Biology subjects

Davis-Gilbert, Z. W.

Publications and source records attributed to Davis-Gilbert, Z. W..

4 recordsLinked to original sources

bioRxiv2026

Development of NanoBRET cellular target engagement assays in primary neurons for activating mutants of p21-activated kinase 1

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

pharmacology and toxicology↗

bioRxiv2025

NUAK2 is a therapeutically tractable regulator of RNA splicing and tumor progression in neuroendocrine prostate cancer

Prostate cancer remains a leading cause of cancer-related mortality in men, with aggressive, treatment-emergent androgen receptor (AR)-indifferent subtypes, including double-negative prostate cancer (DNPC) and neuroendocrine prostate cancer (NEPC), posing major clinical challenges due to limited therapeutic options. NUAK family kinase 2 (NUAK2), an AMPK-related kinase, has been implicated in tumor growth and metastatic progression; however, its functional significance and therapeutic potential in advanced prostate cancer remain largely unexplored. Here, we identify NUAK2 as a therapeutically actionable kinase dependency in AR-indifferent prostate cancer. Transcriptomic analyses across independent patient cohorts demonstrated progressive upregulation of NUAK2 with disease progression, with the highest expression in NEPC. Immunohistochemical analysis of clinical specimens further confirmed elevated NUAK2 protein expression in NEPC relative to prostate adenocarcinoma. Genetic loss- and gain-of-function studies established NUAK2 as a functional dependency that promotes tumor cell proliferation, clonogenic growth, and tumor growth in vivo. Mechanistically, integrated proteomic analyses revealed that NUAK2 associates with spliceosomal and RNA-processing machinery, while NUAK2 perturbation induced widespread alterations in pre-mRNA splicing programs involving genes linked to mitotic regulation and oncogenic signaling. Pharmacologic studies identified trilaciclib (G1T-28), a clinically approved CDK4/6 inhibitor, as a functionally relevant NUAK2 inhibitor that directly engages NUAK2, suppresses tumor growth, and enhances the efficacy of platinum-based chemotherapy across multiple preclinical models. Collectively, these findings uncover NUAK2 as a previously unrecognized regulator of RNA splicing and therapeutic vulnerability in AR-indifferent prostate cancer and provide a rationale for repurposing G1T-28 and developing NUAK2-directed therapeutic strategies for aggressive, treatment-refractory prostate cancer.

cancer biology↗

bioRxiv2025

Identification of Direct-acting nsP2 Helicase Inhibitors with Anti-alphaviral Activity

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/641060v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@856df5org.highwire.dtl.DTLVardef@1f6225borg.highwire.dtl.DTLVardef@4997d5org.highwire.dtl.DTLVardef@18f42f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Structures of the human transcription factor brachyury offer insights into DNA recognition, and identify small molecule binders for the development of degraders for cancer therapy

The transcription factor brachyury is a member of the T-Box family of transcription factors. It is active during embryogenesis and is required for the formation of the posterior mesoderm and the notochord in vertebrates. Aside from its role in embryogenesis, brachyury plays an essential role in tumour growth of the rare chordoma bone cancer and is implicated in other solid tumours. Given that brachyury is minimally expressed in healthy tissues, these findings suggest that brachyury is a potential therapeutic target in cancer. Unfortunately, as a ligandless transcription factor, brachyury has historically been considered undruggable. To investigate direct targeting of brachyury by small molecules, we initially determined the structure of human brachyury both in complex with its cognate DNA and in the absence of DNA. Analysis of these structures provided insights into brachyury DNA binding and the structural context of the G177D variant which is strongly associated with chordoma risk. We used these structures to perform a crystallographic fragment screen of brachyury and identify hotspot regions on numerous pockets on the brachyury surface. Finally, we have performed follow-up chemistry on fragment hits and describe the structure-based progression of a thiazole-containing chemical series. Excitingly, we have produced brachyury binders with low {micro}M potency that can serve as starting point for further medicinal chemistry efforts. These data show that brachyury is ligandable and provides an example of how crystallographic fragment screening may be used to find ligands to target protein classes that are traditionally difficult to address using other approaches.

molecular biology↗