Chemical Tools Research Articles

Discovery and Characterization of a Novel Cereblon-Recruiting PRC1 Bridged PROTAC Degrader.

Bridged PROTAC is a novel protein complex degrader strategy that exploits the target protein's binding partner to degrade undruggable proteins by inducing proximity to an E3 ubiquitin ligase. In this study, we discovered for the first time that cereblon (CRBN) can be employed for the bridged PROTAC approach and report the first-in-class CRBN-recruiting and EED-binding polycomb repressive complex 1 (PRC1) degrader, compound 1 (MS181). We show that 1 induces preferential degradation of PRC1 components, BMI1 and RING1B, in an EED-, CRBN-, and ubiquitin-proteosome system (UPS)-dependent manner. Compound 1 also has superior antiproliferative activity in multiple metastatic cancer cell lines over EED-binding PRC2 degraders and can be efficacious in VHL-defective cancer cells. Altogether, compound 1 is a valuable chemical biology tool to study the role of PRC1 in cancer. Importantly, we show that CRBN can be utilized to develop bridged PROTACs, expanding the bridged PROTAC technology for degrading undruggable proteins.

Discovery of the First-in-Class G9a/GLP PROTAC Degrader.

Aberrantly expressed lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9), have been implicated in numerous cancers. Recent studies have uncovered both catalytic and noncatalytic oncogenic functions of G9a/GLP. As such, G9a/GLP catalytic inhibitors have displayed limited anticancer activity. Here, we report the discovery of the first-in-class G9a/GLP proteolysis targeting chimera (PROTAC) degrader 10 (MS8709), as a potential anticancer therapeutic. 10 induces G9a/GLP degradation in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Futhermore, 10 does not alter the mRNA expression of G9a/GLP and is selective for G9a/GLP over other methyltransferases. Moreover, 10 displays superior cell growth inhibition to the parent G9a/GLP inhibitor UNC0642 in prostate, leukemia, and lung cancer cells and has suitable mouse pharmacokinetic properties for in vivo efficacy studies. Overall, 10 is a valuable chemical biology tool to further investigate the functions of G9a/GLP and a potential therapeutic for treating G9a/GLP-dependent cancers.

Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

Discovery and optimization of narrow spectrum inhibitors of Tousled like kinase 2 (TLK2) using quantitative structure activity relationships

The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was potently inhibited as an off-target kinase. The oxindole has long been considered a promiscuous kinase inhibitor template, but across these 4 specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different ranging from narrow to broad spectrum coverage. We synthesized a large series of analogue, utilizing quantitative structure-activity relationship (QSAR) analysis, water mapping of the kinase ATP binding sites, kinome profiling and small-molecule x-ray structural analysis to optimize TLK2 inhibition and kinome selectivity. This resulted in the identification of several, narrow spectrum, sub-family selective, chemical tool compounds including 128 (UNC-CA2-103) that could enable elucidation of TLK2 biology.

Discovery of a Potent, Selective, and Cell-Active SPIN1 Inhibitor.

The methyl-lysine reader protein SPIN1 plays important roles in various human diseases. However, targeting methyl-lysine reader proteins has been challenging. Very few cellularly active SPIN1 inhibitors have been developed. We previously reported that our G9a/GLP inhibitor UNC0638 weakly inhibited SPIN1. Here, we present our comprehensive structure-activity relationship study that led to the discovery of compound 11, a dual SPIN1 and G9a/GLP inhibitor, and compound 18 (MS8535), a SPIN1 selective inhibitor. We solved the cocrystal structure of SPIN1 in complex with 11, confirming that 11 occupied one of the three Tudor domains. Importantly, 18 displayed high selectivity for SPIN1 over 38 epigenetic targets, including G9a/GLP, and concentration dependently disrupted the interactions of SPIN1 and H3 in cells. Furthermore, 18 was bioavailable in mice. We also developed 19 (MS8535N), which was inactive against SPIN1, as a negative control of 18. Collectively, these compounds are useful chemical tools to study biological functions of SPIN1.

Abstract 3542: The Chemical Probes Portal: An essential resource providing expert advice on chemical tools for cancer research

