Synthesis and anti-inflammatory activity of some new 4,5-dihydro-1,5-diaryl-1H-pyrazole-3-substituted-heteroazole derivatives

A series of 4,5-dihydro-1,5-diaryl-1H-pyrazole-3-substituted-heteroazoles were designed and synthesized in order to obtain new compounds with potential anti-inflammatory activity. The title compounds were screened for in vivo anti-inflammatory activity by using Carrageenan induced rat paw edema method. Diclofenac sodium was used as a standard drug for comparison. Out of the 30 compounds tested, compound 19a, 19b, 25a, 25b exhibited significant anti-inflammatory activity. Selected compounds were also screened for in vitro COX-2 inhibition assay and analgesic activity in the acetic acid induced writhing model.     

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design.

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.     

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.     

High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli

The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 A resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O-acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction.     

Flavonoids from Scutellaria baicalensis inhibit senescence-associated secretory phenotype production by interrupting IκBζ/C/EBPβ pathway: Inhibition of age-related inflammation

BackgroundProlonged exposure to the senescence-associated secretory phenotype (SASP) with age leads to chronic low-grade inflammation in neighboring cells and tissues, causing many chronic degenerative diseases.PurposeThe effects on SASP production of the ethanol extract from Scutellaria radix and 17 isolated flavonoid constituents were examined in vitro and in vivo.MethodsCellular senescence was induced by bleomycin. Expression of the SASP and cell signaling molecules was detected using ELISA, RT-qPCR, Western blotting, and immunofluorescence staining. To investigate the in vivo effects, 21-month-old aged rats were used.ResultsThe ethanol extract and 5 compounds including 1 (Oroxylin A; 5,7-dihydroxy-6-methoxyflavone), 5 (2′,6′,5,7-tetrahydroxy-8-methoxyflavone), 8 (2′,5,7-trihydroxyflavone), 10 (2′,5,7-trihydroxy-8-methoxyflavone) and 11 (2′,5,7-trihydroxy-6-methoxyflavone) potently reduced IL-6 and IL-8 production and gene expression of the SASP, including IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2, and MMP-3. This finding indicates the important role of the B-ring 2′‑hydroxyl group in flavonoid molecules. Furthermore, compounds 8 and 11, the strongest SASP inhibitors, decreased the expression of IκBζ and C/EBPβ protein without affecting either BrdU uptake or the expression of senescence markers, such as pRb and p21. Finally, the oral administration of compound 8 to aged rats at 2 and 4 mg/kg/day for 10 days significantly inhibited the gene expression of SASP and IκBζ in kidneys. This is the first report of the strong SASP inhibitory action of flavonoids from Scutellaria radix on in vitro and in vivo senescence models. The inhibitory action was shown to be mediated mainly by interfering with the IκBζ/C/EBPβ signaling pathway.ConclusionTargeting production of the SASP using flavonoids from Scutellaria radix or its extract might help reduce low-grade sterile inflammation and control age-related diseases.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

A Model for p38MAPK-Induced Astrocyte Senescence

Experimental evidence indicates that aging leads to accumulation of senescent cells in tissues and they develop a secretory phenotype (also known as SASP, for senescence-associated secretory phenotype) that can contribute to chronic inflammation and diseases. Recent results have showed that markers of senescence in astrocytes from aged brains are increased in brains with Alzheimer’s disease. These studies strongly involved the stress kinase p38MAPK in the regulation of the secretory phenotype of astrocytes, yet the molecular mechanisms underlying the onset of senescence and SASP activation remain unclear. In this work, we propose a discrete logical model for astrocyte senescence determined by the level of DNA damage (reparable or irreparable DNA strand breaks) where the kinase p38MAPK plays a central role in the regulation of senescence and SASP. The model produces four alternative stable states: proliferation, transient cycle arrest, apoptosis and senescence (and SASP) computed from its inputs representing DNA damages. Perturbations of the model were performed through gene gain or loss of functions and compared with results concerning cultures of normal and mutant astrocytes showing agreement in most cases. Moreover, the model allows some predictions that remain to be tested experimentally.     

Identification of a small molecule SR9009 that activates NRF2 to counteract cellular senescence

The senescence‐associated secretory phenotype (SASP) is a striking characteristic of senescence. Accumulation of SASP factors causes a pro‐inflammatory response linked to chronic disease. Suppressing senescence and SASP represents a strategy to prevent or control senescence‐associated diseases. Here, we identified a small molecule SR9009 as a potent SASP suppressor in therapy‐induced senescence (TIS) and oncogene‐induced senescence (OIS). The mechanism studies revealed that SR9009 inhibits the SASP and full DNA damage response (DDR) activation through the activation of the NRF2 pathway, thereby decreasing the ROS level by regulating the expression of antioxidant enzymes. We further identified that SR9009 effectively prevents cellular senescence and suppresses the SASP in the livers of both radiation‐induced and oncogene‐induced senescence mouse models, leading to alleviation of immune cell infiltration. Taken together, our findings suggested that SR9009 prevents cellular senescence via the NRF2 pathway in vitro and in vivo, and activation of NRF2 may be a novel therapeutic strategy for preventing cellular senescence.     

