Surface Plasmon Resonance Assay Research Articles

18F]AlF-NOTA-PCP2: a novel PET/CT tracer for enhanced PD-L1 heterogeneity imaging and comparative analysis with [18F]AlF-NOTA-WL12 in glioblastoma xenografts.

The unsatisfactory efficacy of PD-L1 antibodies in glioblastoma (GBM) is largely due to the temporal and spatial heterogeneity of PD-L1 expression. Molecular imaging can enhance understanding of the tumor immune microenvironment and guide immunotherapy. However, highly sensitive imaging agents capable of effectively visualizing PD-L1 heterogeneity are limited. This study introduces a novel PET tracer, offering improved imaging of PD-L1 heterogeneity in GBM xenografts, with a comparative analysis to [18F]AlF-NOTA-WL12. [18F]AlF-NOTA-PCP2 was synthesized with high purity and its affinity for PD-L1 was characterized using surface plasmon resonance (SPR) and cell binding assays. Its specificity for PD-L1 was evaluated both in vitro using various cell lines and in vivo with GBM xenograft models in NOD/SCID mice. PET/CT imaging was conducted to evaluate the tracer's biodistribution, pharmacokinetics, and ability to quantify tumoral spatial heterogeneity of PD-L1 expression. A focused comparative analysis between [18F]AlF-NOTA-PCP2 and [18F]AlF-NOTA-WL12 was conducted, examining binding affinity, biodistribution, pharmacokinetics, and imaging effectiveness in GBM xenografts. Additionally, human radiation dosimetry estimates compared the safety profiles of both tracers. [18F]AlF-NOTA-PCP2 demonstrated high radiochemical purity (> 95%) and a strong affinity for PD-L1, comparable to [18F]AlF-NOTA-WL12. In vitro and in vivo studies confirmed its specificity for PD-L1, with increased uptake in PD-L1 expressing cells and tumors. Toxicological profiles indicated no significant abnormalities in serum biochemical indicators or major organ tissues. MicroPET/CT imaging showed [18F]AlF-NOTA-PCP2's effectiveness in visualizing PD-L1 expression levels and spatial heterogeneity in GBM xenografts. Comparative studies revealed [18F]AlF-NOTA-PCP2's improved pharmacokinetic properties, including higher tumor-to-blood ratios and lower nonspecific liver uptake, as well as reduced radiation exposure compared to [18F]AlF-NOTA-WL12. [18F]AlF-NOTA-PCP2 distinguishes itself as an exceptionally sensitive PET/CT tracer, adept at non-invasively and accurately quantifying PD-L1 expression and its spatial heterogeneity in tumors, especially in GBM. Its favorable pharmacokinetic properties, safety profile, and high affinity for PD-L1 highlight its potential for enhancing the precision of cancer immunotherapy and guiding individualized treatment strategies. While promising, its clinical translation, especially in brain imaging, necessitates further validation in clinical trials.

Creating a more strategic small molecule biophysical hit characterization workflow

To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.

Rational modification of melatonin for broad-spectrum antifungal agents discovery.

Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low-cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen-activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl-substituted derivative Mt-23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one-fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt-23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt-23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt-23 is a commercially valuable, eco-friendly and broad-spectrum antifungal agent to combat crop disease.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt

Ethnopharmacological relevanceLicorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. Aim of the studyThis study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. Materials and methodsSW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. ResultsWighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a “closed” conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. ConclusionWighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Asymmetric Carbene-Alkyne Metathesis-Mediated Cascade: Synthesis of Benzoxazine Polychiral Polyheterocycles and Discovery of a Novel Pain Blocker.

This study introduces a novel approach for synthesizing Benzoxazine-centered Polychiral Polyheterocycles (BPCPHCs) via an innovative asymmetric carbene-alkyne metathesis-triggered cascade. Overcoming challenges associated with intricate stereochemistry and multiple chiral centers, the catalytic asymmetric Carbene Alkyne Metathesis-mediated Cascade (CAMC) is employed using dirhodium catalyst/Brønsted acid co-catalysis, ensuring precise stereo control as validated by X-ray crystallography. Systematic substrate scope evaluation establishes exceptional diastereo- and enantioselectivities, creating a unique library of BPCPHCs. Pharmacological exploration identifies twelve BPCPHCs as potent Nav ion channel blockers, notably compound 8 g. In vivo studies demonstrate that intrathecal injection of 8 g effectively reverses mechanical hyperalgesia associated with chemotherapy-induced peripheral neuropathy (CIPN), suggesting a promising therapeutic avenue. Electrophysiological investigations unveil the inhibitory effects of 8 g on Nav1.7 currents. Molecular docking, dynamics simulations and surface plasmon resonance (SPR) assay provide insights into the stable complex formation and favorable binding free energy of 8 g with C5aR1. This research represents a significant advancement in asymmetric CAMC for BPCPHCs and unveils BPCPHC 8 g as a promising, uniquely acting pain blocker, establishing a C5aR1-Nav1.7 connection in the context of CIPN.

Doxorubicin-based ENO1 targeted drug delivery strategy enhances therapeutic efficacy against colorectal cancer

Alpha-enolase (ENO1), a multifunctional protein with carcinogenic properties, has emerged as a promising cancer biomarker because of its differential expression in cancer and normal cells. On the basis of this characteristic, we designed a cell-targeting peptide that specifically targets ENO1 and connected it with the drug doxorubicin (DOX) by aldehyde–amine condensation. A surface plasmon resonance (SPR) assay showed that the affinity for ENO1 was stronger (KD = 2.5 µM) for the resulting cell-targeting drug, DOX-P, than for DOX. Moreover, DOX-P exhibited acid-responsive capabilities, enabling precise release at the tumor site under the guidance of the homing peptide and alleviating DOX-induced cardiotoxicity. An efficacy experiment confirmed that, the targeting ability of DOX-P toward ENO1 demonstrated superior antitumor activity against colorectal cancer than that of DOX, while reducing its toxicity to cardiomyocytes. Furthermore, in vivo metabolic distribution results indicated low accumulation of DOX-P in nontumor sites, further validating its targeting ability. These results showed that the ENO1-targeted DOX-P peptide has great potential for application in targeted drug-delivery systems for colorectal cancer therapy.

Asymmetric Carbene‐Alkyne Metathesis‐Mediated Cascade: Synthesis of Benzoxazine Polychiral Polyheterocycles and Discovery of a Novel Pain Blocker

AbstractThis study introduces a novel approach for synthesizing Benzoxazine‐centered Polychiral Polyheterocycles (BPCPHCs) via an innovative asymmetric carbene‐alkyne metathesis‐triggered cascade. Overcoming challenges associated with intricate stereochemistry and multiple chiral centers, the catalytic asymmetric Carbene Alkyne Metathesis‐mediated Cascade (CAMC) is employed using dirhodium catalyst/Brønsted acid co‐catalysis, ensuring precise stereo control as validated by X‐ray crystallography. Systematic substrate scope evaluation establishes exceptional diastereo‐ and enantioselectivities, creating a unique library of BPCPHCs. Pharmacological exploration identifies twelve BPCPHCs as potent Nav ion channel blockers, notably compound 8 g. In vivo studies demonstrate that intrathecal injection of 8 g effectively reverses mechanical hyperalgesia associated with chemotherapy‐induced peripheral neuropathy (CIPN), suggesting a promising therapeutic avenue. Electrophysiological investigations unveil the inhibitory effects of 8 g on Nav1.7 currents. Molecular docking, dynamics simulations and surface plasmon resonance (SPR) assay provide insights into the stable complex formation and favorable binding free energy of 8 g with C5aR1. This research represents a significant advancement in asymmetric CAMC for BPCPHCs and unveils BPCPHC 8 g as a promising, uniquely acting pain blocker, establishing a C5aR1‐Nav1.7 connection in the context of CIPN.

Artemyriantholides A–K, guaiane-type sesquiterpenoid dimers from Artemisia myriantha var. pleiocephala and their antihepatoma activity

Artemyriantholides A−K (1−11) as well as 14 known compounds (12−25) were isolated from Artemisia myriantha var. pleiocephala (Asteraceae). The structures and absolute configuration of compounds 2 and 8−9 were confirmed by the single crystal X-ray diffraction analyses, and the others were elucidated by MS, NMR spectral data and electronic circular dichroism calculations. All compounds were chemically characterized as guaiane-type sesquiterpenoid dimers (GSDs). Compound 1 was the first example of the GSD fused via C-3/C-11′ and C-5/C-13′ linkages, and compounds 2 and 5 were rare GSDs containing chlorine atoms. Eleven compounds showed obvious inhibitory activity in HepG2, Huh7 and SK-Hep-1 cell lines by antihepatoma assay to provide the IC50 values ranging from 7.9 to 67.1 μM. Importantly, compounds 5 and 8 exhibited the best inhibitory activity with IC50 values of 14.2 and 18.8 (HepG2), 9.0 and 11.5 (Huh7), and 8.8 and 11.3 μM (SK-Hep-1), respectively. The target of compound 5 was predicted to be MAP2K2 by a computational prediction model. The interaction between compound 5 and MAP2K2 was conducted to give docking score of −9.0 kcal/mol by molecular docking and provide KD value of 43.7 μM by Surface Plasmon Resonance assay.

Discovery of 4-phenyl-1H-indazole derivatives as novel small-molecule inhibitors targeting the PD-1/PD-L1 interaction

The inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway with small molecules is a promising approach for cancer immunotherapy. Herein, novel small molecules compounds bearing various scaffolds including thiophene, thiazole, tetrahydroquinoline, benzimidazole and indazole were designed, synthesized and evaluated for their inhibitory activity against the PD-1/PD-L1 interaction. Among them, compound Z13 exhibited the most potent activity with IC50 of 189.6 nM in the homogeneous time-resolved fluorescence (HTRF) binding assay. Surface plasmon resonance (SPR) assay demonstrated that Z13 bound to PD-L1 with high affinity (KD values of 231 nM and 311 nM for hPD-L1 and mPD-L1, respectively). In the HepG2/Jurkat T co-culture cell model, Z13 decreased the viability rate of HepG2 cells in a concentration-dependent manner. In addition, Z13 showed significant in vivo antitumor efficacy (TGI = 52.6 % at 40 mg/kg) without obvious toxicity in the B16-F10 melanoma model. Furthermore, flow cytometry analysis demonstrated that Z13 inhibited tumor growth in vivo by activating the tumor immune microenvironment. These findings indicate that Z13 is a promising PD-1/PD-L1 inhibitor deserving further investigation.

Compound Shenma Jingfu granule alleviates cerebral ischemia via HIF-1α-mediated promotion of angiogenesis

BackgroundShenma Jingfu Granule, a traditional Chinese medicine formula, has been used clinically for the treatment of cerebral circulation insufficiency. However, the mechanism involved in alleviating cerebral ischemia has not yet been fully elucidated.MethodsAn integrated approach involving network pharmacology and transcriptomics was utilized to clarify the potential mechanisms of SMJF Granule. Molecular docking and surface plasmon resonance (SPR) were employed to identify potential targets and ingredients of SMJF Granule. The anti-CI effect of SMJF Granule was determined on the middle cerebral artery occlusion (MCAO) model by using hematoxylin–eosin (H&E) and Nisslʼs staining, as well as triphenyl tetrazolium chloride (TTC) staining, and the potential targets involved in the mechanisms were validated by RT-qPCR and western blotting.ResultsIntegrated analysis revealed the mechanism of SMJF Granule intervening in CI injury might be related to the HIF-1 signaling pathway and angiogenesis. Molecular docking and SPR assays demonstrated robust binding interactions between key compounds like salvianolic acid A and naringenin with the core target HIF-1α protein. The experiment confirmed that SMJF Granule lowered neurological scores, diminished infarct volume, and alleviated histopathological changes in vivo. The possible mechanism of SMJF Granule was due to regulating HIF-1 pathway, which contributed to up-regulating expression of VEGF and vWF in the penumbral region, showing a significant promotion of angiogenesis.ConclusionSMJF Granule promoted angiogenesis through HIF-1α pathway, thereby alleviating cerebral ischemia injury. In addition, our findings provide some evidence that SMJF Granule is a candidate compound for further investigation in treating CI in the clinical.Graphical

Abstract LB030: Building a platform of validated high throughput screening approaches for molecular glue degrader identification

Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.

