Pub Date : 2025-02-28DOI: 10.1016/j.slasd.2025.100224
Aislinn Mayfield , Xin Zhang, Ivan Efremov, Michael G Kauffman, John F Reilly, Bahareh Eftekharzadeh
Biomolecular condensates (BMCs) are crucial for cellular organization and function, and their dysregulation is linked to neurological, oncologic and inflammatory diseases. This highlights the need for advanced investigative tools leading to targeted BMC therapeutics. To address this need, Nereid Therapeutics uses Corelet™ technology and an automated high-throughput screening (HTS) platform to precisely quantify phase separation events and identify BMC modulators for previously undruggable targets. Hundreds of thousands of small molecules have been screened utilizing Corelet technology, yielding small molecule BMC-modulating compounds which serve as the basis for the development of targeted therapies for diseases with high unmet need.
{"title":"Corelet™ platform: Precision high throughput screening for targeted drug discovery of biomolecular condensates","authors":"Aislinn Mayfield , Xin Zhang, Ivan Efremov, Michael G Kauffman, John F Reilly, Bahareh Eftekharzadeh","doi":"10.1016/j.slasd.2025.100224","DOIUrl":"10.1016/j.slasd.2025.100224","url":null,"abstract":"<div><div>Biomolecular condensates (BMCs) are crucial for cellular organization and function, and their dysregulation is linked to neurological, oncologic and inflammatory diseases. This highlights the need for advanced investigative tools leading to targeted BMC therapeutics. To address this need, Nereid Therapeutics uses Corelet™ technology and an automated high-throughput screening (HTS) platform to precisely quantify phase separation events and identify BMC modulators for previously undruggable targets. Hundreds of thousands of small molecules have been screened utilizing Corelet technology, yielding small molecule BMC-modulating compounds which serve as the basis for the development of targeted therapies for diseases with high unmet need.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100224"},"PeriodicalIF":2.7,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143538195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-23DOI: 10.1016/j.slasd.2025.100222
Thomas S. Dexheimer , Zahra Davoudi , Nathan P. Coussens , Thomas Silvers , Joel Morris , Naoko Takebe , Rabih Said , Jeffrey A. Moscow , James H. Doroshow , Beverly A. Teicher
Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is a key contributor to cancer, making PI3K inhibitors a promising approach for targeted therapy. The selectivity of available inhibitors varies across different PI3K isoforms. Alpelisib and inavolisib are selective for the α-isoform, while duvelisib targets the δ- and γ-isoforms, and copanlisib is a pan-PI3K inhibitor, active against all isoforms. This study investigated the activity of these four PI3K inhibitors in combination with other targeted agents using multi-cell type tumor spheroids composed of 60% malignant cells, 25% endothelial cells, and 15% mesenchymal stem cells. Twenty-nine tumor spheroid models were evaluated, including twenty-six patient-derived cancer cell lines from the NCI Patient-Derived Models Repository and three established cell lines from the NCI-60 human tumor cell line panel. Additive and/or synergistic effects were observed with alpelisib or inavolisib or copanlisib in combination with a RAS/MEK/ERK pathway inhibitor, either selumetinib (MEK), ravoxertinib (ERK 1/2), or tovorafenib (DAY101, RAF). Combinations of each of these three PI3K inhibitors with the KRAS mutation specific inhibitors MTRX1133 (KRAS G12D) or sotorasib (KRAS G12C) had selective activity in cell lines harboring the corresponding target. Lastly, combination effects were observed from vertical inhibition of the PI3K/AKT/mTOR pathway with a PI3K inhibitor in combination with either the mTORC1/2 inhibitor sapanisertib or an AKT inhibitor, ipatasertib or afuresertib.
