On the Mechanism of Action of SJ-172550 in Inhibiting the Interaction of MDM4 and p53

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285∶10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor

Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function

Inhibition of MDMX-p53 peptide binding by compound 1 (IC₅₀ = 3 µM), compound 2 (IC₅₀>100 uM).

<p>Inhibition of MDMX-p53 peptide binding by compound 1 (IC₅₀ = 3 µM), compound 2 (IC₅₀>100 uM).</p

Structures of relevant compounds.

<p><b>Panel A.</b> Structure of SJ-172550 (<b>1</b>) and a non-reactive analog (<b>2</b>). <b>Panel B.</b> The potential mechanism of covalent adduct formation.</p

Formation of covalent adducts between compound 1 and MDMX.

<p><b>Panel a.</b> Mass spectrum arising from unmodified hMDMX (GST-tagged screening construct) showing unmodified mass of the protein. <b>Panel b.</b> Mass spectrum arising from treatment of 20 µM GST-hMDMX with 100 µM of compound <b>1</b> demonstrating multiple alkylation events. Note that 100 µM is well above the solubility limit of compound <b>1</b> and significant aggregation of compound exists. <b>Panel c.</b> Mass spectrum arising from treatment of 1 µM GST-hMDMX with 5 µM of compound <b>1</b> demonstrating no alkylation events. <b>Panel d.</b> Mass spectrum arising from unmodified hMDMX (untagged aa 23 to 111 construct) showing unmodified mass of the protein. <b>Panel e.</b> Mass spectrum arising from treatment of 20 µM hMDMX with 100 µM of compound <b>1</b> demonstrating partial alkylation. <b>Panel f.</b> Mass spectrum arising from treatment of 1 µM hMDMX with 5 µM of compound <b>1</b> demonstrating no alkylation.</p

Thermal stability equilibria of MDMX.

<p><b>Panel a.</b> Thermal shift data for MDMX (23–111) showing a 7 degree stabilization of the protein’s melting point by addition of compound <b>1</b>. The panel shows individual data sampling points from 3 independent experiments from each condition. <b>Panel b.</b> Dose dependency and time dependency of the effect showing an apparent EC₅₀ of roughly 1 µM and minimal time dependency. <b>Panel c.</b> Dose dependent reversal of the effects of compound <b>1</b> by TCEP. <b>Panel d.</b> Dose dependent reversal of the effects of compound <b>1</b> by DTT.</p

Reversibility of the interaction of compound 1 with MDMX.

<p><b>Panel a.</b> SPR study of the binding of <b>1</b> (100 µM) to hMDMX (aa 23–111) under non-reducing conditions. While the off-rate is slow, the interaction is reversible. <b>Panel b.</b> SPR study of the binding of <b>1</b> (100 µM) to hMDMX (aa 23–111) under reducing conditions. No binding is observed.</p

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

<div><p>MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10⁻¹⁶) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.</p></div