Off-target Screening Research Articles

A Pathologist's Guide to Non-clinical Safety Assessment of Adoptive Cell Therapy Products.

Through two decades of research and development, adoptive cell therapies (ACTs) have revolutionized treatment for hematologic malignancies. Many of the seven US Food and Drug Administration (FDA)-approved products are proven to be a curative last line of defense against said malignancies. The ACTs, known more commonly as chimeric antigen receptor (CAR) T-cells, utilize engineered lymphocytes to target and destroy cancer cells in a patient-specific, major histocompatibility complex (MHC)-independent manner, acting as "living drugs" that adapt to and surveil the body post-treatment. Despite their efficacy, CAR T-cell therapies present unique challenges in preclinical safety assessment. The safety and pharmacokinetics of CAR T-cells are influenced by numerous factors including donor and recipient characteristics, product design, and manufacturing processes that are not well-predicted by existing in vitro and in vivo preclinical safety models. The CAR therapy-mediated toxicities in clinical settings primarily arise from unintended targeting of non-tumor cells, potential tumorigenicity, and severe immune activation syndromes like cytokine release syndrome and immune effector cell-associated neurotoxicity. Addressing these issues necessitates a deep understanding of CAR target expression in normal tissues, inclusive of the spatial microanatomical distribution, off-target screening, and a deep understanding CAR cell manufacturing practices and immunopathology.

Inhibitor of the non-structural protein 2 protease shows promising efficacy in mouse models of chikungunya

Chikungunya virus (CHIKV) is responsible for the most endemic alphavirus infections called Chikungunya. The endemicity of Chikungunya has increased over the past two decades, and it is a pathogen with pandemic potential. There is currently no approved direct-acting antiviral to treat the disease. As part of our antiviral drug discovery program focused on alphaviruses and the non-structural protein 2 protease, we discovered that J12 and J13 can inhibit CHIKV nsP2 protease and block the replication of CHIKV in cell cultures. Both compounds are metabolically stable to human liver microsomal and S9 enzymes. J13 has excellent oral bioavailability in pharmacokinetics studies in mice and ameliorated Chikungunya symptoms in preliminary efficacy studies in mice. J13 exhibited an excellent safety profile in in vitro safety pharmacology and off-target screening assays, making J13 and its analogs good candidates for drug development against Chikungunya.

Abstract PR015: Discovery of LY4050784 (FHD-909), a selective BRM (SMARCA2) ATPase inhibitor for the treatment of BRG1(SMARCA4) mutant cancers

Abstract BRM (SMARCA2) and BRG1 (SMARCA4) are highly homologous ATPases that are members of the BAF (also known as the mSWI/SNF) chromatin remodeling complex. BRM and BRG1 are mutually exclusive enzymatic subunits of BAF complexes, and functional genomic screens have shown a synthetic lethal relationship between the two genes. BRG1 is frequently mutated in cancer, including in approximately 10% of non-small cell lung carcinomas. Selective inhibition of BRM is a mechanism by which BRG1-mutated cancer cell growth would be affected by losing BRM function, while normal tissues should be spared as they still express functional BRG1. Given that the ATPase domains of BRM and BRG1 are 92% identical, the identification of selective enzymatic inhibitors of BRM has been challenging. Here, we report the discovery of a novel, highly potent compound that selectively inhibits BRM over BRG1 and that also possesses excellent oral pharmacokinetics. LY4050784 (aka FHD-909) inhibits BRM in cell-based transcriptional and proliferation assays and is greater than 30-fold selective over BRG1. It is also selective against other helicases and does not show any significant activity in off-target screening panels. Dosing of mice carrying BRG1-mutant xenografts causes target gene modulation that is correlated with compound exposure. Treatment of A549 and RERF-LC-AI xenografts with LY4050784 led to tumor growth inhibition of 87% and 96%, respectively, at well-tolerated doses, and significant tumor growth inhibition, including regression, was also achieved in BRG1-mutant models NCI-2126 and NCI-1793. The results suggest that our compound has first-in-class potential as a potent and selective BRM ATPase inhibitor for the treatment of BRG1-mutated cancers, and we are planning to file an IND in Q2 of 2024. Citation Format: Janice Y. Lee, Nathan Brooks, Brandon Antonakos, Bryan Perria, Candace Langan, Bonita D. Jones, Robert Stephen Flack, Zhifang Li, David Terry, Ross Wallace, Robert Bondi, Gereint Sis, Ronee Baracani, Maralee McVean, Gabrielle Kolakowski, Dayo Osimboni, Kevin Wilson. Discovery of LY4050784 (FHD-909), a selective BRM (SMARCA2) ATPase inhibitor for the treatment of BRG1(SMARCA4) mutant cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR015.

