Chimeric Compounds Research Articles

Differential cytotoxicity of novel somatostatin and dopamine chimeric compounds on bronchopulmonary and small intestinal neuroendocrine tumor cell lines.

Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (overall 67%, 5-year survival) in 30 years (1973-2004), whereas the incidence has increased ( approximately 1000%) in the same time frame. No effective or specific antineoplastic agent is available for treatment, although somatostatin analogs inhibit tumor secretion. Given the coexistence of somatostatin and dopamine regulatory receptors on NET cells, the antiproliferative efficacy as well as the signaling and transcriptional targets of their ligands were evaluated. The cytotoxic effects of 12 somatostatin/dopamine compounds were evaluated in 3 NET cell lines, and real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay studies were performed to delineate antiproliferative signaling pathways. The atypical BP-NET, NCI-H720, was most sensitive to the sst(5) analog BIM23206 (half-maximal concentration, 2.4 pM) and demonstrated similar sensitivity to lanreotide and the sst(2) analog BIM23120. The typical BP-NET, NCI-H727, was most sensitive to BIM23120 (0.7 nM) and to the pan-somatostatin receptor analog (BIM23A779). The GI-NET, KRJ-I, was most sensitive to sst(2,5) analogs lanreotide (1 nM) and BIM23244 (7.4 nM). Lanreotide activated extracellular signal regulated kinase-1/2 phosphorylation and p21(WAF1/CIP1) transcription, but inhibited Ki-67 transcription. NCI-H720 was most sensitive to the sst(2,5)- and D(2)-selective compound BIM23A761 (4.2 nM), as was NCI-H727 (5.5 nM). KRJ-I did not respond to any chimeric analog. BIM23A761 activated c-Jun N-terminal kinase signaling and caused inhibition of Ki-67 transcription. P21(WAF1/CIP1) transcription was activated only in NCI-H727 cells. The different responses of each individual cell line suggested that NETs from different locations arising from different neuroendocrine cells may require cell-specific antiproliferative agents based on the unique receptor profile of individual lesions.

Relevance of coexpression of somatostatin and dopamine D2 receptors in pituitary adenomas

Dopamine and somatostatin are both involved in the negative control of normal pituitary cells. Dopamine subtype 2 receptor (D2DR) and somatostatin receptor (sst) agonists, mainly directed to sst2, are used in the treatment of pituitary adenomas. Nevertheless, a majority of corticotroph and gonadotroph adenomas and a third of somatotroph adenomas are still not sufficiently controlled by these treatments. D2DR and sst1, 2, 3 and 5 are present in most pituitary adenomas. These receptors may interact by heterodimerization as shown for sst1-sst5, sst5-D2DR, sst2-sst3 and sst2-D2DR suggesting possible additive effects. D2DR and sst2 agonist cotreatment showed limited additivity on GH secretion in acromegaly. Moreover, new chimeric compounds with sst2, D2DR and sst5 affinity have shown an increased control of secretion and/or proliferation of different types of pituitary adenomas in cell culture. Together with the multi-sst ligand drugs recently developed, these dopamine-somatostatin ligands represent a new opportunity in the combinatory treatment of pituitary adenomas.

Synthesis of a versatile metacyclophane macrolactam

As Hsp90 has emerged as a promising target for the development of cancer chemotherapeutics, so has the need for systematic evaluation of structure–activity relationships between natural product inhibitors and this molecular chaperone. Utilizing our chimera approach, which encompasses the quinone moiety of geldanamycin and the resorcinol moiety of radicicol, molecules have been produced that are highly effective inhibitors of the Hsp90 protein folding machinery. These chimeric inhibitors contain metacyclophane macrolactams that are difficult to cyclize and modify for incorporation of functional diversity. To circumvent this problem, a highly diversifiable α-bromo-α,β-unsaturated ester has been prepared, which allows for the introduction of various functionalities that enable elucidation of structure–activity relationships between chimeric compounds and Hsp90. The rationale, synthesis, and optimization for such a molecule is reported herein.

Control of pituitary adenoma cell proliferation by somatostatin analogs, dopamine agonists and novel chimeric compounds

The antisecretory effects of somatostatin (SRIH) and its analogs are widely recognized and provide the basis for treatment of hormonal hypersecretion in patients with pituitary adenomas, especially in the settings of acromegaly. Dopamine (DA) agonists have also been used for medical treatment of prolactin and/or GH hypersecretion, and recent evidence points to an even greater antisecretory effect for a chimeric molecule, having high affinity for both SRIH and DA receptors. Evidence for an antiproliferative effect of these compounds has also been provided. This review focuses on the antiproliferative effects of SRIH and its analogs, of DA and chimeric compounds on pituitary adenomas, and on the clinical consequences on tumor volume of pharmacological treatment of pituitary adenomas with these drugs.

First evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant

BN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4–6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-β1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 μM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types.

Chimeric peptide nucleic acid compounds modulate splicing of the bcl-x gene in vitro and in vivo

Alternative splicing of the bcl-x gene generates two transcripts: the anti-apoptotic bcl-xL isoform and the pro-apoptotic bcl-xS isoform. The ratio between the two isoforms is a key factor in development and in cancer progression. Here, we show that a short antisense chimeric peptide nucleic acid (PNA) oligonucleotide conjugated to a polypeptide containing eight Ser-Arg repeats (SR)8 can modulate splicing of bcl-x both in vitro and in vivo and induces apoptosis in HeLa cells. The PNA-SR oligo was targeted to a region of bcl-x that does not contain splicing regulatory sequences and was able to override the complex network of splicing enhancers and silencers that regulates the ratio between the two bcl-x isoforms. Thus, PNA-SR oligos are powerful tools that can potentially modulate splice site choice in endogenous genes independent of the presence of other splicing regulatory mechanisms on the target gene.

Somatostatin and dopamine receptor expression in lung carcinoma cells and effects of chimeric somatostatin-dopamine molecules on cell proliferation

To study somatostatin/dopamine (SS/D) synergy in a human cell system constitutively expressing SS and D receptors (SSR and DR, respectively), we characterized the expression of SSR and DR subtypes in the non-small-cell lung cancer line Calu-6, and then we evaluated the effect on cell proliferation of SS/D chimeric molecules (BIM-23A387 and BIM-23A370), which bind with high affinity both sst(2) and D(2)R, and compared the results with those obtained by using SS-14 and subtype-selective SS analogs (SSA) and D agonists (DA). Because Calu-6 cells produce insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) peptides, which play a role in the autocrine/paracrine control of cell growth, we also investigated the effects of chimeric compounds on secretion and expression of IGF system components. Relative high levels of sst(2) and the long isoform of the D(2)R were detected by real-time RT-PCR and Western blot in Calu-6, together with sst(5) and to a lesser extent sst(3) and D(4)R. BIM-23A387 and BIM-23A370 significantly inhibited growth of Calu-6, whereas IGF-IGFBP secretion or expression was unaffected, suggesting a direct inhibitory effect. The inhibition of cell growth, measured by both [(3)H]thymidine incorporation and cell count, was significantly lower when individual SSA and DA control peptides or subtype-specific SSA and DA were tested. BIM-23A370 was more potent than BIM-23A387 (P < 0.001). These findings show that SS/D chimeras can inhibit Calu-6 proliferation in an IGF-independent manner and suggest that this enhanced potency might be because of the induction of SSR/DR dimerization. The Calu-6 cell line, constitutively expressing SSR and DR, provides a suitable model to elucidate the mechanism of action of SSA and DA on regulation of cell growth and to characterize the interaction between SSR and DR.

Efficacy of chimeric molecules directed towards multiple somatostatin and dopamine receptors on inhibition of GH and prolactin secretion from GH-secreting pituitary adenomas classified as partially responsive to somatostatin analog therapy

This study compared the potency of a somatostatin receptor (sstr)2-sstr5 analog, BIM-23244, of an sstr2-dopamine D2 receptor (sstr2-DAD2) molecule, BIM-23A387 and of new somatostatin-dopamine chimeric molecules with differing, enhanced affinities for sstr2, sstr5 and DAD2, BIM-23A758, BIM-23A760 and BIM-23A761, to suppress GH and prolactin (PRL) from 18 human GH adenomas that are partially responsive to octreotide or lanreotide. The sstr2, sstr5 and DAD2 mRNA levels were determined by RT-PCR. The effect of drugs was tested in cell cultures at various concentrations. In all tumors, the sstr2, sstr5 and DAD2 mRNA levels were coexpressed (mean levels+/-s.e.m. 0.4+/-0.1, 5.3+/-1.9 and 2.0+/-0.4 copy/copy beta-glucuronidase). In 13 tumors, the maximal suppression of GH secretion produced by BIM-23A387 (30+/-3%) and BIM-23244 (28+/-3%) was greater than that produced by octreotide (23+/-3%). In six out of 13 tumors, BIM-23A758, BIM-23A760 and BIM- 23A761 produced greater maximal suppression of GH secretion than octreotide (33+/-5, 38+/-2 and 41+/-2 vs 24+/-2%). Their EC(50) values were 10, 2 and 4 pmol/l. BIM-23A761 was more effective than BIM-23A387 in GH suppression (41+/-2 vs 32+/-4%). The new chimeric molecules produced maximal PRL suppression greater than octreotide (62+/-8 to 74+/-5 vs 46+/-11%). Novel dopamine-somatostatin chimeric molecules with differing, enhanced activity at sstr2, sstr5 and DAD2, consistently produced significatly greater suppression of GH and PRL than either octreotide or single-receptor-interacting ligands in tumors from patients classified as only partially responsive to octreotide therapy. The higher efficacy of the chimeric compounds was, at least partially, linked to their high affinity for sstr2 (IC50 1-10 pmol/l). The other mechanisms by which such molecules produce an enhanced inhibition of GH remain to be elucidated.