{"title":"Combinatorial screen of targeted agents with the PI3K inhibitors inavolisib, alpelisib, duvelisib, and copanlisib in multi-cell type tumor spheroids","authors":"Thomas S. Dexheimer , Zahra Davoudi , Nathan P. Coussens , Thomas Silvers , Joel Morris , Naoko Takebe , Rabih Said , Jeffrey A. Moscow , James H. Doroshow , Beverly A. Teicher","doi":"10.1016/j.slasd.2025.100222","DOIUrl":"10.1016/j.slasd.2025.100222","url":null,"abstract":"<div><div>Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is a key contributor to cancer, making PI3K inhibitors a promising approach for targeted therapy. The selectivity of available inhibitors varies across different PI3K isoforms. Alpelisib and inavolisib are selective for the α-isoform, while duvelisib targets the δ- and γ-isoforms, and copanlisib is a pan-PI3K inhibitor, active against all isoforms. This study investigated the activity of these four PI3K inhibitors in combination with other targeted agents using multi-cell type tumor spheroids composed of 60% malignant cells, 25% endothelial cells, and 15% mesenchymal stem cells. Twenty-nine tumor spheroid models were evaluated, including twenty-six patient-derived cancer cell lines from the NCI Patient-Derived Models Repository and three established cell lines from the NCI-60 human tumor cell line panel. Additive and/or synergistic effects were observed with alpelisib or inavolisib or copanlisib in combination with a RAS/MEK/ERK pathway inhibitor, either selumetinib (MEK), ravoxertinib (ERK 1/2), or tovorafenib (DAY101, RAF). Combinations of each of these three PI3K inhibitors with the KRAS mutation specific inhibitors MTRX1133 (KRAS G12D) or sotorasib (KRAS G12C) had selective activity in cell lines harboring the corresponding target. Lastly, combination effects were observed from vertical inhibition of the PI3K/AKT/mTOR pathway with a PI3K inhibitor in combination with either the mTORC1/2 inhibitor sapanisertib or an AKT inhibitor, ipatasertib or afuresertib.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100222"},"PeriodicalIF":2.7,"publicationDate":"2025-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143506646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-11DOI: 10.1016/j.slasd.2025.100221
Nihal Bharath , Emma DiPietro , Olivia Durfee , Ina Kycia , Jennifer Splaine , Praveen Sethupathy , Michael S. Rogers , Khashayar Vakili
Fibrolamellar carcinoma (FLC) is a primary liver cancer with a poor prognosis, primarily due to the lack of effective chemotherapeutic options. The DNAJB1-PRKACA (DP) gene fusion is recognized as the key oncogenic driver in FLC. This fusion arises from a ∼400 kb heterozygous deletion on chromosome 19, which fuses exon 1 of DNAJB1 with exons 2–10 of PRKACA, the gene encoding the catalytic subunit of protein kinase A (PKA). While targeting DP is considered a promising therapeutic approach, attempts to inhibit the kinase function of the DP fusion protein have been largely unsuccessful due to off-target effects on wild-type PKA.
In response to this challenge, we developed a high-throughput screening (HTS) assay to identify inhibitors of DP's downstream signaling pathways involved in transcriptional regulation. Our previous research identified LINC00473 as a transcriptional marker for DP protein expression, and LINC00473 is known to be upregulated in FLC tumors. Additionally, evidence suggests that LINC00473 promotes FLC tumor growth.
Based on the relationship between DP and LINC00473 expression, we engineered the HEK-DP-Luc reporter cell line by modifying HEK293 cells to express DP at the endogenous locus and to express the NanoLuc luciferase gene under the control of the LINC00473 promoter and enhancer. We have optimized the HEK-DP-Luc cells for HTS, and here we present our pipeline for primary screening and counter-screening to identify compounds that inhibit DP's downstream transcriptional activity. This HTS platform provides a novel approach for therapeutic drug discovery in FLC.