Abstract 724: MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling

Abstract The development of novel antibody drugs is a lengthy journey. Many factors can lead to failures during clinical development. According to reports, off-target binding of antibodies could also be a significant cause. It has been stated that about 25% of preclinical candidates might have off-target issues. With the emergence of new modalities like CD3 bispecific antibodies, ADCs, and CAR-T therapies, early-stage off-target screening of antibodies has become even more crucial. Although techniques such as immunohistochemistry, ELISA, and flow cytometry (FACS) have been used for off-target testing of antibodies, these methods still lack sensitivity and specificity, and their throughput is limited. To address the need for antibody target deconvolution and receptor identification, we developed a novel membrane protein screening array-AB5000 (MPSA-AB5000). MPSA-AB5000 is a high-throughput cell-based platform for identifying the targets of antibodies and other ligands that bind to membrane proteins. MPSA-AB5000 is an excellent solution for antibody specificity screening, which enables early off-target screening and can de-risk the antibody drug development program. The MPSA-AB5000 makes use of the full coverage of unique human membrane proteins, including receptors, transporters, enzymes, miscellaneous, and others. Each membrane protein is expressed in live cells to ensure native conformation. In addition, the same membrane protein clones with a C-terminal epitope tag were constructed, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, MPSA-AB5000 uses luciferase reporter cell line detection. The wash-free strategy reduces false negatives, and signal amplification makes remarkable discrimination. Compared to similar technologies, we possess significant advantages in the following aspects: a. High sensitivity and sigh throughput: our technology platform offers not only high sensitivity, capable of detecting subtle interactions but also supports high-throughput screening, allowing for the evaluation of a large number of samples in a short timeframe. b. Avoidance of false negative signals: by eliminating washing steps, our method reduces the risk of false negative signals, ensuring reliable detection of non-specific binding. c. Comprehensive validation approaches: We employ various validation methods such as FACS and ELISA to eliminate false positive signals and enhance the accuracy and credibility of our results. All the advantages ensure that MPSA-AB5000 is more rapid, simple, and highly sensitive than traditional assays. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling [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 724.

