Chemical Ligation of Oligonucleotide Tags to Support Encoded Chemical Library Synthesis.

Chemical ligation can be used to install encoding tags during the synthesis of DNA-encoded chemical libraries and can present a number of advantages. Here we describe methods to generate polymerase-readable oligonucleotide junctions and for the polymerase-mediated amplification of oligonucleotides ligated with these chemistries, including triazole junctions generated from 2'-ribo-3'-propargyl and 5'-azido oligonucleotides and from 2'-deoxy-3'-propargyl and 5'-azido oligonucleotides. We also present methods for the synthesis of phosphorothioate junctions from 3'-thiophospho and 5'-iodo oligonucleotides and for the synthesis of phosphodiester junctions from both 3'-hydroxy and 5'-phospho- and 3'-phospho and 5'-hydroxy oligonucleotides using 1-cyanoimidazole.

Novel Nucleic Acid Binding Small Molecules Discovered Using DNA-Encoded Chemistry.

Inspired by the many reported successful applications of DNA-encoded chemical libraries in drug discovery projects with protein targets, we decided to apply this platform to nucleic acid targets. We used a 120-billion-compound set of 33 distinct DNA-encoded chemical libraries and affinity-mediated selection to discover binders to a panel of DNA targets. Here, we report the successful discovery of small molecules that specifically interacted with DNA G-quartets, which are stable structural motifs found in G-rich regions of genomic DNA, including in the promoter regions of oncogenes. For this study, we chose the G-quartet sequence found in the c-myc promoter as a primary target. Compounds enriched using affinity-mediated selection against this target demonstrated high-affinity binding and high specificity over DNA sequences not containing G-quartet motifs. These compounds demonstrated a moderate ability to discriminate between different G-quartet motifs and also demonstrated activity in a cell-based assay, suggesting direct target engagement in the cell. DNA-encoded chemical libraries and affinity-mediated selection are uniquely suited to discover binders to targets that have no inherent activity outside of a cellular context, and they may also be of utility in other nucleic acid structural motifs.

Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library.

A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.

Chemical ligation methods for the tagging of DNA-encoded chemical libraries.

The generation of DNA-encoded chemical libraries requires the unimolecular association of multiple encoding oligonucleotides with encoded chemical entities during combinatorial synthesis processes. This has traditionally been achieved using enzymatic ligation. We discuss a range of chemical ligation methods that provide alternatives to enzymatic ligation. These chemical ligation methods include the generation of modified internucleotide linkages that support polymerase translocation and other modified linkages that while not supporting the translocation of polymerases can also be used to generate individual cDNA molecules containing encoded chemical information specifying individual library members. We also describe which of these approaches have been successfully utilized for the preparation of DNA-encoded chemical libraries and those that were subsequently used for the discovery of inhibitors.

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags.

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method "relay primer-dependent bypass" utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3'-terminus prior to ligation to adjacent oligonucleotides. The second reading method "repeat-dependent bypass" utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3'-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.

Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display.

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >10(13) different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.

A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene.

Ribozymes are thought to have played a pivotal role in the early evolution of life, but relatively few have been identified in modern organisms. We performed an in vitro selection aimed at isolating self-cleaving RNAs from the human genome. The selection yielded several ribozymes, one of which is a conserved mammalian sequence that resides in an intron of the CPEB3 gene, which belongs to a family of genes regulating messenger RNA polyadenylation. The CPEB3 ribozyme is structurally and biochemically related to the human hepatitis delta virus (HDV) ribozymes. The occurrence of this ribozyme exclusively in mammals suggests that it may have evolved as recently as 200 million years ago. We postulate that HDV arose from the human transcriptome.

Structure-activity relationships of aminoglycoside-arginine conjugates that bind HIV-1 RNAs as determined by fluorescence and NMR spectroscopy.

We present here a new set of aminoglycoside-arginine conjugates (AACs) that are either site-specific or per-arginine conjugates of paromomycin, neamine, and neomycin B as well as their structure-activity relationships. Their binding constants (KD) for TAR and RRE RNAs, measured by fluorescence anisotropy, revealed dependence on the number and location of arginines in the different aminoglycoside conjugates. The binding affinity of the per-arginine aminoglycosides to TAR is higher than to RRE, and hexa-arginine neomycin B is the most potent binder (KD=5 and 23 nM, respectively). The 2D TOCSY NMR spectrum of the TAR monoarginine-neomycin complex reveals binding at the bulge region of TAR.

Stereospecificity of short Rev-derived peptide interactions with RRE IIB RNA.

