Youhoon Chong

Antiviral Activities and Cellular Toxicities of Modified 2′,3′-Dideoxy-2′,3′-Didehydrocytidine Analogues

ABSTRACT The antiviral efficacies and cytotoxicities of 2′,3′- and 4′-substituted 2′,3′-didehydro-2′,3′-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC90s], 0.14 to 5.2 μM), but marked increases in EC90s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC90s, 53 to >100 μM). The β-l-enantiomers of both classes of compounds were more potent than the corresponding β-d-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC90, 0.25 μM). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 μM. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4′-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of β-d-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC90, 0.08 μM) and lamivudine-resistant HIV-1 (EC90 = 0.15 μM) is markedly reduced by introduction of a 3′-fluorine in the sugar (EC90s of compound 2a, 37.5 and 494 μM, respectively).

Peptide-mediated shape- and size-tunable synthesis of gold nanostructures.

While several biological processes have been shown to be useful for the production of well-designed, inorganic nanostructures, the mechanism(s) controlling the size and shape of nano and micron size particles remains elusive. Here we report on the controlled size- and shape-specific production of gold nanostructures under ambient reaction conditions using a dodecapeptide, Midas-2, originally selected from a phage-displayed combinatorial peptide library. Single amino acid changes in Midas-2 greatly influence the size (a few nanometers to approximately 100 microm) and shape (nanoparticles, nanoribbons, nanowires and nanoplatelets) of the gold nanostructures produced, and these are controllable by adjusting the solution pH and gold ion concentration. The ability to control the shape and size of the gold nanostructures by changing the peptide structure and reaction conditions will lead to many potential applications, including nanoelectronics, sensors and optoelectronics, because of their unique size- and shape-dependent optical and electrical properties.

In vitro solubility, stability and permeability of novel quercetin-amino acid conjugates.

In order to discover a quercetin prodrug with improved bioavailability, we synthesized nine quercetin-amion acid conjugates and estimated their pharmacokinetic properties including water solubility, stability against chemical or enzymatic hydrolysis, and cell permeability. Among the synthesized quercetin prodrugs, quercetin-glutamic acid conjugate Qu-E (4g/5g) showed remarkable increases in water solubility, stability, and cell permeability compared with quercetin, which warrants further development as a quercetin prodrug.

Production of a Novel Quercetin Glycoside through Metabolic Engineering of Escherichia coli

ABSTRACT Most flavonoids exist as sugar conjugates. Naturally occurring flavonoid sugar conjugates include glucose, galactose, glucuronide, rhamnose, xylose, and arabinose. These flavonoid glycosides have diverse physiological activities, depending on the type of sugar attached. To synthesize an unnatural flavonoid glycoside, Actinobacillus actinomycetemcomitans gene tll (encoding dTDP-6-deoxy-l-lyxo-4-hexulose reductase, which converts the endogenous nucleotide sugar dTDP-4-dehydro-6-deoxy-l-mannose to dTDP-6-deoxytalose) was introduced into Escherichia coli. In addition, nucleotide-sugar dependent glycosyltransferases (UGTs) were screened to find a UGT that could use dTDP-6-deoxytalose. Supplementation of this engineered strain of E. coli with quercetin resulted in the production of quercetin-3-O-(6-deoxytalose). To increase the production of quercetin 3-O-(6-deoxytalose) by increasing the supplement of dTDP-6-deoxytalose in E. coli, we engineered nucleotide biosynthetic genes of E. coli, such as galU (UTP-glucose 1-phosphate uridyltransferase), rffA (dTDP-4-oxo-6-deoxy-d-glucose transaminase), and/or rfbD (dTDP-4-dehydrorahmnose reductase). The engineered E. coli strain produced approximately 98 mg of quercetin 3-O-(6-deoxytalose)/liter, which is 7-fold more than that produced by the wild-type strain, and the by-products, quercetin 3-O-glucose and quercetin 3-O-rhamnose, were also significantly reduced.

Cation dependent O-methyltransferases from rice

Two lower molecular mass OMT genes (ROMT-15 and -17) were cloned from rice and expressed in Escherichia coli as glutathione S-transferase fusion proteins. ROMT-15 and -17 metabolized caffeoyl-CoA, flavones and flavonols containing two vicinal hydroxyl groups, although they exhibited different substrate specificities. The position of methylation in both luteolin and quercetin was determined to be the 3′ hydroxyl group and myricetin and tricetin were methylated not only at 3′ but also at 5′ hydroxyl groups. ROMT-15 and -17 are cation-dependent and mutation of the predicted metal binding sites resulted in the loss of the enzyme activity, indicating that the metal ion has a critical role in the enzymatic methylation.

