inducible nitric oxide synthase (iNOS) in a post-transcriptional manner. They also inhibited nitric oxide (NO) production but had no effect on the other inflammatory factors studied. More interestingly, a new anti-inflammatory betulin derivative 9 with a wide-spectrum anti-inflammatory activity was discovered. Compound 9 was found to suppress the expression of cytokines interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), as well as that of prostaglandin synthase-2 (COX-2) in addition to iNOS. The in vivo anti-inflammatory effect of compound 9 was indicated via significant suppression of the carrageenan-induced paw inflammation in mice. The results show, for the first time, that the pyrazole-fused betulin derivative (9) and related compounds have anti-inflammatory properties that could be utilized in drug development.

Abietane-Type Diterpenoid Amides with Highly Potent and Selective Activity against Leishmania donovani and Trypanosoma cruzi.

Dehydroabietylamine (1) was used as a starting material to synthesize a small library of dehydroabietyl amides by simple and facile methods, and their activities against two disease-causing trypanosomatids, namely, Leishmania donovani and Trypanosoma cruzi, were assayed. The most potent compound, 10, an amide of dehydroabietylamine and acrylic acid, was found to be highly potent against these parasites, displaying an IC50 value of 0.37 µM against L. donovani axenic amastigotes and an outstanding selectivity index of 63. Moreover, compound 10 fully inhibited the growth of intracellular amastigotes in Leishmania donovani-infected human macrophages with a low IC50 value of 0.06 µM. This compound was also highly effective against T. cruzi amastigotes residing in L6 cells with an IC50 value of 0.6 µM and high selectivity index of 58, being 3.5 times more potent than the reference compound benznidazole. The potent activity of this compound and its relatively low cytotoxicity make it attractive for further development in pursuit of better drugs for patients suffering from leishmaniasis and Chagas disease.

New derivatives of dehydroabietic acid target planktonic and biofilm bacteria in Staphylococcus aureus and effectively disrupt bacterial membrane integrity.

The combination of the dehydroabietic acid scaffold with different amino acids resulted in the discovery of a new class of hybrid compounds that targets both planktonic and biofilms bacteria in Staphylococcus aureus strains and are far more potent anti-biofilm agents than conventional antibiotics. Unlike dehydroabietic acid, these compounds can disrupt biofilms within a short time period and compromise the integrity of the bacterial membrane. Two of the compounds identified in our study are the most potent abietane-type anti-biofilm agents reported so far and display robust activity against pre-formed biofilms at concentrations only 3-6-fold higher than those required to inhibit biofilm formation. Their easy preparation based on proteolysis-resistant d- and unusual amino acids makes them useful chemical probes to gain a deeper understanding of bacterial biofilms and outstanding candidates for further development into new drugs to fight infections.

Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development.

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.

Synthesis and biological evaluation of new oxadiazoline-substituted naphthalenyl acetates as anticancer agents.

A series of new oxadiazoline-substituted naphthalenyl acetates 3a-e and oxadiazoline-substituted 4-methoxynaphthalenyl acetates 7b-e were synthesized and tested by the National Cancer Institute (NCI) for their in vitro anticancer activity. The two derivatives bearing acetoxy groups at the 1 and 3 positions of the phenyl ring 3c and 7c were the most active showing significant anticancer activity against all tested cancer cell lines, with GI50 values ranging from 0.175 to 3.91 µM, and 0.306-11.7 µM, respectively. The selectivity of compound 3c was greater for non-solid tumor cell lines. Computational prediction of molecular and pharmacokinetic properties revealed that both compounds are safe and compound 7c had a good drug-likeness score.