{"title":"A novel high-throughput screening platform to identify inhibitors of DNAJB1-PRKACA-driven transcriptional activity in fibrolamellar carcinoma","authors":"Nihal Bharath , Emma DiPietro , Olivia Durfee , Ina Kycia , Jennifer Splaine , Praveen Sethupathy , Michael S. Rogers , Khashayar Vakili","doi":"10.1016/j.slasd.2025.100221","DOIUrl":"10.1016/j.slasd.2025.100221","url":null,"abstract":"<div><div>Fibrolamellar carcinoma (FLC) is a primary liver cancer with a poor prognosis, primarily due to the lack of effective chemotherapeutic options. The <em>DNAJB1-PRKACA</em> (DP) gene fusion is recognized as the key oncogenic driver in FLC. This fusion arises from a ∼400 kb heterozygous deletion on chromosome 19, which fuses exon 1 of <em>DNAJB1</em> with exons 2–10 of <em>PRKACA</em>, the gene encoding the catalytic subunit of protein kinase A (PKA). While targeting DP is considered a promising therapeutic approach, attempts to inhibit the kinase function of the DP fusion protein have been largely unsuccessful due to off-target effects on wild-type PKA.</div><div>In response to this challenge, we developed a high-throughput screening (HTS) assay to identify inhibitors of DP's downstream signaling pathways involved in transcriptional regulation. Our previous research identified <em>LINC00473</em> as a transcriptional marker for DP protein expression, and <em>LINC00473</em> is known to be upregulated in FLC tumors. Additionally, evidence suggests that <em>LINC00473</em> promotes FLC tumor growth.</div><div>Based on the relationship between DP and <em>LINC00473</em> expression, we engineered the HEK-DP-Luc reporter cell line by modifying HEK293 cells to express DP at the endogenous locus and to express the NanoLuc luciferase gene under the control of the <em>LINC00473</em> promoter and enhancer. We have optimized the HEK-DP-Luc cells for HTS, and here we present our pipeline for primary screening and counter-screening to identify compounds that inhibit DP's downstream transcriptional activity. This HTS platform provides a novel approach for therapeutic drug discovery in FLC.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100221"},"PeriodicalIF":2.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-02-01DOI: 10.1016/j.slasd.2025.100220
Rui Tong Quek , Cyna R. Shirazinejad , Christina L. Young , Kierra S. Hardy , Samuel Lim , Phillip J. Elms , David T. McSwiggen , Timothy J. Mitchison , Pamela A. Silver
Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 h post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.
{"title":"Comparative evaluation of cell-based assay technologies for scoring drug-induced condensation of SARS-CoV-2 nucleocapsid protein","authors":"Rui Tong Quek , Cyna R. Shirazinejad , Christina L. Young , Kierra S. Hardy , Samuel Lim , Phillip J. Elms , David T. McSwiggen , Timothy J. Mitchison , Pamela A. Silver","doi":"10.1016/j.slasd.2025.100220","DOIUrl":"10.1016/j.slasd.2025.100220","url":null,"abstract":"<div><div>Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 h post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100220"},"PeriodicalIF":2.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143082458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-31DOI: 10.1016/j.slasd.2025.100219
Donatella Romaniello , Lorenzo Dall'Olio , Martina Mazzeschi , Anna Francia , Federica Pagano , Valerio Gelfo , Gabriele D'Uva , Enrico Giampieri , Mattia Lauriola
A direct connection between an inflammatory environment and cancer has been extensively proven over the years. We previously reported that the presence of interleukin 1 (IL-1) is responsible for the lack of response to monoclonal antibody targeting epidermal growth factor receptor (EGFR) in colorectal cancer (CRC).
Considering the driver role of NF-kB in controlling the expression of IL-1, herein, we investigate the dynamics of the oscillatory profile of the NF-kB response to monoclonal antibody, on the background of an inflammatory environment. NF-kB is a typical transcription factor that displays intrinsic oscillatory behavior, whose biological relevance in term for example of decoding response to monoclonal antibodies, remains unclear.
Using live cell luciferase techniques, we recorded NF-kB activity over time in response to cetuximab (CTX) alone or in combination with IL-1 cytokines. Our results revealed an additive effect of these two agents on NF-kB activation, which was specific to CTX responsive cells. In contrast, CTX resistant cells did not display a significant change in the NF-kB profile under the IL-1 plus CTX combination. These results suggest an immediate interactive crosstalk between IL-1 and EGFR in the activation of NF-kB signaling pathway, which may lay the basis for the development of drug tolerant persister cells (DTP), leading to CTX resistance.
{"title":"NF-kB oscillation profiles decode response to anti-EGFR monoclonal antibodies","authors":"Donatella Romaniello , Lorenzo Dall'Olio , Martina Mazzeschi , Anna Francia , Federica Pagano , Valerio Gelfo , Gabriele D'Uva , Enrico Giampieri , Mattia Lauriola","doi":"10.1016/j.slasd.2025.100219","DOIUrl":"10.1016/j.slasd.2025.100219","url":null,"abstract":"<div><div>A direct connection between an inflammatory environment and cancer has been extensively proven over the years. We previously reported that the presence of interleukin 1 (IL-1) is responsible for the lack of response to monoclonal antibody targeting epidermal growth factor receptor (EGFR) in colorectal cancer (CRC).</div><div>Considering the driver role of NF-kB in controlling the expression of IL-1, herein, we investigate the dynamics of the oscillatory profile of the NF-kB response to monoclonal antibody, on the background of an inflammatory environment. NF-kB is a typical transcription factor that displays intrinsic oscillatory behavior, whose biological relevance in term for example of decoding response to monoclonal antibodies, remains unclear.</div><div>Using live cell luciferase techniques, we recorded NF-kB activity over time in response to cetuximab (CTX) alone or in combination with IL-1 cytokines. Our results revealed an additive effect of these two agents on NF-kB activation, which was specific to CTX responsive cells. In contrast, CTX resistant cells did not display a significant change in the NF-kB profile under the IL-1 plus CTX combination. These results suggest an immediate interactive crosstalk between IL-1 and EGFR in the activation of NF-kB signaling pathway, which may lay the basis for the development of drug tolerant persister cells (DTP), leading to CTX resistance.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100219"},"PeriodicalIF":2.7,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143076444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-25DOI: 10.1016/j.slasd.2025.100218
Rafael Dariolli , Raphael Nir , Tova Mushlam , Glauco R. Souza , Stephen R. Farmer , Miguel L Batista Jr.