Synthesis and biological activity of mustard derivatives of combretastatins

A series of chimeric compounds bearing the combretastatin and the nitrogen mustard cores were synthesized. All the compounds were cytotoxic and inhibited tubulin polymerization. When combretastatin was joined to chlorambucil via an ester linkage, the resultant compound proved to be significantly more potent than the two compounds put together. When combretastatin was joined to nitrogen mustard via an ether linkage or when a true hybrid was synthesized, loss of potency was observed. Nonetheless, these latter compounds appeared to be more efficacious and surprisingly were able to inhibit tubulin depolymerization at high concentrations.

Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor referred to as N/OFQ peptide (NOP) receptor. NOP receptor activation by N/OFQ modulates several biological functions both at central and peripheral level. Structure activity relationship (SAR) studies demonstrated that the N/OFQ sequence can be divided into a N-terminal tetrapeptide 'message' crucial for receptor activation and a C-terminal 'address' important for receptor binding. On the basis of this message/address concept we synthesized some chimeric compounds in which we substituted the natural message domain with the nonselective nonpeptide NOP ligand (8-Naphthalen-1-yl-methyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4,5]dec-3-yl)-aceticacid methyl ester (NNC 63-0532) and used as address domain the peptide sequences Thr-NH2, N/OFQ(5-9)-NH2, N/OFQ(5-13)-NH2 and N/OFQ(5-17)-NH2. All the compounds were pharmacologically evaluated in the electrically stimulated guinea-pig ileum. NNC 63-0532 produced a concentration-dependent inhibition of the electrically induced twitches showing, in comparison with N/OFQ, lower potency and higher maximal effects. In addition, contrary to N/OFQ, the effects of NNC 63-0532 were insensitive to the NOP selective antagonist [Nphe1, Arg14, Lys15]N/OFQ-NH2 (UFP-101) while prevented by naloxone. Similar results were obtained with NNC 63-0532/Thr-NH2 and NNC 63-0532/N/OFQ(1-9)-NH2. On the contrary, the inhibitory effects of NNC 63-0532/N/OFQ(5-13)-NH2 and NNC 63-0532/N/OFQ(5-17)-NH2 were slightly antagonized by UFP-101 while naloxone prevented the effects of the high but not of the low concentrations of the two ligands. These data indicate that it is possible to functionalize with the N/OFQ address sequence a nonpeptide NOP ligand for increasing its binding to the NOP receptor. Moreover, these results corroborate the idea that the 5-13 sequence represents the crucial core of the N/OFQ address domain.

Can a ‘patch’ in a skipped exon make the pre-mRNA splicing machine run better?

It is becoming clear that exonic sequences can act as determinants of their own fate: the inclusion or exclusion from mature mRNA. Indeed, even silent nucleotide substitutions can cause aberrant exon skipping, resulting in a disease phenotype. It might be possible to restore essential splicing functions, lost through mutations, using molecular therapy at the RNA level. A variety of methods have been attempted, the most promising being the recent use of chimeric compounds that localize splicing-functional peptides by base complementarity.

Coupling of a competitive and an irreversible ligand generates mixed type inhibitors of Trypanosoma cruzi trypanothione reductase.

