Inhibitors of protein-RNA complexation that target the RNA: specific recognition of human immunodeficiency virus type 1 TAR RNA by small organic molecules.

TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model.

Binding of neomycin to the TAR element of HIV-1 RNA induces dissociation of Tat protein by an allosteric mechanism.

Neomycin inhibits the binding of Tat-derived peptides to the trans-activating region (TAR) of HIV-1 RNA. Kinetic studies reveal that neomycin acts as a noncompetitive inhibitor that can bind to the Tat-TAR complex and increase the rate constant (koff) for dissociation of the peptide from the RNA. Neomycin effects a conformational change in the structure of TAR that can be detected by circular dichroism spectroscopy. The increase in ellipticity measured at 265 nm upon binding of the aminoglycoside is opposite to the decrease seen when Tat peptides bind to the RNA. Thus, the structural transition induced by neomycin is apparently incompatible with the binding of Tat and underlies the inhibitory action of the antibiotic. The binding site for neomycin on TAR was identified in ribonuclease protection experiments and is located in the stem immediately below the three-nucleotide bulge that serves as the primary identity element for Tat. Apparent protection of residues in the bulge by neomycin may represent additional contacts to the aminoglycoside, but more likely result from changes in the structure of this region when the ligand binds to the RNA. Binding assays using variants of TAR in which inosine residues were substituted for guanosine residues support the results from the ribonuclease protection experiments. Inosine substitutions in the lower stem, but not the upper stem, decrease the binding constant for neomycin by approximately 100-fold. Neither of these variants affected the binding affinity of Tat peptide. In addition, these latter experiments suggest that the aminoglycoside may be located in the minor groove of the stem. This mode of association may be a critical aspect of neomycin's ability to bind to the Tat-TAR complex and could serve as a guide for the design of other drugs that bind to specific RNA targets as noncompetitive inhibitors.

A combinatorial synthesis of tyrphostins via the "directed sorting" method.

Using solid-phase organic synthesis, we have prepared a 432-member (18 x 8 x 3) sample library based on the AG 490 "tyrphostin" template. By utilizing 432 reactors each equipped with a unique radiofrequency memory ID tag, the 432 products could be obtained as discrete entities (i.e., not as mixtures) via 18 + 8 + 3, or 29 reactions. Reading each ID tag after each reaction step permitted the "directed sorting" of reactors into appropriate reaction vessels containing multiple reactors. After synthesis, all products were cleaved from the solid-phase support and lyophilized to afford powders. Characterization of 5% of the library members by NMR and mass spectrometry provided verification of structure. In addition, TLC analysis of every library member provided evidence that most (or all) are composed of a single major organic compound. Some 88% of these samples were obtained in amounts of between 5 and 19 mg. Using this reaction sequence and the "directed sorting" approach, the synthesis of much larger AG 490-based libraries can be envisioned.

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription.

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.

Discovery of selective, small-molecule inhibitors of RNA complexes--II. Self-splicing group I intron ribozyme.

Self-splicing group I intron RNA was chosen as a potential therapeutic target for small-molecule intervention. High-throughput screening methodologies have been developed to identify small organic molecules that regulate the activities of these catalytic introns. Group introns derived from pathogenic Pneumocystis carinii and phage T4 were used as model systems. Inhibitors identified from a library of approximately equal to 150,000 compounds were shown to regulate biochemical reactions including the two-step intron splicing and an RNA ligation catalyzed by the group I introns. These inhibitors provide a unique opportunity to understand small-molecule recognition of the self-splicing RNA. The methodologies developed for group I introns should be applicable to studies of other RNA systems.

Encoding methods for combinatorial chemistry.

Assuming that the chemical reactions used to synthesize a combinatorial library member are successful, then knowledge of the specific reaction sequence is equivalent to knowing the member's chemical identity. Because the determination of chemical identity is typically not automatable and requires a substantial amount of material, schemes that encode a member's reaction history onto the reaction platform are of value. The primary benefits of encoding are relational nomenclature (all methods) and automated handling (some methods). Encoding methods evaluated to date are spatial, graphical, chemical, spectrometric, electronic, and physical.

Inhibition of self-splicing group I intron RNA: high-throughput screening assays.

High-throughput screening assays have been developed to rapidly identify small molecule inhibitors targeting catalytic group I introns. Biochemical reactions catalyzed by a self-splicing group I intron derived from Pneumocystis carinii or from bacteriophage T4 have been investigated. In vitro biochemical assays amenable to high-throughput screening have been established. Small molecules that inhibit the functions of group I introns have been identified. These inhibitors should be useful in better understanding ribozyme catalysis or in therapeutic intervention of group I intron-containing microorganisms.