Abstract 6023: 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity

Abstract Despite significant developments over the last few decades, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Prostate specific membrane antigen (PSMA), a prostate cancer-specific membrane marker which directly correlates with androgen independence, metastasis, and disease progression, has been well-established as a radioligand target for the diagnosis and treatment of mCRPC. Lutetium-177 (177Lu)-PSMA-617 (PLUVICTOTM) was approved in 2022 for the treatment of progressive PSMA-positive mCRPC. However, only 30% of patients showed a radiological response in the registrational trial. Meanwhile, recent clinical experience has shown very limited efficacy in low PSMA expressing patients. These data collectively request for further improvement in the clinical benefit of PSMA-targeted radiotherapy. Actinium-225 (225Ac), an alpha emitter, is significantly more potent on cancer cell killing and has a shorter range in tissue penetration when compared to the beta emitter 177Lu. Such a profile supports the development of 225Ac based radiotherapies. Using our proprietary Clear-XTM technology platform, we developed 225Ac-FL-020, a novel 225Ac-based PSMA radioligand therapy candidate which has demonstrated prominent pre-clinical properties. We have assessed the binding affinity against PSMA of the non-labeled vector FL-020 in vitro. The in vivo biodistribution profile of FL-020 was characterized by SPECT/CT imaging and biodistribution using Indium-111 (111In)-FL-020 in PSMA high LNCaP tumor-bearing nude mice. In addition, anti-tumor activities of 225Ac-FL-020 were evaluated in the LNCaP xenograft model and directly compared to 225Ac-PSMA-617. FL-020 was found bound to LNCaP cells with an IC50 value of 51.55 nM. Meanwhile, the off-target screening showed that less than 50% inhibition of binding or activity was observed by FL-020 at 10 µM against 85 targets including receptors, ion channels, enzymes, and transporters, indicating the high selectivity of FL-020. Moreover, 111In-FL-020 displayed a very promising in vivo distribution profile with high and sustained tumor uptake and fast systemic clearance. Furthermore, 225Ac-FL-020 exhibited superior anti-tumor activity compared to 225Ac-PSMA-617 at the same dose level (10 KBq/mouse) in the LNCaP xenograft model with a favorable safety profile as indicated by body weight and hematological parameters. A mechanistic study was also conducted in 225Ac-FL-020-treated LNCaP tumor samples where DNA double-strand breaks and tumor cell apoptosis were observed, confirming the MOA of alpha emitters. Taken together, these results collectively demonstrate that 225Ac-FL-020 is a potent and selective PSMA-targeting radioligand therapy candidate with superior anti-tumor activity and a favorable safety profile warranting further clinical development. Citation Format: Fa Liu, Jiangwei Zhang, Junyu Yang, Kirstine T. Thrane, Mathias W. Hallund, Rikke V. Grønlund, Nicholas C. Wong. 225Ac-FL-020 is a novel PSMA-targeting radionuclide drug conjugate (RDC) with superior in vivo anti-tumor activity [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 6023.

Abstract 3230: Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers

Abstract BRM (SMARCA2) and BRG1 (SMARCA4) are highly homologous ATPases that are members of the BAF (also known as the mSWI/SNF) chromatin remodeling complex. BRM and BRG1 are mutually exclusive enzymatic subunits of BAF complexes, and functional genomic screens have shown a synthetic lethal relationship between the two genes. BRG1 is frequently mutated in cancer, including in approximately 10% of non-small cell lung carcinomas. Selective inhibition of BRM is a mechanism by which BRG1-mutated cancer cell growth would be affected by losing BRM function, while normal tissues should be spared as they still express functional BRG1. Given that the ATPase domains of BRM and BRG1 are 92% identical, the identification of selective enzymatic inhibitors of BRM has been challenging. Here, we report the discovery of novel, highly potent compounds that selectively inhibit BRM over BRG1 as seen in cell-based transcription and proliferation assays, that also possess excellent oral pharmacokinetics. Our lead compounds inhibit BRM in a cell-based reporter assay with an unbound IC50 of 1 nM and are greater than 20-fold selective over BRG1. They are also selective against other helicases and do not show any significant activity in off-target screening panels. Dosing of mice carrying BRG1-mutant xenografts causes target gene modulation that is correlated with compound exposure. Treatment of A549 and RERF-LC-AI xenografts with lead compounds led to tumor growth inhibition of 87% and 96%, respectively, at well-tolerated doses, and significant tumor growth inhibition was also achieved in multiple other BRG1-mutant models. The results suggest that our compounds have first-in-class potential as potent and selective BRM ATPase inhibitors for the treatment of BRG1-mutated cancers, and we are planning to file an IND in 2024. Citation Format: Janice Y. Lee, Nathan Brooks, Brandon Antonakos, Bryan Perria, Candace Langan, Bonita D. Jones, Robert Stephen Flack, Zhifang Li, David Terry, Ross Wallace, Robert Bondi, Gereint Sis, Ronee Baracani, Maralee McVean, Gabrielle Kolakowski, Dayo Osimboni, Kevin Wilson. Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers [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 3230.

Comparative safety, pharmacokinetics, and off-target assessment of 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane in mouse and dog: implications for therapeutic development.

