Targeting non-coding viral RNA

Keywords: RNA, non-coding RNA, viral RNA, antivirals, targeting non-coding RNA with small molecule ligands, HIV, human immunodeficiency virus, core packaging signal, Zika virus, exonuclease resistant RNA

The following in silico studies are demonstrating that  the Q-MOL binding sites prediction and ligand docking methodologies, initially developed for protein targets, can be successfully applied "As Is" to polynucleotide-based structures, such as non-coding viral RNA molecules. For details see Q-MOL publication.

Briefly, Q-MOL protein-ligand docking methodology addresses the protein flexibility treatment via computational implementation of certain postulates of energy landscape theory (ELT) of protein folding. To validate the universal nature of ELT implementation and  reparameterized ligand docking OPLS force field, the protein-optimized computational protocols were applied "AS IS" to non-protein polymeric molecules such as ribonucleic acids (RNA).  To this end, two non-coding viral RNA molecules of known structure were investigated: human immunodeficiency virus (HIV) - 1 core packaging signal (PDB 2N1Q, NMR)¹ and exonuclease resistant RNA from Zika virus (PDB 5TPY, X-ray structure)².  The HIV-1 core packaging signal is a cis-acting RNA element located at the 5' leader end of the viral genome.  It directs the packaging of unspliced viral RNA into assembling virus particles by interacting with the Gag protein³. The HIV-1 core packaging signal is highly conserved and represents an amenable target for potential therapeutic intervention aiming to block virus assembly⁴. Zika virus (ZIKV) produces exonuclease-resistant RNAs (xrRNAs) in its 3' untranslated region (UTR) that act as mechanical roadblocks against host cell 5'-3' exoribonucleases (specifically Xrn1)². These structures, specifically xrRNA1 and xrRNA2, utilize a complex, highly stable fold with a molecular "ring" to halt degradation, producing subgenomic flavivirus RNAs (sfRNAs)⁵. sfRNAs are essential for viral pathogenicity and evasion of the host immune system.  The resistance of these sfRNAs to host cell exoribonucleases results in their accumulation.  It has been shown that disrupting these specific RNA structures via mutations can abolish their accumulation, significantly impacting the virus's ability to infect.  Thus, a therapeutic intervention aimed to interfere with flavivirus sfRNAs stability might prove to be a highly effective and precise strategy for treating this viral infection.

The standard Q-MOL drug discovery protocols were applied to both RNA structures.  The probable allosteric binding sites were predicted by applying molecular surface scanning protocol using structures of individual amino acids as docking probes. In both cases, the simulation produced statistically strong signal identifying specific areas on the surface of the molecules (Figure 1 and Figure 2).

Figure 1. Predicted small molecules binding site and primary VLS results against HIV-1 Core Packaging Signal RNA.  A. NMR structure of HIV-1 Core Packaging Signal RNA (PDB 2N1Q). The putative binding site was predicted by Q-MOL molecular surface scanning using individual amino acid structures as docking probes (see Methods). Probabilities of binding across the molecular surface are shown as spheres (red/large – high probability, blue/small – low probability). The nucleotides, forming predicted allosteric site (cluster of high probabilities, indicated with a red arrow), are highlighted yellow.  The following nucleotide stretches define the predicted binding site: U(230)CUCGAC(236) … G(282)GCGAC(287).  The residue numbering corresponds to that of PDB 2N1Q.  B.  Primary Q-MOL VLS results.  The complete NCI DTP SDF library was used as a source of ligands (≈ 275,000 structures). The primary Q-MOL VLS has converged to 162 individual hits. The ligand docking curve is shown.  The docking site was defined around the highlighted yellow residues (indicated with a red arrow).  E, relative binding energy, kcal/mol; Rank, ligand ranking based on the sorting by relative binding energy.

For the HIV-1 core packaging signal (Figure 1), the allosteric binding site is predicted to be formed by the following nucleotide stretches: U(230)CUCGAC(236) … G(282)GCGAC(287).  The residue num­be­ring corresponds to that of PDB 2N1Q.

Figure 2. Predicted small molecules binding site and primary Q-MOL VLS results against an exonuclease resistant RNA from Zika virus.  A. X-ray structure of exonuclease resistant RNA from Zika virus (PDB 5TPY). The putative binding site was predicted by Q-MOL molecular surface scanning using individual amino acid structures as docking probes (see Methods). Probabilities of binding across the molecular surface are shown as spheres (red/large – high probability, blue/small – low probability). The nucleotides, forming predicted allosteric site (cluster of high probabilities, indicated with red arrow), are highlighted yellow.  The following nucleotide stretches define the predicted binding site: U(29)UGGGGAAA(37) … A(52)ACCCC(57).  The residue numbering corresponds to that of PDB 5TPY.  B.  Primary Q-MOL VLS results.  The complete NCI DTP SDF library was used as a source of ligands (≈ 275,000 structures). The primary Q-MOL VLS has converged to 169 individual hits.  The ligand docking curve is shown.  The docking site was defined around the highlighted yellow residues (indicated with a red arrow).  E, relative binding energy, kcal/mol; Rank, ligand ranking based on the sorting by relative binding energy.

In the case of Zika virus exonuclease resistant RNA (Figure 2), the allosteric binding site is predicted to be formed by the following nucleotide stretches: U(29)UGGGGAAA(37) … A(52)ACCCC(57).  The residue numbering corresponds to that of PDB 5TPY.  The primary Q-MOL VLS was performed using the complete NCI DTP SDF library (≈ 275,000 compounds) (Figure 1B, Figure 2B).  The docking sites for RNA-ligand docking simulations were defined around the predicted allosteric binding sites.  The predicted hit structures (162 hits, HIV-1 core packaging signal; 169 hits, Zika virus exonuclease resistant RNA) were bucket-clustered by similarity, and the representative ligands of the most populated cluster buckets were annotated when possible (Table 1, Table 2).

These RNA-ligand docking experiments are obviously lacking in vitro validation, however the limited annotated results demonstrate an excellent correlation between annotated small molecule hits and the viral origins of RNA structures. Moreover, the RNA-ligand docking identified hits that were only relatively recently characterizes as having potent anti-HIV and anti-Zika antiviral properties.  For instance, NSC614929 (Table 1) is a derivative of compounds containing 1,2,4-triazole rings.  The compounds, containing 1,2,4-triazole rings, have shown potential as HIV-1 integrase strand transfer inhibitors and non-nucleoside reverse transcriptase inhibitors^{6, 7}.  The mechanism of action of these compounds might need to be re-visited. 

In case of Zika virus (Table 2), the analogues of compound NSC728370 have been shown to possess effective inhibition against Zika virus^{8, 9}.  Of interest, the cell-penetrating peptide (CPP) (NSC742350, Table 2), derived from HIV-1 TAT protein, has been identified as another binder of Zika exonuclease resistant RNA.  This finding might be of importance for the understanding of Zika virus interactions with host immune systems as recent research indicates that HIV and Zika viruses can interact, particularly in the context of co-infection¹⁰.

Table 1. Selected annotated ligands from NCI DTP library identified as hits by Q-MOL primary VLS against HIV-1 Core Packaging Signal RNA.

