SN-38-A2

Amino Acid Sequence

3D Structure (/structures/AF-P11387-F1-model_v6.cif)

Function

Releases the supercoiling and torsional tension of DNA introduced during the DNA replication and transcription by transiently cleaving and rejoining one strand of the DNA duplex. Introduces a single-strand break via transesterification at a target site in duplex DNA. The scissile phosphodiester is attacked by the catalytic tyrosine of the enzyme, resulting in the formation of a DNA-(3'-phosphotyrosyl)-enzyme intermediate and the expulsion of a 5'-OH DNA strand. The free DNA strand then rotates around the intact phosphodiester bond on the opposing strand, thus removing DNA supercoils. Finally, in the religation step, the DNA 5'-OH attacks the covalent intermediate to expel the active-site tyrosine and restore the DNA phosphodiester backbone (By similarity). Regulates the alternative splicing of tissue factor (F3) pre-mRNA in endothelial cells. Involved in the circadian transcription of the core circadian clock component BMAL1 by altering the chromatin structure around the ROR response elements (ROREs) on the BMAL1 promoter

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To elucidate the pathway through which ATCs promote TOP1 downregulation, we conducted pharmacological inhibitor studies in MIA PaCa-2 cells. Co-treatment with the proteasome inhibitor MG132 (10 μM) prevented SN-38-A2induced reduction of TOP1 protein levels, whereas the lysosomal inhibitor chloroquine (50 μM) had no measurable effect (Figure 4A). This differential sensitivity confirms that ATC-mediated TOP1 degradation proceeds predominantly through the ubiquitin-proteasome system (UPS) rather than autophagic or lysosomal pathways. To further define the role of the cellular protein quality-control (PQC) machinery, we utilized specific chaperone inhibitors. While HSP70 inhibitors (Apoptozole or VER-155008) failed to rescue TOP1 levels, treatment with HSP90 inhibitors (Geldanamycin or Tanespimycin) significantly restored TOP1 expression in the presence of SN-38-A2 (Figure 4B). These results suggest that the HSP90 chaperone system specifically recognizes the adamantane-induced pseudomisfolded state, directing the target protein toward proteasomal clearance (Figure 4C).

Description

Consequently, as our lead carbonate-linked ATC, SN-38-A2 induced pronounced degradation of the TOP1 protein in a concentration-dependent manner. At concentrations as low as 10 nM, SN-38-A2 achieved approximately 61.5% reduction in TOP1 levels relative to untreated controls, as quantified by densitometric analysis normalized to β-actin (Figure 1D,E)

Description

To systematically dissect the structural determinants governing antiproliferative activity and TOP1 degradation efficiency, we designed and synthesized a focused panel of ATCs (SN-38-A3 through SN-38-A8) incorporating systematic variations in the linker composition, length, and flexibility. Notably, SN-38-A8 exhibited the most potent cytotoxicity among the series (IC50 = 14.8 nM), surpassing SN-38 (IC50 = 54.4 nM) but remaining less potent than SN-38-A2 (IC50 = 6.2 nM) (Figure 2B).