核糖核酸酶抑制因子对血管生成素功能的调控

血管生成素（angiogenin,ANG）能有效促进血管生成和肿瘤细胞增殖,在肿瘤发生发展中起重要作用.其主要分子机制是通过核转位和激活PI3K/AKT/mTOR信号通路,刺激rRNA转录和核糖体生成.ANG也被发现在肌萎缩侧索硬化症（ALS）和帕金森病（PD）患者中存在基因编码区的功能突变,表明其在运动神经元生理方面发挥作用,其缺陷是神经退行性疾病的一个危险因素.核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞内酸性蛋白质,由460个氨基酸残基组成,分子质量约为50 kD,当其与核糖核酸酶A（RNaseA）结合形成复合物后,可抑制RNaseA 的90％以上活性,从而有效调节细胞内RNA水平. ANG具有低核糖核酸酶活性, 是RNase超家族一员,与RNase A有着高度保守的同源顺序. 序列、结构和酶学等分析表明,RI也能够与ANG紧密结合,且得到体外实验的证明. 研究发现,RI具抑癌基因功能;RI与ANG在细胞内共定位;Co IP和GST pull down证实其相互作用,获取了RI与ANG在体内结合的直接证据;RI与AKT磷酸化表达负相关.在膀胱癌细胞及临床标本中证实了RI与 ANG和PI3K/AKT通路分子表达的相关性及与肿瘤细胞生长与转移的关系．在细胞和动物模型研究表明,RI调节ANG活性的功能及其分子机制,即RI通过结合ANG而封锁其核转位和调控PI3K/AKT/mTOR信号通路及其相关通路交互应答（cross talk）的能力,从而抑制肿瘤生长及转移. RI是一个有希望的抗肿瘤蛋白新药和血管生成抑制剂,可望成为基因治疗的靶基因.     

Inhibition of monoamine oxidase by (E)-styrylisatin analogues

Previous studies have shown that (E)-8-(3-chlorostyryl)caffeine (CSC) is a specific reversible inhibitor of human monoamine oxidase B (MAO-B) and does not bind to human MAO-A. Since the small molecule isatin is a natural reversible inhibitor of both MAO-B and MAO-A, (E)-5-styrylisatin and (E)-6-styrylisatin analogues were synthesized in an attempt to identify inhibitors with enhanced potencies and specificities for MAO-B. The (E)-styrylisatin analogues were found to exhibit higher binding affinities than isatin with the MAO preparations tested. The (E)-5-styrylisatin analogues bound more tightly than the (E)-6 analogue although the latter exhibits the highest MAO-B selectivity. Molecular docking studies with MAO-B indicate that the increased binding affinity exhibited by the (E)-styrylisatin analogues, in comparison to isatin, is best explained by the ability of the styrylisatins to bridge both the entrance cavity and the substrate cavity of the enzyme. Experimental support for this model is shown by the weaker binding of the analogues to the Ile199Ala mutant of human MAO-B. The lower selectivity of the (E)-styrylisatin analogues between MAO-A and MAO-B, in contrast to CSC, is best explained by the differing relative geometries of the aromatic rings for these two classes of inhibitors.     

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose–response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.     

Dehydroquinate synthase: the use of substrate analogues to probe the early steps of the catalyzed reaction