The essential HIV-1 regulatory protein Rev binds to the Rev responsive element (RRE) of the HIV-1 mRNA. A short alpha-helical peptide derived from Rev (Rev 34-50) and a truncated form of the RRE sequence (RRE IIB) provide a useful in vitro system to study the interactions between Rev and RRE. The current studies focus on evaluating the specificity of the binding interactions between Rev 34-50 and RRE IIB. The binding of L- and D-Rev peptides to natural and enantiomeric RRE IIB RNA was studied by fluorescence spectroscopy. D-Rev and L-Rev peptides bind to RRE IIB with similar affinities. CD measurements are consistent with a nonhelical, probably beta-hairpin, conformation for D-Rev in the complex. The binding affinities of D/L Rev peptides to L-RRE IIB RNA are also similar to those with natural D-RRE IIB. Furthermore, the conformations of L- and D-peptides when bound to L-RRE are reciprocal to the conformations of these peptides in complex with D-RRE. RNA footprinting studies show that L- and D-Rev peptides bind to the same site on RRE IIB. Our results demonstrate lack of stereospecificity in RRE RNA-Rev peptide interactions. However, it is quite possible that the interactions between full-length Rev protein and RRE are highly specific.

The Tat antagonist neomycin B hexa-arginine conjugate inhibits gp-120-induced death of human neuroblastoma cells.

Several patients with acquired immunodeficiency syndrome (AIDS) develop neurological complications, which are referred to as human immunodeficiency virus (HIV)-associated dementia (HAD). The HIV-1 coat glycoprotein gp-120 has been proposed as the major etiologic agent for neuronal loss reported postmortem in the brain of AIDS patients. Chemokine receptors may play a role in gp-120-triggered neurotoxicity, both in vitro and in vivo, thus being an intriguing target for developing therapeutic strategies aimed to prevent or reduce neuronal damage occurring during HIV infection. We have previously shown that human CHP100 neuroblastoma cells express CXCR4 and CCR5 chemokine receptors and that interaction between gp-120 and these receptors contributes to cytotoxicity elicited by the protein. Here, we examined the neuroprotective potential of neomycin B hexa-arginine conjugate (NeoR), a recently synthesized compound with anti-HIV activity. We found that gp-120-triggered death is significantly reduced by NeoR, and this protective effect seems related to the ability of NeoR to interact with CXCR4 receptors. The ability of NeoR to cross the blood-brain barrier, as demonstrated in mice by systemic administration of the fluorescein conjugate drug, makes this compound a powerful and attractive therapeutic agent.

Stereospecificity of aminoglycoside-ribosomal interactions.

Aminoglycoside antibiotics bind to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination. Aminoglycoside binding to A-site RNA decoding region constructs is established here to be only weakly stereospecific. Mirror-image prokaryotic A-site decoding region constructs were prepared in the natural D-series and the enantiomeric L-series and tested for binding to a series of aminoglycosides. In general, aminoglycosides bind to the D-series decoding region constructs with 2-3-fold higher affinities than they bind to the enantiomeric L-series. Moreover, L-neamine, the enantiomer of naturally occurring D-neamine, was prepared and shown to bind approximately 2-fold more weakly than D-neamine to the natural series decoding region construct, a result consistent with weakly stereospecific binding. The binding of naturally occurring D-neamine and its synthetic L-enantiomer was further evaluated with respect to binding to prokaryotic and eukaryotic ribosomes. Here, weak stereospecifcity was again observed with L-neamine being the more potent binder by a factor of approximately 2. However, on a functional level, unnatural L-neamine proved to inhibit in vitro translation with significantly lower potency (approximately 5-fold) than D-neamine. In addition, both L- and D-neamine are bacteriocidal toward Gram-(-) bacteria. L-Neamine inhibits the growth of E. coli and P. aeruginosa with 8- and 3-fold higher MIC than D-neamine. Interestingly, L-neamine also inhibits the growth of aminoglycoside-resistant E. coli, which expresses a kinase able to phosphorylate and detoxify aminoglycosides of the D-series. These observations suggest that mirror-image aminoglycosides may avoid certain forms of enzyme-mediated resistance.

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates.

The potential of RNAs and RNA-protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA (trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.

Anti-HIV activity of a novel aminoglycoside-arginine conjugate.

We have previously described conjugates of L-arginine with aminoglycosides (AAC) that have shown anti-human immunodeficiency virus type 1 (HIV-1) activity in in vitro cell culture systems. Here, we extend our report to a novel neomycin B-arginine conjugate (NeoR) that has shown up to 30-fold increased potency over previous AAC compounds. NeoR inhibited the replication of both R5 and X4 strains of HIV-1 in cells expressing the appropriate coreceptor or peripheral blood mononuclear cells. In lymphoid tissue ex vivo, NeoR blocked the replication of the dualtropic strain 89.6 suggesting anti-HIV activity of AAC on the site of in vivo virus replication. NeoR blocked the binding of HIV particles to lymphoid cells and was also able to antagonize the activity of the CXCR4 receptor so it may prevent the emergence of X4 HIV-1 strains. Nevertheless, in a cellular assay, we were unable to detect anti-Tat dependent transactivation activity as previously suggested for this family of compounds.