Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-06-28
2003-11-04
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S013600, C536S013200, C536S016600, C514S025000, C514S039000, C514S041000
Reexamination Certificate
active
06642365
ABSTRACT:
REFERENCE TO RELATED APPLICATIONS
The present application is the national stage under 35 U.S.C. 371 of international application PCT/IL99/00704, filed Dec. 28, 1999 which designated the United States, and which international application was published under PCT Article 21 (2) in the English language.
FIELD OF THE INVENTION
The present invention relates to antiviral compounds, more particularly to peptidomimetic conjugates of saccharides, such as aminoglycoside antibiotics, with acetamidino and guanidino compounds, and to antiviral, including antiretroviral, pharmaceutical compositions comprising them.
ABBREVIATIONS AND DEFINITION
HIV: human immunodeficiency virus; RT: reverse transcriptase; RNAse: ribonuclease; UC781: a non-nucleoside RT inhibitor; AZT: azidothymidine; KS: Kaposi sarcoma; AIDS: acquired immunodeficiency syndrome; Tat: trans-activator of transcription; TAR: trans-activation responsive RNA region; LTR: long terminal repeat; P-TEFb: positive transcription elongation factor b; CDK9: cyclin-dependent kinase; ALX40-4C: D-arginine nonapeptide; CGP64222: peptide peptoid mimetic of Tat basic domain; HeLa: human epithelial cell line derived from cervical cancer; CXCR4: CXC (&agr;-chemotactic cytokines related to interleukin-8, containing C-X-C motif in their sequence, e.g. SDF-1&agr;) chemokine receptor 4; CD4: cluster of differentiation 4 (characteristic receptor of T-helper cells); CCR5: CC (&bgr;-chemotactic cytokines, containing CC motif in their sequence) chemokine receptor 5; PBMC: peripheral blood mononuclear cells; T22: octadeca peptide, CXCR4 antagonist; AAC: aminoglycoside-arginine conjugates; R52: Tat-derived model undeca peptide, containing a single arginine moiety at position 52 of native Tat protein, in the strand of lysines; R4K: tetra-argininamido kanamycin A conjugate; R3G: tri-argininamido gentamicin C conjugate; MMP: &agr;-methyl D-mannopyranoside RMMP: mono-argininamido MMP conjugate; R4GC1a: tetra-argininamido gentamicin C1a isomer conjugate; GABA: &ggr;-aminobutyric acid; GB4K: tetra-&ggr;-(N-guanidino) butyramido-kanamycin A conjugate; NeoR: hexa-argininamido neomycin B conjugate; EIAV: equine infectious anemia virus; ED: equine dermal fibroblasts; DMF: dimethyl formamide; DCC: dicyclohexyl carbodiimide; M.p.: melting point; Pd/C: palladium on charcoal catalyst; TFA: trifluoro acetic acid; FABHRMS: fast atom bombardment high resolution mass spectroscopy; HSQC: heteronuclear single-quantum coherence; TOCSY: total correlation spectroscopy; RRE: Rev responsive RNA element; CAT: chloramphenicol acetyl transferase; DTT: dithiotreitol; EDTA: ethylenediamine tetraacetic acid; CI
50
: concentration of compound, that causes 50% inhibition of Tat-TAR interaction; CE
50
: concentration of 50% elution from affinity column; CD
50
: 50% binding concentration, related to K
d
; K
d
: dissociation constant; LAN-1: human neurioblastoma cell line; MPC-11: murine plasmocytoma cell line; MT-2, MT-4: human T-lymphoma cell lines, transfected with HTLV-I; HTLV-I, HTLV-II: Human-T-lymphoma virus type I or II; DMEM: Dulbecco modified Eagle's medium; FCS: fetal calf serum; polybrene: hexadimetrine bromide; pfu: plaque forming unit; ELISA: enzyme-linked immuno sorption assay; P4-CCR5 MAGI: human cell line of monocyte/macrophages origin; HUVEC: human umbilical vascular endothelial cells; SUP-T1: human T-cell line; cpe: cytopathic effect; IC
50
: 50% inhibitory concentration; CC
50
: 50% cytotoxic concentration; EC
50
: 50% effective concentration; TI
50
: 50% in vitro therapeutic index (ratio CC
50
/EC
50
); SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel-electrophoresis; TLC: thin layer chromatography; HRP: horseradish peroxidase; SDF-1&agr;: stromal cell derived factor 1, subtype &agr;, the natural ligand of CXCR4; IL2: interleukin 2; IgG: immunoglobulin G; mAb: monoclonal antibody; 12G5: anti-CXCR4 mAb; 2D7: anti-CCR5 mAb; Leu3a: anti-CD4 mAb; PE: phycoerythrin; FITC: fluorescein isothiocyanate; RANTES: regulated on activation normal T-cell expressed and secreted chemokine; MPD: methyl pentandiol; SIR: single isomorphus replacement; SIRA: single anomalous replacement; MAD: multiple anomalous diffraction.
