Inhibition of HIV-1 Rev-RRE interaction by diphenylfuran derivatives.

The interactions between RNA structures, such as RRE in the HIV-1 genome, and proteins, such as Rev of HIV-1, are essential for efficient viral replication. Compounds that bind specifically to such RNAs and disrupt their protein complexes offer a novel mechanism for inhibition of replication of the virus. As a step in this approach, we have designed and characterized a series of synthetic diphenylfuran cations that selectively inhibit Rev binding to RRE. Fluorescence titrations and gel band-shift results indicate that the diphenylfurans bind to RRE and inhibit Rev complex formation in a structure-dependent manner. The derivative with the greatest affinity for RRE has an association constant of greater than 10(7) M-1 and inhibits formation of the Rev--RRE complex at concentrations below 1 microM. It binds to RRE considerably more strongly than it binds to simple RNA duplexes. Spectral changes and energy transfer results on complex formation suggest that the compound has a nonclassical intercalation binding mode. CD studies with modified RRE hairpins indicate that the active diphenylfurans bind at the structured internal loop of RRE and cause a conformational change. The most active diphenylfurans are tetracations that appear to bind to RRE by a threading intercalation mode and cause a conformational change in the RNA that is essential for inhibition of Rev complex formation with RRE.

Design and analysis of RNA structure-specific agents as potential antivirals.

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants > 10(7)M-1), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.

An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.

The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.

Small changes in cationic substituents of diphenylfuran derivatives have major effects on the binding affinity and the binding mode with RNA helical duplexes.

The interactions of dicationic, 2-4, and tetracationic, 5-7, diphenylfuran analogs of 1 (furamidine) with RNA have been analyzed by thermal melting, spectroscopic, viscometric, kinetic and molecular-modeling techniques. The results of these studies indicate that most of the furan derivatives bind to RNA duplexes by intercalation in contrast to their minor-groove binding mode in AT sequences of DNA, but similar to their binding mode in GC rich regions of DNA. The highest affinity for RNA is found for an imidazoline dication, 2. With some substituents which inhibit formation of a strong intercalation complex, the results suggest a non-intercalative type of binding occurs. The non-intercalative binding probably occurs through a complex with the furan derivative bound in the narrow, deep major groove of A-form RNA helices.

Oligonucleotide N3'-->P5' phosphoramidates.

Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 3'-NHP(O)(O-)O-5' internucleoside linkages and the study of their hybridization properties. Thermal dissociation experiments show that these compounds form very stable duplexes with single-stranded DNA, RNA, and with themselves following Watson-Crick base pairing. The duplex thermal stability was enhanced by 2.2-2.6 degrees C per modified linkage compared with phosphodiesters. The structure of complexes formed by phosphoramidates closely resembles that of RNA oligomers and corresponds to an A form, as judged by CD spectroscopy. N3'-->P5' phosphoramidates also form stable triplexes with double-stranded DNA under near-physiological conditions when natural phosphodiesters fail to do so. Physicochemical characteristics of the amidates are similar to those of RNA oligomers, even though they are composed of 2'-deoxyfuranose-based nucleosides.

Sequence specific thermodynamic and structural properties for DNA.RNA duplexes.

DNA.RNA hybrid duplexes are found in many important biological processes and are involved in developing modes of disease treatment, such as antisense therapy, yet little is known about the sequence dependence of their structure and stability. The structure and thermodynamic stability of DNA.RNA hybrid model systems corresponding in composition and length and containing (1) all purine or all pyrimidine bases on each strand or (2) mixed purine and pyrimidine bases on each strand have been evaluated relative to pure RNA and DNA duplexes by thermal melting, CD, and electrophoresis analyses. The spread in free energies of denaturation of the homopurine.homopyrimidine systems covers over 14 kcal/mol of single strands, while the mixed sequence free energies vary by less than 4 kcal/mol. The RNA-homopurine.DNA-homopyrimidine hybrid resembles a corresponding pure RNA duplex in both structure and stability, whereas the DNA-homopurine.RNA-homopyrimidine hybrid resembles a corresponding pure DNA duplex. The mixed sequence hybrids show intermediate structure between the corresponding pure RNA and pure DNA duplexes and a stability closer to that of the pure DNA duplex. From these results and the evaluation of published hybrid data [Hall, K. B., & McLaughlin, L. W. (1991) Biochemistry 30, 10606-10613; Roberts, W. R., & Crothers, D. M. (1992) Science 258, 1463-1466], it can be predicted that a hybrid duplex containing more RNA purine bases will have a CD spectrum, and probably conformation, resembling that of A-form duplexes and will be more stable than a corresponding hybrid duplex with fewer RNA purine bases.

The search for structure-specific nucleic acid-interactive drugs: effects of compound structure on RNA versus DNA interaction strength.

The RNA genomes of a number of pathogenic RNA viruses, such as HIV-1, have extensive folded conformations with imperfect A-form duplexes that are essential for virus function and could serve as targets for structure-specific antiviral drugs. As an initial step in the discovery of such drugs, the interactions with RNA of a wide variety of compounds, which are known to bind to DNA in the minor groove, by classical or by threading intercalation, have been evaluated by thermal melting and viscometric analyses. The corresponding sequence RNA and DNA polymers, poly(A).poly(U) and poly(dA).poly(dT), were used as test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA (e.g., netropsin, distamycin, and a zinc porphyrin derivative) do not have significant interactions with RNA. Compounds that bind in the minor grove in AT sequences of DNA but have other favorable interactions in GC sequences of DNA (e.q., Hoechst 33258, DAPI, and other aromatic diamidines) can have very strong RNA interactions. A group of classical intercalators and a group of intercalators with unfused aromatic ring systems contain compounds that intercalate and have strong interactions with RNA. At this time, no clear pattern of molecular structure that favors RNA over DNA interactions for intercalators has emerged. Compounds that bind to DNA by threading intercalation generally bind to RNA by the same mode, but none of the threading intercalators tested to date have shown selective interactions with RNA.

DAPI (4',6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites.

The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.