Integrated analyses for identification of a three-gene signature associated with Chaihu Shugan San formula for hepatocellular carcinoma treatment.

Chaihu Shugan San (CSS) is a well-known traditional herbal formula that has the potential to ameliorate hepatocellular carcinoma (HCC); however, its mechanism of action remains unknown. Here, we identified the key targets of CSS against HCC and developed a prognostic model to predict the survival of patients with HCC. The effect of CSS plus sorafenib on HCC cell proliferation was evaluated using the MTT assay. LASSO-Cox regression was used to establish a three-gene signature model targeting CSS. Correlations between immune cells, immune checkpoints and risk score were determined to evaluate the immune-related effects of CSS. The interactions between the components and targets were validated using molecular docking and Surface Plasmon Resonance (SPR) assays. CSS and sorafenib synergistically inhibited HCC cell proliferation. Ten core compounds and 224 targets were identified using a drug compound-target network. The prognostic model of the three CSS targets (AKT1, MAPK3 and CASP3) showed predictive ability. Risk scores positively correlated with cancer-promoting immune cells and high expression of immune checkpoint proteins. Molecular docking and SPR analyses confirmed the strong binding affinities of the active components and the target genes. Western blot analysis confirmed the synergistic effect of CSS and sorafenib in inhibiting the expression of these three targets. In conclusion, CSS may regulate the activity of immune-related factors in the tumour microenvironment, reverse immune escape, enhance immune responses through AKT1, MAPK3, and CASP3, and synergistically alleviate HCC. The co-administration of sorafenib with CSS has a strong clinical outlook against HCC.

A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella

ABSTRACT Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.

Abstract 4599: A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most prevalent malignant childhood brain tumour. Although prognosis has improved over time, 95% of children with high-risk or relapsed MB eventually succumb to the disease. A key driver gene in high-risk MB is the c-MYC oncogene, for which there are currently no approved inhibitors. Histone deacetylases (HDACs) are transcriptional repressors that are dysregulated in many cancers, making them an attractive therapeutic target. Although several HDAC inhibitors have been FDA-approved for cancer treatment, they have been associated with high toxicity and limited efficacy as single agents. Therefore, simultaneously targeting c-MYC and HDACs may result in synergistic effects that could overcome drug resistance in high-risk MB. Methods and Results: We previously reported a novel pyrido-benzimidazole analogue, SE486-11, which enhanced the therapeutic effect of HDAC inhibitors in MYCN-driven cancers. We recently developed analogues (UNSW-SC compounds) with more potent activity (IC50: 0.017 to 3.70 µM). Here, we showed that these compounds significantly reduced cell viability and induced apoptosis in MB cells. MYC status was a key determinant of sensitivity of UNSW-SC compounds, with more than 3-fold higher cytotoxicity in high MYC compared to low MYC MB cells. The lead compound, UNSW-SC-22, was shown to reduce MYC protein expression and was found to be highly synergistic in enhancing the efficacy of HDAC inhibitors in MB cells. Most importantly, we demonstrated that UNSW-SC-22 was able to freely cross the blood brain barrier, reaching a concentration of 10.8 μM, with a half-life of 1.22 hours. UNSW-SC-22 directly bound to c-MYC and MYCN proteins at low micromolar equilibrium dissociation constant (KD) values which was demonstrated by surface plasmon resonance assay. In a MYCN-driven MB mouse model, we found that UNSW-SC-22 as a single agent at 90 mg/kg decreased tumor growth and prolonged survival. Through RNA-sequencing, we identified 512 differentially expressed genes after in vitro treatment with UNSW-SC-22. Importantly, GSEA analysis revealed MYC targets and cell cycle pathways as highly downregulated pathways, and upregulation in the p53 pathway. Several genes, such as MELK and USP1 were validated as down-stream targets of UNSW-SC-22. As UNSW-SC-22 is a first-in-class anticancer drug, which we are currently testing extensively in other MYC-driven MB mice models. Conclusion: Collectively, our data strongly suggest that c-Myc and MYCN are the molecular targets of UNSW-SC compounds, and these compounds have strong potential to serve as specific targeted therapy to treat subgroup 3 and 4 of MB patients with c-Myc and MYCN overexpression and amplification. Citation Format: Sin Wi Ng, Satyanarayana Gadde, Qian Wang, Belamy B. Cheung, Glenn M. Marshall. A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4599.