Obesity and type 2 diabetes (T2D) are strongly linked to abnormal adipocyte metabolism and adipose tissue (AT) dysfunction. However, existing adipose tissue models have limitations, particularly in the stable culture of fat cells that maintain physiologically relevant phenotypes, hindering a deeper understanding of adipocyte biology and the molecular mechanisms behind differentiation. Current model systems fail to fully replicate In vivo metabolism, posing challenges in adipose tissue research. Three-dimensional (3D) AT organoids, although promising, present significant handling challenges during long-term culture. As adipocytes maturate and accumulate fat, they develop organotypic characteristics, increasing the buoyancy effect, which causes the organoids to oscillate, complicating culture manipulation and rendering multiple handling steps difficult.
Due to these challenges, most adipose spheroid and organoid models are scaffold-based, despite many cell types' ability to secrete extracellular matrix (ECM) components and self-assemble into aggregates. Scaffold-free 3D organoids have been less explored. To address the shortage of affordable and reliable AT models, we utilized magnetic bioprinting technology to develop a human-derived 3D model of adipose tissue. This system incorporates a magnetic holder that restrains organoids, preventing them from floating and minimizing the risk of loss during manipulation.
This study outlines a protocol for generating In vitro AT-derived organoid using 3D magnetic bioprinting, with a focus on manufacturing, culturing, and assessing the morpho-functional characteristics of late-stage AT organoids. Magnetic bioprinting allows for the replication of tissue structure and function In vitro without the risk of organoid loss, making it an ideal method for high-throughput AT organoid culture. Additionally, the combination of 3D scaffold-free manufacturing with In vitro disease modeling offers a valuable tool for discovering treatments for metabolic diseases such as obesity and T2D.
{"title":"Optimized scaffold-free human 3D adipose tissue organoid culture for obesity and disease modeling","authors":"Rafael Dariolli , Raphael Nir , Tova Mushlam , Glauco R. Souza , Stephen R. Farmer , Miguel L Batista Jr.","doi":"10.1016/j.slasd.2025.100218","DOIUrl":"10.1016/j.slasd.2025.100218","url":null,"abstract":"<div><div>Obesity and type 2 diabetes (T2D) are strongly linked to abnormal adipocyte metabolism and adipose tissue (AT) dysfunction. However, existing adipose tissue models have limitations, particularly in the stable culture of fat cells that maintain physiologically relevant phenotypes, hindering a deeper understanding of adipocyte biology and the molecular mechanisms behind differentiation. Current model systems fail to fully replicate <em>In vivo</em> metabolism, posing challenges in adipose tissue research. Three-dimensional (3D) AT organoids, although promising, present significant handling challenges during long-term culture. As adipocytes maturate and accumulate fat, they develop organotypic characteristics, increasing the buoyancy effect, which causes the organoids to oscillate, complicating culture manipulation and rendering multiple handling steps difficult.</div><div>Due to these challenges, most adipose spheroid and organoid models are scaffold-based, despite many cell types' ability to secrete extracellular matrix (ECM) components and self-assemble into aggregates. Scaffold-free 3D organoids have been less explored. To address the shortage of affordable and reliable AT models, we utilized magnetic bioprinting technology to develop a human-derived 3D model of adipose tissue. This system incorporates a magnetic holder that restrains organoids, preventing them from floating and minimizing the risk of loss during manipulation.</div><div>This study outlines a protocol for generating <em>In vitro</em> AT-derived organoid using 3D magnetic bioprinting, with a focus on manufacturing, culturing, and assessing the morpho-functional characteristics of late-stage AT organoids. Magnetic bioprinting allows for the replication of tissue structure and function <em>In vitro</em> without the risk of organoid loss, making it an ideal method for high-throughput AT organoid culture. Additionally, the combination of 3D scaffold-free manufacturing with <em>In vitro</em> disease modeling offers a valuable tool for discovering treatments for metabolic diseases such as obesity and T2D.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100218"},"PeriodicalIF":2.7,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-18DOI: 10.1016/j.slasd.2025.100216
Stacey E. Chin , Pablo Gallego , Anna Aagaard , Sara Carmen , Nathalie Barrett , Marcin Wolny , Sophie Cloarec , Judy Paterson , Rohan Sivapalan , James Hunt , Thomas V. Murray , Tracy Delaney , Tove Sjögren , Frances Neal