Labeling of cysteine-containing peptides with 2-nitro-5-thiobenzoic acid

A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB). The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm. By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified. The stability of the derivative was tested using glutathione-TNB as a model compound. Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage. The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices. This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.     

The oxygen-centered radicals scavenging activity of sulfasalazine and its metabolites. A direct protection of the bowel.

Oxygen-centered radicals, such as superoxide (O2-) and hydroxyl radicals (.OH) generated by phagocytes have been suggested to be involved in the pathogenesis of chronic inflammations of the bowel, such as Crohn's disease and colitis ulcerosa. Recently, sulfasalazine (SASP) and its metabolites have been reported to exert their effects as a direct scavenger of oxygen-centered radicals in the bowel. To scavenge oxygen-centered radicals in vivo, however, SASP and its metabolites have to react with O2- and/or .OH in vitro very rapidly, furthermore they have to reach an appropriate (possible millimolar) concentration range at the site of inflammation. To test this possibility, we investigated the direct O2- and .OH scavenging activity of SASP and its metabolites using the specific electron paramagnetic resonance/spin trapping method, and we compared the 50% inhibition rates of SASP and its metabolites with their known concentrations in the bowel and in the human plasma. It was found that SASP and its metabolites, such as 5-amino-salicylic acid (5-ASA), and acetyl-5-amino-salicylic acid (AC-5-ASA), but not sulfapyridine (SP) and acetyl-sulfapyridine (Ac-SP) have a direct O2- and .OH scavenging activity in vitro systems. Among the compounds, SASP and 5-ASA can reach a concentration which is appropriate to scavenge oxygen-centered radicals in the bowel but not in the human plasma. It was concluded that the in vivo antiinflammatory effects of SASP and its metabolites are, at least partly, due to the direct oxygen-centered scavenging activity of these drugs.     

Ethylene Production by Hevea brasiliensis Tissues Treated with Latex Yield-stirnulatory Compounds

Leaflets and pieces of immature stem of Hevea brasiliensis weretreated with a range of compounds, and the subsequent ratesof release of ethylene compared with the reported efficacy ofthe individual substances as stimulants of latex yield. Themost effective ethylene inducers were also the best stimulants.Amongst the 53 compounds tested, no compound which was not ayield stimulant induced the formation of significant amountsof ethylene. Of all the known stimulants tested, only acetylenewas neither converted to, nor induced the formation of ethylene.     

Design,synthesis, and evaluation of pyrrolo[2,1-f][1,2,4]triazine derivatives as novel hedgehog signaling pathway inhibitors

A novel series of Hh signaling pathway inhibitors were designed by replacing the pyrimidine skeleton of our earlier reported lead compound 1 with pyrrolo[2,1-f][1,2,4]triazine scaffold. Starting from this new scaffold, SAR exploration was investigated based on structural modification on A-ring, C-ring and D-ring. And several much potent compounds were studies in vivo to profile their pharmacokinetic properties. Finally, optimization leads to the identification of compound 19a, a potent Hh signaling pathway inhibitor with superior potency in vitro and satisfactory pharmacokinetic properties in vivo.     

Antimicrobial screening involving Helicobacter pylori of nano-therapeutic compounds based on the amoxicillin antibiotic drug

Nano-structure Cu(II) complex [Cu(AMAB)₂]Cl₂ with Schiff base (AMAB) derived from the condensation between 4-(dimethylamino)benzaldehyde and amoxicillin trihydrate was prepared. (AMAB) Schiff base and its Cu(II) complex were identified and confirmed by different physicochemical techniques. The Schiff base (AMAB) was coordinated to copper ion through carbonyl oxygen and imine nitrogen donor sites. X-ray powder diffraction shows a cubic crystal system of the Cu(II) complex. The density functional theory was used to optimize the structure geometries of the investigated compounds. The molecular docking of the active amino acids of the investigated proteins' interactions with the tested compounds was evaluated. The bactericidal or bacteriostatic effect of the compounds was screened against some bacterial strains. The activity of Cu-chelate against Gram-negative bacteria was mainly more effective than its (AMAB) ligand and vice versa in the case of Gram-positive bacteria. The biological activity of the prepared compounds with biomolecules calf thymus DNA (CT-DNA) was determined by electronic absorption spectra and DNA gel electrophoresis technique. All studies revealed that the Cu-chelate derivative exhibited better binding affinity to both CT-DNA than the AMAB and amoxicillin itself. The anti-inflammatory effect of the designed compounds was determined by testing their protein denaturation inhibitory activity spectrophotometrically. All obtained data supported that the designed nano-Cu(II) complex with Schiff base (AMAB) is a potent bactericide against H. pylori, and exhibits anti-inflammatory activity. The dual inhibition effects of the designed compound represent a modern therapeutic approach with extended spectrum of action. Therefore, it can act as good drug target in antimicrobial and anti-inflammtory therapies. Finally, H. pylori resistance to amoxicillin is absent or rare in many countries, thus amoxicillin nanoparticles may be beneficial for countries where amoxicillin resistance is reported.     