The early steps of the proposed mechanistic pathway for dehydroquinate synthase have been probed with a series of substrate analogues. These analogues, 3-9, are structurally prohibited from undergoing the beta-elimination of inorganic phosphate that represents the committed step in the conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). In agreement with previous observations, the analogues that possess shortened side chains (3,5, and 6) bind more tightly to the enzyme than those (4 and 7-9) that are more nearly isosteric with the substrate. Two hitherto unrecognized factors that influence binding have been identified: (i) carbacylic analogues bind 25-100 times more tightly than the corresponding oxacyclic materials (indeed, the carbacyclic phosphonate 5 has a Ki value of 8 x 10(-10)M) and (ii) the side chain appears to be bound in a gauche conformation similar to the most stable conformation of the cis-vinylhomophosphonate 8. These trends in binding can be rationalized by considering the behavior of the analogues in the first two chemical steps of the mechanism: NAD+-mediated oxidation at C-5 and enolization at C-6 (the first part of the E1cB elimination of inorganic phosphate). Direct spectrophotometric determination of the equilibrium level of enzyme-bound NADH indicates that the carbacyclic analogues are more readily oxidized than the oxacyclic compounds, and this predictable difference in redox behavior is reflected in the observed differences in binding. The gauche conformation of the C-7 side chain appears to be required for proton abstraction from C-6, since only those analogues that can adopt this conformation undergo enzyme-catalyzed exchange of the C-6 proton with the solvent. This conformation positions one of the peripheral oxygens of the phosphate (or phosphonate) group close to the C-6 proton. Taken together with other data, these results suggest that the enzyme exploits this substrate base in the enolization, which occurs through an intramolecular proton transfer. The loss of Pi then completes the beta-elimination.     

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNA^Asp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNA^Asp with a K_d value that is comparable to their IC₅₀ value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.     

Respective role of each of the purine N7 nitrogens of 5'-O-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine in binding to and activation of the RNase L of mouse cells

Through a combination of chemical and enzymatic approaches a series of sequence-specific tubercidin-substituted ppp5'A2'p(5'A2'p)n5'A (n = 1 to about 10; 2-5A) analogues were generated. In addition to the previously developed methodology of Imai and Torrence [Imai, J., & Torrence, P.F. (1985) J. Org. Chem. 50, 1418-1420], a new approach to synthesis of 2',5'-linked oligonucleotides utilized adenosine in 3',5' linkage as a precursor to the targeted 5'-terminus of the desired product. For instance, A3'p5'A could be condensed under conditions of lead ion catalysis with tubercidin 5'-phosphate to give A3'p5'A2'p5'(c7A). Treatment with the 3',5'-specific nuclease P1 led to p5'A2'p5'(c7A). The combined use of the above procedures led to the synthesis of p5'(c7A)2'p5'A2'p5'A, p5'A2'p5'(c7A)2'p5'A, p5'A2'p5'A2'p5'(c7A), and p5'A2p5'(c7A)2'p5'(c7A), which were converted to their corresponding 5'-triphosphates by the usual methods. Evaluation of these analogues for their ability to bind to and activate the 2-5A-dependent endonuclease (RNase L) of mouse L cells showed that there were small changes (less than or equal to 10-fold) in the ability of the four tubercidin analogues to bind to RNase L. However, whenever the first and/or third adenosine nucleotide units were replaced by tubercidin, a dramatic decrease in ability to activate RNase L occurred. Only the second (from the 5'-terminus) adenosine residue could be replaced by tubercidin without any effect on RNase L activation ability.     

The ribonucleolytic activity of angiogenin.

Angiogenin (ANG), a homologue of bovine pancreatic ribonuclease A (RNase A), promotes the growth of new blood vessels. The biological activity of ANG is dependent on its ribonucleolytic activity, which is far lower than that of RNase A. Here, the efficient heterologous production of human ANG in Escherichia coli was achieved by replacing two sequences of rare codons with codons favored by E. coli. Hypersensitive fluorogenic substrates were used to determine steady-state kinetic parameters for catalysis by ANG in continuous assays. The ANG pH-rate profile is a classic bell-shaped curve, with pK(1) = 5.0 and pK(2) = 7.0. The ribonucleolytic activity of ANG is highly sensitive to Na(+) concentration. A decrease in Na(+) concentration from 0.25 to 0.025 M causes a 170-fold increase in the value of k(cat)/K(M). Likewise, the binding of ANG to a tetranucleotide substrate analogue is dependent on [Na(+)]. ANG cleaves a dinucleotide version of the fluorogenic substrates with a k(cat)/K(M) value of 61 M(-1) s(-1). When the substrate is extended from two nucleotides to four or six nucleotides, values of k(cat)/K(M) increase by 5- and 12-fold, respectively. Together, these data provide a thorough picture of substrate binding and turnover by ANG.     

Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors

An expansion of structure–activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor of PDE4 is presented. The development of SAR included strategic incorporation of known substituents on the critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From these studies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A inhibitors. Each of these analogues was submitted across a panel of 21 PDE family members and was shown to be highly selective for PDE4 isoforms (PDE4A, PDE4B, PDE4C, PDE4D). Both 10 and 18 were then evaluated in divergent cell-based assays to assess their relevant use as probes of PDE4 activity. Finally, docking studies with selective ligands (including 10 and 18) were undertaken to better understand this chemotypes ability to bind and inhibit PDE4 selectively.     

Design,synthesis and biological evaluation of cyclic angiotensin II analogues with 3,5 side-chain bridges. Role of C-terminal aromatic residue and ring cluster for activity and implications in the drug design of AT1 non-peptide antagonists

The novel amide linked angiotensin II (ANG II) cyclic analogues: gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Ile(8)] ANG II (I) and gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Phe(8)] ANG II (II) have been designed, synthesized and bioassayed in anesthetized rabbits in order to unravel structural ring cluster characteristics important for receptor activation. Analogue I with Ile at position 8 was an inhibitor of Angiotensin II while analogue II with Phe at position 8 was found to be an agonist. Similar results were reported for cyclic compounds that have reversed the linking between positions 3 and 5. The overall results show that positions 3 and 5 do not govern the biological activity of the synthetic analogues. It also appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo-electronic feature for Angiotensin II to exert its biological activity. A non-peptide mimetic of ANG II, 1-[2'-[(N-benzyl)tetrazol-5-yl]biphenyl-4-yl]methyl]-2-hydroxymethylbenzimidazole (BZI8) has been designed and synthesized. This molecule is more rigid and much less active than AT(1) non-peptide mimetic losartan probably because it lacks to mimic the orientation of tetrazole and the pharmacophore segments of butyl chain and imidazole ring.     

5-Cyano-6-oxo-1,6-dihydro-pyrimidines as potent antagonists targeting exchange proteins directly activated by cAMP

Exchange proteins directly activated by cAMP (Epac) are a family of guanine nucleotide exchange factors that regulate a wide variety of intracellular processes in response to second messenger cAMP. To explore the structural determinants for Epac antagonist properties of high throughput screening (HTS) hit ESI-08, pyrimidine 1, a series of 5-cyano-6-oxo-1,6-dihydro-pyrimidine analogues have been synthesized and evaluated for their activities for Epac inhibition. Structure-activity relationship (SAR) analysis led to the identification of three more potent Epac antagonists (6b, 6g, and 6h). These inhibitors may serve as valuable pharmacological probes for further elucidation of the physiological functions and mechanisms of Epac regulation. Our SAR results and molecular docking studies have also revealed that further optimization of the moieties at the C-6 position of pyrimidine scaffold may allow us to discover more potent Epac-specific antagonists.     

Sar1Ile7]angiotensin III, a new selective antagonist of the pressor effect of angiotensin III in conscious rats

Two analogues of angiotensin III were compared as antagonists of the pressor response to angiotensin II (ANG II) and angiotensin III (ANG III) in conscious, unrestrained rats. Dose-mean arterial pressure (MAP) response curves were obtained for ANG II and ANG III in the absence or presence of [Ile7]ANG III (1.3 x 10(-7) mol/kg) or [Sar1 Ile7]ANG III (1.2 x 10(-7) mol/kg). In the presence of [Ile7]ANG III, the dose-MAP response curves for ANG II and ANG III were significantly displaced to the right. [Ile7]ANG III behaved as a partial agonist on ANG II but not ANG III receptors. In the presence of [Sar1 Ile7]ANG III, the dose-MAP response curve for ANG III but not ANG II was significantly displaced to the right. This suggests that [Sar1 Ile7]ANG III is a selective antagonist of ANG III in the vasculature. [Ile7]ANG III, on the other hand, antagonizes both ANG II and ANG III receptors. Our results support the hypothesis of the existence of a sub-class of angiotensin receptors activated by ANG III in the vascular smooth muscle.     

