Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

Zinc coupling potentiates anti-HIV-1 activity of baicalin

Baicalin (BA) has been shown with anti-HIV-1 activity. Zinc is a nutrient element. The anti-HIV-1 activity of zinc complex of baicalin (BA-Zn) in vitro was studied and compared with the anti-HIV-1 activities between BA and BA-Zn in the present study. Our results suggested that BA-Zn has lower cytotoxicity and higher anti-HIV-1 activity compared with those of BA in vitro. The CC50s of BA-Zn and BA were 221.52 and 101.73 microM, respectively. The cytotoxicity of BA-Zn was about 1.2-fold lower than that of BA. The BA and BA-Zn inhibited HIV-1 induced syncytium formation, HIV-1 p24 antigen and HIV-1 RT production. The EC50s of BA-Zn on inhibiting HIV-1 induced syncytium formation (29.08 microM) and RT production (31.17 microM) were lower than those of BA (43.27 and 47.34 microM, respectively). BA-Zn was more effective than BA in inhibiting the activities of recombinant RT and HIV-1 entry into host cells. Zinc coupling enhanced the anti-HIV-1 activity of baicalin.     

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.     

Semi-synthesis of oxygenated dolabellane diterpenes with highly in vitro anti-HIV-1 activity

Research on dolabellane diterpenes of brown algae Dictyota spp. has shown that these diterpenoids have strong anti-HIV-1 activity, but there are not data about antiviral activity of dolabellane diterpenes isolated from octocorals, which are antipodes of those isolated from the brown algae. Dolabellanes 13-keto-1(R),11(S)-dolabella-3(E),7(E),12(18)-triene (1) and β-Araneosene (2) were isolated from the Caribbean octocoral Eunicea laciniata, and both showed low anti-HIV-1 activity and low toxicity. Since it was shown that oxygenated dolabellanes from algae have better anti-HIV-1 activity, in this work some derivatives of the main dolabellane of E. laciniata 1 were obtained by epoxidation (3), epoxide opening (4), and allylic oxidation (5). The derivatives showed significant improvement in the anti-HIV-1potency (100-fold), being compounds 3 and 5 the most active ones. Their high antiviral activities, along with their low cytotoxicity, make them promissory antiviral compounds; and it is worth noting that the absolute configuration at the ring junction in the dolabellane skeleton does not seem to be determinant in the antiviral potency of these diterpeneoids.     

Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity.

F11.2.32, a monoclonal antibody directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross-reacts with peptides 36-46 and 36-57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36-46 and 36-57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.     

Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate

The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.     

Antisense oligonucleotides with antitumor activity

Antisense oligonucleotides (AONs) provide an efficient approach for developing target-selective anticancer drugs, because they can inhibit gene expression sequence specifically. To improve the therapeutic effenciency of AONs, two new types of the compounds have been developed. The first group of antisense oligodeoxynucleotides investigated contains base modified nucleotide units. Incorporation of 5-substituted pyrimidines into AONs increases cell membrane permeability (a), duplex stability (b), and nuclease resistance (c). These properties were studied using a large number of model oligonucleotides. The application of 5-(1-hexynyl)dU has been found to be the best modification. Application of MMP-9 collagenase inhibitor oligonucleotides (potential metastasis inhibitors) containing these nucleotide units instead of thymidines increased the collagenase inhibition potency by one order of magnitude compared to that of parental oligonucleotide including thymine bases. The second group of the compounds investigated represents a new type of antisense oligonucleotide synthesized by the antisense directed prodrug therapy (ADPT) conception. According to this principle, a telomerase inhibitor AON was conjugated with 5-fluoro-2'-deoxyuridine (FdU) and oligo-FdUs by phosphodiester bond at the 3'-terminus. The antitumor activities of conjugates in comparison with that of FdU were tested in HT1080 human fibrosarcoma and HT29 human colon adenocarcinoma cell lines. In HT29 cell culture the antiproliferative activity of prodrugs significantly increased with increasing length of the 3'-(FdU)n tail. The conjugate with one FdU unit was about 5 times, while the AON-(FdU)3 analogue was almost 19 times more active than FdU. Antitumor activity of the prodrug containing six FdU units was extremely high (relative efficiency = 26.6), therefore, in vivo testing of this analogue seems to be reasonable and promising. Antiproliferative activity of (FdU)n conjugated with a telomerase inhibitor increased by 5-13 times in HT1080 cells as compared to FdU administered in nucleoside form.     

Chemoenzymatic syntheses and anti-HIV-1 activity of glucose-nucleoside conjugates as prodrugs

Phosphodiester linked conjugates of various nucleosides such as d4U, d4T, IdUrd, ddI, ddA, virazole, ara-A, and ara-C containing a glucosyl moiety have been described. These compounds were designed to act as prodrugs, where the corresponding 5'-monophosphates may be generated intracellularly. The synthesis of the glycoconjugates was achieved in good yields by condensation of a glucosyl phosphoramidite 7 with nucleosides in the presence of an activating agent. It was demonstrated that the glucose conjugates improve the water solubility of the nucleoside analogues, for example, up to 31-fold for the ara-A conjugate compared to that of ara-A alone. The new conjugates were tested for their anti-HIV-1 activity in human lymphocytes. These derivatives offer a convenient design for potential prodrug candidates with the possibility of improving the physicochemical properties and therapeutic activity of nucleoside analogues.     

Design of oligonucleotides for sequence-specific triple-helix formation. Properties of oligonucleotides containing 2'-deoxyxanthosine.