Abstract 3134: SDP03923-000-9106, a novel and optimized LIV-1 ADC with superior anti-tumor efficacy in breast cancer xenograft models

Abstract Background: LIV-1, also known as SLC39A6 or ZIP6, is a transmembrane protein belonging to the zinc transporter family. LIV-1 is a promising target for antibody-drug conjugate (ADC) therapy due to its broad expression in tumors and limited normal tissue expression. Ladiratuzumab vedotin, a LIV-1 targeting ADC with payload of MMAE, is now in phase I clinical trial stage and is the only LIV-1 ADC under clinical development worldwide. The preliminary clinical results demonstrated promising efficacy in TNBC (ORR=32%) but no response in HR+/HER2- breast cancer (ORR=0%), and typical MMAE-related adverse events were observed. Herein, we presented a novel LIV-1 directed ADC, SDP03923-000-9106, consisting of a LIV-1 mAb developed in house conjugated to a proprietary topoisomerase I inhibitor via a cleavable linker with an optimized average drug-to-antibody ratio (DAR) of 6. Method: The in vitro binding affinity of SDP03923-000-9106 against LIV-1 protein was measured by Surface Plasmon Resonance (SPR) assays. The expression levels of LIV-1 in different cancer cell lines were detected by flow cytometry and IHC staining. In vitro activity against cancer cell lines was analyzed by CellTiter Glo assays following 10 days of treatment. The in vivo anti-tumor activities were investigated in both CDX and PDX models of breast cancer. Results: SDP03923-000-9106 is a novel anti-LIV-1 ADC comprised of a humanized LIV-1 monoclonal antibody conjugated to the potent topoisomerase I inhibitor (a novel exatecan derivative designed by Hengrui with a better liposolubility and cellular permeability) by a cleavable linker. SDP03923-000-9106 had an optimal drug-to-antibody ratio (DAR) of 6 and showed strong binding affinity to LIV-1 and favorable internalization. SDP03923-000-9106 exhibited robust anti-tumor activity in cell lines across breast cancer, esophageal squamous cancer, gastric cancer, prostate cancer and non-small cell lung cancer. In the LIV-1 highly expressed breast cancer cell line of MCF-7, SDP03923-000-9106 demonstrated a strong inhibitory effect with IC50 of 5.36 nM. Moreover, in a LIV-1 high-expressing patient-derived xenograft model of breast cancer, SDP03923-000-9106 showed pronounced tumor regression at both doses of 3 and 10 mg/kg and showed stronger anti-tumor activity (TGI: 109%) at the same dose of 3 mg/kg versus BMK-MMAE (TGI: 75%; BMK-MMAE is synthesized by Hengrui using the published structure of Ladiratuzumab vedotin). Conclusion: In summary, SDP03923-000-9106 is a novel LIV-1 targeted ADC with a highly permeable payload demonstrating great anti-tumor activity across multiple types of tumors in preclinical studies. The promising preclinical data support advancing SDP03923-000-9106 into clinical testing, and Investigational New Drug (IND) application to NMPA has been submitted. Citation Format: Yanling Gong, Xing Sun, Changyong Yang, Simeng Chen, Wei Zhang, Cheng Liao. SDP03923-000-9106, a novel and optimized LIV-1 ADC with superior anti-tumor efficacy in breast cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3134.