Binding of non-natural 3'-nucleotides to ribonuclease A

2'-Fluoro-2'-deoxyuridine 3'-phosphate (dU(F)MP) and arabinouridine 3'-phosphate (araUMP) have non-natural furanose rings. dU(F)MP and araUMP were prepared by chemical synthesis and found to have three- to sevenfold higher affinity than uridine 3'-phosphate (3'-UMP) or 2'-deoxyuridine 3'-phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3'-UMP, dU(F)MP, and araUMP (pK(a) = 5.9) being more anionic than that of dUMP (pK(a) = 6.3). The three-dimensional structures of the crystalline complexes of RNase A with dUMP, dU(F)MP and araUMP were determined at < 1.7 A resolution by X-ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3'-UMP and dU(F)MP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A.araUMP complex, the 2'-hydroxyl group is exposed to the solvent. All four 3'-nucleotides bind more tightly to wild-type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3'-nucleotides, and indicate that non-natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.     

High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3',5'-ADP, 2',5'-ADP, 5'-ADP, U-2'-p and U-3'-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5'-ADP (Ki = 1.2 microM), adopts a syn conformation (in contrast to 3',5'-ADP and 2',5'-ADP, which adopt an anti), and it is the beta- rather than the alpha-phosphate group that binds to P1. 3',5'-ADP binds with the 5'-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2',5'-ADP. This inhibitor binds with either the 3' or the 5' phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2' versus 3') has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.     

Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2

Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2), which releases arachidonic acid (AA). ANG II also generates H2O2 and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca2+ depletion and calcium chelator EGTA and inhibitors of CaM [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII [(2-[N-(2-hydroxyethyl)]-N-(4-me-thoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine)]. cPLA2 inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 +/- 27.91% with ANG II and 110.18 +/- 22.40% with ETYA + ANG II; 405.00 +/- 86.22% with AA and 153.97 +/- 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5(S)-, 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H2O2 production but not Akt phosphorylation. Oleic acid, which also increased H2O2 production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA2 and independent of H2O2 production.     

Identification of potent Yes1 kinase inhibitors using a library screening approach

Yes1 kinase has been implicated as a potential therapeutic target in a number of cancers including melanomas, breast cancers, and rhabdomyosarcomas. Described here is the development of a robust and miniaturized biochemical assay for Yes1 kinase that was applied in a high throughput screen (HTS) of kinase-focused small molecule libraries. The HTS provided 144 (17% hit rate) small molecule compounds with IC₅₀ values in the sub-micromolar range. Three of the most potent Yes1 inhibitors were then examined in a cell-based assay for inhibition of cell survival in rhabdomyosarcoma cell lines. Homology models of Yes1 were generated in active and inactive conformations, and docking of inhibitors supports binding to the active conformation (DFG-in) of Yes1. This is the first report of a large high throughput enzymatic activity screen for identification of Yes1 kinase inhibitors, thereby elucidating the polypharmacology of a variety of small molecules and clinical candidates.     

Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).     