Neurotrophic factor 3 (NTF3) is a cysteine knot protein and a member of the nerve growth factor (NGF) family of cytokines. NTF3 engages the Trk family of receptor tyrosine kinases, playing a pivotal role in the development and function of both the central and peripheral nervous systems. Its involvement in neuronal survival, differentiation, and growth links NTF3 to a spectrum of neurodegenerative diseases. Consequently, targeting NTF3 with antibodies holds promise as a first in class therapeutic opportunity for a wide range of conditions.
Specific and neutralizing antibodies against NTF3 were successfully isolated using phage display. Initial phage display selections revealed a preference of hits for a longer than average complementarity-determining region 3 (CDR3) in the heavy chain variable domain (VH). To investigate this further we developed a long loop length VH CDR3 antibody library that demonstrated increased hit rates versus a standard antibody library and allowed the isolation of IgG that demonstrated inhibition of functional activity, coupled with a favourable kinetic profile.
Structural analysis of the Fab/NTF3 interaction, via X-ray crystallography, unveiled an unconventional interaction wherein regions beyond the longer CDR loops of the Fab induced ordering in a flexible loop on NTF3, which remained disordered in its free antigenic state. This comprehensive approach not only sheds light on the therapeutic potential of NTF3-specific antibodies but also provides critical structural details that enhance our understanding of the complex NTF3-Fab interaction thus offering valuable insights for future antibody design and therapeutic development.
{"title":"Identification of unique binding mode anti-NTF3 antibodies from a novel long VH CDR3 phage display library","authors":"Stacey E. Chin , Pablo Gallego , Anna Aagaard , Sara Carmen , Nathalie Barrett , Marcin Wolny , Sophie Cloarec , Judy Paterson , Rohan Sivapalan , James Hunt , Thomas V. Murray , Tracy Delaney , Tove Sjögren , Frances Neal","doi":"10.1016/j.slasd.2025.100216","DOIUrl":"10.1016/j.slasd.2025.100216","url":null,"abstract":"<div><div>Neurotrophic factor 3 (NTF3) is a cysteine knot protein and a member of the nerve growth factor (NGF) family of cytokines. NTF3 engages the Trk family of receptor tyrosine kinases, playing a pivotal role in the development and function of both the central and peripheral nervous systems. Its involvement in neuronal survival, differentiation, and growth links NTF3 to a spectrum of neurodegenerative diseases. Consequently, targeting NTF3 with antibodies holds promise as a first in class therapeutic opportunity for a wide range of conditions.</div><div>Specific and neutralizing antibodies against NTF3 were successfully isolated using phage display. Initial phage display selections revealed a preference of hits for a longer than average complementarity-determining region 3 (CDR3) in the heavy chain variable domain (VH). To investigate this further we developed a long loop length VH CDR3 antibody library that demonstrated increased hit rates versus a standard antibody library and allowed the isolation of IgG that demonstrated inhibition of functional activity, coupled with a favourable kinetic profile.</div><div>Structural analysis of the Fab/NTF3 interaction, via X-ray crystallography, unveiled an unconventional interaction wherein regions beyond the longer CDR loops of the Fab induced ordering in a flexible loop on NTF3, which remained disordered in its free antigenic state. This comprehensive approach not only sheds light on the therapeutic potential of NTF3-specific antibodies but also provides critical structural details that enhance our understanding of the complex NTF3-Fab interaction thus offering valuable insights for future antibody design and therapeutic development.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100216"},"PeriodicalIF":2.7,"publicationDate":"2025-01-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-17DOI: 10.1016/j.slasd.2025.100215
Kasper P. Lundquist , Isabella Romeo , Raffaele B. Puglielli , Maëlle Pestalozzi , Marie L. Gram , Emily S. Hudson , Ofri Levi , Yoav S. Arava , Charlotte H. Gotfredsen , Mads H. Clausen
Fragment-based screening is an efficient method for early-stage drug discovery. In this study, we aimed to create a fragment library optimized for producing high hit rates against RNA targets. RNA has historically been an underexplored target, but recent research suggests potential for optimizing small molecule libraries for RNA binding. We extended this concept to fragment libraries to produce an RNA optimized fluorinated fragment library. We then screened this library, alongside two non-RNA optimized fragment libraries, against three RNA targets: the human cytoplasmic A-site and the S. cerevisiae tRNAAsp anticodon stem loop with and without nucleobase modifications. The screens yielded 24, 31, and 20 hits against the respective targets. Importantly, statistical analysis confirmed a significant overrepresentation of hits in our RNA optimized library. Based on these findings, we propose guidelines for developing RNA optimized fragment libraries. We hope the guidelines will help expediting fragment-based ligand discovery for RNA targets and contribute to presenting RNA as a promising target in drug discovery.