NCI DTP NSC	Structure	Comments
NSC607319		Name: Phylloflavan It has shown potential in laboratory studies for selectively inhibiting HIV infection. Phylloflavan and other related compounds have been investigated in extracts from plants like Smilax corbularia, which is used in Thai folk medicine for various ailments, including AIDS      treatment 11, 12.
NSC172865		Name: 1-[2,2-bis(methylsulfonyl)ethenyl]-2-chlo­ro­­benzene It is primarily referenced in patent literature and pharmaceutical research as an intermediate or building block in the synthesis of HIV-1 capsid inhibitors 13, 14.
NSC614929		Name: glycine-N,N-bis(1H-1,2,4-triazol-1-ylme­thyl)­ethyl ester It has been synthesized as a derivative of a class of compounds with demonstrated anti-HIV activity. Related molecules with 1,2,4-triazole rings have shown potential as HIV-1 integrase strand transfer inhibitors and non-nucleoside reverse transcriptase inhibitors (NNRTIs) 6, 7.
NSC724853		Name: 6,8-Dichloro-7-methyl-7H-[1,2,3,4,5]­penta­thiepino[6,7-c] pyrrole It is related to a class of compounds investigated as potential anti-HIV agents 15, 16.
NSC726550		Name: bis {2 - (2'-imino-4-oxo thiazol-5-yl)­thia­dia­zolo[2,3-c]triazol-5-yl}disulfide Thiazolidinone derivatives are recognized as a class of potent non-nucleoside reverse tran­scriptase inhibitors (NNRTIs) 17.
NSC664909		Name: 1,4-bis-[N-Tricysteinylethylenediamino]-5,8­-di­hydroxy anthraquinone Various anthraquinones substituted with hydro­xyl, amino, or sulfonate groups have demon­strated in vitro activity against HIV-1. Some active anthraquinones are known to inhibit the HIV-1 reverse transcriptase enzyme, which is crucial for the virus's replication process 18. Hypericin is a naturally occurring anthraquinone dimer has been shown to exhibit anti-HIV-1 activity in human lymphocytes 19.
NSC15257	Related compound:	Related compound name: sodium diethyl­dithiocar­bamate (DDTC).  DDTC was used in clinical trials for HIV-1 and showed a significant delay in progression to AIDS 20.

Table 2. Selected annotated ligands from NCI DTP library identified as hits in Q-MOL primary VLS against exonuclease resistant RNA from Zika virus.

NCI DTP NSC	Structure	Comments
NSC725729		Name: bis-triazole Numerous triazole derivatives have been in­ves­ti­gated for their ability to inhibit the Zika virus 21, 22.
NSC742350	Peptide: RKKRRQRRRGG	Name: Cell-penetrating peptide (CPP) or protein transduction domain (PTD) The peptide is derived from the HIV-1 TAT protein, often extended with a glycine spacer (GG). It facilitates the rapid, efficient, and direct uptake of cargo (proteins, peptides, or DNA) into cells, bypassing typical endocytic pathways, often used for delivery of therapeutic agents and in molecular imaging. Recent research indicates that HIV and Zika viruses can interact, particularly in the context of co-infection 10.
NSC728370		Name: 2,4-Di-( 4,6-dimethylythio pyrazolo [3,4-d] pyrimidine-1-yl)-6-methyle pyrimidine Studies have shown that 1H-pyrazolo[3, 4-d]pyrimidine derivatives, as part of a series of 4,7-disubstituted 7H-pyrrolo[2, 3-d]pyrimidine analogs, possess effective inhibition against Zika virus and dengue virus. These compounds have been demonstrated to act as entry inhibitors, blocking Zika virus infection during the viral entry and fusion steps of the virus life cycle 8, 9.
NSC730504		Name: 7-[4-[[5-Chloro-2-oxo-3-(4-pyrimidin-2-ylsulfonylphenyl)iminoindol-1-yl]methyl]piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxoquinoline-3-carboxylic acid This compound and related derivatives were identified as potent inhibitors of Zika virus.  They act as Zika entry inhibitors, hindering the virus's ability to enter host cells.  Beyond Zika, these compounds show inhibitory effects against influenza A and coronaviruses 23, 24.
NSC723641		Name: 2-Ethylthio-5-(p-chloro­phenoxy)­methy­lene triazolo[3,4-b]thiadiazole Triazolo[3,4-b]thiadiazole scaffolds are being explored as potential inhibitors of the Zika virus 21.
NSC710505		Name: 4-amino-1-{[(1-(2-hydroxy­ethoxy)­methyl­)­-­1,2,3-trizol-5-yl]methyl}-1H-pyrazolo­[3,4-d]pyrimidine Derivatives of this scaffold have been investi­gated as potential inhibitors of the Zika virus 9.

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