Homopyrimidine-homopurine sequences are targeted by homopyrimidine oligodeoxynucleotides to form triple helix structure. Xanthine was introduced to the third strand of pyrimidine-rich 15-mer oligodeoxy-nucleotides to recognize a cytosine in the purine strand in a homopyrimidine-homopurine duplex target.     

Synthesis and anti-HIV-1 activity of a novel series of 1,4,2-benzodithiazine-dioxides

Previously, we discovered a series of novel benzodithiazines-dioxides with both antiviral and anticancer activities. In order to design compounds with distinct antiviral properties, we prepared new compounds with modifications on the imidazole and pyrimidine rings. Herein, we present the synthesis and antiviral activity of 8-chloro-2,3-dihydroimidazo[1,2-b][1,4,2]benzodithiazine 5,5-dioxides (22, 23, 30, and 31) and 9-chloro-2,3,4-trihydropyrimido[1,2-b][1,4,2]benzodithiazine 6,6-dioxides (14, 24, 25, and 27). We successfully identified a lead compound with remarkable anti-HIV-1 activity (EC(50)=0.09microM). These compounds showed minimal cytotoxicity and are therefore suitable for antiviral development.     

Cationic polypeptides are required for anti-HIV-1 activity of human vaginal fluid

Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.     

Synthesis of novel 6-substituted acyclouridine derivatives and their anti-HIV-1 activity

To improve the anti-HIV activity of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), a variety of its analogues were synthesized. Introduction of SR group to the C-6 position was carried out based on LDA lithiation followed by the reaction of aryl- or alkyl disulfide. An addition-elimination reaction of a 6-phenylsulfinyl derivative was used for synthesizing the analogues having OR or NHR group at the C-6 position. The C-5 modified derivatives were synthesized mainly based on LTMP lithiation of a 6-phenylthio derivative. Modification at the 2- or 4-position was also carried out. Some compounds prepared in the present study showed higher activity than HEPT.     

Aryl nucleoside H-phosphonates. Part 16: Synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least partially, to intracellular conversion into the corresponding nucleoside 5′-monophosphates, and their efficiency correlated well with the pK_a values of the aryloxy groups present.     

Low-molecular-weight anti-HIV-1 peptides from the amino-terminal sequence of RANTES: possible lead compounds for coreceptor-directed anti-HIV-1 agents.

A series of small peptides corresponding to the amino-terminal sequence of RANTES were synthesized, and their anti-HIV-1 activity was evaluated. Pentapeptides, H-(10Ala-RANTES 6-10)-OH and Ac-(10Ala-RANTES 6-10)-NH2, were the smallest anti-HIV-1 peptides so far developed, and would be potentially important lead compounds for coreceptor-directed anti-HIV-1 drugs.     

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.     

Induction of ribozyme activity by anti-ribozyme oligonucleotides.

A new type of hammerhead ribozyme, with cleavage activity enhanced by oligonucleotides, was constructed. Stem II of the ribozyme was substituted with a non complementary loop (loop II). The modified ribozyme exhibited negligible cleavage of a target RNA; however, it was converted to an active molecule in the presence of oligonucleotides which were complementary to loop II. The oligonucleotide compensated for the disabled stem II by binding with the ribozyme. The induction of the cleavage activity was sequence-specific and the oligonucleotides containing a purine base as the 3'-dangling end were able to induce the cleavage activity of the ribozyme most efficiently. A photo-crosslinking experiment proved that a pseudo-half-knot structure was formed in the active molecule. The cleavage of two kinds of substrate RNAs with different sequences was controlled by the corresponding ribozymes activated by specific oligonucleotides.     

2'-Deoxy-8-(propyn-1-yl)adenosine-containing oligonucleotides: effects on stability of duplex and quadruplex structures

2'-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its influence on thermal stability of duplex and quadruplex structures investigated by UV, CD and 1H NMR. The obtained results seem to indicate that the presence of the modified base negatively affects the stability of double stranded DNA whereas remarkably increases the stability of parallel quadruplex structures.     

Structure-activity relationship study of flavone compounds with anti-HIV-1 integrase activity: a density functional theory study

Human immunodeficiency virus type-1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Flavone compounds have been very much studied due to their activity during the inhibition process of HIV-1 IN. In this study, we employed density functional theory (DFT) using the B3LYP hybrid functional to calculate a set of molecular properties for 32 flavonoid compounds with anti-HIV-1 IN activity. The stepwise discriminant analysis (SDA), principal component analysis (PCA) and hierarchical cluster analysis (HCA) methods were employed to reduce dimensionality and investigate possible relationship between the calculated properties and the anti-HIV-1 IN activity. These analyses showed that the molecular hydrophobicity (ClogP), charge on atom 11 and electrophilic index (omega) are responsible for the separation between anti-HIV-1 IN active and inactive compounds.     

Induction of anti-HIV-1 immune responses by retroviral vectors.

Retroviral vectors encoding HIV-1 proteins, in particular, the envelope from HIV-1 IIIB, have been constructed and used to generate infectious vector particles. Murine cells transduced with these vectors express HIV proteins. Vector-transduced cells, when injected into syngeneic BALB/c mice, induce potent CD8+, class I MHC-restricted cytotoxic T-lymphocyte responses and elicit the production of neutralizing antibody specific for HIV-1. The induction of similar responses in primates may provide the basis for considering the use of these vectors as immunostimulants in humans. The retroviral vectors or vector-transduced cells would probably be first employed as an immunotherapeutic for HIV-infected individuals.