The modified phytochemical derivative, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12), has been identified as a potential therapeutic platform based on its capacity to improve disease outcomes in models of neurodegeneration and cancer. However, comprehensive safety studies investigating pathology and off-target binding have not been conducted. To address this, we administered C-DIM12 orogastrically to outbred male CD-1 mice for 7days (50mg/kg/day, 200mg/kg/day, and 300mg/kg/day) and investigated changes in hematology, clinical chemistry, and whole-body tissue pathology. We also delivered a single dose of C-DIM12 (1mg/kg, 5mg/kg, 25mg/kg, 100mg/kg, 300mg/kg, 1,000mg/kg) orogastrically to male and female beagle dogs and investigated hematology and clinical chemistry, as well as plasma pharmacokinetics over 48-h. Consecutive in-vitro off-target binding through inhibition was performed with 10μM C-DIM12 against 68 targets in tandem with predictive off-target structural binding capacity. These data show that the highest dose C-DIM12 administered in each species caused modest liver pathology in mouse and dog, whereas lower doses were unremarkable. Off-target screening and predictive modeling of C-DIM12 show inhibition of serine/threonine kinases, calcium signaling, G-protein coupled receptors, extracellular matrix degradation, and vascular and transcriptional regulation pathways. Collectively, these data demonstrate that low doses of C-DIM12 do not induce pathology and are capable of modulating targets relevant to neurodegeneration and cancer.

Evobrutinib pathway to its major metabolite M463-2 and insights from a biotransformation and DDI perspective

Evobrutinib is a highly selective, covalent, central nervous system-penetrant Bruton’s tyrosine kinase (BTK) inhibitor, currently in Phase III trials for the treatment of relapsing multiple sclerosis. One major circulating metabolite of evobrutinib has been previously identified as the racemic dihydro-diol M463-2 (MSC2430422) in a Phase I human mass balance study. Phenotyping experiments were conducted to confirm the metabolic pathway of evobrutinib to M463-2. Ratio of the enantiomers was determined by enantioselective liquid chromatography with tandem mass spectrometry analysis of plasma samples from humans and preclinical species. Drug-drug interaction (DDI) characterisation, evaluation of pharmacological activity on BTK, and off-target screening experiments followed assessing safety of the metabolite. The biotransformation of evobrutinib to M463-2 was determined to be a two-step process with a CYP-mediated oxidation acting to form an epoxide intermediate, which was further hydrolysed by soluble and mitochondrial epoxide hydrolase. Only the (S)-enantiomer was determined to be a major metabolite, the (R)-enantiomer was minor. In vitro studies demonstrated the (S)-enantiomer lacked clinically relevant pharmacological activity, off-target effects and DDIs. The biotransformation of evobrutinib to its major metabolite has been elucidated, with the major (S)-enantiomer being shown to pose no on/off target or DDI risks.

Identification and validation of fusidic acid and flufenamic acid as inhibitors of SARS-CoV-2 replication using DrugSolver CavitomiX

In this work, we present DrugSolver CavitomiX, a novel computational pipeline for drug repurposing and identifying ligands and inhibitors of target enzymes. The pipeline is based on cavity point clouds representing physico-chemical properties of the cavity induced solely by the protein. To test the pipeline’s ability to identify inhibitors, we chose enzymes essential for SARS-CoV-2 replication as a test system. The active-site cavities of the viral enzymes main protease (Mpro) and papain-like protease (Plpro), as well as of the human transmembrane serine protease 2 (TMPRSS2), were selected as target cavities. Using active-site point-cloud comparisons, it was possible to identify two compounds—flufenamic acid and fusidic acid—which show strong inhibition of viral replication. The complexes from which fusidic acid and flufenamic acid were derived would not have been identified using classical sequence- and structure-based methods as they show very little structural (TM-score: 0.1 and 0.09, respectively) and very low sequence (~ 5%) identity to Mpro and TMPRSS2, respectively. Furthermore, a cavity-based off-target screening was performed using acetylcholinesterase (AChE) as an example. Using cavity comparisons, the human carboxylesterase was successfully identified, which is a described off-target for AChE inhibitors.

