A Novel Tricyclic Ligand-Containing Nonpeptidic HIV-1 Protease Inhibitor,GRL-0739, Effectively Inhibits the Replication of Multidrug-Resistant HIV-1 Variants and Has a Desirable Central Nervous System Penetration Property In Vitro

We report here that GRL-0739, a novel nonpeptidic HIV-1 protease inhibitor containing a tricycle (cyclohexyl-bis-tetrahydrofuranylurethane [THF]) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC₅₀], 0.0019 to 0.0036 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC₅₀], 21.0 μM). GRL-0739 blocked the infectivity and replication of HIV-1_NL4-3 variants selected by concentrations of up to 5 μM ritonavir or atazanavir (EC₅₀, 0.035 to 0.058 μM). GRL-0739 was also highly active against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, as well as against the HIV-2_ROD variant. The development of resistance against GRL-0739 was substantially delayed compared to that of amprenavir (APV). The effects of the nonspecific binding of human serum proteins on the anti-HIV-1 activity of GRL-0739 were insignificant. In addition, GRL-0739 showed a desirable central nervous system (CNS) penetration property, as assessed using a novel in vitro blood-brain barrier model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and make greater van der Waals contacts with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has desirable features as a compound with good CNS-penetrating capability for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene confers highly desirable anti-HIV-1 potency in the design of novel protease inhibitors with greater CNS penetration profiles.     

A Conserved Hydrogen-Bonding Network of P2 bis-Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC₅₀s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIV_A02) compared to ritonavir (RTV; EC₅₀, >1.0 μM) and tipranavir (TPV; EC₅₀, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIV_DRV^R_P20), with a 2.6-fold increase in its EC₅₀ (0.097 μM) compared to its corresponding EC₅₀ (0.038 μM) against wild-type HIV-1_NL4-3 (HIV_WT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30′ of HIV_A02 protease (PR_A02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30′ due to an absence of polar groups. The P2′ thiazolyl moiety of RTV showed two conformations in the crystal structure of the PR_A02-RTV complex, one of which showed loss of contacts in the S2′ binding pocket of PR_A02, supporting RTV''s compromised antiviral activity (EC₅₀, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30′ most likely contributes to its persistently greater antiviral activity against HIV_WT, HIV_A02, and HIV_DRV^R_P20.     

P2′ Benzene Carboxylic Acid Moiety Is Associated with Decrease in Cellular Uptake: Evaluation of Novel Nonpeptidic HIV-1 Protease Inhibitors Containing P2 bis-Tetrahydrofuran Moiety

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2′ moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR^WT). Both GRL007 (K_i of 12.7 pM with PR^WT) and GRL008 (K_i of 8.9 pM) inhibited PR^WT with high potency in vitro. X-ray crystallographic analysis of PR^WT in complex with GRL007 or GRL008 showed that the bis-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR^WT. The P2′ moiety of both compounds showed one direct contact with the backbone of Asp30′ and a bridging polar contact with Gly48′ through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC₅₀] of >1 μM) while GRL008 was highly active (EC₅₀ of 0.04 μM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2′ benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2′ moiety may improve its antiretroviral features.     

Synthesis and antibacterial and antiviral activities of myricetin derivatives containing a 1,2,4-triazole Schiff base

