RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Toll-like receptor 3 activation selectively reverses HIV latency in microglial cells.

BACKGROUND: Multiple toll-like receptors (TLRs) are expressed in cells of the monocytic lineage, including microglia, which constitute the major reservoir for human immunodeficiency virus (HIV) infection in the brain. We hypothesized that TLR receptor mediated responses to inflammatory conditions by microglial cells in the central nervous system (CNS) are able to induce latent HIV proviruses, and contribute to the etiology of HIV-associated neurocognitive disorders. RESULTS: Newly developed human microglial cell lines (hµglia), obtained by immortalizing human primary microglia with simian virus-40 (SV40) large T antigen and the human telomerase reverse transcriptase, were used to generate latently infected cells using a single-round HIV virus carrying a green fluorescence protein reporter (hµglia/HIV, clones HC01 and HC69). Treatment of these cells with a panel of TLR ligands showed surprisingly that two potent TLR3 agonists, poly (I:C) and bacterial ribosomal RNA potently reactivated HIV in hµglia/HIV cells. LPS (TLR4 agonist), flagellin (TLR5 agonist), and FSL-1 (TLR6 agonist) reactivated HIV to a lesser extent, while Pam3CSK4 (TLR2/1 agonist) and HKLM (TLR2 agonist) only weakly reversed HIV latency in these cells. While agonists for TLR2/1, 4, 5 and 6 reactivated HIV through transient NF-κB induction, poly (I:C), the TLR3 agonist, did not activate NF-κB, and instead induced the virus by a previously unreported mechanism mediated by IRF3. The selective induction of IRF3 by poly (I:C) was confirmed by chromatin immunoprecipitation (ChIP) analysis. In comparison, in latently infected rat-derived microglial cells (hT-CHME-5/HIV, clone HC14), poly (I:C), LPS and flagellin were only partially active. The TLR response profile in human microglial cells is also distinct from that shown by latently infected monocyte cell lines (THP-1/HIV, clone HA3, U937/HIV, clone HUC5, and SC/HIV, clone HSCC4), where TLR2/1, 4, 5, 6 or 8, but not for TLR3, 7 or 9, reactivated HIV. CONCLUSIONS: TLR signaling, in particular TLR3 activation, can efficiently reactivate HIV transcription in infected microglia, but not in monocytes or T cells. The unique response profile of microglial cells to TLR3 is fundamental to understanding how the virus responds to continuous microbial exposure, especially during inflammatory episodes, that characterizes HIV infection in the CNS.

Circulating nucleic acids in type 1 diabetes may modulate the thymocyte turnover rate.

The autoimmunity of type 1 diabetes is associated with T-cell hyperactivity. Current study was designed to examine the effect of circulating ribonucleic acids (RNAs), isolated from type 1 diabetic patients on proliferative, apoptotic and inflammatory potential of rat thymocytes. Rat thymocytes were assayed for proliferating nuclear cell antigen (PCNA), Bcl-2, Bax and NF-κB level, using the flow cytometric and fluorometric assays. Cells were allocated into groups, treated with RNAs purified from plasma of juvenile diabetics, adult type 1 diabetic patients, control healthy children, healthy adult persons, nucleic acids and polynucleotide standards (RNA, polyC, PolyA, PolyIC, and CpG). The upregulation of PCNA and Bcl-2 protein and downregulation of Bax protein and NF-κB was shown when the thymocytes where incubated with RNA purified from plasma of juvenile type 1 diabetic patients. The dysregulation of inflammatory cascade and central tolerance may be a defect in autoimmune diseases related to innate immunity leading to corresponding alteration in adaptive immune response.

The use of a MITO-Porter to deliver exogenous therapeutic RNA to a mitochondrial disease's cell with a A1555G mutation in the mitochondrial 12S rRNA gene results in an increase in mitochondrial respiratory activity.

We report on validating a mitochondrial gene therapeutic strategy using fibroblasts derived from patients with an A1555G point mutation in mitochondrial DNA coding 12S ribosomal RNA (rRNA (12S)). Wild-type rRNA (12S) as a therapeutic RNA was encapsulated in a mitochondrial targeting liposome, a MITO-Porter (rRNA-MITO-Porter), and an attempt was made to deliver the MITO-Porter to mitochondria of the diseased cells. It was confirmed that the rRNA-MITO-Porter treatment significantly decreased the ratio of the mutant rRNA content. Moreover, it was shown that the mitochondrial respiratory activities of the diseased cells were improved as the result of the mitochondrial transfection of the rRNA-MITO-Porter.