Enhanced Stability and Intracellular Accumulation of Quercetin by Protection of the Chemically or Metabolically Susceptible Hydroxyl Groups with a Pivaloxymethyl (POM) Promoiety

In order to increase stability of quercetin, its metabolically and chemically susceptible hydroxyl groups 7-OH and 3-OH respectively were transiently blocked with a pivaloxymethyl (POM) promoiety to provide two novel quercetin conjugates [7-O-POM-Q, 3-O-POM-Q]. In the absence of stabilizer (ascorbic acid), the synthesized conjugates showed significantly increased stability in cell culture media [t(½) = 4 h, 52 h] compared with quercetin (t(½) < 30 min) and quercetin prodrug 1 (t(½) = 0.8 h). In addition, the quercetin conjugate 2 underwent efficient cellular uptake and intracellular levels of its hydrolysis product, quercetin, were maintained up to 12 h. Stability and intracellular accumulation of were demonstrated by its stabilizer-independent cytostatic effect and induction of apoptotic cell death. Even though was more stable than, it failed to penetrate cell membranes. However, the remarkable stability of warrants further investigation of quercetin conjugates with various promoieties at the 3-OH position.

Benzimidazole Derivatives as Potent JAK1-Selective Inhibitors.

The Janus kinase (JAK) family comprises four members (JAK1, JAK2, JAK3, and Tyk2) that play a key role in mediating cytokine receptor signaling. JAK inhibition thus modulates cytokine-mediated effects. In particular, selective inhibition of JAK1 or JAK3 may provide an efficient therapeutic agent for the treatment of inflammatory diseases, with minimized side effects. In this study, as part of our continued efforts to develop a selective JAK1 inhibitor, a series of 1,2-disubstituted benzimidazole-5-carboxamide derivatives was prepared and their inhibitory activities against all four JAK isozymes were evaluated. A clear structure-activity relationship was observed with respect to JAK1 selectivity; this highlighted the importance of hydrogen bond donors at both N(1) and R2 positions located within a specific distance from the benzimidazole core. One of the synthesized compounds, 1-(2-aminoethyl)-2-(piperidin-4-yl)-1H-benzo[d]imidazole-5-carboxamide (5c), showed remarkable JAK1 selectivity (63-fold vs JAK2, 25-fold vs JAK3, and 74-fold vs Tyk2). Molecular docking revealed that the 2-aminoethyl and piperidin-4-yl substituents of 5c function as probes to differentiate the ATP-binding site of JAK1 from that of JAK2, resulting in preferential JAK1 binding. A kinase panel assay confirmed the JAK1 selectivity of 5c, which showed no appreciable inhibitory activity against 26 other protein kinases at 10 μM.

Water-soluble and cleavable quercetin-amino acid conjugates as safe modulators for P-glycoprotein-based multidrug resistance.

Quercetin-amino acid conjugates with alanine or glutamic acid moiety attached at 7-O and/or 3-O position of quercetin were prepared, and their multidrug resistance (MDR)-modulatory effects were evaluated. A quercetin-glutamic acid conjugate, 7-O-Glu-Q (3a), was as potent as verapamil in reversing MDR and sensitized MDR MES-SA/Dx5 cells to various anticancer drugs with EC50 values of 0.8-0.9 μM. Analysis on Rh-123 accumulation confirmed that 3a inhibits drug efflux by Pgp, and Pgp ATPase assay showed that 3a interacts with the drug-binding site of Pgp to stimulate its ATPase activity. Physicochemical analysis of 3a revealed that solubility, stability, and cellular uptake of quercetin were significantly improved by the glutamic acid promoiety, which eventually dissociates from 3a to produce quercetin and quercetin metabolites in intracellular milieu. Taken together, potent MDR-modulating activity along with intracellular conversion into the natural flavonoid quercetin warrants development of the quercetin-amino acid conjugates as safe MDR modulators.

