The Mpro structure-based modifications of ebselen derivatives for improved antiviral activity against SARS-CoV-2 virus.

The main protease (Mpro or 3CLpro) of SARS-CoV-2 virus is a cysteine enzyme critical for viral replication and transcription, thus indicating a potential target for antiviral therapy. A recent repurposing effort has identified ebselen, a multifunctional drug candidate as an inhibitor of Mpro. Our docking of ebselen to the binding pocket of Mpro crystal structure suggests a noncovalent interaction for improvement of potency, antiviral activity and selectivity. To test this hypothesis, we designed and synthesized ebselen derivatives aimed at enhancing their non-covalent bonds within Mpro. The inhibition of Mpro by ebselen derivatives (0.3 µM) was screened in both HPLC and FRET assays. Nine ebselen derivatives (EBs) exhibited stronger inhibitory effect on Mpro with IC50 of 0.07-0.38 µM. Further evaluation of three derivatives showed that EB2-7 exhibited the most potent inhibition of SARS-CoV-2 viral replication with an IC50 value of 4.08 µM in HPAepiC cells, as compared to the prototype ebselen at 24.61 µM. Mechanistically, EB2-7 functions as a noncovalent Mpro inhibitor in LC-MS/MS assay. Taken together, our identification of ebselen derivatives with improved antiviral activity may lead to developmental potential for treatment of COVID-19 and SARS-CoV-2 infection.

Dual Cathepsin B and Glutathione-Activated Dimeric and Trimeric Phthalocyanine-Based Photodynamic Molecular Beacons for Targeted Photodynamic Therapy.

Two dual stimuli-activated photosensitizers were developed, in which two or three glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS)-substituted zinc(II) phthalocyanine units were connected via one or two cathepsin B-cleavable Gly-Phe-Leu-Gly peptide linker(s). These dimeric and trimeric phthalocyanines were fully quenched in the native form due to the photoinduced electron transfer to the DNBS substituents and the self-quenching of the phthalocyanine units. In the presence of GSH and cathepsin B, or upon internalization into A549 and HepG2 cancer cells, these probes were activated through the release of free phthalocyanine units. The intracellular fluorescence intensity was increased upon post-incubation with GSH ester or reduced upon pre-treatment with a cathepsin B inhibitor. Upon light irradiation, these photosensitizers became highly cytotoxic with IC50 values of 0.21-0.39 µM. The photocytotoxicity was also dependent on the intracellular GSH and cathepsin B levels. The results showed that these conjugates could serve as smart photosensitizers for targeted photodynamic therapy.

New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (-)-fenchone hydrazonesv.

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76-118. In addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation between the structure of the synthesised compounds and the antiviral activity was established.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

New insight into the role of glutathione reductase in glutathione peroxidase-like activity determination by coupled reductase assay: Molecular Docking Study.

Previously we have shown that among 15 substituted salicyloyl (2-hydroxybenzoyl) 5-seleninic acids (SSAs) 4 compounds with longer side chains or a cyclohexyl group exhibit no glutathione peroxidase (GPx)-like activity in the coupled reductase assay. Experimental inhibition of glutathione reductase (GR) by the selenenylsulfide (a main intermediate in the catalytic cycle for GPx-like activity determination) of one of the inactive compounds led us to assess the interactions between 15 selenenylsulfide compounds and the active site of GR by molecular docking. Docking results showed that S and Se atoms in selenenylsulfides of the compounds with no GPx-like activity were beyond 5 Šfrom S atom of Cys-58 or N atom of imidazole ring of His-467 (Root Mean Square Distances for general assessment of 3 major distances were over 4.8 Å) in the active site, so that they could not be catalyzed to be reduced by GR. Furthermore, their docking scores over 89 Kcal/mol meant that the selenenylsulfides were bound too strongly to the active site to leave it, leading eventually to inhibition of GR. We also applied the molecular docking to other GPx mimics such as ebselen, cyclic seleninate esters and di(propylaminomethylphenyl) diselenides to explain the differences in their GPx-like activity depending to the assays used. Our results suggest that the reduction of a selenenylsulfide by GR plays a positive role in GPx-like activity of GPx mimics in the coupled assay and recommended the prediction of possibility and strength of GPx-like activity by molecular docking before entering experimental research.

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2.

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PLpro ) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PLpro . Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PLpro are valuable starting points for the development of new pan-coronaviral inhibitors.

