Akiyoshi Matsumoto

Anti-human immunodeficiency virus activity of a novel synthetic peptide, T22 ([Tyr-5,12, Lys-7]polyphemusin II): a possible inhibitor of virus-cell fusion.

More than 40 peptides associated with tachyplesin and polyphemusin, which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus, were synthesized. Among these peptides, we found that a novel compound, which was called T22 ([Tyr-5,12, Lys-7]polyphemusin II), strongly inhibited the human immunodeficiency virus type 1 (HIV-1)-induced cytopathic effect and viral antigen expression. Its 50% effective concentration was 0.008 micrograms/ml, while its 50% cytotoxic concentration was 54 micrograms/ml. The anti-HIV activity of T22 was observed with several strains of HIV-1, including zidovudine-resistant strains, and with HIV-2 within the concentration range of 0.006 to 0.071 microgram/ml. T22 efficiently inhibited giant cell formation on the cocultivation of MOLT-4/HIV and MOLT-4 cells but modestly inhibited direct HIV binding. T22 did not inhibit reverse transcriptase activity. A time-of-addition study, which involved monitoring of the appearance of proviral DNA by using the polymerase chain reaction technique, found that T22 exerted its effect on a process, most probably virus-cell fusion or uncoating, immediately after virus adsorption. Images

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.

Inhibitory Mechanism of the CXCR4 Antagonist T22 against Human Immunodeficiency Virus Type 1 Infection

We recently reported that a cationic peptide, T22 ([Tyr(5,12), Lys(7)]-polyphemusin II), specifically inhibits human immunodeficiency virus type 1 (HIV-1) infection mediated by CXCR4 (T. Murakami et al., J. Exp. Med. 186:1389-1393, 1997). Here we demonstrate that T22 effectively inhibits replication of T-tropic HIV-1, including primary isolates, but not of non-T-tropic strains. By using a panel of chimeric viruses between T- and M-tropic HIV-1 strains, viral determinants for T22 susceptibility were mapped to the V3 loop region of gp120. T22 bound to CXCR4 and interfered with stromal-cell-derived factor-1alpha-CXCR4 interactions in a competitive manner. Blocking of anti-CXCR4 monoclonal antibodies by T22 suggested that the peptide interacts with the N terminus and two of the extracellular loops of CXCR4. Furthermore, the inhibition of cell-cell fusion in cells expressing CXCR4/CXCR2 chimeric receptors suggested that determinants for sensitivity of CXCR4 to T22 include the three extracellular loops of the coreceptor.

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.

Pharmacophore identification of a chemokine receptor (CXCR4) antagonist, T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II), which specifically blocks T cell-line-tropic HIV-1 infection

We have previously found that T22 ([Tyr(5,12), Lys7]-polyphemusin II) has strong anti-human immunodeficiency virus (HIV) activity, and that T22 inhibits T cell-line-tropic HIV-1 infection mediated by CXCR4/fusin. T22 is an 18-residue peptide amide, which takes an antiparallel beta-sheet structure that is maintained by two disulfide bridges. Structure-activity relationship (SAR) studies on T22 have disclosed the contributions of each region of T22 to activity or cytotoxicity, and have provided the following useful information to develop new CXCR4 antagonists: The number of Arg residues in the N-terminal and C-terminal regions of T22 is closely related to anti-HIV activity. Addition of a variety of functional groups at the N-terminal end results in increases in activity. Disulfide rings, especially the major disulfide loop, are indispensable for anti-HIV activity and maintenance of the beta-sheet structure. Trp3 can be replaced by other aromatic residues (Tyr, Phe and L-2-naphthylalanine). Between two repeats of Tyr-Arg-Lys, which are a characteristic structure in T22, Tyr-Arg-Lys in the N-terminal portion is more closely associated with anti-HIV activity and maintenance of the beta-sheet structure. A positive charge in the side chain at the (i + 1) position of the beta-turn region is necessary for strong activity. Through these studies, we have found several compounds having higher selectivity indexes (50% cytotoxic concentration/50% effective concentration) than that of T22.

High-level production of alternatively spliced soluble interleukin-6 receptor in serum of patients with adult T-cell leukaemia/HTLV-I-associated myelopathy.