A series of novel myricetin derivatives containing a 1,2,4-triazole Schiff base were designed and synthesized. Their structures were systematically characterized using ¹H NMR, ¹³C NMR, and HRMS. During antibacterial bioassays, 6f, 6i, and 6q demonstrated a good inhibitory effect against Xanthomonas axonopodis pv. citri (Xac), with half-maximal effective concentration (EC₅₀) values of 10.0, 9.4, and 8.8 μg mL⁻¹, respectively, which were better than those of bismerthiazol (54.9 μg mL⁻¹) and thiodiazole copper (61.1 μg mL⁻¹). Note that 6w demonstrated a good inhibitory effect against Ralstonia solanacearum (Rs) with and EC₅₀ value of 15.5 μg mL⁻¹, which was better than those of bismerthiazol (55.2 μg mL⁻¹) and thiodiazole copper (127.9 μg mL⁻¹). Similarly, 6a, 6d, and 6e demonstrated a good inhibitory effect against Xanthomonas oryzae pv. oryzae (Xoo) with EC₅₀ values of 47.1, 61.2, and 61.0 μg mL⁻¹, respectively, which were better than those of bismerthiazol (148.2 μg mL⁻¹) and thiodiazole copper (175.5 μg mL⁻¹). Furthermore, we used scanning electron microscopy (SEM) to study the possible sterilization process of the target compound 6q against Xac. The results indicated the possibility of destroying the bacterial cell membrane structure, resulting in an incomplete bacterial structure, and thus achieving inhibition. Furthermore, antiviral bioassays revealed that most compounds exhibited excellent antiviral activity against tobacco mosaic virus (TMV) at a concentration of 500 μg mL⁻¹. The results of the molecular docking studies for 6g with TMV-CP (PDB code: 1EI7) showed that compound 6g had partially interacted with TMV-CP. Therefore, mechanistic studies of the action of compound 6g could be further studied based on that.

The myricetin derivatives containing a 1,2,4-triazole Schiff base were designed and synthesized. Antibacterial mechanism was investigated through SEM.     

Novel chalcone derivatives containing a 1,2,4-triazine moiety: design,synthesis, antibacterial and antiviral activities

A series of novel chalcone derivatives containing the 1,2,4-triazine moiety were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and elemental analyses. Antiviral bioassays revealed that most of the compounds exhibited good antiviral activity against tobacco mosaic virus (TMV) at a concentration of 500 μg mL⁻¹. The designated compound 4l was 50% effective in terms of curative and protective activities against TMV with 50% effective concentrations (EC₅₀) of 10.9 and 79.4 μg mL⁻¹, which were better than those of ningnanmycin (81.4 and 82.2 μg mL⁻¹). Microscale thermophoresis (MST) also showed that the binding of compound 4l to coat protein (TMV-CP) yielded a K_d value of 0.275 ± 0.160 μmol L⁻¹, which was better than that of ningnanmycin (0.523 ± 0.250 μmol L⁻¹). At the same time, molecular docking studies for 4l with TMV-CP (PDB code:1EI7) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP. Meanwhile, compound 4a demonstrated excellent antibacterial activities against Ralstonia solanacearum (R. solanacearum), with an EC₅₀ value of 0.1 μg mL⁻¹, which was better than that of thiodiazole-copper (36.1 μg mL⁻¹) and bismerthiazol (49.5 μg mL⁻¹). The compounds act by causing folding and deformation of the bacterial cell membrane as observed using scanning electron microscopy (SEM). The chalcone derivatives thus synthesized could become potential alternative templates for novel antiviral and antibacterial agents.

A series of novel chalcone derivatives containing the 1,2,4-triazine moiety were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and elemental analyses.     

An investigation of the metabolic activity,isozyme contribution,species differences and potential drug–drug interactions of PI-103, and the identification of efflux transporters for PI-103-O-glucuronide in HeLa1A9 cells

PI-103 is a phosphatidylinositol 3-kinase inhibitor that includes multiple receptor affinity modifications, and it is also a therapeutic drug candidate primarily for human malignant tumors. However, its metabolic fate and potential drug–drug interactions involving human cytochrome P450 (CYP) and UDP-glucuronosyltransferases (UGT) enzymes remain unknown. In this study, our results demonstrated that the intrinsic clearance (CL_int) values of oxidated metabolite (M1) in human liver microsomes (HLM) and human intestine microsomes (HIM) were 3.10 and 0.08 μL min⁻¹ mg⁻¹, respectively, while PI-103 underwent efficient glucuronidation with CL_int values of 15.59 and 211.04 μL min⁻¹ mg⁻¹ for mono-glucuronide (M2) by HLM and HIM, respectively. Additionally, reaction phenotyping results indicated that CYP1A1 (51.50 μL min⁻¹ mg⁻¹), 1A2 (46.96 μL min⁻¹ mg⁻¹), and UGT1A1 (18.80 μL min⁻¹ mg⁻¹), 1A7 (8.52 μL min⁻¹ mg⁻¹), 1A8 (8.38 μL min⁻¹ mg⁻¹), 1A9 (34.62 μL min⁻¹ mg⁻¹), 1A10 (107.01 μL min⁻¹ mg⁻¹) were the most important contributors for the oxidation and glucuronidation of PI-103. Chemical inhibition assays also suggest that CYP1A2 and UGT1A1, 1A9 play a predominant role in the metabolism of PI-103 in HLM. Significant activity correlations were detected between phenacetin-N-deacetylation and M1 (r = 0.760, p = 0.004) as well as β-estradiol-3-O-glucuronide and M2 (r = 0.589, p = 0.044), and propofol-O-glucuronidation and M2 (r = 0.717, p = 0.009). Furthermore, the metabolism of PI-103 revealed marked species differences, and dogs, rats, mice and mini-pigs were not the appropriate animal models. Gene silencing of breast cancer resistance protein (BCRP) or multidrug resistance-associated protein (MRPs) transporter results indicated that M2 was mainly excreted by BCRP, MRP1 and MRP4 transporters. Moreover, PI-103 displayed broad-spectrum inhibition towards human CYPs and UGTs isozymes with IC₅₀ values ranging from 0.33 to 6.89 μM. Among them, PI-103 showed potent non-competitive inhibitory effects against CYP1A2, 2C19, 2E1 with IC₅₀ and K_i values of less than 1 μM. In addition, PI-103 exhibited moderate non-competitive inhibition against UGT1A7, 2B7, and moderate mixed-type inhibition towards CYP2B6, 2C9 and UGT1A3. Their IC₅₀ and K_i values were 1.16–6.89 and 0.56–5.64 μM, respectively. In contrast, PI-103 could activate the activity of UGT1A4 in a mechanistic two-site model with a K_i value of 13.76 μM. Taken together, PI-103 was subjected to significant hepatic and intestinal metabolism. CYP1A1, 1A2 and UGT1A1, 1A7, 1A8, 1A9, 1A10 were the main contributing isozymes, whereas BCRP, MRP1 and MRP4 contributed most to the efflux excretion of M2. Meanwhile, PI-103 had a potent and broad-spectrum inhibitory effect against human CYPs and UGTs isozymes. These findings could improve understanding of the metabolic fates and efflux transport of PI-103. The inhibited human CYP and UGT activities could trigger harmful DDIs when PI-103 is co-administered with clinical drugs primarily cleared by these CYPs or UGTs isoforms. Additional in vivo studies are required to evaluate the clinical significance of the data presented herein.

Metabolic activity and disposition characteristics of PI-103.     

Biological activity evaluation and action mechanism of chalcone derivatives containing thiophene sulfonate

A series of novel chalcone derivatives containing a thiophene sulfonate group were designed and synthesized. The structures of all title compounds were determined by ¹H-NMR, ¹³C-NMR and HRMS. Antibacterial bioassays indicated that, compound 2l demonstrated excellent antibacterial activities against Xanthomonas axonopodis pv. citri (Xac), with an EC₅₀ value of 11.4 μg mL⁻¹, which is significantly superior to those of bismerthiazol (BT) (51.6 μg mL⁻¹) and thiodiazole-copper (TC) (94.7 μg mL⁻¹). Meanwhile, the mechanism of action of compound 2l was confirmed by using scanning electron microscopy (SEM). In addition, compound 2e showed remarkable inactivation activity against Tobacco mosaic virus (TMV), with an EC₅₀ value of 44.3 μg mL⁻¹, which was superior to that of ningnanmycin (120.6 μg mL⁻¹). Microscale thermophoresis (MST) also showed that the binding of compounds 2e and 2h to Tobacco mosaic virus coat protein (TMV-CP) yielded K_d values of 0.270 and 0.301 μmol L⁻¹, which are better than that of ningnanmycin (0.596 μmol L⁻¹). At the same time, molecular docking studies for 2e and 2h with TMV-CP (PDB code: 1EI7) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP in each case. These results suggested that chalcone derivatives containing a thiophene sulfonate group may be considered as activators in the design of antibacterial and antiviral agents.

