Biology subjects

Yazdani, K.

Publications and source records attributed to Yazdani, K..

4 recordsLinked to original sources

bioRxiv2023

Competitive Microarray Screening Reveals Functional Ligands for the DHX15 RNA G-quadruplex

RNAs are increasingly considered valuable therapeutic targets, and in turn the development of methods to identify and validate both RNA targets and RNA-binding compounds is more important than ever. In this study, we utilized a bioinformatic approach to identify a hairpin-containing RNA G-quadruplex (rG4) in the 5' UTR of DHX15 mRNA. By using a competitive small molecule microarray (SMM) approach, we identified a compound that specifically binds to the DHX15 rG4 with a KD of 12.6 {+/-} 1 {micro}M. This rG4 directly impacts translation of a DHX15 reporter mRNA in vitro, and binding of our compound (F1) to the structure inhibits translation up to 57% with an IC50 of 22.9 {+/-} 3.8 {micro}M. The DHX15 protein is an "undruggable" helicase associated with several types of cancer progression, and our data represent the first published effort to target the rG4 in DHX15 mRNA to inhibit its translation. Overall, our work is informative for the development of novel small molecule cancer therapeutics for RNA targets starting from target identification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/550542v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@19eb40borg.highwire.dtl.DTLVardef@3e6a17org.highwire.dtl.DTLVardef@1a71f9eorg.highwire.dtl.DTLVardef@10ce4df_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

DECODING COMPLEXITY IN BIOMOLECULAR RECOGNITION OF DNA I-MOTIFS

DNA i-motifs (iMs) are non-canonical C-rich secondary structures implicated in numerous cellular processes. Though iMs exist throughout the genome, our understanding of iM recognition by proteins or small molecules is limited to a few examples. We designed a DNA microarray containing 10,976 genomic iM sequences to examine the binding profiles of four iM-binding proteins, mitoxantrone, and the iMab antibody. iMab microarray screens demonstrated that pH 6.5, 5% BSA buffer was optimal, and fluorescence was correlated with iM C-tract length. hnRNP K broadly recognizes diverse iM sequences, favoring 3-5 cytosine repeats flanked by thymine-rich loops of 1-3 nucleotides. Array binding mirrored public ChIP-Seq datasets, in which 35% of well-bound array iMs are enriched in hnRNP K peaks. In contrast, other reported iM-binding proteins had weaker binding or preferred G-quadruplex (G4) sequences instead. Mitoxantrone broadly binds both shorter iMs and G4s, consistent with an intercalation mechanism. These results suggest that hnRNP K may play a role in iM-mediated regulation of gene expression in vivo, whereas hnRNP A1 and ASF/SF2 are possibly more selective in their binding preferences. This powerful approach represents the most comprehensive investigation of how biomolecules selectively recognize genomic iMs to date.

biochemistry↗

bioRxiv2022

Machine Learning Informs RNA-Binding Chemical Space

Small molecule targeting of RNA has emerged as a new frontier in medicinal chemistry, but compared to the protein targeting literature our understanding of chemical matter that binds to RNA is limited. In this study, we report Repository Of BInders to Nucleic acids (ROBIN), a new library of nucleic acid binders identified by small molecule microarray (SMM) screening. The complete results of 36 individual nucleic acid SMM screens against a library of 24,572 small molecules are reported (including a total of 1,627,072 interactions assayed). A set of 2,003 RNA-binding small molecules is identified, representing the largest fully public, experimentally derived library of its kind to date. Machine learning is used to develop highly predictive and interpretable models to characterize RNA-binding molecules. This work demonstrates that machine learning algorithms applied to experimentally derived sets of RNA binders are a powerful method to inform RNA-targeted chemical space.

biochemistry↗

bioRxiv2022

Investigating the NRAS 5' UTR as a Target for Small Molecules

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. Constitutively activated NRAS induces the MAPK and AKT signaling pathways and leads to uncontrolled proliferation and cell growth, making it an attractive target for small molecule inhibition. Like all RAS-family proteins, it has proven difficult to identify small molecules that directly inhibit the protein. An alternative approach would involve targeting the NRAS mRNA. The 5' untranslated region (5' UTR) of the NRAS mRNA is reported to contain a G-quadruplex (G4) that regulates translation of NRAS mRNA. Stabilizing the G4 structure with small molecules could reduce NRAS protein expression in cancer cells by impacting translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5' UTR of the NRAS mRNA. We used a small molecule microarray (SMM) screen to identify molecules that selectively bind to the NRAS-G4. Biophysical studies demonstrated that compound 18 binds reversibly to the NRAS-G4 structure with submicromolar affinity. A Luciferase based reporter assay indicated that 18 inhibits the translation of NRAS via stabilizing the NRAS-G4 in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends (RACE), RT-PCR analysis on 14 different NRAS-expressing cell lines, coupled with analysis of publicly available CAGE seq experiments, revealed that predominant NRAS transcript does not possess the G4 structure. Further analysis of published rG4 and G4 sequencing data indicated the presence of G4 structure in the promoter region of NRAS gene (DNA) but not in the mRNA. Thus, although many NRAS transcripts lack a G4 in many cell lines the broader concept of targeting folded regions within 5 UTRs to control translation remains a highly attractive strategy and this work represents an intriguing example of transcript heterogeneity impacting targetability.

biochemistry↗