9-Aminoacridines and (terpyridine)platinum(II) complexes are competitive and irreversible inhibitors, respectively, of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. Four chimeric compounds in which 2-methoxy-6-chloro-9-aminoacridine was covalently linked to the (2-hydroxyethanethiolate)(2,2':6',2' '-terpyridine)platinum(II) complex were synthesized and studied as inhibitors of the parasite enzyme. The derivatives differed by the nature and/or the length of the spacer connecting the two aromatic systems. All four compounds were effective mixed type inhibitors of trypanothione reductase with K(i) and K(i)' values of 0.3-4 and 2-11 microM, respectively. The most potent inhibitor had an ethylthioether linkage between the two aromatic ring systems, and the other compounds contained an alkyl ether group with 4-6 methylene groups. In contrast to the parasite enzyme, human glutathione reductase, the closest related host enzyme was not inhibited by these compounds. The finding that the conjugation of a competitive and an irreversible inhibitor can give rise to reversible mixed type inhibitors underlines the difficulties associated with inhibitor design based on the three-dimensional structure of trypanothione reductase.

Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.

Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.

Synthesis of chimeric 7alpha-substituted estradiol derivatives linked to cholesterol and cholesterylamine.

We report the synthesis of 7alpha-substituted beta-estradiol derivatives bearing side chains terminated with cholesterol and 3beta-cholesterylamine. These chimeric compounds were designed to exhibit high affinity for estrogen receptors (ERs) and cellular plasma membranes to potentially enable regulated uptake of ERs by mammalian cells. Evaluation with recombinant yeast reporting compound-mediated ER dimerization revealed potencies similar to the antiestrogen ICI 182780. Compounds that efficiently deliver dominant negative ERs into cells may provide novel therapeutics against breast cancers.

Carbopeptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template.

Multifunctional, topological template molecules such as linear and cyclic peptides have been used for the attachment of peptide strands to form novel protein models of, for example, 4-alpha-helix bundles. The concept of carbohydrates as templates for de novo design of potential protein models has been previously described and these novel chimeric compounds were termed carbopeptides. Here, a second generation strategy in which carbopeptides are synthesized by chemoselective ligation of a peptide aldehyde to an aminooxy-functionalized alpha-D-galactopyranoside is described. This template was prepared by per-O-acylation of methyl alpha-D-galactopyranoside with N,N-Boc2-aminooxyacetic acid to form a tetra-functionalized template, followed by treatment with TFA-CH2Cl2 to release the aminooxy functionality. The peptide aldehydes Fmoc-Ser-Gly-Gly-H and H-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H were synthesized by a BAL strategy. Four identical copies of peptide aldehyde were smoothly attached to the template by chemoselective ligation to form a 2.1 and a 2.9 kDa carbopeptide, respectively.

Antimicrobial effects of novel siderophores linked to β-lactam antibiotics

As a strategy to increase the penetration of antibiotic drugs through the outer membrane of Gram-negative pathogens, facilitated transport through siderophore receptors has been frequently exploited. Hydroxamic acids, catechols, or very close isosteres of catechols, which are mimics of naturally occurring siderophores, have been used successfully as covalently linked escorting moieties, but a much wider diversity of iron binding motifs exists. This observation, coupled to the relative lack of specificity of siderophore receptors, prompted us to initiate a program to identify novel, noncatechol siderophoric structures. We screened over 300 compounds for their ability to (1) support growth in low iron medium of a Pseudomonas aeruginosa siderophore biosynthesis deletion mutant, or (2) compete with a bactericidal siderophore-antibiotic conjugate for siderophore receptor access. From these assays we identified a set of small molecules that fulfilled one or both of these criteria. We then synthesized these compounds with functional groups suitable for attachment to both monobactam and cephalosporin core structures. Siderophore-β-lactam conjugates then were tested against a panel of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Although several of the resultant chimeric compounds had antimicrobial activity approaching that of ceftazidime, and most compounds demonstrated very potent activity against their cellular targets, only a single compound was obtained that had enhanced, siderophore-mediated antibacterial activity. Results with tonB mutants frequently showed increased rather than decreased susceptibilities, suggesting that multiple factors influenced the intracellular concentration of the drugs.

Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells.

