Hideki Nakashima

Regioselective syntheses of sulfated polysaccharides: specific anti-HIV-1 activity of novel chitin sulfates.

A novel and convenient method for the regioselective syntheses of sulfated analogs of chitin and chitosan is described in relation to studies on structure-biological activity. Fully protected, soluble derivatives of chitosan were found to be useful intermediates for the syntheses of a novel class of sulfated polysaccharides, 2-acetamido-2-deoxy-3-O-sulfo-(1-->4)-beta-D-glucopyranan (3-sulfate, 3S, 4) and (1-->4)-2-deoxy-2-sulfoamido-3-O-sulfo-(1-->4)-beta-D-glucopyranan (2,3-disulfate, 23-S, 3). These compounds were tested for their activities in (i) inhibiting HIV-1 replication in vitro and (ii) inhibiting blood coagulation. The results reveal that the selective sulfation at O-2 and/or O-3 affords potent antiretroviral agents showing a much higher inhibitory effect on the infection of AIDS virus in vitro than that by the known 6-O-sulfated derivative (6-sulfate, 6S). Moreover, the 23-S product completely inhibited the infection of AIDS virus to T lymphocytes at concentrations as low as 0.28 microgram/mL without significant cytotoxicity. The regioselective introduction of sulfate group(s) at O-2 and/or O-3 had little effect on generating anticoagulant activity, whereas 6-O-sulfated chitin strongly inhibits blood coagulation. These results suggest that the specific interaction of these new types of chitin sulfates with gp 120 of the AIDS virus depends significantly on the sites of sulfation rather than on the total degree of substitution on sugar residues.

Inhibition of human immunodeficiency viral replication by tannins and related compounds.

Among 87 chemically defined tannins and related compounds, several hydrolyzable tannins, but not condensed tannins or other lower molecular weight polyphenols, significantly inhibited both the cytopathic effect of human immunodeficiency virus (HIV) and the expression of HIV antigen in human lymphotropic virus type I (HTLV-1)-positive MT-4 cells. The 50% effective concentrations (2.0-4.8 micrograms/ml) of the active compounds were 13- to 15-fold lower than their 50% cytotoxic concentrations. Their anti-HIV activity was demonstrated to be mediated, at least in part, by inhibition of HIV adsorption to the cells.

A novel anti-HIV synthetic peptide, T-22 ([Tyr5,12,Lys7]-polyphemusin II)

Tachyplesin and polyphemusin are antimicrobial peptides recently isolated from the hemocytes of horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus). We synthesized them and their analogs and examined their antiviral activity against human immunodeficiency virus (HIV) type 1 in vitro. The infection of human T cells with the virus was markedly inhibited by some of them at low concentrations. In this structure-activity study, we found that [Tyr5,12, Lys7]-polyphemusin II, which was designated as T22, had extremely high anti-HIV activity. Its 50% inhibitory concentration (EC50) was 0.008 micrograms/ml, while its 50% cytotoxic concentration (CC50) was 54 micrograms/ml and these values were comparable to those of AZT. This result indicates that T22 would be a potential candidate for the therapy of HIV infection.

A comparative study of the solution structures of tachyplesin I and a novel anti-HIV synthetic peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II), determined by nuclear magnetic resonance

The solution structure of tachyplesin I, which was isolated from membrane acid extracts of the hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus), was determined by nuclear magnetic resonance (NMR) and distance geometry calculation. Tachyplesin I takes an antiparallel beta-sheet structure with a type-II beta-turn. Recently, among more than 20 synthetic peptides associated with tachyplesin and its isopeptide (polyphemusin), we found that a novel compound, which we designated as T22 ([Tyr5,12, Lys7]-polyphemusin II), strongly inhibited the human immunodeficiency virus (HIV)-1-induced cytopathic effect and viral antigen expression. The solution structure of T22 was investigated using NMR, and its secondary structure was confirmed to be similar to that of tachyplesin I. The anti-parallel beta-sheet structure and the several amino-acid side chains on the plane of the beta-sheet of T22 are thought to be associated with the expression of anti-HIV activity.

Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV-1 and HIV-2

Nucleoside analogues such as azidothymidine (AZT), dideoxyinosine (ddI), and dideoxycytidine (ddC) [1] inhibit the viral reverse transcriptase (RT) of human immunodeficiency virus (HIV) and terminate DNA chain synthesis from viral RNA inside the infected cells. However, these nucleosides manifest serious side-effects such as bonemarrow toxicity and the appearance of AZT-resistant viruses upon long-term treatment [2]. It is difficult to make an effective vaccine owing to the multiplicity of AIDS viruses. Therefore, anti-AIDS compounds having a different blocking mechanism are of interest. We have synthesized sulfated polysaccharides demonstrating high anti-AIDS virus activity by sulfation of synthetic and natural-occurring polysaccharides [3-6]. The mechanism of action of sulfated polysaccharides is assumed to be different from that of the nucleosides, that is, they bind to HIV virions and prevent them from penetrating into target cells [7-9]. However, sulfated polysaccharides such as dextran sulfate have high blood anticoagulant activity, leading to undesirable side-effects when used as anti-AIDS drugs [10].

HIV-1 reproduction is inhibited by peptides derived from the N- and C-termini of HIV-1 protease

Two octapeptides derived from the sequence of the N- and C-termini of HIV-1 protease were tested for their ability to inhibit HIV-1 reproduction. Weak inhibitory activity was found with each of the two peptides. It is assumed that HIV-1 protease is the target of the inhibitory action. In spite of the moderate inhibitory activity the results are encouraging since they may be improved by various means.

Synthesis of sulfated alkyl malto- and laminara-oligosaccharides with potent inhibitory effects on AIDS virus infection

A series of sulfated alkyl oligosaccharides, including a sulfate dodecyl laminarapentaoside and a sulfated octadecyl maltohexaoside with potent anti-human immunodeficiency virus (HIV) activity, has been synthesized. An alkyl oligosaccharide in which a long alkyl group is bonded to the reducing end of the oligosaccharide was first synthesized in high yield. Peracetylated oligosaccharides reacted with such aliphatic alcohols as 1-decyl and 1-dodecyl alcohols with Lewis acids as catalysts. As in the glycosylation of the alpha and beta peracetylated glycosides, the beta anomer reacted exclusively, the acetylation was carried out with a sodium acetate-acetic anhydride at high temperatures to maximize the proportion of the beta anomer.

Mechanisms of the inhibition of reverse transcription by unmodified and modified antisense oligonucleotides

We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment bound to the antisense oligonucleotide by the reverse transcriptase‐associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization by binding to the AMV RT rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription.

Phosphorothioate analogues of oligodeoxyribonucleotide: Synthesis and activity as inhibitors of replication of human immunodeficiency virus

The modifications of oligodeoxyribonucleotides include replacement of the other chain either all-PS (S-ODNs), or end-capped with several PS (SO-ODNs) groups at both 3- and 5-ends. A general synthesis of phosphorothioate analogues of oligodeoxyribonucleotides is described using the new phosphite. In assays of HIV, oligomers (S-ODNs) with complete replacement of phosphodiesters with phosphorothioate groups were found to be very active. Finally of particular interest is S-ODNs-rev or tat (20mers) which possessed slightly higher anti-HIV activity than S-dC28 itself. By contrast, the unmodified oligomers (ODNs) as well as SO-ODNs did not have any inhibitory effect.

Synthesis of sulfated derivatives of curdlan and their anti-HIV activity

Abstract Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N -sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO 3 -pyridine complex in a heterogeneous phase. It was shown from 13 C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure.