Biology subjects

Fitzgerald, M. C.

Publications and source records attributed to Fitzgerald, M. C..

5 recordsLinked to original sources

bioRxiv2026

The KRAS G12C Inhibitor Divarasib Stabilizes RBM39 and Antagonizes Aryl-Sulfonamide Degraders

KRAS G12C inhibitors have demonstrated meaningful clinical benefit in KRAS G12C-mutant non-small cell lung cancer (NSCLC), yet responses remain heterogeneous and treatment-associated toxicities persist for reasons that are incompletely understood. Cysteine profiling indicates that these covalent inhibitors are highly selective for mutant KRAS; however, such approaches cannot detect noncovalent engagement of additional non-RAS proteins. Here, we used a protein-folding stability profiling technique, stability of proteins from rates of oxidation (SPROX), to identify protein targets of the clinical KRAS G12C inhibitor, divarasib (GDC-6036), in KRAS-mutant NSCLC lysates. SPROX revealed a focused set of candidate interactors, including the essential splicing factor RBM39, which was reproducibly stabilized at both divarasib concentrations tested. We subsequently confirmed that divarasib directly and noncovalently binds to RBM39 protein. In NSCLC cells, divarasib increased RBM39 protein abundance and antagonized RBM39 degradation induced by the aryl-sulfonamide molecular glue indisulam through a post-transcriptional mechanism. Divarasib and RBM39 degraders reciprocally antagonized each other's cytotoxicity, and RBM39 knockdown modestly reduced divarasib-induced cell death. Mechanistically, divarasib-mediated RBM39 stabilization regulated both INSR expression and alternative splicing, altered downstream insulin receptor signaling, and contributed to divarasib-associated cytotoxicity. Consistent with these findings, RBM39 and INSR expression were positively correlated across multiple human cancer types. Collectively, these findings identify RBM39 as a previously unrecognized noncovalent target of divarasib and uncover an RBM39-INSR signaling axis that modulates cellular responses to both divarasib and RBM39 degraders.

pharmacology and toxicology↗

bioRxiv2025

Discovery of Host-Directed Small Molecules with Broad Anti-Leishmanial Efficacy

Leishmaniasis, a neglected tropical disease affecting nearly 10% of the global population, suffers from limited therapeutic options and rising drug resistance. To address this, we developed 343 analogs of AR-12, a compound that has previously illustrated host-directed anti-leishmanial effects. Primary screening using a luminescence-based assay revealed 66 analogs with greater selectivity than the parent compound, AR-12. Sixteen promising candidates, selected for high potency (IC < 1 {micro}M) or high selectivity (>15), underwent secondary screening via Giemsa staining. Four lead compounds (53, 134, 197, and 354) demonstrated therapeutic indices greater than 40. Tertiary assays confirmed their broad in vitro efficacy against both Leishmania donovani and L. mexicana. Notably, 197 exhibited potent host-directed activity and proteomic analysis identified lysozyme as a mechanistic target, implicating it in the host-mediated clearance of intracellular parasites. These findings highlight the dual host- and pathogen-directed mechanisms of these compounds and support their potential as the basis for new therapeutic strategies. Further optimization and clinical exploration of these leads are warranted to meet the urgent need for effective leishmaniasis treatments. AUTHOR SUMMARYLeishmaniasis is a parasitic infectious disease with limited therapeutic options and rising drug resistance. Drugs that act directly against Leishmania can further drive drug resistance. Host-directed therapies work to enable the host responses to enhance pathogen clearance and reduce disease progression. Host-directed therapies both limit the emergence of new drug resistance and combat drug resistant infections. In this work we screened a library of 343 AR-12 analogs for host-directed anti-leishmanial activity. This work identified 4 hit compounds. We then did proteomic analysis to understand the mechanism of action of the primary lead compound, identifying the protein lysozyme as having a role in host-directed activity against Leishmania infection.

biochemistry↗

bioRxiv2025

Small molecules reveal differential shifts in stability and protein binding for G-quadruplex RNA

The potential of therapeutically targeting RNA with small molecules continues to grow yet progress is hindered by difficulties in determining specific mechanisms of action, including impacts on RNA-protein binding. RNA G-quadruplexes (rGQs) are a particularly promising target due to their range of biological functions, structural stability, and hydrophobic surfaces, which promote small molecule and protein interactions alike. Challenges arise due to 1) the low structural diversity among rGQs, thereby limiting binding selectivity, and 2) a lack of knowledge regarding how small molecules can manipulate rGQ-protein binding on a global scale. We first leveraged a small molecule library privileged for RNA tertiary structures that displayed differential binding to rGQs based on loop length, consistent with computational predictions for DNA GQs. We next utilized an RT-qPCR-based assay to measure stability against enzymatic readthrough, expected to be a common mechanism in rGQ function. We discovered small molecules with significant, bidirectional impacts on rGQ stability, even within the same scaffold. Using Stability of Proteins from Rates of Oxidation (SPROX), a stability-based proteomics method, we then elucidated proteome level impacts of both stabilizing and destabilizing rGQ-targeting molecules on rGQ-protein interactions. This technique revealed small molecule-induced impacts on a unique subset of rGQ-binding proteins, along with proteins that exhibited differential changes based on the identity of the small molecule. The domain and peptide-level insights resulting from SPROX allow for the generation of specific hypotheses for both rGQ function and small molecule modulation thereof. Taken altogether, this methodology helps bridge the gap between small molecule-RNA targeting and RNA-protein interactions, providing insight into how small molecules can influence protein binding partners through modulation of target RNA structures.

biochemistry↗

bioRxiv2021

Sex differences in migraine: A twin study

Migraine is a neurological disorder with a prominent sex difference such that two thirds of sufferers are female. The mechanisms behind the preponderance of migraine in women have yet to be elucidated. With data on 51,872 participants from the Swedish Twin Registry, we report results from two distinct analyses intended to clarify the degree to which genetic and environmental factors contribute to sex differences in migraine. First, we fit a sex-limitation model to determine if quantitative genetic differences (i.e., is migraine equally heritable across men and women) and/or qualitative genetic differences (i.e., are different genes involved in migraine across men and women) were present. Next, we used a multilevel logistic regression model to compare the prevalence of migraine in individuals from opposite-sex and same-sex twin pairs to determine whether differences in the prenatal hormone environment contribute to migraine risk. In the final analytic sample, women were found to have a significantly higher rate of migraine without aura relative to men (17.6% vs 5.5%). The results from an ADE sex-limitation model indicate that migraine is equally heritable in men and women, with a broad sense heritability of .45, (95% CI = .40 - .50), while results from a reduced AE sex-limitation model provide subtle evidence for differences in the genes underlying migraine across men and women. The logistic regression analysis revealed a significant increase in migraine risk for females with a male co-twin relative to females with a female co-twin (OR = 1.51, 95% CI = 1.26 - 1.81). These results suggest that the prominent sex difference in migraine prevalence is not entirely accounted for by genetic factors, while demonstrating that masculinization of the prenatal environment may increase migraine risk for females. This effect points to a potential prenatal neuroendocrine factor in the development of migraine.

genetics↗

bioRxiv2021

Chemoproteomic-enabled characterization of small GTPase Rab1a as a target of an N-arylbenzdiimidazole ligand's rescue of Parkinson's-associated cell toxicity

The development of phenotypic models of Parkinsons disease (PD) has enabled screening and identification of phenotypically active small molecules that restore complex biological pathways affected by PD toxicity. While these phenotypic screening platforms are powerful, they do not inherently enable direct identification of the cellular targets of promising lead compounds. To overcome this, chemoproteomic platforms like Thermal Proteome Profiling (TPP) and Stability of Proteins from Rates of Oxidation (SPROX) can be implemented to reveal protein targets of biologically active small molecules. Here we utilize both of these chemoproteomic strategies to identify targets of an N-arylbenzdiimidazole compound, NAB2, which was previously identified for its ability to restore viability in cellular models of PD-associated -synuclein toxicity. The combined results from our TPP and SPROX analyses of NAB2 and the proteins in a neuroblastoma-derived SHSY5Y cell lysate reveal a previously unrecognized protein target of NAB2. This newly recognized target, Rab1a, is a small GTPase that acts as a molecular switch to regulate ER-to-Golgi trafficking, a process that is disrupted by -synuclein toxicity and restored by NAB2 treatment. Further validation reveals that NAB2 binds to Rab1a with selectivity for its GDP-bound form and that NAB2 treatment phenocopies Rab1a overexpression in alleviation of -synuclein toxicity. Finally, we conduct a preliminary investigation into the relationship between Rab1a and the E3 ubiquitin ligase, Nedd4, a previously identified NAB2 target. Together, these efforts expand our understanding of the mechanism of NAB2 in the alleviation of -synuclein toxicity and reinforce the utility of chemoproteomic identification of the targets of phenotypically active small molecules that regulate complex biological pathways.

biochemistry↗