Ribozymes as potential anti-HIV-1 therapeutic agents.

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.

[Antiherpetic effect of RNA-tilorone molecular complex in cell culture].

The authors studied the antiviral effect of an interferonogenic yeast RNA-tilorone molecular complex (MC) compared to the Virolex, videly used antiherpetic drug, and standard interferon (IFN) alpha/beta inducer poly(I)poly(C) in Vero cells culture infected with herpes simplex type I virus (HSV-1). The tilorone contained by MC has been shown to be twice less toxic and twice more active against HSV than its free molecules. The value of chemotherapeutic index (CI) of Virolex in experiments with Vero cells reaches 2500, CI poly(I)poly(C) and MC being 324; the last value meets the requirements for promising drugs.

The influence of rRNA and tRNA on the translation of avian globin mRNA in cell-free systems of protein synthesis.

Cell-free synthesis of globin chains in the presence of globin mRNA in an Ehrlich ascites-cell-free system is further stimulated by addition of 18- and 28-S rRNA but not of 4-S tRNA and 5-S rRNA. This stimulation can not be observed in the wheat germ cell-free system. When 125I-labelled globin mRNA was incubated in the two systems we have found after 60 min a 75% decrease of trichloroacetic acid precipitable polynucleotides in the ascites but only a 20% decrease in the wheat germ system. The RNAase action on mRNA can be reduced by the addition of 18- and 28-S rRNA but not by 5-S rRNA and 4-S tRNA. We suggest that the stimulating effect of the two rRNA species in the ascites cell-free system is due to a higher activity of a specific RNAase in this system and a competitive protection of mRNA from RNAase action.

Biosynthesis of vitamin B-12. Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis.

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K12 and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E.coli 113-3 bind Ado[58Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as l3 protein isolated from E.coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is preformed by protein L18. 4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.

Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro.

Incubation medium II causes release of ribosomal subunits from isolated prelabeled nuclei of regenerating rat liver in vitro (Sato, T., Ishikawa, K. and Ogato, K. (1976) Biochim. Biophys. Acta 000, 000-000). The effects of individual components of this medium on release of subunits were studied and the following results were obtained. 1. Dialyzed cytosol was effective in causing release of total labeled RNA, but its effect on release of labeled ribosomal subunits was rather lower than that of low molecular yeast RNA. Spermidine inhibited the release of total labeled RNA as well as that of labeled ribosomal subunits. 2. Low molecular yeast RNA was the most effective component for inducing release of labeled ribosomal subunits. Homologous ribosomal RNA was as effective as yeast RNA. Cytoplasmic ribosomes, prepared by washing with solution of high salt concentration, and their subunits were also effective. 3. Transfer RNA was not so effective as yeast RNA and ribosomal RNA and even after heat treatment it had little effect. 4. Among the homopolyribonucleotides tested, polyuridylic acid had a strong effect but polyadenylic acid, polycytidylic acid and polyinosinic acid had no effect. 5. The effects of yeast RNA and polyuridylic acid in causing release of labeled ribosomal subunits were dependent upon their concentrations in the reaction mixture. The characteristics of the factors which cause release of labeled ribosomal subunits in vitro are discussed on the basis of the results.

Interaction of Cibacron blue F3GA and polynucleotides with ricin A-chain, 60 S ribosomal subunit-inactivating protein.