Abstract Background: The Chemical Probes Portal (Portal) serves as a vital resource in the realm of cancer research and biomedical science generally - offering expert guidance on high-quality chemical tools, usually protein inhibitors, crucial for studying physiological and pathological processes and validating therapeutic targets. Researchers often grapple with selecting suitable chemical probes, inadvertently utilizing tools ill-suited for their purpose, and not following best practice, thereby contaminating the scientific literature and wasting valuable time and resources. To address this challenge, the Portal (https://www.chemicalprobes.org) was established as a non-profit, independent online platform, meticulously curating, annotating, and scoring the quality of chemical probes and providing associated information and advice. Results: The Portal stands apart as the sole expert-curated public resource evaluating the quality and utility of small-molecule chemical tool compounds for use in cancer research and wider biomedical research. More than 250 experts, comprising scientific leaders in chemical biology, medicinal chemistry, pharmacology, and related fields, contribute their experience and insights to assess featured chemical probes. Since 2021, a progressively updated and enhanced version of the Portal has been operational, housing over 1000 compounds targeting 500+ proteins across diverse families, now including PROTACs and molecular glues. We also flag 254 ‘Unsuitables’, which are compounds that should not be used to interrogate a specific protein target. Additionally, the Portal now offers informative pages, providing advice delineating guidelines for selecting well-validated compounds, including those suitable for rodent studies, necessitating specific pharmacokinetic and pharmacodynamic properties. Best practice use requires employing high-quality chemical probes from more than one distinct chemical class that target the same protein, incorporating an inactive control compound, and following the recommended concentration range - all aimed at mitigating off-target risks. Conclusion: The enhanced Chemical Probes Portal provides a vital platform for researchers to navigate and evaluate chemical probes, easily and effectively. It employs a rigorous expert review to ensure the quality of the probes listed. The resource not only aids high-quality probe selection but also provides advice through an Information Centre, offering guidelines, FAQs, and additional information on probe criteria and use. The continuous expansion and outreach efforts aim to make the Portal an indispensable tool for cancer researchers and the broader biomedical research community. Researchers are encouraged to participate in the Portal's development and contribute to the robustness of chemical probe use in scientific endeavors. Citation Format: Domenico Sanfelice, Albert Antolin, Alisa Crisp, Yi Chen, Paul Brennan, Susanne Müller, Bissan Al-Lazikani, Aled Edwards, Paul Workman. The Chemical Probes Portal: An essential resource providing expert advice on chemical tools for cancer research [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 3542.

Abstract 3047: Interrogating TEAD protein interactions in cancer

Abstract The Hippo signaling pathway is a major regulator of cellular growth and is frequently deregulated in cancer. The main effectors of the Hippo pathway are the TEAD transcription factors which function by interacting with other co-effector proteins to modulate transcriptional outputs. Notably YAP and TAZ interact with TEAD to mediate oncogenic transcriptional programs, and blocking interaction of TEAD with YAP and TAZ is a sought-after mechanism for small molecule TEAD inhibitors. However, the interaction network of TEAD extends well beyond YAP and TAZ to a variety of additional cofactors, including chromatin remodelers, epigenetic factors, and other transcriptional regulators. Thus, the impact of inhibiting TEAD could extend to unanticipated protein interactions and cellular functions. The purpose of this study is to investigate, define, and interrogate TEAD interaction networks, providing foundational knowledge for interrogating TEAD biological functions and pharmacological effects. We are employing mass spectrometry-based technologies combined with chemical and genetic tools and biochemistry to interrogate TEAD protein interaction networks. We have performed affinity purification coupled with mass spectrometry for TEAD and have identified dozens of significant interacting proteins. TEAD interactions include expected proteins, such as known Hippo pathway members, as well as unexpected protein interactions, such as DNA damage response proteins. Cellular assays probing these DNA damage protein interactions implicate TEAD as a crucial factor for efficient DNA repair mechanisms and maintaining genomic stability. These results raise questions towards a combinatorial effect for TEAD inhibitors and DNA damaging chemotherapeutic agents. Altogether, these ongoing proteomic studies are providing insights into TEAD interaction networks, leading to a deeper understanding of TEAD biology, and providing foundational knowledge that can inform the development of TEAD inhibitors as effective cancer therapies. Citation Format: Alissa Guarnaccia, Jennie Lill, Anwesha Dey. Interrogating TEAD protein interactions in cancer [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 3047.

Abstract 680: Leveraging an RNA-targeting platform for the discovery of cell-active c-MYC mRNA-binding small molecules

