Biology subjects

Kovachka, S.

Publications and source records attributed to Kovachka, S..

3 recordsLinked to original sources

bioRxiv2026

Structural and cellular insights into the inhibition of the drug efflux activity of the HEDGEHOG receptor PATCHED1

PTCH1, the receptor for the Sonic Hedgehog morphogen, mediates cholesterol transport across the plasma membrane by harnessing the proton motive force. In cancer, PTCH1 is frequently overexpressed and promotes chemoresistance by transporting drugs such as doxorubicin (dxr) out of cells. Among the inhibitors identified, PAH stands out for its ability to significantly enhance the efficacy of several chemotherapeutic drugs on melanoma and breast cancer cells. To investigate PTCH1s structure in complex with its inhibitor PAH, we overexpressed a construct spanning residues 1-619 and 720-1305 in HEK293 cells. The protein localized to the membrane, and transfected cells exhibited reduced sensitivity to dxr compared to control cells. Additionally, we observed a pH-dependent efflux of dxr, which was reversed by PAH, confirming that the PTCH1 construct used in this study functions as an active drug-efflux pump. In the structure of PTCH1 bound to PAH determined using cryo-electron microscopy, PAH occupies a hydrophobic cavity in an extracellular domain which is normally occupied by cholesterol in other PTCH1 structures, and engages in a key hydrogen bond via one of its hydroxyl groups, a feature previously established as essential for its inhibitory function. These findings not only clarify the molecular basis of PAHs action but also provide a structural roadmap for rational drug design, enabling the development of next-generation inhibitors with enhanced potency.

biochemistry↗

bioRxiv2025

Live-Cell Covalent Profiling Reveals Principles of RNA-Small Molecule Recognition across the Human Transcriptome

RNA folds are abundant in mammalian cells yet poorly characterized as small-molecule targets. We present a scalable, unbiased live-cell pipeline that maps where small molecules bind RNA across the human transcriptome and convert those binders into selective degraders. A 200-member fragment library bearing diazirine/alkyne handles yielded 23 RNA-binding candidates. Chem-CLIP-Map-Seq in MDA-MB-231 cells identified 723 RNA targets and their binding sites, revealing a strong bias toward 5' and 3' untranslated regions (UTRs) in mRNAs and enrichment at thermodynamically stable structures, with limited binding to non-coding RNAs. Expression level and local stability contributed to engagement. An integrated machine-learning model trained on multiple fingerprints distinguished binders from non-binders, and highlighted chemotypes and physicochemical features that favor RNA recognition. Four fragments were converted to RiboTACs; despite broad binding, cleavage was highly selective, with X1-RiboTAC degrading MPP7 and SSC4D mRNAs in an RNase L-dependent manner and reducing their protein levels. A competitive profiling workflow quantified in-cell target occupancy and guided optimization of the RNA-binding module to reprogram selectivity: an X1 derivative produced an MPP7-selective RiboTAC that lowered MPP7 mRNA levels and suppressed breast-cancer cell migration, while sparing SSC4D transcripts. This end-to-end framework, including transcriptome-wide mapping, data-driven rules, and tunable degradation, establishes practical principles for ligandable RNA sites in cells and enables rational design of RNA-targeted small molecules and degraders. TEASERLive-cell mapping reveals ligandable RNA sites and guides design of selective RNA degraders. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/686775v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@a216c8org.highwire.dtl.DTLVardef@185c59eorg.highwire.dtl.DTLVardef@17131e2org.highwire.dtl.DTLVardef@81ffa2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Inhibition of PTCH1 drug efflux activity enhances chemotherapy efficacy against triple negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subgroup characterized by a high risk of resistance to chemotherapies and high relapse potential. High levels of mRNA from the Hedgehog receptor PTCH1 are associated with poor prognosis in TNBC. PTCH1 is overexpressed in many aggressive cancers. We previously reported that PTCH1 is a multidrug transporter that triggers resistance to chemotherapy of adrenocortical carcinoma and melanoma cells, and that inhibiting PTCH1 drug efflux strongly enhanced chemotherapy efficacy on these cell lines both in vitro and in vivo. In the present study, we found that PTCH1 inhibition also significantly inhibited doxorubicin efflux in three TNBC cell lines leading to a strong increase of the cytotoxic effect of doxorubicin and docetaxel, and an inhibition of cell migration. Altogether, our data highlight the therapeutic potential of targeting PTCH1 drug efflux activity using drug association strategies for the treatment of TNBC patients.

cancer biology↗