Cibacron blue F3GA, a sulfonated polyaromatic blue dye, inhibited the ability of ricin A-chain to inactivate ribosomes. Difference-spectroscopic study revealed that the dye bound to the A-chain (Kd = 0.72 microM), producing a difference spectrum with a single maximum at 688 nm and two minima at 585 and 628 nm. Such a significant difference spectrum was not observed in the presence of ricin B-chain or intact ricin, neither of which can inactivate ribosomes. Modification of arginine residues in the A-chain with phenylglyoxal showed a correlation between the loss of inhibitory activity on protein synthesis and the loss of difference absorbance produced by the dye-A-chain interaction. Both losses occurred significantly at an early stage of the modification. Furthermore, the dye protected the A-chain against a loss of its inhibitory activity resulting from the modification of arginine residues. These results suggest that the same arginine residues participate both in the interaction with the dye and in the inactivation of ribosomes. Based on these data, the dye appears to interact with the active site of the A-chain. Addition of several polynucleotides, namely rRNA, tRNA, poly(U) and DNA, to the dye-A-chain complex resulted in a marked displacement of the dye, whereas mono- and dinucleotides had little or no effect on the dye-A-chain interaction. These findings indicate the possible existence of a polynucleotide binding site in the active site of the A-chain. A combination of these and other results suggests that the A-chain recognizes and acts on some part of RNA of the 60 S ribosomal subunit.

RNA-protein interactions in the ribonucleoprotein T-complexes in a mitochondrial extract from Leishmania tarentolae.

We have investigated protein-RNA interactions and the incorporation of [alpha-32P]UTP into the guide RNA and mRNA components of the 'T-complexes' in a mitochondrial extract from Leishmania tarentolae. The terminal uridylyl transferase-containing complex T-IV is probably involved in the maturation of the 3'-oligo(U) tail of the gRNAs, but the biological function and biochemical nature of the remaining T-complexes is not known. We have found that the relative extent of labeling of the RNA components is dependent on the UTP concentration: at low levels, the main endogenous RNA components labeled are the gRNAs in T-IV; at higher levels, the mRNAs in all of the T-complexes are preferentially labeled. We also show a tentative correlation in the migration pattern of UTP-labeled T-complexes and complexes which bind exogenous labeled RNA. The relative extent of binding to specific complexes is dependent upon the type of RNA. Most of the interactions between the labeled RNAs and proteins can be disrupted by heparin or a large excess of rRNA, but two labeled complexes were resistant to competition. Most of the binding of labeled exogenous gRNA is disrupted by competition with a large excess of rRNA, but predigestion of the extract with micrococcal nuclease and saturation with rRNA uncovered a high affinity complex, which involves at least two proteins interacting with the bound gRNAs. A knowledge of the RNA and protein components may aid in understanding the biological roles of these RNP complexes.

Inhibition of globin synthesis by rabbit reticulocyte rRNAs.

The effect of concentration of rabbit reticulocyte rRNAs on the translation of rabbit globin mRNAs was studied by using a rabbit reticulocyte lysate system. Globin synthesis was studied using L-[U-14C]leucine. Both the 18S and 28S rRNA inhibited globin synthesis. The 18S rRNA inhibited the synthesis of alpha-globin chain more than that of beta chain, but the 28S rRNA inhibited the synthesis of alpha- and beta-chains almost equally. Nascent chains prelabelled with [14C]Leu or f[35S]Met were released at various concentrations of rRNAs. Release of the nascent chains was not inhibited at various concentrations of rRNAs. The ratios [14C]alpha/[14C]beta and [35S]alpha/[35S]beta in the released chains were almost constant at various concentrations of rRNAs. It therefore appears that the inhibition of globin synthesis by these rRNAs was at the step of initiation.

Effect of polycations and polyanions on cell division of cultured mammalian cells.

The polycations (H1 histone and polylysine) and polyanions (heparin and various RNA preparations) stimulate cell division of cultured mammalian cells. The mechanisms by which both polycations (H1 histone and polylysine) and polyanions (heparin and RNA) may increase the rate of cell division are discussed.

[Inhibitory effect of 18S ribosomal RNA of different origin on a wheat embryo cell-free translation system]. / Ingibiruiushchee deistvie 18S ribosomnykh RNK razlichnogo proiskhozhdeniia na beskletochnuiu sistemu transliatsii iz zarodyshei pshenitsy.

The action of 18S rRNAs from three different sources--mouse reticulocytes, Ehrlich ascite carcinoma cells and wheat embryo--on cell-free translational system derived from wheat embryo was investigated. It is shown that all rRNA preparations have similar inhibitory action on translational activity of the system. This action is not dependent on the source of rRNA and mRNA by which the system is primed. Such a similarity of action strongly suggests a nonspecific mechanism of inhibition which may be based on polyanionic properties of RNA molecules.