Examination of in vitro and in vivo antitumor activity of Menin-MLL inhibitor, D0060-319.

e15119 Background: Rearrangements of the mixed-lineage leukemia (MLL) gene result in the production of MLL fusion proteins, which occurs in 5-10% of acute leukemia in adults and > 70% of infant leukemia, respectively. Menin protein, encoded by the MEN1 gene, interacts with MLL fusion proteins to induce leukemia. D0060-319, a Menin-MLL inhibitor, shows a significant antitumor effect in vivo and in vitro. Methods: The fluorescence polarization (FP) binding assay to assess inhibition of D0060-319 to Menin-MLL interaction. The cross reactivity of 44 molecular targets was tested to assess their off-target binding potential. The anti-proliferative ability of D0060-319 was evaluated by CCK-8 in different cell lines. The in vivo anti-tumor effect of D0060-319 was evaluated in a human MV4-11 and MOLM-13 cell mouse xenograft tumor model. Briefly, five million MV4-11 cells or 500 thousand MOLM-13 cells were subcutaneously implanted the right flank of CB-17 SCID mice to establish cell mouse xenograft tumor model. When tumor volume reached 100-120mm3, the animals were grouped and administrated with D0060-319 at the dose of 25, 50, 75, 100 mg/kg or vehicle at twice daily for 21days. Tumor measurements and body weights were performed twice weekly to assess the antitumor efficacy and tolerability of D0060-319. Results: D0060-319 competitively binds Menin to inhibit the interaction with MLL fusion protein with IC50 of 7.46nM, has high antiproliferative activity in MV4-11 and MOLM-13 cell lines containing MLL fusion proteins with IC50 values of 4.0nM and 1.7nM, respectively. However, in Kasumi-1, K562, HL-60 and KG-1 cell lines lacking MLL rearrangement, D0060-319 antiproliferative activity with IC50 > 10 μM, indicating selectivity for leukemia cell lines containing MLL fusion proteins active. In the MV4-11 cell mouse xenograft tumor model, D0060-319 significantly inhibited tumor growth at the dose of 25mg/kg, with a tumor growth inhibition (TGI) rate of 82.97%, and other doses (50, 75 and 100mg/kg) tumor completely regressed. In the MOLM-13 cell mouse xenograft tumor model, a dose-dependent anti-tumor effect was observed after 14 days of administration, with TGIs of 81.08%, 88.47% and 90.26% at the dose of 50, 75 or 100 mg/kg, respectively. In both CDX models, no bodyweight loss was observed, suggesting that D0060-319 was well tolerated. Furthermore, off-target screening assays showed that D0060-319 did not cross-react with any of the 44 molecular targets at 10 μM, indicating that D0060-319 is highly selective, high-affinity for the MLL-binding pocket on Menin. Conclusions: In MLL-rearranged leukemia, D0060-319 showed high antitumor activity and good tolerability both in vitro and in vivo.

CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B.

Hemophilia B (HB) is an X-linked recessive disease caused by F9 gene mutation and functional coagulation factor IX (FIX) deficiency. Patients suffer from chronic arthritis and death threats owing to excessive bleeding. Compared with traditional treatments, gene therapy for HB has obvious advantages, especially when the hyperactive FIX mutant (FIX-Padua) is used. However, the mechanism by which FIX-Padua works remains ambiguous due to a lack of research models. Here, in situ introduction of F9-Padua mutation was performed in human induced pluripotent stem cells (hiPSCs) via CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs). The hyperactivity of FIX-Padua was confirmed to be 364% of the normal level in edited hiPSCs-derived hepatocytes, providing a reliable model for exploring the mechanism of the hyperactivity of FIX-Padua. Moreover, the F9 cDNA containing F9-Padua was integrated before the F9 initiation codon by CRISPR/Cas9 in iPSCs from an HB patient (HB-hiPSCs). Integrated HB-hiPSCs after off-target screening were differentiated into hepatocytes. The FIX activity in the supernatant of integrated hepatocytes showed a 4.2-fold increase and reached 63.64% of the normal level, suggesting a universal treatment for HB patients with various mutations in F9 exons. Overall, our study provides new approaches for the exploration and development of cell-based gene therapy for HB.