Isoindoline scaffold-based dual inhibitors of HDAC6 and HSP90 suppressing the growth of lung cancer in vitro and in vivo.

This study reports the synthesis of a series of 2-aroylisoindoline hydroxamic acids employing N-benzyl, long alkyl chain and acrylamide units as diverse linkers. In-vitro studies led to the identification of N-benzyl linker-bearing compound (10) and long chain linker-containing compound (17) as dual selective HDAC6/HSP90 inhibitors. Compound 17 displays potent inhibition of HDAC6 isoform (IC50 = 4.3 nM) and HSP90a inhibition (IC50 = 46.8 nM) along with substantial cell growth inhibitory effects with GI50 = 0.76 µM (lung A549) and GI50 = 0.52 µM (lung EGFR resistant H1975). Compound 10 displays potent antiproliferative activity against lung A549 (GI50 = 0.37 µM) and lung H1975 cell lines (GI50 = 0.13 µM) mediated through selective HDAC6 inhibition (IC50 = 33.3 nM) and HSP90 inhibition (IC50 = 66 nM). In addition, compound 17 also modulated the expression of signatory biomarkers associated with HDAC6 and HSP90 inhibition. In the in vivo efficacy evaluation in human H1975 xenografts, 17 induced slightly remarkable suppression of tumor growth both in monotherapy as well as the combination therapy with afatinib (20 mg/kg). Moreover, compound 17 could effectively reduce programmed death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose dependent manner suggesting that dual inhibition of HDAC6 and HSP90 can modulate immunosuppressive ability of tumor area.

Integrating Molecular Network and Culture Media Variation to Explore the Production of Bioactive Metabolites by Vibrio diabolicus A1SM3.

Vibrio diabolicus A1SM3 strain was isolated from a sediment sample from Manaure Solar Saltern in La Guajira and the produced crude extracts have shown antibacterial activity against methicillin-resistant Staphylococcus aureus and cytotoxic activity against human lung cell line. Thus, the aim of this research was to identify the main compound responsible for the biological activity observed and to systematically study how each carbon and nitrogen source in the growth media, and variation of the salinity, affect its production. For the characterization of the bioactive metabolites, 15 fractions obtained from Vibrio diabolicus A1SM3 crude extract were analyzed by HPLC-MS/MS and their activity was established. The bioactive fractions were dereplicated with Antibase and Marinlit databases, which combined with nuclear magnetic resonance (NMR) spectra and fragmentation by MS/MS, led to the identification of 2,2-di(3-indolyl)-3-indolone (isotrisindoline), an indole-derivative antibiotic, previously isolated from marine organisms. The influence of the variations of the culture media in isotrisindoline production was established by molecular network and MZmine showing that the media containing starch and peptone at 7% NaCl was the best culture media to produce it. Also, polyhydroxybutyrates (PHB) identification was established by MS/MS mainly in casamino acids media, contributing to the first report on PHB production by this strain.

Synthesis, antitumor activity and DNA binding features of benzothiazolyl and benzimidazolyl substituted isoindolines.

In this paper novel isoindolines substituted with cyano and amidino benzimidazoles and benzothiazoles were synthesized as new potential anti-cancer agents. The new structures were evaluated for antiproliferative activity, cell cycle changes, cell death, as well as DNA binding and topoisomerase inhibition properties on selected compounds. Results showed that all tested compounds exerted antitumor activity, especially amidinobenzothiazole and amidinobenzimidazole substituted isoindolin-1-ones and benzimidazole substituted 1-iminoisoindoline that showed antiproliferative effect in the submicromolar range. Moreover, the DNA-binding properties of selected compounds were evaluated by biophysical and biochemical approaches including thermal denaturation studies, circular dichroism spectra analyses and topoisomerase I/II inhibition assays and results identified some of them as strong DNA ligands, harboring or not additional topoisomerase II inhibition and able to locate in the nucleus as determined by fluorescence microscopy. In conclusion, we evidenced novel cyano- and amidino-substituted isoindolines coupled with benzimidazoles and benzothiazoles as topoisomerase inhibitors and/or DNA binding compounds with potent antitumor activities.

Sulfatase activity assay using an activity-based probe by generation of N-methyl isoindole under reducing conditions.

Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. This is an important step in several biological processes such as cellular degradation, hormone regulation, and cell signaling. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions, containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), exhibiting little change in kinetic parameters compared to non-reducing conditions. Probe 1 displayed reasonable kinetic parameters under both non-reducing and reducing conditions, among which the use of Tris buffer and Tris buffer containing GSH appeared to be appropriate conditions for inhibitor screening. Probe 1 showed stronger intensity upon treatment with sulfatase under neutral conditions than under acidic conditions, but it still has limitations in the selectivity for a specific sulfatase. Nevertheless, the fluorescent signal generated as a result of the release of N-methylisoindole after treatment of probe 1 with sulfatase provides a new assay for measuring sulfatase activity that could be adapted for high throughput screening.

Design, synthesis, and biological evaluation of structurally modified isoindolinone and quinazolinone derivatives as hedgehog pathway inhibitors.

The Hedgehog (Hh) signaling pathway is associated with diverse aspects of cellular events, such as cell migration, proliferation, and differentiation throughout embryonic development and tissue patterning. An abnormal Hh signaling pathway is linked to numerous human cancers, including basal cell carcinoma (BCC), medulloblastoma (MB), lung cancer, prostate cancer, and ovarian cancer, and it is therefore a promising target in cancer therapy. Using a structure-hopping approach, we designed new Hh signaling pathway inhibitors with isoindolinone or quinazolinone moieties, which were synthesized and biologically evaluated using an 8xGli-luciferase (Gli-Luc) reporter assay in NIH3T3 cells. Compounds 9-11 and 14 with isoindolinone scaffolds demonstrated moderate Hh inhibitory activity; whereas quinazolinone derivatives 24, 29, 32, 34, and 35 exhibited good potency with submicromolar IC50 values and the analog 28 showed nanomolar IC50 value. Although sonidegib shows a decrease in inhibitory effect on vismodegib resistance-conferring Smo mutants, the structurally modified new compounds not only possess the pharmacophoric properties of Hh pathway inhibition but also preserve the suppressive potency in drug-resistant Smo mutants. Mechanistically, quinazolinone derivatives 28 and 34 suppress Hh signaling by blocking Smo and Gli translocation into the cilia, similar to vismodegib and sonidegib. Additionally, the human microsomal stability of the representative analogs 28 and 34 were determined to be comparable to that of the reference compound sonidegib. Thus, these new scaffolds can serve as a platform for the development of novel cancer therapeutics targeting the Hh pathway.

Thalidomide (5HPP-33) suppresses microtubule dynamics and depolymerizes the microtubule network by binding at the vinblastine binding site on tubulin.

Thalidomides were initially thought to be broad-range drugs specifically for curing insomnia and relieving morning sickness in pregnant women. However, its use was discontinued because of a major drawback of causing teratogenicity. In this study, we found that a thalidomide derivative, 5-hydroxy-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33), inhibited the proliferation of MCF-7 with a half-maximal inhibitory concentration of 4.5 ± 0.4 µM. 5HPP-33 depolymerized microtubules and inhibited the reassembly of cold-depolymerized microtubules in MCF-7 cells. Using time-lapse imaging, the effect of 5HPP-33 on the dynamics of individual microtubules in live MCF-7 cells was analyzed. 5HPP-33 (5 µM) decreased the rates of growth and shortening excursions by 34 and 33%, respectively, and increased the time microtubules spent in the pause state by 92% as compared to that of the vehicle-treated MCF-7 cells. 5HPP-33 (5 µM) reduced the dynamicity of microtubules by 62% compared to the control. 5HPP-33 treatment reduced the distance between the two poles of a bipolar spindle, induced multipolarity in some of the treated cells, and blocked cells at mitosis. In vitro, 5HPP-33 bound to tubulin with a weak affinity. Vinblastine inhibited the binding of 5HPP-33 to tubulin, and 5HPP-33 inhibited the binding of BODIPY FL-vinblastine to tubulin. Further, a molecular docking analysis suggested that 5HPP-33 shares its binding site on tubulin with vinblastine. The results provided significant insight into the antimitotic mechanism of action of 5HPP-33 and also suggest a possible mechanism for the teratogenicity of thalidomides.

In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5.

The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important therapeutic target.

MDM2-p53 protein-protein interaction inhibitors: a-ring substituted isoindolinones.

