Synthesis and biological evaluation of novel imidazole nucleosides as potential anti-dengue virus agents

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-β-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4′-thio and 4′-seleno derivatives of EICAR and EICNR 3–6 were prepared. The resulting 4′-thioEICAR and 4′-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

New cationic polyprenyl derivative proposed as a lipofecting agent

Cationic linear poly-cis-isoprenoid prepared from natural plant polyprenol in a mixture with dioleyl phosphatidylethanolamine was found to be an effective lipofection agent for eukaryotic cells. The transfecting activity is related to the poly-cis structure of the polyprenyl chain.     

Synthesis, biological evaluation and radiochemical labeling of a dansylhydrazone derivative as a potential imaging agent for apoptosis

To develop a small molecule-based tracer for in vivo apoptosis imaging, dansylhydrazone (DFNSH) was synthesized in 93% yield in less than 30 min. The biological evaluation showed that DFNSH selectively binds to paclitaxel-induced apoptotic cancer cells. The high magnification fluorescent images demonstrate that DFNSH is localized within the cytoplasm of cells that bound Alexa 488 labeled annexin V on the plasma membrane. [(18)F]-DFNSH ([(18)F]-3) was synthesized and isolated in 50-60% radiochemical yields, based on [K/K(222)](18)F, with a synthesis time of 50 min (EOB). The straightforward preparation of fluorine-18 labeled 3 makes it a promising tracer for PET imaging of apoptosis.     

A new naphthalene derivative with anti-amyloidogenic activity as potential therapeutic agent for Alzheimer's disease

The aggregation of β-amyloid peptides is associated to neurodegeneration in Alzheimer’s disease (AD) patients. Consequently, the inhibition of both oligomerization and fibrillation of β-amyloid peptides is considered a plausible therapeutic approach for AD. Herein, the synthesis of new naphthalene derivatives and their evaluation as anti-β-amyloidogenic agents are presented. Molecular dynamic simulations predicted the formation of thermodynamically stable complexes between the compounds, the Aβ_1-42 peptide and fibrils. In human microglia cells, these compounds inhibited the aggregation of Aβ_1-42 peptide. The lead compound 8 showed a high affinity to amyloid plaques in mice brain ex vivo assays and an adequate log P_oct/PBS value. Compound 8 also improved the cognitive function and decreased hippocampal β-amyloid burden in the brain of 3xTg-AD female mice. Altogether, our results suggest that 8 could be a novel therapeutic agent for AD.     

GIP as a potential therapeutic agent?

Glucose-dependent insulinotropic polypeptide (GIP) is released from K-cells in the gut after meal ingestion, and acts in concert with glucagon-like peptide 1 (GLP-1) to augment glucose-stimulated insulin secretion. While derivatives of GLP-1 are under active investigation for the treatment of type 2 diabetes, the case is different for GIP. Indeed, the insulinotropic effect of GIP is almost absent in patients with type 2 diabetes. In addition, the unfavourable pharmacokinetic profile of native GIP obviates its clinical application. Different analogues of GIP exhibiting prolonged stability and enhanced biological potency have been generated in order improve the anti-diabetic properties of GIP. However, glucose-normalisation, as is typically observed during the intravenous administration of GLP-1 in patients with type 2 diabetes, has not yet been achieved with GIP or its derivatives. Since GIP appears to play a role in lipid physiology and elevated levels of GIP have been associated with obesity, antagonising GIP action has been proposed as a therapeutic strategy for obesity. This concept has recently been reinforced by the observation that GIP receptor knock-out mice are protected from high-fat diet-induced obesity. However, eliminating the effect of endogenous GIP may at the same time impair postprandial insulin secretion, thereby severely disturbing glucose homeostasis. Therefore, therapeutic strategies based on either augmenting or antagonising GIP action are far from being established alternatives for the future therapy of type 2 diabetes or obesity.     

Bis(3-hydroxy-4-pyridinone)-EDTA derivative as a potential therapeutic Al-chelating agent. Synthesis, solution studies and biological assays

The 3-hydroxy-4-pyridinones are chelating agents of current interest due to their high affinity for hard metal ions and potential clinical applications as metal-decorporation agents. A new bis-(3-hydroxy-4-pyridinone) derivative of EDTA have been developed, and herein we describe the results of solution studies to determine the protonation constants and the partition coefficient. Biodistribution studies, performed with 67Ga-overload mice, showed rapid clearance of the radiotracer from the body, thus indicating that the new ligand should be a quite effective agent for the in vivo aluminium removal.     

Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1

Heparan sulfate (HS) is a highly sulfated polysaccharide and is present in large quantities on the cell surface and in the extracellular matrix. Herpes simplex virus type 1 (HSV-1) utilizes a specialized cell surface HS, known as 3-O-sulfated HS, as an entry receptor to establish infection. Here, we exploit an approach to inhibiting HSV-1 infection by using a 3-O-sulfated octasaccharide, mimicking the active domain of the entry receptor. The 3-O-sulfated octasaccharide was synthesized by incubating a heparin octasaccharide (3-OH octasaccharide) with HS 3-O-sulfotransferase isoform 3. The resultant 3-O-sulfated octasaccharide has a structure of Delta UA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS3S6S-IdoUA2S-GlcNS6S (where Delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is D-glucosamine, and IdoUA is L-iduronic acid). Results from cell-based assays revealed that the 3-O-sulfated octasaccharide has stronger activity in blocking HSV-1 infection than that of the 3-OH octasaccharide, suggesting that the inhibition of HSV-1 infection requires a unique sulfation moiety. Our results suggest the feasibility of inhibiting HSV-1 infection by blocking viral entry with a specific oligosaccharide.     

Selection of predicted siRNA as potential antiviral therapeutic agent against influenza virus

Influenza virus A (IVA) infection is responsible for recent death worldwide. Hence, there is a need to develop therapeutic agents against the virus. We describe the prediction of short interfering RNA (siRNA) as potential therapeutic molecules for the HA (Haemagglutinin) and NA (Neuraminidase) genes. We screened 90,522 siRNA candidates for HA and 13,576 for NA and selected 1006 and 1307 candidates for HA and NA, respectively based on the proportion of viral sequences that are targeted by the corresponding siRNA, with complete matches. Further short listing to select siRNA with no off-target hits, fulfilling all the guidelines mentioned in approach, provided us 13 siRNAs for haemagglutinin and 13 siRNAs for neuraminidase. The approach of finding siRNA using multiple sequence alignments of amino acid sequences has led to the identification of five conserved amino acid sequences, three in hemagglutinin i.e. RGLFGAIAGFIE, YNAELLV and AIAGFIE and two in neuraminidase i.e. RTQSEC and EECSYP which on reveres translation provided siRNA sequences as potential therapeutic candidates. The approaches used during this study have enabled us to identify potentially therapeutic siRNAs against divergent IVA strains.     

Quantum dots encapsulated with amphiphilic alginate as bioprobe for fast screening anti-dengue virus agents

The increasing threats of viral diseases have gained worldwide attention in recent years. Quite a few infectious diseases are still lacking effective prevention or treatment. The pace of developing antiviral agents could be expedited by the availability of quick and efficient drug screening platforms. In this study, quantum dot (QD), an emerging probe for biological imaging and medical diagnostics, was employed to form complexes with virus and used as fluorescent imaging probes for exploring potential antiviral therapeutics. Inorganic CdSe/ZnS QDs synthesized in organic phase were encapsulated by amphiphilic alginate to attain biocompatible water-soluble QDs via phase transfer. Virus employed for this study was dengue virus which is a notorious one in tropical and subtropical regions of the world. To construct a QD-virus imaging modality capable of providing meaningful information, preservation of viral infectivity after tagging virus with QDs is of utmost importance. In order to form colloidal complexes of QD-virus, electrostatic repulsion force generated from both negatively charged virus and QDs was neutralized by various concentrations of cationic polybrene. Results showed that BHK-21 cells infected with dengue viruses incorporated with QDs exhibited bright fluorescence intracellularly within 30 min. To demonstrate the potency of QD-virus complexes as bioprobes for screening antiviral agents, BHK-21 cells were incubated for one hour with allophycocyanin purified from blue-green algae and then infected with QD-virus complexes. Based on the developed cell-based imaging assay, allophycocyanin with concentration of 125 microg/mL led to extremely weak intracellular fluorescence post-infection of QD-virus complexes for 30 min. That is, the efficacy of anti-dengue viral activity of the algae extract was clearly illustrated by the inorganic-organic hybrid platform constructed in current study.     

Discovery of a quinoline-based phenyl sulfone derivative as an antitrypanosomal agent

A series of natural products-based phenyl sulfone derivative and their property-based analogues were investigated as potential growth inhibitors of Trypanosoma brucei. Trypanosoma brucei is a kinetoplastid protozoan parasite that causes trypanosomiasis. In this work, we found that nopol- and quinoline-based phenyl sulfone derivative were the most active and selective for T. brucei, and they were not reactive towards the active thiol of T. brucei’s cysteine protease rhodesain. A thiol reactive variant of the quinoline-based phenyl sulfone was subsequently investigated and found to be a moderate inhibitor of rhodesain. The quinoline-based compound that is not reactive towards rhodesain can serve a template for phenotypic-based lead discovery while its thiol-active congener can serve as template for structure-based investigation of new antitrypanosomal agents.     

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀ ∼ 20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀ ∼ 7.2 μM) as compared to diospyrin (IC₅₀ ∼ 12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀ ∼ 0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2 mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.     

Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with ^99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding ^99mTc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.     

Structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga

A sulfated polysaccharide, named fucoidan, from the marine alga Cladosiphon okamuranus is comprised of carbohydrate units containing glucuronic acid and sulfated fucose residues. Here we found this compound potently inhibits dengue virus type 2 (DEN2) infection. Viral infection was inhibited when DEN2, but not other serotypes, was pretreated with fucoidan. A carboxy-reduced fucoidan derivative in which glucuronic acid was converted to glucose did not inhibit viral infection. Elimination of the sulfated function group from fucoidan significantly attenuated the inhibitory activity on DEN2 infection with <1% fucoidan. DEN2 particles bound exclusively to fucoidan, indicating that fucoidan interacts directly with envelope glycoprotein (EGP) on DEN2. Structure-based analysis suggested that Arg323 of DEN2 EGP, which is conformationally proximal to one of the putative heparin binding residues, Lys310, is critical for the interaction with fucoidan. In conclusion, both the sulfated group and glucuronic acid of fucoidan account for the inhibition of DEN2 infection.     

Synthesis and biological evaluation of technetium-labeled d-glucose-MAG3 derivative as agent for tumor diagnosis

A d-glucose-MAG₃ derivative was successfully synthesized and radiolabeled in high labeling yield. Biodistribution studies in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle ratio and moderate tumor-to-blood ratio. Thus, d-glucose-MAG₃ is a potential agent for tumor diagnosis.     

Semi-synthesis of a 3-O-sulfated red seaweed galactan-derived disaccharide alditol

Beta-D-Galp3-SO3-(1-->4)-3,6-anhydro-L-GalOH (agarobiitol 3(2)-sulfate, 4) was semi-synthetically prepared as follows: production of agarobiitol (1) from agarose by partial reductive hydrolysis, protection of the primary hydroxyl groups of 1 with trityl groups to produce the 1(1),6(2)-di-O-tritylated derivative (2), regioselective dibutylstannylene-mediated sulfation of 2 to give the 3(2)-O-sulfated-1(1),6(2)-di-O-tritylated compound (3), and detritylation of compound 3 to give the final product (4). This semi-synthetic route allowed the preparation of a red seaweed galactan-derived disaccharide alditol with sulfate group located at C-3 of the galactopyranosidic ring. Because red seaweed galactans are glycosidically linked at C-3 of the beta-D-Galp unit, a sulfated derivative with this structure could not be obtained by partial reductive hydrolysis of sulfated red seaweed galactans.     

A 3-O-sulfated heparan sulfate binding peptide preferentially targets herpes simplex virus 2-infected cells

Herpes simplex virus 2 (HSV-2) is the primary cause of genital herpes, which is one of the most common sexually transmitted viral infections worldwide and a major cofactor for human immunodeficiency virus infection. The lack of an effective vaccine or treatment and the emergence of drug-resistant strains highlight the need for developing new antivirals for HSV-2. Here, we demonstrate that a low-molecular-weight peptide isolated against 3-O-sulfated heparan sulfate (3-OS HS) can efficiently block HSV-2 infection. Treatment with the peptide inhibited viral entry and cell-to-cell spread both in vitro and in vivo using a mouse model of genital HSV-2 infection. Quite interestingly, the peptide showed a preferential binding to HSV-2-infected cells, with more than 200% increased binding compared to uninfected cells. Our additional results show that heparan sulfate expression is upregulated by 25% upon HSV-2 infection, which is a significant new finding that could be exploited for designing new diagnostic tests and treatment strategies against HSV-2-infected cells. In addition, our results also raise the possibility that 3-OS HS modifications within HS may be upregulated even more to accommodate for a significantly higher increase in the peptide binding to the infected cells.     

Polyvalent saccharide-functionalized generation 3 poly(amidoamine) dendrimer-methotrexate conjugate as a potential anticancer agent

A saccharide-terminated generation 3 (G3) polyamidoamine (PAMAM) dendrimer was synthesized as a drug carrier. Utilizing this dendritic platform, we have successfully synthesized polyvalent conjugates (G3-MTX) containing the drug methotrexate (MTX). Surface Plasmon Resonance (SPR) results showed that G3-MTX presented three orders of magnitude enhancement in binding avidity to folate-binding protein (FBP) as compared to the free folic acid (FA). Flow cytometric and confocal microscopic analysis showed that conjugate (G3-MTX-FI) containing imaging agent fluorescein-5(6)-carboxamidohexanoic acid (FI) was internalized into folate receptor (FR)-expressing KB cells in dose-dependent and receptor-mediated fashion. The G3-MTX induced a dose-dependent cytotoxicity in the KB cells. Therefore, the polyvalent G3-MTX may have potential as an anticancer nanodevice for the specific targeting and killing of FR-expressing tumor cells.     

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.