Differences in breast cancer survival and stage by age in off-target screening groups: a population-based retrospective study

Age is an important prognostic factor in breast cancer. The target age to screen is under debate. This study aimed to assess the influence of age on the diagnosis and survival among women with breast cancer. This was a retrospective cohort study of the Population-Based Cancer Registry of Campinas, Brazil, and included all women diagnosed from 2010 to 2014. The outcomes assessed were overall survival and stage. For statistical analyses, the Kaplan-Meier method, log-rank tests, and chi-square tests were used. The sample comprised 1741 women aged 40 to 79 years. Diagnoses at stages 0 to II were the more frequent. In the 40 to 49 years and 50 to 59 years age groups, the frequency of stage 0 (in situ) was 20.5% and 14.9% (P=.022), respectively, and the frequency of stage I was 20.2% and 25.8% (P=.042), respectively. The mean overall survival was 8.9 years (8.6-9.2) in the 40 to 49 years age group and 7.7 years (7.3-8.1) in the 70 to 79 years age group. The 5-year overall survival was higher in the 40 to 49 years age group than in the 50 to 59 years age group for stage 0 (in situ) (100.0% vs 95.0%; P=.036) and stage III (77.4% vs 66.2%; P=.046) diagnoses. The 5-year overall survival was higher in 60 to 69 years age group than in the 70 to 79 years age group for stages I (94.6% vs 86.5%; P=.002) and III (83.5% vs 64.9%; P=.010). In all age groups, significant differences in survival were not observed for stage 0 (in situ) vs stage I diagnoses, stage 0 vs stage II diagnoses, and stage I vs stage II diagnoses. Women aged 40 to 49 years had the highest proportion of in situ tumors, and stages III and IV accounted for about one-third of the cases in all age groups. There was no difference in the overall survival for stage 0 (in situ) vs stage I or II diagnoses in all age groups.

Abstract 2645: A high-throughput platform for targets screening by membrane protein library

Abstract The specificity of antibodies, CAR-T cells and other biologic therapeutics to their binding targets is important to evaluate drug quality. The non-specific binding may lead serious toxic side effects. Therefore, specificity detection is an important step in the evaluation process of drug development. At present, the non-specific binding of antibody drugs is often verified only after serious adverse reactions founding in clinical trials, which may result in drug safety accidents and the abandonment of previous studies. To address the need for antibody target deconvolution and receptor identification, we developed a novel approach “AB5000”. AB5000 is an excellent solution for antibody specificity screening which enable early off-target screening and can de-risk the antibody drug development program. The AB5000 makes use of the full coverage membrane protein, representing over 5465 unique human membrane proteins including receptors, transporters, enzymes, miscellaneous and other membrane protein. Gene vector was transfected into target cells. Each membrane protein introduces a C-terminal epitope tag, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, we use luciferase reporter cell line system which is more rapid, simple and highly sensitive than flow cytometry or other binding assays. The AB5000 has widely application in testing the specificity of antibodies, deconvolution of orphan antibodies and ligands, detection of CAR-T proteins. The AB5000 represents a rapid, comprehensive, and highly sensitive platform for identifying the membrane protein targets of a wide variety of antibodies, protein ligands, and functional interactions. It is a promising powerful tool for biotherapeutic modalities development. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. A high-throughput platform for targets screening by membrane protein library [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2645.

Identification of a chemical probe for lipid kinase phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ)

Phosphatidylinositol-5-phosphate 4-kinase gamma (PI5P4Kγ), which phosphorylates phosphatidylinositol-5-monophosphate (PI(5)P), is a human lipid kinase with intriguing roles in inflammation, T cell activation, autophagy regulation, immunity, heart failure, and several cancers. To provide a high-quality chemical tool that would enable additional characterization of this protein, we designed and evaluated a potent, selective, and cell-active inhibitor of human PI5P4Kγ. We describe the use of the PI5P4Kγ NanoBRET assay to generate structure–activity relationships (SAR), support chemical probe (2) design, and identify a structurally related negative control (4). We have characterized the binding of our chemical probe to PI5P4Kγ using orthogonal assay formats reliant on competition with an ATP-competitive reagent. Based on our results in these assays, additional ATP titration studies, and published co-crystal structures with structurally related compounds, we hypothesize that 2 binds in the ATP active site of PI5P4Kγ. Kinome-wide profiling complemented by further off-target screening confirmed the selectivity of both our chemical probe and negative control. When a breast cancer cell line (MCF-7) was treated with compound 2, increased mTORC1 signaling was observed, demonstrating that efficacious binding of 2 to PI5P4Kγ in cells results in activation of a negative feedback loop also reported in PI5P4Kγ knockout mice.