Synthesis, antibacterial, antiviral activities and action mechanism of chalcone derivatives containing thiophene sulfonate.     

Combined algicidal effect of urocanic acid,N-acetylhistamine and l-histidine to harmful alga Phaeocystis globosa

The algicidal compounds produced by Bacillus sp. strain B1 against Phaeocystis globosa, one of the main red-tide algae, were isolated and identified in a previous study as urocanic acid (uro), l-histidine (his) and N-acetylhistamine (ace). The 96 h median effective concentration EC₅₀ values indicated the algicidal effect order of uro (8 μg mL⁻¹) > ace (16 μg mL⁻¹) > his (23 μg mL⁻¹). The interaction between uro and ace had a synergistic effect on Phaeocystis globosa, accelerated the increase in its intracellular reactive oxygen species (ROS) levels, and further decreased the activities of antioxidases after 96 h, causing destruction of cell membrane integrity and nuclear structure. However, the other two binary mixtures uro + his and ace + his were both antagonistic to Phaeocystis globosa. The increase in the level of ROS indicated that the algal cells suffered from oxidative damage. The surplus ROS induced the increase in malondialdehyde (MDA) content and activities of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), all of which reached maxima after 72 h treatment. Transmission electron microscopy (TEM) analysis revealed that these nitrogen-containing compounds caused destruction of cell membrane integrity, chloroplasts and nuclear structure. The present study will provide useful information for the combined effect of algicidal compounds on the harmful alga Phaeocystis globosa. This is the first report to explore single and combined algicidal effects of three nitrogen-containing compounds against the harmful alga Phaeocystis globosa.

Ultrastructure of Phaeocystis globosa cells after treatment with EC₅₀ value for 72 h: (a) control, (b) ace (16 μg mL⁻¹), (c) uro (8 μg mL⁻¹), (d) uro + ace (1 : 1 TU, 8 : 16 μg mL⁻¹) Chl, chloroplast; CW, cell wall; N, nucleus; PM, plasma membrane.     

New chalcone derivatives: synthesis,antiviral activity and mechanism of action

In this work, twenty-eight chalcone derivatives containing a purine (sulfur) ether moiety were synthesized and their antiviral activities were evaluated. Biological results showed that compound 5d exhibited outstanding inactive activity against tobacco mosaic virus (TMV) in vivo (EC₅₀ = 65.8 μg mL⁻¹), which is significantly superior to that of ribavirin (EC₅₀ = 154.3 μg mL⁻¹). Transmission electron microscopy indicated that compound 5d can break the integrity of TMV particles. The results of microscale thermophoresis, fluorescence titration and molecular docking showed that compound 5d had stronger combining affinity (K_a = 1.02 ×10⁵ L mol⁻¹, K_d = 13.4 μmol L⁻¹) with TMV coat protein (TMV-CP), which is due to the formation of five hydrogen bonds between compound 5d and the amino-acid residues of TMV-CP. These findings revealed that compound 5d can effectively inhibit the infective ability of TMV. This work provides inspiration and reference for the discovery of new antiviral agents.

The chalcone derivatives containing a purine (sulfur) ether moiety were synthesized. The antiviral mechanism suggested that the antiviral activity of compound 5d may depend on its stronger binding affinity with TMV-CP.     

Chemical composition,nutraceuticals characterization,NMR confirmation of squalene and antioxidant activities of Basella rubra L. seed oil

Basella rubra (Malabar spinach) is a commonly consumed green leafy vegetable in southern parts of India. The chemical composition, nutraceuticals characterization, squalene Nuclear Magnetic Resonance (NMR), in vitro antioxidant activities and cytotoxicity of B. rubra seed oil (33.08%) was investigated. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of palmitic (27.21 μmol%), oleic (33.83 μmol%) and linoleic acid (26.02 μmol%) with a total of 64.38 μmol% unsaturated fatty acids respectively. HPLC nutraceutical characterization showed a major constituent of gallic acid (11.23 mg%), γ-tocopherols (17.74 mg%), cycloartenylferulate (1.7 mg%), and squalene (1 g%). Squalene was further recovered (98%), purified (99.9%), and confirmed through ¹H and ¹³C NMR. The in vitro antioxidant activities recorded by using 2,2-diphenyl-1-picrylhydrazyl (EC₅₀ = 6 mg mL⁻¹), ferric reducing antioxidant power (361.85 mM of Trolox Eq./100 g) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (EC₅₀ = 56.19 mg mL⁻¹) scavenging activity. In vitro cytotoxicity assessed on 3T3-L1 showed good cell survival without any toxicity (upto 400 μg mL⁻¹). B. rubra seed oil has proven nutraceuticals and antioxidant potentials with least toxicity which can be recommended for functional foods applications.