{"title":"Design, synthesis, and screening of an RNA optimized fluorinated fragment library","authors":"Kasper P. Lundquist , Isabella Romeo , Raffaele B. Puglielli , Maëlle Pestalozzi , Marie L. Gram , Emily S. Hudson , Ofri Levi , Yoav S. Arava , Charlotte H. Gotfredsen , Mads H. Clausen","doi":"10.1016/j.slasd.2025.100215","DOIUrl":"10.1016/j.slasd.2025.100215","url":null,"abstract":"<div><div>Fragment-based screening is an efficient method for early-stage drug discovery. In this study, we aimed to create a fragment library optimized for producing high hit rates against RNA targets. RNA has historically been an underexplored target, but recent research suggests potential for optimizing small molecule libraries for RNA binding. We extended this concept to fragment libraries to produce an RNA optimized fluorinated fragment library. We then screened this library, alongside two non-RNA optimized fragment libraries, against three RNA targets: the human cytoplasmic A-site and the <em>S. cerevisiae</em> tRNA<sup>Asp</sup> anticodon stem loop with and without nucleobase modifications. The screens yielded 24, 31, and 20 hits against the respective targets. Importantly, statistical analysis confirmed a significant overrepresentation of hits in our RNA optimized library. Based on these findings, we propose guidelines for developing RNA optimized fragment libraries. We hope the guidelines will help expediting fragment-based ligand discovery for RNA targets and contribute to presenting RNA as a promising target in drug discovery.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100215"},"PeriodicalIF":2.7,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-16DOI: 10.1016/j.slasd.2025.100214
Martin P. Schwalm , Stefan Knapp
Cancer research, cancer treatment, and the field of chemical biology are examples which heavily rely on the discovery of selective kinase inhibitors. While determining on-target potency is often feasible for most laboratories, the equally critical but frequently neglected selectivity screening remains less accessible to the broader scientific community. This limitation can stem from various factors, such as the unavailability of a large number of purified kinases or the costs associated with commercial screening systems. To address these challenges and enable a wider range of scientists, this protocol leverages a commercial kinome selectivity screen to facilitate a low-cost, two-day, single-plate selectivity screen against 192 kinases.
{"title":"Single-plate kinome screening in live-cells to enable highly cost-efficient kinase inhibitor profiling","authors":"Martin P. Schwalm , Stefan Knapp","doi":"10.1016/j.slasd.2025.100214","DOIUrl":"10.1016/j.slasd.2025.100214","url":null,"abstract":"<div><div>Cancer research, cancer treatment, and the field of chemical biology are examples which heavily rely on the discovery of selective kinase inhibitors. While determining on-target potency is often feasible for most laboratories, the equally critical but frequently neglected selectivity screening remains less accessible to the broader scientific community. This limitation can stem from various factors, such as the unavailability of a large number of purified kinases or the costs associated with commercial screening systems. To address these challenges and enable a wider range of scientists, this protocol leverages a commercial kinome selectivity screen to facilitate a low-cost, two-day, single-plate selectivity screen against 192 kinases.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100214"},"PeriodicalIF":2.7,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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