Low Micromolar Concentration Range Research Articles

Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

Solid-Phase Parallel Synthesis of Dual Histone Deacetylase-Cyclooxygenase Inhibitors.

Multi-target drugs (MTDs) are emerging alternatives to combination therapies. Since both histone deacetylases (HDACs) and cyclooxygenase-2 (COX-2) are known to be overexpressed in several cancer types, we herein report the design, synthesis, and biological evaluation of a library of dual HDAC-COX inhibitors. The designed compounds were synthesized via an efficient parallel synthesis approach using preloaded solid-phase resins. Biological in vitro assays demonstrated that several of the synthesized compounds possess pronounced inhibitory activities against HDAC and COX isoforms. The membrane permeability and inhibition of cellular HDAC activity of selected compounds were confirmed by whole-cell HDAC inhibition assays and immunoblot experiments. The most promising dual inhibitors, C3 and C4, evoked antiproliferative effects in the low micromolar concentration range and caused a significant increase in apoptotic cells. In contrast to previous reports, the simultaneous inhibition of HDAC and COX activity by dual HDAC-COX inhibitors or combination treatments with vorinostat and celecoxib did not result in additive or synergistic anticancer activities.

Stafiba: A STAT5-Selective Small-Molecule Inhibitor.

The transcription factors STAT5a and STAT5b are constitutively active in many human tumors. Combined inhibition of both STAT5 proteins is a valuable approach with promising applications in tumor biology. We recently reported resorcinol bisphosphate as a moderately active inhibitor of the protein-protein interaction domains, the SH2 domains, of both STAT5a and STAT5b. Here, we describe the development of resorcinol bisphosphate to Stafiba, a phosphatase-stable inhibitor of STAT5a and STAT5b with activity in the low micromolar concentration range. Our data provide insights into the structure-activity relationships of resorcinol bisphosphates and the corresponding bisphosphonates for use as inhibitors of both STAT5a and STAT5b.

6‐Methylquinazolin‐4(3H)‐one Based Compounds as BRD9 Epigenetic Reader Binders: A Rational Combination of in silico Studies and Chemical Synthesis

Abstract6‐methylquinazolin‐4(3H)‐one‐based compounds were here identified and synthesized as novel binders of bromodomain‐containing protein 9 (BRD9) epigenetic reader. Accounting a fast and efficient synthetic route aimed to easily obtain differently 2‐ and 8‐disubstituted 6‐methylquinazolin‐4(3H)‐one derivatives, a virtual library of synthesizable items was built and submitted to molecular docking experiments. Based on two 3D structure‐based pharmacophore models recently developed by us on BRD9, 16 compounds were selected and synthesized, using mild conditions with good yields in relatively short reaction times. Among them, 14, 16, 18, 22, and 26 emerged as the most potent compounds of these series, able to bind BRD9 at the low micromolar range of concentrations. These molecules also showed a promising selective behavior when tested against BRD4 bromodomain. These results highlighted the quinazolin‐4(3H)‐one chemical core as a valuable scaffold for developing promising BRD9 binders.

Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 μM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

Heteroleptic Pd(II) and Pt(II) Complexes with Redox-Active Ligands: Synthesis, Structure, and Multimodal Anticancer Mechanism.

A series of heteroleptic square-planar Pt and Pd complexes with bis(diisopropylphenyl) iminoacenaphtene (dpp-Bian) and Cl, 1,3-dithia-2-thione-4,5-dithiolate (dmit), or 1,3-dithia-2-thione-4,5-diselenolate (dsit) ligands have been prepared and characterized by spectroscopic techniques, elemental analysis, X-ray diffraction analysis, and cyclic voltammetry (CV). The intermolecular noncovalent interactions in the crystal structures were assessed by density functional theory (DFT) calculations. The anticancer activity of Pd complexes in breast cancer cell lines was limited by their solubility. Pd(dpp-Bian) complexes with dmit and dsit ligands as well as an uncoordinated dpp-Bian ligand were devoid of cytotoxicity, while the [Pd(dpp-Bian)Cl2] complex was cytotoxic. On the contrary, all Pt(dpp-Bian) complexes demonstrated anticancer activity in a low micromolar concentration range, which was 8-20 times higher than the activity of cisplatin, and up to 2.5-fold selectivity toward cancer cells over healthy fibroblasts. The presence of a redox-active dpp-Bian ligand in Pt and Pd complexes resulted in the induction of reactive oxygen species (ROS) in cancer cells. In addition, these complexes were able to intercalate into DNA, indicating the dual mechanism of action.