Basella rubra (Malabar spinach) is a commonly consumed green leafy vegetable in southern parts of India.     

Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding Gene UL89

Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC₅₀] = 0.34 μM) than ganciclovir (EC₅₀ = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC₅₀ = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC₅₀ = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC₅₀ for wild-type HCMV = 0.25 ± 0.04 μM, EC₅₀ for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).     

Antibiotic activities of propanolamine containing 1,4-benzoxazin-3-ones against phytopathogenic bacteria

Various 1,4-benzoxazin-3-one derivatives containing propanolamine groups have been shown to exhibit good antibacterial activity against Pseudomonas syringae pv actinidiae (Psa), X. axonopodis pv citri (Xac) and Xanthomonas oryzae pv oryzae (Xoo). 1,4-benzoxazin-3-one 4n showed the best inhibitory effects against Psa, Xac and Xoo, exhibiting in vitro EC₅₀ values of 4.95, 4.71 and 8.50 μg mL⁻¹, respectively. These potencies were superior to the corresponding EC₅₀ values of the commercial antibiotics bismerthiazol (BT, 89.10, and 116.90 μg mL⁻¹) and thiodiazole copper (TC, 127.30, 82.73 and 87.50 μg mL⁻¹). Treatment on the bacterial leaf blight of rice revealed that compound 4n displayed better curative (51%) and protective (48%) activities for reducing rice BLB than either BT (41%, 39%) or TC (43%, 41%). Scanning electron microscopy (SEM) imaging of Xoo that had been treated with 1,4-benzoxazin-3-one 4n (50–100 μg mL⁻¹) revealed that the bacterial cells had experienced extensive cell wall damage, which is the likely cause of its antimicrobial activity and bacterial death.

Various 1,4-benzoxazin-3-one derivatives containing propanolamine groups have been shown to exhibit good antibacterial activity against Pseudomonas syringae pv actinidiae (Psa), X. axonopodis pv citri (Xac) and Xanthomonas oryzae pv oryzae (Xoo).     

Biochemical Characterization of Recombinant Enterovirus 71 3C Protease with Fluorogenic Model Peptide Substrates and Development of a Biochemical Assay

Enterovirus 71 (EV71), a primary pathogen of hand, foot, and mouth disease (HFMD), affects primarily infants and children. Currently, there are no effective drugs against HFMD. EV71 3C protease performs multiple tasks in the viral replication, which makes it an ideal antiviral target. We synthesized a small set of fluorogenic model peptides derived from cleavage sites of EV71 polyprotein and examined their efficiencies of cleavage by EV71 3C protease. The novel peptide P08 [(2-(N-methylamino)benzoyl) (NMA)-IEALFQGPPK(DNP)FR] was determined to be the most efficiently cleaved by EV71 3C protease, with a kinetic constant k_cat/K_m of 11.8 ± 0.82 mM⁻¹ min⁻¹. Compared with literature reports, P08 gave significant improvement in the signal/background ratio, which makes it an attractive substrate for assay development. A Molecular dynamics simulation study elaborated the interactions between substrate P08 and EV71 3C protease. Arg39, which is located at the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a Z′ factor of 0.87 ± 0.05 was developed. A novel compound (compound 3) (50% inhibitory concentration [IC₅₀] = 1.89 ± 0.25 μM) was discovered using this assay, which effectively suppressed the proliferation of EV 71 (strain Fuyang) in rhabdomyosarcoma (RD) cells with a highly selective index (50% effective concentration [EC₅₀] = 4.54 ± 0.51 μM; 50% cytotoxic concentration [CC₅₀] > 100 μM). This fast and efficient assay for lead discovery and optimization provides an ideal platform for anti-EV71 drug development targeting 3C protease.     