Abstract 3105: Computational integration with phage library to optimize clones of DEFB4A antibodies for cancer diagnostics and therapeutics

Abstract DEFB4A, a human beta-defensin, functions as a potential cancer biomarker, with elevated expression observed in various cancers, making it a candidate for diagnostic and prognostic applications. Additionally, its role in immune modulation suggests that DEFB4A may hold promise as a target for novel cancer treatments, particularly in the context of immunotherapy. This study aimed to develop a stable and high-affinity DEFB4A antibody for clinical and pre-clinical use. To achieve this, a combination of high-diversity phage libraries and computation-assisted analysis was employed to generate single-chain variable fragments (ScFv) clones specifically targeting DEFB4A. This research presents a robust approach for the generation of DEFB4A-targeting single-chain variable fragments (ScFv) from a phage library characterized by a high diversity of 109 unique clones and high titer of 1013 cfu/mL. Computational optimization was then applied to enhance the affinity, stability, and expression of these candidates through rational design and virtual mutagenesis programs. The binding activity of the optimized mutants was validated using biophysical methods such as surface plasmon resonance (SPR) and thermal stability assays. The process showed a remarkable 100-fold increase in affinity and stability. Additionally, the selected ScFv clone was further employed in immunohistochemistry (IHC) to detect DEFB4A in breast, prostate, and gastric cancers, showing higher expression levels compared to initial clones. This study highlights the synergy of high-diversity phage libraries and computational methods in crafting DEFB4A antibodies with enhanced affinity, stability, and expression. The findings advance the field of antibody engineering, emphasizing the crucial integration of experimental and computational approaches. This work lays the groundwork for the development of high-performance biologics with diagnostic and prognostic applications across diverse cancer types. Furthermore, it demonstrates the possibility of integrating computational approaches in antibody development, offering the promise of expediting the process in the future. Citation Format: Yichen Guo, Jina Yom, Andy (Xi) Han, Zhaoying Guo, Eden Zewdu, Rachel Gonzalez, Tianli Qu, Bailey Gilmore, Xuan Liu, Wei Fu, Xiaomin Hu. Computational integration with phage library to optimize clones of DEFB4A antibodies for cancer diagnostics and therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3105.

Abstract 3109: Development of novel mono and bivalent nanobodies against EGFR for targeted cancer therapy

Abstract Introduction: The epidermal growth factor receptor (EGFR) plays a central role in the tumorigenesis of various solid tumors, rendering it a noteworthy target for cancer therapy. Nanobodies (Nbs) offer an appealing therapeutic approach due to their low immunogenicity, rapid clearance, and high targeting specificity. The aim of this study is to isolate, design, and characterize Nbs directed against EGFR. Methods: The Nb library was constructed following llama immunization with cells overexpressing EGFR. After three rounds of panning the phage-display library (both in solid phase and solution), individual clones were screened for EGFR reactivity using ELISA against Virus-Like Particles. Positive clones were sequenced, cloned into an expression vector, and then purified by immobilized metal affinity chromatography (IMAC). The Nbs were characterized through immunofluorescence (IF), flow cytometry (FACS), and surface plasmon resonance (SPR). The most promising candidates were used to design bivalent constructs, which were subsequently characterized by SPR and in a cell proliferation assay. Results: We successfully constructed an EGFR-targeted Nb library, which allowed the selection of multiple EGFR-specific Nbs following the optimization of the panning strategy. Two of the candidates exhibit strong binding to native EGFR in cells and outperform the gold standard anti-EGFR Nb 7D12. They demonstrate enhanced binding parameters in surface plasmon resonance (SPR) assays, with fast association and slow dissociation rates. The homo/heterodimeric bivalent constructs show improved affinity and the ability to inhibit cancer cell proliferation. Conclusion: We were able to develop novel mono and bivalent nanobodies with enhanced binding properties which can be used for targeted cancer therapy. Citation Format: Diana C. Aguilar Cortes, Mauro O. Heitrich, Marisa M. Fernandez, Santiago Werbajh, Gabriela Canziani, Vanesa Zylberman, Emilio L. Malchiod, Osvaldo L. Podhajcer, Sabrina E. Vinzón. Development of novel mono and bivalent nanobodies against EGFR for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3109.

Preclinical Characterization of DPI-4452: A 68Ga/177Lu Theranostic Ligand for Carbonic Anhydrase IX.