Inhibition of Microtubule Dynamics in Cancer Cells by Indole-Modified Latonduine Derivatives and Their Metal Complexes

Indolo[2,3-d]benzazepines (indololatonduines) are rarely discussed in the literature. In this project, we prepared a series of novel indololatonduine derivatives and their RuII and OsII complexes and investigated their microtubule-targeting properties in comparison with paclitaxel and colchicine. Compounds were fully characterized by spectroscopic techniques (1H NMR and UV–vis), ESI mass-spectrometry, and X-ray crystallography, and their purity was confirmed by elemental analysis. The stabilities of the compounds in DMSO and water were confirmed by 1H and 13C NMR and UV–vis spectroscopy. Novel indololatonduines demonstrated anticancer activity in vitro in a low micromolar concentration range, while their coordination to metal centers resulted in a decrease of cytotoxicity. The preliminary in vivo activity of the RuII complex was investigated. Fluorescence staining and in vitro tubulin polymerization assays revealed the prepared compounds to have excellent microtubule-destabilizing activities, even more potent than the well-known microtubule-destabilizing agent colchicine.

Triapine Analogues and Their Copper(II) Complexes: Synthesis, Characterization, Solution Speciation, Redox Activity, Cytotoxicity, and mR2 RNR Inhibition.

Three new thiosemicarbazones (TSCs) HL1–HL3 as triapine analogues bearing a redox-active phenolic moiety at the terminal nitrogen atom were prepared. Reactions of HL1–HL3 with CuCl2·2H2O in anoxic methanol afforded three copper(II) complexes, namely, Cu(HL1)Cl2 (1), [Cu(L2)Cl] (2′), and Cu(HL3)Cl2 (3), in good yields. Solution speciation studies revealed that the metal-free ligands are stable as HL1–HL3 at pH 7.4, while being air-sensitive in the basic pH range. In dimethyl sulfoxide they exist as a mixture of E and Z isomers. A mechanism of the E/Z isomerization with an inversion at the nitrogen atom of the Schiff base imine bond is proposed. The monocationic complexes [Cu(L1–3)]+ are the most abundant species in aqueous solutions at pH 7.4. Electrochemical and spectroelectrochemical studies of 1, 2′, and 3 confirmed their redox activity in both the cathodic and the anodic region of potentials. The one-electron reduction was identified as metal-centered by electron paramagnetic resonance spectroelectrochemistry. An electrochemical oxidation pointed out the ligand-centered oxidation, while chemical oxidations of HL1 and HL2 as well as 1 and 2′ afforded several two-electron and four-electron oxidation products, which were isolated and comprehensively characterized. Complexes 1 and 2′ showed an antiproliferative activity in Colo205 and Colo320 cancer cell lines with half-maximal inhibitory concentration values in the low micromolar concentration range, while 3 with the most closely related ligand to triapine displayed the best selectivity for cancer cells versus normal fibroblast cells (MRC-5). HL1 and 1 in the presence of 1,4-dithiothreitol are as potent inhibitors of mR2 ribonucleotide reductase as triapine.

Type and position of linkage govern the cytotoxicity of oleanolic acid rhodamine B hybrids

Oleanolic acid/rhodamine B hybrids exhibit different cytotoxicity depending on the way these two structural elements are linked. While a hybrid holding a piperazinyl spacer at C-28 proved to be cytotoxic in the nano-molar concentration range, hybrids with a direct linkage of the Rho B residue to C-3 of the triterpenoid skeleton are cytotoxic only in the low micro-molar concentration range without any selectivity. This once again underlines the importance of selecting the right spacer and the most appropriate position on the skeleton of the triterpene to achieve the most cytotoxic hybrids possible.

Structure-Based Identification of Potent Lysine-Specific Demethylase 1 Inhibitor Peptides and Temporary Cyclization to Enhance Proteolytic Stability and Cell Growth-Inhibitory Activity.

Peptides are attractive drug candidates, but their utility is greatly limited by their inherent susceptibility to proteolytic degradation and their inability to pass through the cell membrane. Here, we employ a strategy of temporary cyclization to develop a cell-active lysine-specific demethylase 1 (LSD1/KDM1A) inhibitor peptide. We first identified a highly potent LSD1-inhibitory linear peptide, with the assistance of X-ray crystal structure data of inhibitor peptide-bound LSD1·CoREST. The peptide was converted to a redox-activatable cyclic peptide incorporating cell-penetrating peptide (CPP), expecting selective activation under intracellular reducing conditions. The cyclic peptide moiety exhibited enhanced stability to protease and was converted to the linear, unmodified LSD1 inhibitor peptide under reducing conditions. The cyclic peptide with CPP inhibited the proliferation of human acute myeloid leukemia cells (HL-60) in the low micromolar concentration range.