Novel 4-(2-pyrimidinylamino)benzamide derivatives as potent hedgehog signaling pathway inhibitors

A series of novel hedgehog signaling pathway inhibitors have been designed and synthesized based on our previously reported scaffold of 4-(2-pyrimidinylamino)benzamide. The Hh signaling pathway inhibitory activities were evaluated by Gli-luciferase reporter method and most compounds showed more potent inhibitory activities than vismodegib. Three compounds were picked out to evaluated in vivo for their PK properties, and compound 23b bearing a 2-pyridyl A-ring and (morpholin-4-yl)methylene at 3-position of D-ring demonstrated satisfactory PK properties. This study suggested the 4-(2-pyrimidinylamino)benzamides were a series of potent Hh signaling pathway inhibitors, deserving to further structural optimization.     

Imidazopyridazinones as novel PDE7 inhibitors: SAR and in vivo studies in Parkinson's disease model

The synthesis and structure-activity relationship studies of a series of compounds from imidazopyridazinone scaffold as PDE7 inhibitors are disclosed. Potent analogs such as compounds 7 (31nM), 8 (27nM), and 9 (12nM) were identified. The PDE selectivity and pharmacokinetic profile of compounds 7, 8 and 9 are also disclosed. The adequate CNS penetration of compound 7 in mice allowed it to be tested in the MPTP induced PD model and haloperidol induced catalepsy model to probe the differential pharmacology of PDE7 in the striatal pathway.     

Synthesis, in silico docking experiments of new 2-pyrrolidinone derivatives and study of their anti-inflammatory activity

A new class of 2-pyrrolidinone derivatives was designed, synthesized, and tested for their antioxidant and anti-inflammatory activities. The compounds were evaluated for their inhibitory activity against LOX. The most potent among them, 14d [IC(50) 0.08 (±0.005)mM], and 14e [IC(50) 0.0705 (±0.003)mM], were also tested in vivo. The compound 14d induced equipotent inhibition against rat paw edema, which is very close to the effect produced by the commonly used standard, namely indomethacin (47%). The LOX inhibitory activity of the compound 14e proceeds in parallel to the % inhibitory value of lipid peroxidation meaning that this LOX inhibitory activity is supported by the lipid peroxidation inhibition. The molecular features that govern their bioactivity were explored through in silico docking experiments. The results showed that acidic moieties must be placed in certain distance and orientation in the active site of LOX enzyme in order to productively exhibit inhibitory activity. In addition, the 2-pyrrolidinone template significantly contributes in the inhibitory properties of the new compounds.     

Sampling and energy evaluation challenges in ligand binding protein design

The steroid hormone 17α‐hydroxylprogesterone (17‐OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17‐OHP containing an extended, nonpolar, shape‐complementary binding pocket for the four‐ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17‐OHP with micromolar affinity. A co‐crystal structure of one of the designs revealed that 17‐OHP is rotated 180° around a pseudo‐two‐fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same “flipped” orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two‐fold symmetry of the molecule.     

Symmetry-based ligand design and evaluation of small molecule inhibitors of programmed cell death-1/programmed death-ligand 1 interaction

The development of small molecule inhibitors of PD-1/PD-L1 is eagerly anticipated for treatment of cancer. We focused on the symmetry of the ternary complex structure of reported small molecule ligands and hPD-L1 homodimers, and designed partially- or fully-symmetric compounds for more potent inhibitors. The design of the new compounds was guided by our hypothesis that the designed symmetric compound would induce a flip of sidechain of _ATyr56 protein residue to form a new cavity. The designed compound 4 exhibited substantially increased binding affinity to hPD-L1, as well as PD-1/PD-L1 inhibitory activity in physiological conditions. Compound 4 also showed a dose-dependent increase in IFN-γ secretion levels in a mixed lymphocyte reaction assay. These results not only indicate the feasibility of targeting the PD-1/PD-L1 pathway with small molecules, but illustrate the applicability of the symmetry-based ligand design as an attractive methodology for targeting protein-protein interaction stabilizers.