BACKGROUND OF THE INVENTION
The transactivation responsive RNA (TAR) region of human immunodeficiency virus (HIV) long terminal repeat (LTR) regulates the viral gene expression via interaction with the HIV transactivator protein, Tat, and thus is an attractive target for drug design strategies (Gait and Karn, 1995). TAR is found at the 5′ end of all HIV-1 transcripts. It adopts a hairpin secondary structure consisting of a highly conservative hexanucleotide loop and a three-nucleotide bulge flanked by two double-stranded stems (Calnan et al., 1991 a, b). TAR is a positive enhancer that stimulates the synthesis of productive transcripts. It is unique in terms of eukariotic transcription control because it only functions as an RNA element. The activation by Tat is entirely dependent on the presence of the TAR RNA sequence. Tat activates expression by specific binding to TAR, which increases viral mRNA production several hundred-fold by stimulation of the elongation capacity of RNA polymerase II (Kingsman and Kingsman, 1996). HIV Tat binds the cyclin T subunit of P-TEFb and recruits P-TEFb to the HIV-1 LTR promoter. This process requires binding of Tat to the TAR bulge and of cyclin T to the TAR loop. The cyclin T associated CDK9 kinase then induces phosphorylation of the C-terminal domain of RNA polymerase II, and of other polymerase II-associated proteins, leading to the transition from non-processive to processive transcription (Cullen, 1998).
Binding of Tat protein to TAR is mediated by the nine amino acid region RKKRRQRRR (residues 49-57) of the protein (e.g. Calnan et al., 1991 a, b; Churcher et al., 1993). The nona-arginine peptide (R
9
) binds to TAR with the same affinity and specificity as the above wild-type Tat peptide, whereas the nona-lysine peptide (K
9
) binds to TAR non-specifically and with a ten-fold lower affinity. The R
9
-containing Tat mutant protein gives wild-type trans-activation activity and is 100-fold more active than the K
9
-containing protein. Insertion of a single arginine moiety at position 52 or 53 (synthetic peptides R52 of the sequence YKKKRKKKKA or R53) restores RNA-binding affinity and specificity of the peptide as well as its trans-activation potency (Calnan et al., 1991 b). Mutagenesis studies on TAR RNA demonstrated that the bulge (U23-C24-U25) and two base pairs at both sides of the bulge (e.g. Cordingley et al,. 1990; Roy et al, 1990; Delling et al., 1992) are important for Tat binding. Full length Tat protein binds TAR with only moderate affinity and specificity in vitro. The first 37 N-terminal amino acids of the Tat protein decrease its affinity to TAR in comparison to the specific recognition Tat (38-72) peptide (Rana and Jeang, 1999). It was shown that human cyclin T1 promotes cooperative binding of Tat protein to TAR RNA in vitro and mediates trans-activation in vivo. Although cyclin T1 does not bind TAR RNA, it may interact with the TAR loop in a ternary complex of cyclin T1-Tat-TAR (Wei et al., 1998).
NMR structures of HIV TAR bound to different ligands, e.g. as peptides that mimic the basic region of Tat, arginine and arginineamide, show that the ligands bind to the TAR RNA major groove (Puglisi et al., 1992, 1993; Aboul-ela et al., 1995, 1996). The bulge structure allows ligands to access the major groove of TAR, which induces folding in the bulge and formation of unusual base-triples (Puglisi et al., 1992, 1993, Aboul-ela et al., 1995, 1996). The TAR RNA hairpin can adopt two major conformations. In the absence of ligands, the bulge nucleotides stack within the RNA stem, severely distorting its helical continuity (Puglisi et al., 1992; Aboul-ela et al., 1996). When either L-arginineamide or the Tat peptide bind to TAR, the bulge nucleotides loop out of the remaining stem, allowing the upper and the lower stem helices to stack coaxially (Puglisi el al., 1992, 1993). The NMR structure of L-arginineamide bou
Evdokimov Artem G.
Lapidot Aviva
Litovchick Alexander
Browdy and Neimark
Khare Devesh
Wilson James O.
Yeda Research and Development Co. Ltd.
LandOfFree
Anti-(retro)viral conjugates of saccharides and acetamidino... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Anti-(retro)viral conjugates of saccharides and acetamidino..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Anti-(retro)viral conjugates of saccharides and acetamidino... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3166002