Structural insights into long-distance signal transduction pathways mediated by plant glutamate receptor-like channels.

In recent years, studies have shed light on the physiological role of plant glutamate receptor-like channels (GLRs). However, the mechanism by which these channels are activated, and in particular, what is the physiological role of their binding to amino acids, remains elusive. The first direct biochemical demonstration that the Arabidopsis thaliana GLR3.3 isoform binds glutamate and other amino acids in a low micromolar range of concentrations was reported only recently. The first crystal structures of the ligand-binding domains of AtGLR3.3 and AtGLR3.2 isoforms also have been released. We foresee that these new experimental pieces of evidence provide the basis for a better understanding of how GLRs are activated and modulated in different physiological responses.

Mimetics of extra virgin olive oil phenols with anti-cancer stem cell activity.

The extra virgin olive oil (EVOO) dihydroxy-phenol oleacein is a natural inhibitor of multiple metabolic and epigenetic enzymes capable of suppressing the functional traits of cancer stem cells (CSC). Here, we used a natural product-inspired drug discovery approach to identify new compounds that phenotypically mimic the anti-CSC activity of oleacein. We coupled 3D quantitative structure-activity relationship-based virtual profiling with phenotypic analysis using 3D tumorsphere formation as a gold standard for assessing the presence of CSC. Among the top 20 computationally-predicted oleacein mimetics, four fulfilled the phenotypic endpoint of specifically suppressing the tumorsphere-initiating capacity of CSC, in the absence of significant cytotoxicity against differentiated cancer cells growing in 2D cultures in the same low micromolar concentration range. Of these, 3,4-dihydrophenetyl butyrate –a lipophilic ester conjugate of the hydroxytyrosol moiety of oleacein– and (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide) –an inhibitor of Trypanosoma cruzi triosephosphate isomerase– were also highly effective at significantly reducing the proportion of aldehyde dehydrogenase (ALDH)-positive CSC-like proliferating cells. Preservation of the mTOR/DNMT binding mode of oleacein was dispensable for suppression of the ALDH+-CSC functional phenotype in hydroxytyrosol-unrelated mimetics. The anti-CSC chemistry of complex EVOO phenols such as oleacein can be phenocopied through the use of mimetics capturing its physico-chemical properties.

Triphenylphosphonium conjugates of 1,2,3-triazolyl nucleoside analogues. Synthesis and cytotoxicity evaluation

A series of triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl analogues of several pyrimidine nucleosides was synthesized and evaluated for the in vitro cytotoxicity against human cancer cell lines M-HeLa, MCF-7, PANC-1, PC-3, DU145, SKOV-3, A275, and normal human cell line WI-38. In these TPP-conjugates triphenylphosphonium cation was attached via a tetramethylene chain to the N-3 atom of the heterocycle moiety (uracil, thymine, quinazoline-2,4-dione), which was coupled with the D-ribofuranosyl-1,2,3-triazol-4-yl fragment via methylene or tetramethylene linker. It was shown for the first time that the conjugation of 1,2,3-triazolyl derivatives of uridine, its analogues featuring quinazoline-2,4-dione fragment as well as uracil and thymine derivatives, having propargyl or a 1,2,3-triazolyl substituent at the N-1 atom, with a TPP-butyl cation endowed some of them with cytotoxic activity against human cancer cells. Among all human cancer cell lines used, DU-145 and A375 cells were the most sensitive to these TPP conjugates. At the same time, all tested compounds did not inhibit growth of normal cells WI-38. Propargyl containig TPP-conjugates of uracil 4f, 4j, and thymine 5f showed the highest cytotoxicity with IC50 values in the low micromolar concentration range. The present findings suggest that TPP-conjugates of uracil and thymine derivatives would be promising for further development as an anticancer agent.

MiRNA Modulation and Antitumor Activity by the Extra-Virgin Olive Oil Polyphenol Oleacein in Human Melanoma Cells.

