Among brain tumors, glioblastoma (GBM) is widely recognized as the most aggressive and lethal primary brain malignancy which is characterized by a with a 3-year survival rate of only around 10 % [1]. The median survival time remains short due to the aggressive nature of the GBM and its resistance to traditional treatments as well as the immunosuppressive microenvironment [2]. The current standard treatment for GBM includes maximal surgical resection followed by radiation therapy and temozolomide(TMZ) [3,4] chemotherapy which is often combined with tumor-treating fields. Surgical resection aims to remove as much tumor tissue as possible, but microscopic infiltrative cells often persist which often leads to recurrence [5,6].

Despite advances, GBM therapy is hindered by multiple factors such as the limited systemic drug penetration by the BBB where only about 20 % of systemically administered temozolomide reaches the cerebral parenchyma [7,8]. The efflux pumps like ABC transporters [9,10] further reduce intracellular drug concentrations. Moreover, the recurrence of GBM is highly recurrent due to the presence of glioma stem cells (GSCs), which possess high migratory potential, resistance to chemotherapy and radiation in addition to the ability to form recurrent tumors [11,12]. Immunotherapies struggle in GBM's “cold tumor” microenvironment, characterized by low T-cell infiltration and high levels of immunosuppressive cytokines like TGF-β. Even with aggressive treatments, median survival remains 12–18 months, underscoring the need for urgent and critical need to develop novel therapeutic strategies for GBM that can overcome the limitations of existing treatment modalities.

Among the molecular targets implicated in glioblastoma (GBM), chitinase-3-like protein 1 (CHI3L1, also known as YKL-40) has emerged as a key factor in GBM pathogenesis [13,14]. CHI3L1 is a secreted glycoprotein that lacks enzymatic chitinase activity but still plays a fundamental role in promoting tumor progression by enhancing cell survival, migration, invasion, and angiogenesis [[14], [15], [16], [17], [18]]. In the brain, CHI3L1 is primarily expressed by astrocytes and microglia under the transcriptional control of regulators such as NFI-X3 and STAT3 [19]. CHI3L1 exerts its effects through interactions with a network of receptors including CRTH2 [20], RAGE [21], IL-13 receptor α2 (IL-13Rα2) [22], PAR-2 [23], and CD44 [24]. The interaction of CHI3L1 with its receptors triggers the activation of key oncogenic signaling pathways—including ERK1/2, PI3K/Akt, Wnt/β-catenin, JNK, and NF-κB—which collectively govern critical processes such as inhibition of apoptosis, modulation of immune responses, extracellular matrix remodeling, and inflammasome activation [25,26].

Studies have shown that CHI3L1 is highly expressed in glioblastoma [27] compared to other cancers and normal tissues. Additionally, studies haves shown that the expression of CHI3L1 correlates with clinical and molecular features of malignancy where high CHI3L1 expression have been associated with poor patient prognosis, resistance to chemotherapy and radiotherapy [28]. Additionally, Chi3L1 expression levels have been established as a key marker for the mesenchymal subtype of glioblastoma [29]. At the molecular level, CHI3L1 has been found to regulate glioma cell invasion, migration, growth, and tumor vascularization [30]. Furthermore, recent studies have indicated that CHI3L1 plays important roles in the glioblastoma immune microenvironment where it closely associated with immune responses, inflammatory activities, and immunosuppression [1,31]. Meanwhile, inhibition of CHI3L1 have been reported to reduce immunosuppression, decrease the mesenchymal signature of glioblastoma, and restrict GSC plasticity, potentially overcoming immunotherapy resistance [32]. Together, these findings suggest that CHI3L1-targeted immunotherapy, either alone or in combination with other immunotherapies such as immune checkpoint inhibitors, represents a promising new strategy for treating GBM by modulating cellular plasticity and reducing tumor burden.

To date, only a few small molecules CHI3L1 modulators have been reported; K284 [33], G721–0282 [34] (G721), and CHI3L1-IN-1 [35] (Fig. 1). CHI3L1-IN-1 was identified via an indirect AlphaScreen assay, which is effective for identifying compounds that compete with a known probe but does not confirm direct binding or reveal binding modes. K284 was discovered through pull-down assays and has been shown to significantly reduced inflammatory responses, organ damage, and memory impairment by targeting CHI3L1-regulated pathways such as CXCL3 and PTX3 [[36], [37], [38]]. Meanwhile, G721–0282 (G721) was discovered based on docking studies [34] and has been shown to inhibit lung metastasis [33], osteosarcoma (OS) cell proliferation, migration, and invasion by disrupting the STAT3 signaling pathway and promoting apoptosis, both in vitro and in vivo [34].

While these discoveries have highlighted the importance of CHI3L1 in the modulation of different diseases, important gaps remain in our understanding of their mechanisms of action and their capacity to modulate CHI3L1 function effectively. Furthermore, assessment of the direct binding of these modulators using microscale thermophoresis (MST) resulted in a varied activity profile with G721 exhibiting no detectable dose-dependent binding. Meanwhile, CHI3L1-IN-1 exhibited weak binding with a dissociation constant (Kd) in the millimolar range. Lastly, K284 demonstrated measurable binding with a Kd of 152 μM.

Given the weak to moderate activity of current CHI3L1 inhibitors, the discovery of more potent CHI3L1 modulators represents a critical unmet need. While traditional drug discovery pathways are resource-intensive, virtual screening (VS) offers an efficient alternative for early-stage hit identification [39,40]. Moreover, with recent crystallization of CHI3L1 [35] reported the presence of a distinct binding pocket, offering an opportunity to apply structure-based drug discovery approaches to identify more potent and selective modulators. Accordingly, we planned to carry out a VS assay to identify novel small molecules capable of modulating CHI3L1 with improved potency in comparison to current modulators. Moreover, given that ideal therapeutic candidates for GBM must combine biochemical potency with blood-brain barrier (BBB) penetration and favorable pharmacokinetic (PK) properties we planned for a PK assay for the top identified molecules.

Herein, we present a CHI3L1-targeted drug discovery pipeline that integrates pharmacophore-based virtual screening (VS) of an in-house small-molecule library composed of 16,922 prepared small molecules to identify novel modulators of CHI3L1. The identified candidates were subjected to a side-by-side evaluation of CHI3L1-binding potency along with previously reported modulators using microscale thermophoresis (MST). The most promising hit from the VS campaign underwent detailed pharmacokinetic (PK) profiling and was further evaluated for efficacy in GBM using three-dimensional (3D) spheroid models. 3D spheroid models were employed for their enhanced physiological relevance which offers a more accurate representation of the tumor microenvironment (TME) compared to traditional two-dimensional (2D) monolayer cultures [41]. The PK and GBM spheroid models were extended to all known CHI3L1 modulators, establishing the first standardized comparison of their binding affinities and functional effects in disease-relevant systems. This integrated approach establishes a robust platform for rational CHI3L1 modulators discovery with high translational potential. Structural information for all screened compounds are provided in the supplementary file, while Tables S1 and S2 summarize the top virtual screening hits selected for experimental validation.