bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.07.31.742089

Fluorescence polarization-based fragment screen identifies inhibitors of APOBEC3A and APOBEC3B

Brunello, J. S.·Allegakoen, H. R.·Li, W.·Revalde, J. L.·Bhadoria, R.·Sanders, C. M.·Arkin, M. R.·Ostrem, J. M. L.
View original source ↗Export metadata ↓

Abstract

APOBEC3A and APOBEC3B are antiviral cytidine deaminases found to drive cancer-associated mutagenesis, contributing to tumor evolution and therapeutic resistance across multiple cancer types. Inhibiting these enzymes holds promise for prolonging response to a wide range of cancer therapies by delaying development of resistance. However, APOBEC3A and APOBEC3B remain challenging drug targets, with no potent and selective small molecule inhibitors reported. Here, we use a fluorescence polarization-based assay to identify small molecules inhibitors of the APOBEC3A-single-stranded DNA interaction. From a library of 2,400 disulfide compounds, we identified 64 hits (mean polarization +/- 3 sigma, hit rate of 2.7%). Intact protein mass spectrometry revealed that a subset of compounds covalently engages A3A at cysteine 64, including Compounds 1 and 2. Compounds 1 and 2 disrupt APOBEC3A/APOBEC3B-single-stranded DNA interactions and inhibit APOBEC3A/APOBEC3B deaminase activity in a dose-dependent manner, with micromolar IC50. Surprisingly, inhibition of APOBEC3A/APOBEC3B by Compounds 1 and 2 is independent of covalent tethering to cysteine, suggesting a predominantly non-covalent mode of binding. Together, these studies establish an integrated workflow for APOBEC ligand discovery and identify Compounds 1 and 2 as starting points for developing chemical probes to investigate APOBEC-driven mutagenesis and therapeutic resistance.

Explore related subjects

cancer biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Brunello, J. S., Allegakoen, H. R., Li, W., Revalde, J. L., Bhadoria, R., Sanders, C. M., Arkin, M. R., Ostrem, J. M. L.. 2026-08-03. Fluorescence polarization-based fragment screen identifies inhibitors of APOBEC3A and APOBEC3B. https://doi.org/10.64898/2026.07.31.742089

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

MYC-Hyperactivated Osteosarcoma Models Exhibit Resistance to Cabozantinib plus TIGIT Blockade

Background: Relapsed and refractory osteosarcoma (OS) remains a major therapeutic challenge, with fewer than 20% of patients surviving beyond 3 years. Increasing evidence indicates that MYC amplification/overexpression is associated with inferior survival. Small molecule inhibitors and immunotherapies have limited single-agent efficacy in pediatric solid tumors. Using syngeneic cell lines derived from p53-driven and MYC-hyperactivated genetically engineered mouse models (GEMMs) of OS, we tested cabozantinib, a multi-tyrosine kinase inhibitor with immunomodulatory properties, with TIGIT immune checkpoint blockade and investigated mechanisms underlying therapeutic response and resistance. Methods: In vitro cabozantinib sensitivity was established in GEMM-derived cell lines. Mice bearing tibial tumors were randomized to vehicle control, cabozantinib, anti-TIGIT antibody, or combination therapy, and tumor growth and survival assessed after a 3-week treatment period. Temporal RNA sequencing was performed at early (8-15 days) and late (18-24 days) time points to characterize transcriptomic changes associated with efficacy. Results: MYC-hyperactivated cell lines were more resistant to cabozantinib in vitro than p53-driven lines (mean IC50 5.51 vs 0.65 mciroM, p=0.0016). In p53-driven orthotopic models, combination therapy significantly decreased tumor growth and improved survival compared to solvent and cabozantinib alone, while in MYC-hyperactivated models cabozantinib-containing regimens delayed tumor progression relative to control or anti-TIGIT monotherapy, however the addition of anti-TIGIT did not significantly improve survival over cabozantinib alone. Temporal transcriptomics revealed upregulated anti-tumor immune-response pathways and decreased M2 macrophages only with combination treatment in the p53-driven model. In contrast, combination-treated MYC-hyperactivated models demonstrated increased TNF signaling and elevated Cxcl5 and Ccr2 expression, indicative of increased myeloid cell recruitment, and upregulation of extracellular matrix (ECM) remodeling pathways suggest a therapy-induced stress adapted state that propagates treatment resistance over time. Conclusion: New therapies are needed for patients with relapse or refractory OS. By targeting tumor-intrinsic resistance mechanisms and modulating the tumor microenvironment using cabozantinib and anti-TIGIT therapy, improved tumor control and survival was achieved in p53-driven orthotopic OS models. MYC-hyperactivated models were able to overcome therapeutic pressure and employ myeloid recruitment and ECM remodeling programs to achieve treatment resistance. Targeting of these programs should be considered in future studies investigating therapeutic strategies in relapsed and refractory OS.

cancer biology↗
bioRxiv2026

Mitochondrial priming in human germ cell tumors is dependent on MCL1 and BCL2L1

Germ cell tumors (GCTs) are highly sensitized to cell death in response to DNA damaging agents, a property that underlies the success of current chemotherapeutic regimens. To address the molecular basis for this, known as apoptotic priming, we evaluated how different BCL2 family members modulate the heightened sensitivity of GCTs to therapy. Our analysis of human GCTs finds consistently high expression of the pro-survival factors MCL1 and BCL2L1 (BCLX) in a cohort of primary tumors and in their embryonic precursor cells, frequently accompanied by copy number gains of these loci and reciprocal losses of their pro-apoptotic interaction partners and inhibitors, PMAIP1 (NOXA) and BAD. We find that co-inhibition of MCL1 and BCLX using selective BH3 mimetics results in a potent synthetic lethality in multiple GCT embryonal carcinoma cell lines. When these cell lines were cultured with the DNA damaging agents cisplatin or etoposide, inhibition of MCL1 or BCLX potentiated their apoptotic effect in undifferentiated embryonal carcinoma cell lines, but not in retinoic acid-differentiated cells. The inhibition of MCL1 also heightened cisplatin sensitivity in p53-deficient or -mutant cell lines, which is associated with resistance to therapy. Employing an in ovo human xenograft model, we validate that the combination of cisplatin and MCL1 inhibition enhanced the therapeutic response by eliminating tumor cells. Our findings identify MCL1 and BCLX as critical factors to maintain GCT viability and as putative therapeutic targets to further augment GCT responsiveness to DNA damaging agents.

cancer biology↗
bioRxiv2026

β3-Integrin controls pericyte metabolic states and shapes tumour-stromal metabolic crosstalk in breast cancer

Pericytes are emerging as dynamic regulators of the tumour microenvironment. Yet, their role in tumour metabolism remains elusive. Here, we investigate whether {beta}3-integrin regulates pericyte metabolic state and shapes stromal-tumour metabolic interactions in breast cancer. By integrating spatial and single-cell transcriptomics from human breast tumours with multi-omics profiling of tumour-derived pericytes in vitro, we identify two {beta}3-integrin-dependent metabolic states. {beta}3-integrin-high pericytes display a metabolically active phenotype characterised by increased glycolysis and enhanced de novo serine/glycine synthesis, supporting collagen production. In contrast, {beta}3-integrin loss induces a lipid-associated state, marked by neutral lipid accumulation and lipid droplets. Mechanistically, {beta}3-integrin regulates this metabolic switch via mTOR signalling. Importantly, these states extend beyond pericytes, with adjacent cancer cells shifting towards fatty acid oxidation and lipid use near {beta}3-integrin-low pericytes. Together, our findings establish {beta}3-integrin as a key metabolic switch in pericytes and highlight their role in driving tumour metabolic plasticity.

cancer biology↗