Evaluation of the antioxidant activities of fatty polyhydroquinolines synthesized by Hantzsch multicomponent reactions

Polyhydroquinolines (PHQs) are the unsymmetrical Hantzsch derivatives of 1,4-dihydropyridines with several biological applications. In this work, new fatty 2- and 3-substituted PHQ derivatives from different fatty acids and fatty alcohol feedstocks were synthesized at good yields via a four-component reaction (4CR). The antioxidant activities of fatty PHQs were investigated using three different antioxidant methods. The experiments showed that the compounds derived from 2-nitrobenzaldehyde and fatty palmitic (C16:0) and oleic (C18:1) chains showed better antioxidant activity. This revealed that combining the ortho NO₂ group in the aromatic ring with the insertion of fatty chains in the PHQ core contributed to the antioxidant activity. However, among all the fatty PHQs tested, the fatty 2-substituted compound derived from oleyl alcohol and 2-nitrobenzaldehyde showed the highest antioxidant activity (EC₅₀, 2.11–4.69 μM), which was similar to those of the antioxidant standards butylated hydroxytoluene (EC₅₀, 1.98–6.47 μM) and vitamin E (EC₅₀, 1.19–5.88 μM). In addition, this lipophilic compound showed higher antioxidant activity than the antihypertensive drug nifedipine (EC₅₀, 49.25–126.86 μM). These results indicate that the new fatty PHQs may find novel applications as antioxidant additives.

The insertion of a fatty long chain into polyhydroquinoline (PHQ) core contributes to antioxidant potential, the new fatty PHQs showed activities similar to commercial antioxidants.     

GRL-02031, a novel nonpeptidic protease inhibitor (PI) containing a stereochemically defined fused cyclopentanyltetrahydrofuran potent against multi-PI-resistant human immunodeficiency virus type 1 in vitro

We generated a novel nonpeptidic protease inhibitor (PI), GRL-02031, by incorporating a stereochemically defined fused cyclopentanyltetrahydrofuran (Cp-THF) which exerted potent activity against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) isolates, including multidrug-resistant HIV-1 variants. GRL-02031 was highly potent against laboratory HIV-1 strains and primary clinical isolates, including subtypes A, B, C, and E (50% effective concentration [EC(50)] range, 0.015 to 0.038 microM), with minimal cytotoxicity (50% cytotoxic concentration, >100 microM in CD4(+) MT-2 cells), although it was less active against two HIV-2 strains (HIV-2(EHO) and HIV-2(ROD)) (EC(50), approximately 0.60 microM) than against HIV-1 strains. GRL-02031 at relatively low concentrations blocked the infection and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to 5 microM of saquinavir, amprenavir, indinavir, nelfinavir, or ritonavir and 1 microM of lopinavir or atazanavir (EC(50) range, 0.036 to 0.14 microM). GRL-02031 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to the conventional antiretroviral regimens that then existed, with EC(50)s ranging from 0.014 to 0.042 microM (changes in the EC(50)s were less than twofold the EC(50) for wild-type HIV-1). Upon selection of HIV-1(NL4-3) in the presence of GRL-02031, mutants carrying L10F, L33F, M46I, I47V, Q58E, V82I, I84V, and I85V in the protease-encoding region and G62R (within p17), L363M (p24-p2 cleavage site), R409K (within p7), and I437T (p7-p1 cleavage site) in the gag-encoding region emerged. GRL-02031 was potent against a variety of HIV-1(NL4-3)-based molecular infectious clones containing a single primary mutation reported previously or a combination of such mutations, although it was slightly less active against HIV-1 variants containing consecutive amino acid substitutions: M46I and I47V or I84V and I85V. Structural modeling analysis demonstrated a distinct bimodal binding of GRL-02031 to protease, which may provide advantages to GRL-02031 in blocking the replication of a wide spectrum of HIV-1 variants resistant to PIs and in delaying the development of resistance of HIV-1 to GRL-02031. The present data warrant the further development of GRL-02031 as a potential therapeutic agent for the treatment of infections with primary and multidrug-resistant HIV-1 variants.     