Structure-activity relationships for the MDM2-p53 inhibitory activity of a series of A-ring substituted 2-N-benzyl-3-(4-chlorophenyl)-3-(1-(hydroxymethyl)cyclopropyl)methoxy)isoindolinones have been investigated, giving rise to compounds with improved potency over their unsubstituted counterparts. Isoindolinone A-ring substitution with a 4-chloro group for the 4-nitrobenzyl, 4-bromobenzyl and 4-cyanobenzyl derivatives (10a-c) and substitution with a 6-tert-butyl group for the 4-nitrobenzyl derivative (10j) were found to confer additional potency. Resolution of the enantiomers of 10a showed that potent MDM2-p53 activity resided in the (-)-enantiomer ((-)-10a; IC(50)=44 ± 6 nM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compounds 10a and (-)-10a increase p53 protein levels, activate p53-dependent MDM2 and p21 transcription in MDM2 amplified cells, and show improved selectivity for growth inhibition in wild type p53 cell lines over the parent compound.

Discovery and SAR of substituted 3-oxoisoindoline-4-carboxamides as potent inhibitors of poly(ADP-ribose) polymerase (PARP) for the treatment of cancer.

Through conformational restriction of a benzamide by formation of a seven-membered hydrogen-bond with an oxindole carbonyl group, a series of PARP inhibitors was designed for appropriate orientation for binding to the PARP surface. This series of compounds with a 3-oxoisoindoline-4-carboxamide core structure, displayed modest to good activity against PARP-1 in both intrinsic and cellular assays. SAR studies at the lactam nitrogen of the pharmacophore have suggested that a secondary or tertiary amine is important for cellular potency. An X-ray structure of compound 1e bound to the protein confirmed the formation of a seven-membered intramolecular hydrogen bond. Though revealed previously in peptides, this type of seven-membered intramolecular hydrogen bond is rarely observed in small molecules. Largely due to the formation of the intramolecular hydrogen bond, the 3-oxoisoindoline-4-carboxamide core structure appears to be planar in the X-ray structure. An additional hydrogen bond interaction of the piperidine nitrogen to Gly-888 also contributes to the binding affinity of 1e to PARP-1.

Probing the binding sites and mechanisms of action of two human ether-a-go-go-related gene channel activators, 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD307243).

We studied the mechanisms and sites of activator actions of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid [PD307243 (PD)] and 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea [NS1643 (NS)] on the human ether-a-go-go-related gene (hERG) channel expressed in oocytes and COS-7 cells. PD and NS affected hERG in a concentration-dependent manner, reaching a maximal increase in current amplitude by 100% and > or = 300% (1-s test pulse to 0 mV), with apparent K(d) values of 3 and 20 microM, respectively. Both drugs slowed hERG inactivation. NS additionally shifted the activation curve in the negative direction, accelerated activation, and slowed deactivation. Kinetic model simulations suggested that the activator effects of PD and NS could be largely accounted for by their effects on the hERG gating kinetics. Both drugs worked from outside the cell membrane but their binding sites seemed to be distinctly different. Perturbing the conformation of outer vestibule/external pore entrance (by cysteine substitution at high-impact positions or cysteine side chain modification at intermediate-impact positions) prevented the activator effect of NS but not that of PD. Furthermore, the peptide toxin BeKm-1, which bound to the outer mouth of the hERG channel, suppressed NS effect but potentiated PD effect. We propose that NS is a "gating-modifier": it binds to the outer vestibule/pore entrance of hERG and increases current amplitudes by promoting channel activation while retarding inactivation. The activator effect of PD was prevented by external quaternary ammonium cations or dofetilide, which approached the hERG selectivity filter from opposite sides of the membrane and depleted K(+) ions in the selectivity filter. We suggest that PD may work as a "pore-modifier" of the hERG channel.

Evaluation of 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones as inhibitors of human protein kinase CK2.

Protein kinase CK2 (Casein Kinase 2) is an extremely pleiotropic Ser/Thr kinase with high constitutive activity. The observation of CK2 deregulations in various pathological processes suggests that CK2 inhibitors may have a therapeutic value, particularly as anti-neoplastic and antiviral drugs. Here, we present the 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-diones as a novel potent class of CK2 inhibitors. We identified this class of inhibitors by high-throughput docking of a compound collection in the ATP-binding site of human CK2. The most active compounds are 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanoic acid and 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)acetic acid with IC(50) values of 0.15 microM and 0.3 microM, respectively. These inhibitors are ATP-competitive and they only minimally inhibit the activities of protein kinases DYRK1a, MSK1, GSK3 and CDK5. Binding modes for the most active inhibitors are proposed.