The membrane protein carbonic anhydrase IX (CAIX) is highly expressed in many hypoxic or von Hippel-Lindau tumor suppressor-mutated tumor types. Its restricted expression in healthy tissues makes CAIX an attractive diagnostic and therapeutic target. DPI-4452 is a CAIX-targeting cyclic peptide with a DOTA cage, allowing radionuclide chelation for theranostic purposes. Here, we report CAIX expression in multiple tumor types and provide invitro and invivo evaluations of 68Ga-labeled DPI-4452 ([68Ga]Ga-DPI-4452) and 177Lu-labeled DPI-4452 ([177Lu]Lu-DPI-4452). Methods: CAIX expression was assessed by immunohistochemistry with a panel of tumor and healthy tissues. The molecular interactions of complexed and uncomplexed DPI-4452 with CAIX were assessed by surface plasmon resonance and cell-binding assays. In vivo characterization of radiolabeled and nonradiolabeled DPI-4452 was performed in HT-29 colorectal cancer (CRC) and SK-RC-52 clear cell renal cell carcinoma (ccRCC) human xenograft mouse models and in healthy beagle dogs. Results: Overexpression of CAIX was shown in several tumor types, including ccRCC, CRC, and pancreatic ductal adenocarcinoma. DPI-4452 specifically and selectively bound CAIX with subnanomolar affinity. In cell-binding assays, DPI-4452 displayed comparably high affinities for human and canine CAIX but a much lower affinity for murine CAIX, demonstrating that the dog is a relevant species for biodistribution studies. DPI-4452 was rapidly eliminated from the systemic circulation of beagle dogs. The highest uptake of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 was observed in the small intestine and stomach, 2 organs known to express CAIX. Uptake in other organs (e.g., kidneys) was remarkably low. In HT-29 and SK-RC-52 xenograft mouse models, both [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 showed tumor-selective uptake; in addition, [177Lu]Lu-DPI-4452 significantly reduced tumor growth. These results demonstrated the theranostic potential of DPI-4452. Conclusion: DPI-4452 selectively targets CAIX. [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 localized to tumors and were well tolerated in mice. [177Lu]Lu-DPI-4452 demonstrated strong tumor growth inhibition in 2 xenograft mouse models. Thus, the 2 agents potentially provide a theranostic approach for selecting and treating patients with CAIX-expressing tumors such as ccRCC, CRC, and pancreatic ductal adenocarcinoma.

Dauricine attenuates ovariectomized-induced bone loss and RANKL-induced osteoclastogenesis via inhibiting ROS-mediated NF-κB and NFATc1 activity

BackgroundOsteoclast plays an important role in maintaining the balance between bone anabolism and bone catabolism. The abnormality of osteoclast is closely related to osteolytic bone diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastasis. PurposeWe aim to search for natural compound that may suppress osteoclast formation and function. Study designIn this study, we assessed the impact of Dauricine (Dau) on the formation and function of osteoclasts in vitro, as well as its potential in preventing bone loss in an ovariectomy mouse model in vivo. MethodsMultiple in vitro experiments were carried out, including osteoclastogenesis, podosomal belt formation, bone resorption assay, RNA-sequencing, real-time quantitative PCR, ROS level detection, surface plasmon resonance assay, luciferase assay and western blot. To verify the effect in vivo, an ovariectomized mouse model (OVX model) was constructed, and bone parameters were measured using micro-CT and histology. Furthermore, metabolomics analysis was performed on blood serum samples from the OVX model. ResultsIn vitro experiments demonstrated that Dau inhibits RANKL-induced osteoclastogenesis, podosomal belt formation, and bone resorption function. RNA-sequencing results revealed that Dau significantly suppresses genes related to osteoclast. Functional enrichment analysis indicated that Dau's inhibition of osteoclasts may be associated with NF-κB signaling pathway and reactive oxygen metabolism pathway. Molecular docking, surface plasmon resonance assay and western blot analysis further confirmed that Dau inhibits RANKL-induced osteoclastogenesis by modulating the ROS/NF-κB/NFATc1 pathway. Moreover, administration of Dau to OVX-induced mice validated its efficacy in treating bone loss disease. ConclusionDau prevents OVX-induced bone loss by inhibiting osteoclast activity and bone resorption, potentially offering a new approach for preventing and treating metabolic bone diseases such as osteoporosis. This study provides innovative insights into the inhibitory effects of Dau in an in vivo OVX model and elucidates the underlying mechanism.