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor.

The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

Simultaneous Global Phase I Studies of a Differentiated BTK Inhibitor, Dtrmwxhs-12, in Relapsed/Refractory Patients with Chronic Lymphocytic Leukemia and B-Cell Lymphomas

Simultaneous Global Phase I Studies of a Differentiated BTK Inhibitor, Dtrmwxhs-12, in Relapsed/Refractory Patients with Chronic Lymphocytic Leukemia and B-Cell Lymphomas

Multifunctional Enkephalin Analogs with a New Biological Profile: MOR/DOR Agonism and KOR Antagonism.

In our previous studies, we developed a series of mixed MOR/DOR agonists that are enkephalin-like tetrapeptide analogs with an N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety at the C-terminus. Further SAR study on the analogs, initiated by the findings from off-target screening, resulted in the discovery of LYS744 (6, Dmt-DNle-Gly-Phe(p-Cl)-Ppp), a multifunctional ligand with MOR/DOR agonist and KOR antagonist activity (GTPγS assay: IC50 = 52 nM, Imax = 122% cf. IC50 = 59 nM, Imax = 100% for naloxone) with nanomolar range of binding affinity (Ki = 1.3 nM cf. Ki = 2.4 nM for salvinorin A). Based on its unique biological profile, 6 is considered to possess high therapeutic potential for the treatment of chronic pain by modulating pathological KOR activation while retaining analgesic efficacy attributed to its MOR/DOR agonist activity.

A practical guide to secondary pharmacology in drug discovery

Secondary pharmacological profiling is increasingly applied in pharmaceutical drug discovery to address unwanted pharmacological side effects of drug candidates before entering the clinic. Regulators, drug makers and patients share a demand for deep characterization of secondary pharmacology effects of novel drugs and their metabolites. The scope of such profiling has therefore expanded substantially in the past two decades, leading to the implementation of broad in silico profiling methods and focused in vitro off-target screening panels, to identify liabilities, but also opportunities, as early as possible. The pharmaceutical industry applies such panels at all stages of drug discovery routinely up to early development. Nevertheless, target composition, screening technologies, assay formats, interpretation and scheduling of panels can vary significantly between companies in the absence of dedicated guidelines. To contribute towards best practices in secondary pharmacology profiling, this review aims to summarize the state-of-the art in this field. Considerations are discussed with respect to panel design, screening strategy, implementation and interpretation of the data, including regulatory perspectives. The cascaded, or integrated, use of in silico and off-target profiling allows to exploit synergies for comprehensive safety assessment of drug candidates.

New Advances in Cell Microarray Technology to Expand Applications in Target Deconvolution and Off-Target Screening.

Identifying the cell surface receptors of natural ligands, and deconvoluting the receptor targets of candidate drug leads, presents a challenge in medical research and drug discovery. Traditionally, success rates have been low, and screening efforts often generate numerous false-positive hits that require extensive follow-up to either validate or disregard. If successful, receptor identification enables the discovery of previously unknown, disease-relevant targets, provides critical insights into biological pathways and disease processes, and allows for secondary targets to be uncovered. By expressing the majority of the human plasma membrane proteome in human cells on glass slides in situ, human cell microarray technology provides a powerful approach for identifying receptor target interactions. This approach significantly increases the success rates in identifying specific primary receptor targets and off-targets while limiting the number of false-positive hits. Here we describe cell microarray technology, focusing on new advances including the use of whole cells as bait for receptor interactions, and the inclusion of secreted proteins that widens the utility of the technology in off-target screening.

Knowledge-Based Approaches to Off-Target Screening.

Selective binding of a drug for its target vs other proteins is an important consideration in designing compounds with minimal risk of toxicity and therefore a key aspect of lead optimization. Screening all compounds against all known off-target proteins would be prohibitively expensive, so project teams must decide which compounds to screen in which assays. This chapter describes informatics-based methods that help prioritize testing, including screening minipanels and prioritization using predictive models (e-counterscreening).