Tetrodotoxin (TTX) is a lethal neurotoxin produced by the endosymbiotic bacteria in the gut of puffer fish. Currently, there is no effective and economical method to detect TTX, so it is very interesting to develop low-cost and high-sensitivity detection methods by using nucleic-acid aptamers as the recognition molecules. However, traditional SELEX screening of aptamers for targeting small molecules such as TTX is labor-intensive, and usually the success rate is low. Here, we employed a strategy of “repurposing old aptamers for new uses” to develop high-affinity aptamers for TTX. To this end, we first collected thermally stable DNA aptamers and predicted their affinities for TTX by molecular docking; then, we identified high-affinity candidates and verified them by microscale thermophoresis (MST) experiments. In this way, two thermally stable aptamers (Tv-51 and AI-57) were found to possess nanomolar affinities for TTX. Moreover, we performed spontaneous binding simulations to reveal their binding mechanisms to TTX and thereby identified the key bases for the binding. Guided by these, two variants (Tv-46 and AI-52) with higher affinities and specificities were subsequently engineered and confirmed by the MST experiments. So, this study not only provides potential recognition molecules for the technology developments of TTX detection, but also demonstrates an effective repurposing approach to the discovery of high-affinity aptamers for new target molecules. 

https://doi.org/10.1016/j.csbj.2022.04.033

Repurposing of thermally stable nucleic-acid aptamers for targeting tetrodotoxin (TTX)

De novo design of DNA aptamers that target okadaic acid (OA) by docking-then-assembling of single nucleotides.

Effective treatment or vaccine is not yet available for combating SARS coronavirus 2 (SARS-CoV-2) that caused the COVID-19 pandemic. Recent studies showed that two drugs, Camostat and Nafamostat, might be repurposed to treat COVID-19 by inhibiting human TMPRSS2 required for proteolytic activation of viral spike (S) glycoprotein. However, their molecular mechanisms of pharmacological action remain unclear. Here, we perform molecular dynamics simulations to investigate their native binding sites on TMPRSS2. We revealed that both drugs could spontaneously and stably bind to the TMPRSS2 catalytic center, and thereby inhibit its proteolytic processing of the S protein. Also, we found that Nafamostat is more specific than Camostat for binding to the catalytic center, consistent with reported observation that Nafamostat blocks the SARS-CoV-2 infection at a lower concentration. Thus, this study provides mechanistic insights into the Camostat and Nafamostat inhibition of the SARS-CoV-2 infection, and offers useful information for COVID-19 drug development.

Spontaneous binding of potential COVID-19 drugs (Camostat and Nafamostat) to human serine protease TMPRSS2.

A major obstacle of the selective inhibitor design for specific human phosphodiesterase (PDE) is that highly conserved catalytic pockets are difficult to be distinguished by inhibitor molecules. To overcome this, a feasible path is to understand the molecular determinants underlying the selectivity of current inhibitors. BAY60-7550 (BAY for short; IC50 = 4.7 nM) is a highly selective inhibitor targeting PDE2A which is a dual-specificity PDE and an attractive target for therapeutic intervention of the central nervous system (CNS) disorders. Recent studies suggest that molecular determinants may be in binding processes of BAY. However, a detailed understanding of these processes are still lacking. To explore these processes, High-Throughput Molecular Dynamics (HTMD) simulations were performed to reproduce the spontaneous association of BAY with catalytic pockets of 4 PDE isoforms; Ligand Gaussian Accelerated Molecular Dynamics (LiGaMD) simulations were performed to reproduce the unbinding-rebinding process of FKG and MC2, two pyrazolopyrimidinone PDE2A selective inhibitors, in the PDE2A system. The produced molecular trajectories were analyzed by the Markov state model (MSM) and the molecular mechanics/generalized Born surface area (MM/GBSA). The results showed that the non-covalent interactions between the non-conserved residues and BAY, especially the hydrogen bonds, determined the unique binding pathways of BAY on the surface of PDE2A. These pathways were different from those of BAY on the surface of the other three PDE isoforms and the binding pathways of the other two PDE2A inhibitors in PDE2A systems. These differences were ultimately reflected in the high selectivity of this inhibitor for PDE2A. As a result, this study demonstrates the critical role of the binding processes in the selectivity of BAY, and also identifies the key non-conserved residues affecting the binding processes of BAY. Thus, this study provides a new perspective and data support for the further development of BAY-derived inhibitors targeting PDE2A 

https://doi.org/10.1016/j.csbj.2022.09.013

Long-Timescale Simulations Revealed Critical Non-Conserved Residues of Phosphodiesterases Affecting Selectivity of BAY60-7550

The invention belongs to the technical field of nucleotide tertiary structure prediction, and particularly relates to a G-quadruplex nucleic acid aptamer tertiary structure prediction method of targeted biotoxin. The method comprises the following steps: predicting a G-quadruplex secondary structure; predicting a G-quadruplex tertiary topological configuration; carrying out nucleic acid aptamer tail end complementation and structure optimization; carrying out temperature-dependent molecular dynamics simulation (comprising two processes: constant-temperature molecular dynamics simulation and heating molecular dynamics simulation); and obtaining a most stable nucleic acid tertiary structure model by adopting an RMSD analysis method. The method disclosed by the invention is already used for predicting a tertiary structure of a specific nucleic acid aptamer sequence GO18-T-d of the targeted gonyautoxin; the sequence is a known G-quadruplex aptamer sequence obtained by screening through anexponential enrichment ligand evolution technology, the G-quadruplex configuration is detected to be a parallel G-quadruplex configuration through a circular dichroism experiment, a prediction resultis completely matched with a detection result of the circular dichroism experiment, and it is proved that the prediction method has good accuracy.

G-quadruplex nucleic acid aptamer tertiary structure prediction method of targeted biotoxin

As human phosphodiesterase (PDE) proteins are attractive drug targets, a large number of selective PDE inhibitors have been developed. However, since the catalytic sites of PDE isoforms are conserved in sequence and structure, it remains unclear how these inhibitors discriminate PDE isoforms in a selective manner. Here we perform long-time scale molecular dynamics (MD) simulations to investigate the spontaneous association processes of a highly selective PDE2A inhibitor (BAY60-7550) with the catalytic pockets of six PDE isoforms. We found that the free-energy landscapes of PDE:BAY60-7550 interactions on the PDE surfaces are very different between various PDE isoforms; and the free-energy landscape of PDE2A forms a favorable low-energy pathway that not only drives BAY60-7550 toward the target binding site, but also guides BAY60-7750 to adopt its native binding conformation known from crystal structure. Thus, this study reveals that the inhibitor interactions with the PDE surface residues play an important role in its high selectivity for PDE2A, and thereby provides new fundamental insights into the PDE isoform-specific inhibitor selectivity.

Surface Binding Energy Landscapes Affect Phosphodiesterase Isoform-Specific Inhibitor Selectivity.

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Surface Binding Energy Landscapes Affect Phosphodiesterase Isoform-Specific Inhibitor Selectivity.