Extra-virgin olive oil (EVOO) polyphenols contribute to Mediterranean diet health-promoting properties. One of the most abundant secoiridoid present in EVOO, Oleacein (OA), demonstrated anticancer activity against several tumors. Nevertheless, its role against melanoma has not still investigated. This study aimed at determining in vitro the antimelanoma activity of OA and the relative mechanism of action. OA induced cell growth inhibition in 501Mel melanoma cells with an IC50 in the low micromolar range of concentrations. Moreover, an OA concentration approximating the IC50 induced G1/S phase arrest, DNA fragmentation, and downregulation of genes encoding antiapoptotic (BCL2 and MCL1) and proproliferative (c-KIT, K-RAS, PIK3R3, mTOR) proteins, while increased transcription levels of the proapoptotic protein BAX. Concordantly, OA increased the levels of miR-193a-3p (targeting MCL1, c-KIT and K-RAS), miR-193a-5p (targeting PIK3R3 and mTOR), miR-34a-5p (targeting BCL2 and c-KIT) and miR-16-5p (miR-16-5p targeting BCL2, K-RAS and mTOR), while decreased miR-214-3p (targeting BAX). These modulatory effects might contribute to the inhibition of 501Mel melanoma cell growth observed after treatment with an olive leaves-derived formulation rich in OA, with potential application against in situ cutaneous melanoma. Altogether, these results demonstrate the ability of OA to contrast the proliferation of cutaneous melanoma cells through the transcriptional modulation of relevant genes and microRNAs, confirming the anticancer potential of EVOO and suggesting OA as a chemopreventive agent for cancer disease therapy.

Identification of the subtype-selective Sirt5 inhibitor balsalazide through systematic SAR analysis and rationalization via theoretical investigations.

We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and balsalazide with Sirt5 in the presence of NAD+. Based on the evidence gathered here, we designed and synthesized 13 analogues of balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the N-aroyl-β-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part minimally altered potency. Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD+ or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking studies pave the way for further optimization.

Aromatic Cytokinin Arabinosides Promote PAMP-like Responses and Positively Regulate Leaf Longevity.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

Alternarin A, a Drimane Meroterpenoid, Suppresses Neuronal Excitability from the Coral-Associated Fungi Alternaria sp. ZH-15.

Alternarin A (1), a rearranged drimane meroterpenoid characterized by a thioglycerate moiety, was isolated together with two known analogues from the coral-associated fungi Alternaria sp. ZH-15. Its structure was determined based on spectroscopic analysis, modified Mosher's method, and TDDFT/ECD calculations. In a primary cultured cortical neuronal network, compound 1 effectively inhibited the activity of spontaneous synchronous Ca2+ oscillations and 4-aminopyridine induced epileptic discharges in the low micromolar concentration range.

Synthesis and Cytotoxic Activity of New Vindoline Derivatives Coupled to Natural and Synthetic Pharmacophores.

New Vinca alkaloid derivatives were synthesized to improve the biological activity of the natural alkaloid vindoline. To this end, experiments were performed to link vindoline with various structural units, such as amino acids, a 1,2,3-triazole derivative, morpholine, piperazine and N-methylpiperazine. The structure of the new compounds was characterized by NMR spectroscopy and mass spectrometry (MS). Several compounds exhibited in vitro antiproliferative activity against human gynecological cancer cell lines with IC50 values in the low micromolar concentration range.

Gold-catalyzed cyclization in the synthesis of antimitotic 2,3-dihydrobenzo[b]oxepine derivatives of colchicine

New allocolchicine derivatives bearing 2,3-dihydrobenzo[b]oxepine moiety were synthesized via gold-catalyzed cyclization as a key synthetic step. The obtained 2,3-dihydrobenzo[b]oxepine-containing allocolchicinoids possess cytotoxic activity against HEK293, PANC-1, COL0357, HeLa, and Colon26 cancer cell lines at low micromolar range of concentrations.

Novel Inhibitors of DNA Repair Enzyme TDP1 Combining Monoterpenoid and Adamantane Fragments.

The DNA repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a current inhibition target to improve the efficacy of cancer chemotherapy. Previous studies showed that compounds combining adamantane and monoterpenoid fragments are active against TDP1 enzyme. This investigation is focused on the synthesis of monoterpenoid derived esters of 1-adamantane carboxylic acid as TDP1 inhibitors. New esters were synthesized by the interaction between 1-adamantane carboxylic acid chloride and monoterpenoid alcohols. The esters were tested against TDP1 and its binding to the enzyme was modeling. 13 Novel ester-based TDP1 inhibitors were synthesized with yields of 21-94%; of these, nine esters had not been previously described. A number of the esters were found to inhibit TDP1, with IC50 values ranging from 0.86-4.08 µM. Molecular modelling against the TDP1 crystal structure showed a good fit of the active esters in the catalytic pocket, explaining their potency. A non-toxic dose of ester, containing a 3,7- dimethyloctanol fragment, was found to enhance the cytotoxic effect of topotecan, a clinically used anti-cancer drug, against the human lung adenocarcinoma cell line A549. The esters synthesized were found to be active against TDP1 in the lower micromolar concentration range, with these findings being corroborated by molecular modeling. Simultaneous action of the ester synthesized from 3,7-dimethyloctanol-1 and topotecan revealed a synergistic effect.