Chelation-enhanced fluorescence chemosensing of Pb(II), an inherently quenching metal ion.

Pb(II) ion serves as a quencher of anthracene fluorescence both intermolecularly and in intracomplex systems reported to date. The advantages of intensimetric analyses showing increasing signal require the design of mechanistically novel fluorescent chemosensors capable of yielding enhanced fluorescence upon chelation of inherently quenching metals. We synthesized both 2- and 9-derivatives of anthracene bearing the N-methylthiohydroxamate ligand, which is capable of quenching fluorescence in the uncomplexed form and which shows some selectivity for Pb(II). Complexation of the 2-derivative to Pb(II) results in rapid metal ion-catalyzed hydrolysis, rendering this compound useless as a sensor with real-time response. However, complexation of the 9-derivative with Pb(II) results in a 13-fold fluorescence increase, reversible upon dissociation of the metal ion. While blood lead analysis is a major potential application for fluorescent chemosensors, the present compound is insufficiently selective for this use.

The impact of polystyrene resins in solid-phase organic synthesis.

A major objective of the DIVERSOMER technology is to provide pure and characterized compounds for biological testing in order to prevent 'false negatives' in our libraries. On several occasions, analysis of the final products by 1H-NMR and MS, has revealed by-products from the polystyrene solid support. Subsequently, three alternative methods were studied to remove polystyrene by-products; (i) prewashing of the resin prior to execution of the synthesis; (ii) pretreatment of the resin with the cleavage conditions consistent with the solid-phase synthesis reaction scheme; and (iii) parallel purification.

Desperately seeking sensors.

The list of biologically active small molecules for which fluorescent sensors would be desirable is enormous. The list of sensors actually available is surprisingly small. The reason derives in part from a lack of communication between chemistry inventors and biology end-users. A new World Wide Web board has been created to address this need.

Synthesis and transacylating reactivity of beta-cyclodextrin ethylenediamines.

The synthesis of the ethylenediamine-connected cyclodextrin dimer is reported, together with the syntheses of several reference cyclodextrinylamines. Each compound displayed enhanced transacylation or transphosphorylation of activated substrates, with the primary amine-bearing monocyclodextrin compound showing the greatest activity. No special rate advantage was observed for this cyclodextrin dimer, although such effects do exist in other cycloextrin dimers reported previously.

Intramolecular carboxylate catalysis in the depurination of a 7-methylguanosine derivative.

We have compared the pH-independent rates of glycosidic hydrolysis in a carboxylate-bearing 7-methylguanosine derivative with those of a reference compound and with that of 7-methylguanosine itself. A syn-oriented carboxylate group affords catalysis in the hydrolysis reaction, although the instability of 7-alkylguanosines above pH 7 severely limits the useful pH range that could be studied. The effect of the carboxylate near neutral pH can be viewed in three different ways: it provides a 3-fold acceleration as compared to underivatized 7-methylguanosine, an approximately 30-fold acceleration when the decelerating effect of the ketal group is considered, and because of slow decomposition of the reference compound under the reaction conditions, we conclude that the carboxylate provides an acceleration of > or = 43-fold as compared to the protio reference compound.

Fluorescent chemosensing of catechol and catecholamines in water.

It has been known for almost 35 years that catechol complexes reversibly to boronic acids. We observe that 2-anthrylboronic acid complexes catechol in water with Kd 330 microM and concomitant 20-fold reduction in fluorescence intensity. L-DOPA and dopamine behave similarly, suggesting a mechanism for the development of real-time sensing schemes.

Structural requirements for efficient photoinduced electron transfer (PET) quenching in 9-aminoalkylanthracenes.

The structure-quenching relationship in 9-aminoalkylanthracenes is examined by the synthesis and fluorescence evaluation of five derivatives; it is observed that benzylic 2° and 3° amines lead to ≥95% quenching of anthracene fluorescence in water, while other amines afford substantially lower quenching efficiencies.

Fluorimetric metal ion sensing usingN-methyl-9-anthrylhydroxamic acid.

We have synthesizedN-methyl-9-anthrylhydroxamic acid, which is a fluorescent analogue ofN-methylbenzohydroxamic acid. Complexation with various di- and trivalent metal ions occurs (logK from 4 to 5) in water with resulting fluorescence quenching. Because the Fe(III) and Al(III) complexes substituted rather slowly, the addition of EDTA provides a temporal method for obtaining some selectivity in the chemosensor.