Uracil derivatives as HIV-1 capsid protein inhibitors: design,in silico,in vitro and cytotoxicity studies

A series of novel uracil derivatives such as bispyrimidine dione and tetrapyrimidine dione derivatives were designed based on the existing four-point pharmacophore model as effective HIV capsid protein inhibitors. The compounds were initially docked with an HIV capsid protein monomer to rationalize the ideas of design and to find the potential binding modes. The successful design and computational studies led to the synthesis of bispyrimidine dione and tetrapyrimidine dione derivatives from uracil and aromatic aldehydes in the presence of HCl using novel methodology. The in vitro evaluation in HIV p24 assay revealed five potential uracil derivatives with IC₅₀ values ranging from 191.5 μg ml⁻¹ to 62.5 μg ml⁻¹. The meta-chloro substituted uracil compound 9a showed promising activity with an IC₅₀ value of 62.5 μg ml⁻¹ which is well correlated with the computational studies. As expected, all the active compounds were noncytotoxic in BA/F3 and Mo7e cell lines highlighting the thoughtful design. The structure activity relationship indicates the position priority and lower log P values as the possible cause of inhibitory potential of the uracil compounds.

The paper describes the design, synthesis, computational and biological validation of a series of novel uracil derivatives as effective HIV capsid protein inhibitors.     

Inhibition of L5178Y Cells in Culture by Methotrexate and Antibiotics

Methotrexate, tetracycline, gentamicin, streptomycin, and penicillin inhibited the growth of L5178Y murine leukemia cells in culture with I₅₀ (concentration of drug that caused a 50% inhibition of growth at 72 h) values of 0.0028 μg/ml (6.2 × 10⁻⁹ M), 7.9 μg/ml, 200 μg/ml, 1,700 μg/ml, and 3,000 μg/ml (5,000 U/ml), respectively. At concentrations achieved clinically or utilized in the laboratory, the antibiotics did not alter the I₅₀ of methotrexate.     

Nitro/Nitrosyl-Ruthenium Complexes Are Potent and Selective Anti-Trypanosoma cruzi Agents Causing Autophagy and Necrotic Parasite Death

cis-[RuCl(NO₂)(dppb)(5,5′-mebipy)] (complex 1), cis-[Ru(NO₂)₂(dppb)(5,5′-mebipy)] (complex 2), ct-[RuCl(NO)(dppb)(5,5′-mebipy)](PF₆)₂ (complex 3), and cc-[RuCl(NO)(dppb)(5,5′-mebipy)](PF₆)₂ (complex 4), where 5,5′-mebipy is 5,5′-dimethyl-2,2′-bipyridine and dppb is 1,4-bis(diphenylphosphino)butane, were synthesized and characterized. The structure of complex 2 was determined by X-ray crystallography. These complexes exhibited a higher anti-Trypanosoma cruzi activity than benznidazole, the current antiparasitic drug. Complex 3 was the most potent, displaying a 50% effective concentration (EC₅₀) of 2.1 ± 0.6 μM against trypomastigotes and a 50% inhibitory concentration (IC₅₀) of 1.3 ± 0.2 μM against amastigotes, while it displayed a 50% cytotoxic concentration (CC₅₀) of 51.4 ± 0.2 μM in macrophages. It was observed that the nitrosyl complex 3, but not its analog lacking the nitrosyl group, releases nitric oxide into parasite cells. This release has a diminished effect on the trypanosomal protease cruzain but induces substantial parasite autophagy, which is followed by a series of irreversible morphological impairments to the parasites and finally results in cell death by necrosis. In infected mice, orally administered complex 3 (five times at a dose of 75 μmol/kg of body weight) reduced blood parasitemia and increased the survival rate of the mice. Combination index analysis of complex 3 indicated that its in vitro activity against trypomastigotes is synergic with benznidazole. In addition, drug combination enhanced efficacy in infected mice, suggesting that ruthenium-nitrosyl complexes are potential constituents for drug combinations.