We have previously shown, using human T‐cell lymphocytotrophic virus‐I (HTLV‐I)‐infected cell lines, that soluble interleukin‐6 receptor (sIL‐6R) is generated through an alternative splicing mechanism. In this study, we examined human sera for the presence of alternatively spliced soluble IL‐6R (AS‐sIL‐6R). We produced a monoclonal antibody (mAb) recognizing the unique sequence of AS‐sIL‐6R peptide, generated by an altered reading frame. We also made recombinant AS‐sIL‐6R protein in Spodoptera frugiperda‐9 (Sf‐9) cells carrying baculovirus, which encoded altered sIL‐6R or conventional IL‐6R cDNA. mAbs specifically recognized AS‐sIL‐6R, but not conventional IL‐6R, as demonstrated by Western blot analyses, fluorescence‐activated cell sorter, immunofluorescence analyses and enzyme‐linked immunosorbent assay (ELISA). We adapted an ELISA system and used it for detection of altered sIL‐6R in sera from 23 healthy persons, 12 patients with adult T‐cell leukaemia (ATL) and 13 patients with HTLV‐I‐associated myelopathy (HAM). Serum levels of AS‐sIL‐6R were 6·4 or 6·1 times greater in ATL (28·7±20·4 ng/ml, P<0·0001) and in HAM patients (27·5±12·1 ng/ml, P<0·0001) than in healthy individuals (4·5±2·1 ng/ml). High levels of AS‐sIL‐6R were also observed in plasma from rheumatoid arthritis patients and in persons with elevated levels of alanine aminotransferase (ALT), antinuclear antibody (ANA), or α‐fetoprotein (AFP). However, in human immunodeficiency virus‐1 (HIV‐1), hepatitis B virus (HBV) or hepatitis C virus (HCV)‐infected individuals, AS‐sIL‐6R levels were not elevated. In this study, we confirmed that AS‐sIL‐6R is indeed present in human sera. These observations suggest that alternative splicing of IL‐6R mRNA is of consequence in ATL, HAM and in some autoimmune diseases. The HTLV‐I‐infected T cells appeared to play an important role in AS‐sIL‐6R production.

Altered interleukin-2 receptor alpha-chain is expressed in human T-cell leukaemia virus type-I-infected T-cell lines and human peripheral blood mononuclear cells of adult T-cell leukaemia patients through an alternative splicing mechanism.

A polymerase chain reaction (PCR) method was used to detect the interleukin‐2 receptor α‐chain (IL‐2Rα) chain which lacks the conventional transmembrane (TM) domain in mRNA from human T‐cell leukaemia virus type‐I (HTLV‐I) ‐infected cell lines or peripheral blood mononuclear cells (PBMC) isolated from adult T‐cell leukaemia (ATL) patients. Primer pairs encompassing the TM domain were selected to generate a 357‐base pair (bp) fragment. A 146‐bp PCR product was observed consistently in addition to the target 357‐bp PCR product in mRNA from HTLV‐I‐infected cell lines, such as MT‐1, MT‐2, MT‐4 and in PBMC isolated from ATL patients. However, this 146‐bp PCR product was undetectable in HTLV‐I‐negative cell lines. The product was also detected in PBMC from normal individuals if activated in vitro with phytohaemagglutinin but not without stimulation. DNA sequence analyses revealed that exons from 5 to 7, which define a 211‐bp region containing the conventional TM domain, were deleted in the 146‐bp PCR product. The C‐terminal amino acid sequence starting from Gly174 of the 211‐bp‐deleted molecule was distinct from that of conventional IL‐2Rα as a result of an altered reading frame. We identified a 45 000 MW peptide generated from IL‐2Rα mRNA through this exon skip in cell lysate of MT‐1 and MT‐2 by Western blot analyses using an antibody raised against the peptides specific to an altered IL‐2Rα. Our results indicate that an altered IL‐2Rα chain is expressed in HTLV‐I‐infected T lymphocytic cell lines and in ATL patients.

Convenient one-pot synthesis of cystine-containing peptides using the trimethylsilyl chloride–dimethyl sulfoxide/trifluoroacetic acid system and its application to the synthesis of bifunctional anti-HIV compounds1

A one-pot synthesis of cystine-containing peptides is achieved by treatment of protected peptidyl resins with trimethylsilyl chloride–dimethyl sulfoxide/trifluoroacetic acid in the presence of anisole. This methodology has been successfully applied to the synthesis of highly active anti-HIV peptides conjugated with 3′-azido-3′-deoxythymidine.