Significant enhancement in radical-scavenging activity of curcuminoids conferred by acetoxy substituent at the central methylene carbon

For a compound to be a radical-trapping antioxidant, the antioxidant-derived radical must be sufficiently inert to molecular oxygen as this would generate harmful chain-propagating peroxyl radicals. Curcumin has a unique structure with phenolic hydroxyl group as well as β-diketone moiety in the same molecule, both of which are able to donate electrons to free radicals. However, due to the reactivity toward molecular oxygen, the carbon-centered radical derived from β-diketone moiety do not serve as radical-trapping antioxidants. In this study, we reasoned that stabilization of the carbon-centered radical through substitution with an electron-withdrawing group would enhance the radical-scavenging antioxidative activity of the resulting curcuminoids. Thus, various substituents (methyl, allyl, methoxy, xanthate, and acetoxy) covering broad spectrum of the polar substituent effect were introduced to the central methylene position of both phenolic and non-phenolic curcuminoids. With the free phenolic hydroxyl groups present, the methylene-substituent did not exert significant effect on the antioxidant activity of the curcuminoids (EC(50)=23.2-30.3 μM) with the exception of the acetoxy-substituted derivative (EC(50)=8.7 μM) which showed more potent activity than curcumin (EC(50)=22.6 μM). When substituted to the non-phenolic curcumin scaffold, however, the methylene-substituent enhanced antioxidant activity of the otherwise inactive curcuminoids in the increasing order of methyl

Bacterial Synthesis of a Flavonoid Deoxyaminosugar Conjugate in Escherichia coli Expressing a Glycosyltransferase of Arabidopsis thaliana

Many natural compounds are attached to sugars, and this influences their biological activities by altering molecular and cellular specificities. Among the different sugar moieties found in natural compounds, deoxyand aminosugars display the most variation. Here, we report that flavonoid deoxyaminosugar conjugates, which are rare in nature can be produced in an engineered Escherichia coli strain expressing a glycosyltransferase from Arabidopsis thaliana. Flavonoids are synthesized mainly in plants and have diverse physiological impacts on humans. Most natural flavonoids are conjugated to sugars. The sugar moiety affects the absorption of flavonoids in the small intestine. Naturally occurring flavonoid sugar conjugates include glucoside, galactoside, glucuronide, rhamnoside, xyloside, and arabinoside. Flavonoid deoxyaminosugar conjugates have not been found in nature. The biosynthesis of flavonoid–sugar conjugates utilizes uridine-diphosphate-dependent glycosyltransferases (UGTs). UGTs from various plants and microorganisms have been characterized. UGTs from plants show sugar–donor and sugar–acceptor selectivity. Thus, regioselectivity is an important advantage of in vitro enzymatic synthesis or in vivo synthesis by using bacteria expressing UGTs over chemical synthesis. However, it is difficult to synthesize unusual sugar conjugates by these routes without understanding the specificity of UGTs. To synthesize a flavonoid deoxyaminosugar conjugate, we examined UDP–sugar metabolism in Escherichia coli and the sugar specificity of UGTs. Several nucleotide sugars, including UDP-glucose, UDP-glucuronic acid, TDP-rhamnose, or UDP-N-acetylglucosamine, are present naturally in E. coli. Among them is the unusual deoxyamino sugar, UDP-4-deoxy-4-formamido-l-arabinose (UDP-l-Ara4FN). Starting from UDP-glucose, three genes, ugd (UDP-glucose dehydrogenase), arnA (UDP-l-Ara4N formyltransferase/ UDP-GlcA C-4“-decarboxylase) and arnB (UDP-l-Ara4O C-4” transaminase), are involved in the biosynthesis of UDP-lAra4FN (Scheme 1). ArnA converts UDP–glucuronic acid into UDP-4’’-ketopentose, which is the first committed step for UDP–4-amino-4-deoxy-arabinoside. ArnB transfers an amino group to form UDP–4-amino-4-deoxy-l-arabinose (UDP-Ara-4N). ArnA further converts UDP–Ara-4-N to UDP–l-Ara4FN. It is assumed that this pathway could provide the sugar donor for the biosynthesis of a flavonoid–Ara4FN conjugate. UDP-l-Ara4FN is a derivative of UDP-arabinose. Most plant UGTs have selectivity for UDP–glucose. However, a UGT from Arabidopsis thaliana, AtUGT78D3, has specificity for UDP-arabinose and uses quercetin as a sugar acceptor to make quercetin–3-O-arabinoside. To examine whether AtUGT78D3 could use UDP–l-Ara4FN, we carried out molecular docking with the modeled structure of AtUGT78D3. We used this approach because UDP–l-Ara4FN is not commercially available. The structure of AtUGT78D3 was built by using molecular modeling. UDP–l-Ara4FN or UDP–arabinose was docked into the mod-