Computational Modeling of Virally-encoded Ion Channel Structure.

Viroporins are ion channels encoded within a virus's genome, that facilitate a range of devastating infectious diseases such as COVID-19, HIV, and rotavirus. The non-structural protein 4 (NSP4) from rotavirus includes a viroporin domain that disrupts cellular Ca2+ homeostasis, initiating viral replication, and leading to life-threatening vomiting and diarrhea. Though the structure of soluble segments of NSP4 has been determined, membrane-associated regions, including the viroporin domain, remain elusive when utilizing well-established available experimental methods such as x-ray crystallography. However, two recently published protein folding algorithms, AlphaFold2 and trRosetta, demonstrated a high degree of accuracy, when determining the structure of membrane proteins from their primary amino acid sequences, though their training datasets are known to exclude proteins from viral systems. We tested the ability of these non-viral algorithms to predict functional molecular structures of the full-length NSP4 from SA11 rotavirus. We also compared the accuracy of these structures to predictions of other experimental structures of eukaryotic proteins from the Protein Data Banks (PDB), and show that the algorithms predict models more similar to corresponding experimental data than what we saw for the viroporin structure. Our data suggest that while AlphaFold2 and trRosetta each produced distinct NSP4 models, constructs based on either model showed viroporin activity when expressed in E. coli, consistent with that seen from other historical NSP4 sequences.

Cytosine-Based TET Enzyme Inhibitors.

DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here, we describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based lead compound, Bobcat339, that has mid-µM inhibitor activity against TET1 and TET2, but does not inhibit the DNA methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and serve as a starting point for new therapeutics that target DNA methylation and gene transcription.