It is widely accepted that most cell types efficiently exclude oligonucleotides in vitro and require specific delivery systems, such as cationic lipids, to enhance uptake and subsequent antisense effects. Oligonucleotides are not readily transfected into leukaemia cell lines using cationic lipid systems and streptolysin O (SLO) is used to effect their delivery. We wished to investigate the optimal oligonucleotide composition for antisense efficacy and specificity following delivery into leukaemia cells using SLO. For this study the well characterised chronic myeloid leukaemia cell line KYO-1 was selected and oligonucleotides (20mers) were targeted to an empirically identified accessible site of c- myc mRNA. The efficiency and specificity of antisense effect was measured 4 and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne phosphodiester and phosphorothioate compounds were found to present substantial non-specific effects at 20 microM but were inactive at 0.2 microM. Indeed, no antisense-specific effect was noted at any concentration at either time. All of the other oligonucleotides tested induced some measurable antisense effect, except 7 (chimeric, all-phosphorothioate, 2'-methoxyethoxy termini) which was essentially inactive at 20 microM. The rank efficiency order of the remaining antisense compounds was 4 = 3 >> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects induced by the chimeric methylphosphonate-phosphodiester compounds were found to be highly specific. Increased phosphorothioate content in the oligonucleotide backbone correlated with reduced antisense activity (efficacy: 2'-methoxyethoxy series 9 >> 8 >> 7, 2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained for increased activity correlating with increased oligonucleotide-mRNA heteroduplex thermal stability. In conclusion, the chimeric methylphosphonate-phosphodiester oligodeoxynucleotides present the most favourable characteristics of the compounds tested, for efficient and specific antisense suppression of gene expression following SLO-mediated delivery.

Plasmodium falciparum:Effect of Chemical Structure on Efficacy and Specificity of Antisense Oligonucleotides against Malariain Vitro

Antisense oligodeoxynucleotides (AS ODNs) have shown promise both as potential anti-malarial chemotherapeutic agents and as a means for identifying genes critical for parasite survival. Because conventional ODNs containing phosphodiester (PO) groups are subject to rapid nuclease degradation, ODNs with phosphorothioate (PS) groups are commonly used. However, at high concentration, these lose target specificity, and in some animal models, they become toxic. We compared a variety of chemical modifications (PO, PS, PO-PS hybrids, 2′-O-methyl-2′-deoxy chimeras) and structural modifications (sequence alterations favoring self-stabilizing loop formation) for their ability to inhibitPlasmodium falciparummalaria culturedin vitro.All modifications were done using an AS ODN sequence targeted against dihydrofolate reductase thymidylate synthase (DHFR). Inhibition by PO-PS hybrids containing as few as three PS groups at the 3′- and 5′-ends did not differ significantly from that obtained using compounds containing all-PS groups. Similarly, inhibition by PS chimeric compounds containing 2′-O-methyl modifications did not differ significantly from that of conventional PS compounds. In contrast, while inhibition by PO-PS hybrid chimeras did not differ significantly from that of all-PS compounds at low concentrations, at 1 μMthey inhibited parasite growth 25% less (P< 0.001) than all-compounds or PS 2′-O-methyl-2′-deoxy chimeras. Extension of the nucleotide sequence to increase stem—loop formation yielded two compounds which inhibited parasite growth about 20% more than unmodified compounds, though this difference was not significant. Furthermore, most of this increase appears to correlate with the greater number of PS groups associated with the increased ODN length. We conclude that limiting the number of PS groups and inclusion of PO 2′-O-methyl groups may yield compounds with high antisense activity but low non-sequence-dependent effects. Such compounds are currently being testedin vivo.

Optimization of Antisense Oligodeoxynucleotide Structure for Targeting bcr-abl mRNA

Antisense oligodeoxynucleotides targeted to bcr-abl are potential ex vivo purging agents for use with autologous bone marrow transplantation in the treatment of chronic myeloid leukemia (CML). We investigated, in a cell-free system, the activity and nuclease resistance of phosphodiester, phosphorothioate, chimeric methylphosphonate/phosphodiester, and chimeric methylphosphonate/phosphorothioate antisense octadecamers directed against either b2a2 or b3a2 bcr-abl breakpoint RNAs. Certain chimeric compounds were shown to possess targeted activity broadly equal to the parent phosphodiester or phosphorothioate forms and greater resistance to the nucleases present in cell extracts. Selected chimeric structures were compared with phosphodiester and phosphorothioate analogues for antisense activity in human CML cells containing either b2a2 or b3a2 bcr-abl breakpoint mRNAs. We present results showing that all four structures can suppress bcr-abl mRNA level in vivo. The rank of in vivo activity is chimeric methylphosphonate/phosphodiester > or = phosphodiester > phosphorothioate > methylphosphonate/phosphorothioate. We show that b2a2 breakpoint RNAs can be more effectively targeted than b3a2 sequence RNAs both in vitro and in vivo and suggest that RNA secondary structure may be a possible explanation for this phenomenon.

Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds.

Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent inhibitors of normal transcription. Using the linker between the two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be approximately 15 A. A reactive side group placed in the linker next to Rif crosslinks to the template strand of DNA at the -2 or -3 position of the promoter. Thus, bound Rif is juxtaposed to DNA immediately upstream of the start site, suggesting that Rif plugs the channel leading RNA out of the active center.