Biology subjects

Schmoker, A. M.

Publications and source records attributed to Schmoker, A. M..

4 recordsLinked to original sources

bioRxiv2026

Glycogen Synthase Kinase-3β Regulates Cellular Prion Protein Levels

The normal cellular prion protein (PrPC) is an essential substrate in all forms of prion diseases and a receptor for A{beta} oligomers in Alzheimers disease. However, it is not fully understood how cells regulate PrPC levels. Recently, we identified glycogen synthase kinase-3{beta} (GSK-3{beta}) as a potential regulator of PrPC levels in a whole genome knockout screen. Here, we show that both cell surface and total PrPC levels can be reduced either by siRNA-mediated Gsk3b (but not Gsk3a) knockdown or by CRISPR-mediated Gs3b knockout. Whole cell mass spectrometric analysis showed that PrPC was the 60th most significantly reduced protein (out of 7227 total proteins detected) in Gsk3b knockout cells, compared to wild-type cells. Two different GSK-3 inhibitors, laduviglusib (CHIR-99021) and AZD-1080, reduced PrPC levels in mouse CAD5 and human BE(2)-C cells, both in undifferentiated and differentiated states. PrPC levels were similarly reduced by cycloheximide treatment in both Gsk3b knockout and WT cells, indicating that GSK-3{beta} regulates PrPC levels through a post-translational mechanism. Finally, treatment with either laduviglusib or AZD-1080 reduced PrPSc levels in CAD5 cells infected with three different rodent prion strains. Overall, the results reveal that GSK-3{beta} activity controls PrPC levels in living cells, revealing a novel regulatory mechanism and promising therapeutic target.

cell biology↗

bioRxiv2025

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

biochemistry↗

bioRxiv2023

Exploration of the Tunability of BRD4 Degradation by DCAF16 Trans-labelling Covalent Glues

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named Template-assisted covalent modification, where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

pharmacology and toxicology↗

bioRxiv2021

A global map of the Zika virus phosphoproteome reveals host-driven regulation of viral budding

Flaviviruses are enveloped, positive-strand RNA viruses that cause millions of infections in the human population annually. Although Zika virus (ZIKV) had been detected in humans as early as the 1950s, its reemergence in South America in 2015 resulted in a global health crisis. While flaviviruses encode 10 proteins that can be post-translationally modified by host enzymes, little is known regarding post-translational modifications (PTMs) of the flavivirus proteome. We used mass spectrometry to comprehensively identify host-driven PTMs on the ZIKV proteome. This approach allowed us to identify 43 PTMs across 8 ZIKV proteins, including several that are highly conserved within the Flavivirus genus. Notably, we found two phosphosites on the ZIKV envelope protein that are functionally important for viral propagation. Both appear to regulate viral budding, while one also impacts ZIKV cytopathogenicity. Additionally, we discovered host kinases that interact with ZIKV proteins and determined that Bosutinib--an FDA-approved tyrosine kinase inhibitor that targets some of these host kinases--impairs ZIKV growth, in part by blocking phosphorylation of a tyrosine residue on the envelope protein. Thus, we have defined a high-resolution map of host-driven PTMs on ZIKV proteins as well as cellular interacting kinases, uncovered novel mechanisms of host driven-regulation of ZIKV budding and cytopathogenicity, and identified an FDA-approved inhibitor of ZIKV growth.

microbiology↗