Sugar recognition by the glucose and mannose permeases of Escherichia coli. Steady-state kinetics and inhibition studies

The glucose (EII(Glc)) and mannose (EII(Man)) permeases of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Escherichia coli belong to structurally different families of PTS transporters. The sugar recognition mechanism of the two transporters is compared using as inhibitors and pseudosubstrates all possible monodeoxy analogues, monodeoxyfluoro analogues, and epimers of D-glucose. The analogues were tested as phosphoryl acceptors in vitro and as uptake inhibitors with intact cells. Both EII have a high K(m) of phosphorylation for glucose modified at C-4 and C-6, and these analogues also are weak inhibitors of uptake. Conversely, modifications at C-1 (and also at C-2 with EII(Man)) were well tolerated. OH-3 is proposed to interact with hydrogen bond donors on EII(Glc) and EII(Man), since only substitution by fluorine was tolerated. Glucose-6-aldehydes, which exist as gem-diols in aqueous solution, are potent and highly selective inhibitors of "nonvectorial" phosphorylation by EII(Glc) (K(I) 3-250 microM). These aldehydes are comparatively weak inhibitors of transport by EII(Glc) and of phosphorylation and transport by EII(Man). Both transporters display biphasic kinetics (with glucose and some analogues) but simple Michaelis-Menten kinetics with 3-fluoroglucose (and other analogues). Kinetic simulations of the phosphorylation activities measured with different substrates and inhibitors indicate that two independent activities are present at the cytoplasmic side of the transporter. A working model that accounts for the kinetic data is presented.     

Structure-aided optimization of 3-O-β-chacotriosyl ursolic acid as novel H5N1 entry inhibitors with high selective index

Currently, entry inhibitors contribute immensely in developing a new generation of anti-influenza virus drugs. Our earlier studies have identified that 3-O-β-chacotriosyl ursolic acid (1) could inhibit H5N1 pseudovirus by targeting hemagglutinin (HA). In the present study, a series of C-28 modified pentacyclic triterpene saponins via conjugation with a series of amide derivatives were synthesized and their antiviral activities against influenza A/Duck/Guangdong/99 virus (H5N1) in MDCK cells were evaluated. The SARs analysis of these compounds revealed that introduction of certain amide structures at the 17-COOH of ursolic acid could significantly enhance both their antiviral activity and selective index. This study indicated that the attachment of the methoxy group or Cl atom to the phenyl ring at the ortho- or para-position was crucial to improve inhibitory activity. Mechanism studies demonstrated that these title triterpenoids could bind tightly to the viral envelope HA to block the attachment of viruses to host cells, which was consistent with docking studies.     

Mutational analysis of the hexose transporter of Plasmodium falciparum and development of a three-dimensional model

Plasmodium falciparum infection kills more than 1 million children annually. Novel drug targets are urgently being sought as multidrug resistance limits the range of treatment options for this protozoan pathogen. PfHT1, the major hexose transporter of P. falciparum is a promising new target. We report detailed structure-function studies on PfHT1 using site-directed mutagenesis approaches on residues located in helix V (Q169N) and helix VII ((302)SGL --> AGT). Studies with hexose analogues in these mutants have established that hexose recognition and permeation are intimately linked to these helices. A "fructose filter" effect results from the Q169N mutation (abolishing fructose uptake but preserving affinity and transport of glucose, as reported in Woodrow, C. J., Burchmore, R. J. S., and Krishna, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9931-9936). Associated changes in competition for glucose uptake by C-2, C-3, and C-6 glucose analogues compared with native PfHT1 indicate subtle alterations in substrate interaction in this mutant. The K(m) values for glucose uptake in helix VII mutants are also similar to native PfHT1. Hydrogen bonding to positions C-5 and C-6 in glucose analogues becomes relatively more important in these mutants compared with native PfHT1. To increase understanding of hexose permeation pathways in PfHT1, we have developed the first three-dimensional model for PfHT1. As predicted for GLUT1, the principal mammalian glucose transporter, PfHT1 contains a main and an auxiliary channel. After modeling, the Q169N mutation leads predominantly to local structural changes, including displacement of neighboring helix IV. The (302)SGL position in helix VII lies in the same plane as Gln-169 in helix V but is also adjacent to the main hexose permeation pathway, consistent with results from experiments mutating this triplet motif. Furthermore, there are obvious structural and functional differences between GLUT1 and PfHT1 that can now be explored in detail using the approaches presented here. The development of specific inhibitors for PfHT1 will also be aided by these insights.