Abstract The MYC gene, with its oncogenic potential, has long been a formidable challenge in conventional drug discovery efforts, and its critical role in cancer progression and resistance has underscored the need for innovative therapeutic strategies. Here we demonstrate the capabilities of the Ribometrix RNA-targeting platform to modulate the c-MYC mRNA with small molecules with the aim of reducing c-MYC protein levels. Using our comprehensive platform of RNA-targeting drug discovery tools and analyses including chemical probing, structure modeling, high-purity RNA production, high-throughput screening, and biophysical characterization, we evaluated the potential to directly target c-MYC mRNA using small molecules. Our analysis revealed six high-confidence structured elements throughout the c-MYC mRNA expected to harbor tertiary structures amenable to drug-like ligand binding. Leveraging the multifaceted chemical probing and structure modeling components of our platform, we confirmed that our in vitro-transcribed RNA screening constructs adopt the same structures found in endogenous cellular c-MYC transcripts. After large-scale in vitro RNA preparation, we subjected four of these RNA elements to high-throughput screens of chemically diverse drug-like libraries using mass spectrometry affinity-selection that identified multiple chemical series with sub-micromolar affinity. We validated binding affinity using orthogonal methods including isothermal titration calorimetry and NMR. To eliminate pan-binding ligands, we evaluated of selectivity against a panel of non-target mRNA structures. Importantly, these compounds lead to rapid (4 hr) reduction of MYC protein levels in a small cell lung cancer cell line, DMS-273, with no cellular toxicity at 72 hr suggesting an on-target effect. Employing our suite of chemical probing tools, we confirmed that compounds from multiple series engage the c-MYC mRNA in cells. Medicinal chemistry efforts are ongoing to increase potency and define the precise mechanism of action in clinically relevant models. The discovery of these c-MYC mRNA-binding small molecules not only validates the utility of the Ribometrix RNA-targeted small molecule discovery platform but also showcases its potential in tackling traditionally 'undruggable' targets and provides a promising avenue for developing novel anti-cancer therapies. As we learn more about the intricacies of c-MYC mRNA biology and further refine effective small molecules, our results provide practical insights into previously-unknown vulnerabilities of MYC and present tangible opportunities for advancing targeted cancer therapies. Citation Format: Matthew J. Smola, Krista Marran, Sarah E. Thompson, Brittani Patterson, Roheeth K. Pavana, Caleb Sutherland, Jessica A. Sorrentino, Katherine D. Warner. Leveraging an RNA-targeting platform for the discovery of cell-active c-MYC mRNA-binding small molecules [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 680.

Abstract 7114: Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays

Abstract The 17-member poly(ADP-ribose) polymerase (PARP) family of proteins mediate a number of cellular processes including gene transcription, cell-division and DNA-repair. PARPs catalyze the addition of poly- or mono- ADP-ribose chains on target proteins, modifying their function and/or recruitment in various pathways. Poly(ADP-ribose) Glycohydrolase (PARG) catabolizes PAR units and facilitates the recycling of PARP and other modified effector proteins. A number of PARPS are implicated in human disease, and PARP inhibitors (PARPi’s) have seen major clinical success, however, many PARPs (especially mono PARPs) are understudied and chemical tools for understanding cellular PARPi selectivity and potency across the entire family are lacking. Similarly, several PARG inhibitors have been developed to date, but tools for directly assessing target engagement in a cellular context are sorely needed. Here we describe the development and application of target engagement assays for characterizing PARP and PARG inhibitor selectivity and affinity in live cells. This method is based on the NanoBRET™ assay platform, which is a proximity-based probe displacement assay that operates in live cells. Using NanoBRET™ technology, we have developed a single probe that covers 12 out of 17 PARP family members. We explored the family-wide selectivity of a diverse panel of clinical and preclinical PARP inhibitors in live cells, representing the first family-wide, cell-based method for profiling PARP selectivity. We also describe the development of a second probe that reports on PARG target engagement. Target engagement of PARG was observed for three out of five tested inhibitors, suggesting a potential indirect mode of action for certain inhibitors on PARG glycohydrolase activity. These assays offer a complementary approach for studying target engagement within the PARylation cycle. Citation Format: Ani Michaud, Kelly Teske, Jennifer Wilkinson, Chad Zimprich, James Vasta, Cesear Corona, Min Zhou, Elizabeth Dominguez, Kaitlin Dunn-Hoffman, Matthew Robers. Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays [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 7114.

Abstract 5813: AI-driven identification and validation of novel synthetic lethal gene pairs through deep mining of cancer dependency data

Abstract The targeting of synthetic lethal (SL) gene pairs is emerging as a new paradigm in precision oncology, with the potential to deliver treatments with higher efficacy and fewer side-effects. However, existing approaches have focused on a few well-validated pairs, and there is a clear need to expand the toolbox of potential SL targets. We have performed deep mining of cancer dependency data using a suite of machine learning tools and AI algorithms underpinned by robust statistical analysis to identify the next generation of SL gene pairs. To ensure robust, high confidence validation of multiple SL pairs simultaneously, we have used three cell lines and orthogonal assay formats. This has led to the confirmation of new druggable SL genes suitable for clinical development. We have identified the cancer subtypes with the strongest evidence of SL vulnerabilities and quantified patient populations who would most benefit from novel therapeutic agents. For one gene pair, we have generated novel best-in-class inhibitors of the target of interest, with exceptional selectivity over the SL partner, and used these chemical tools to validate the SL relationship in 2D and 3D models. Citation Format: Daniel S. Miller, Oliver Vipond, Alfie Brennan, Anna Hercot. AI-driven identification and validation of novel synthetic lethal gene pairs through deep mining of cancer dependency data [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 5813.

Abstract 4511: Discovery of the first-in-class G9a/GLP PROTAC degrader

Abstract G9a and GLP are lysine methyltransferases that catalyze dimethylation of histone H3 lysine 9 (H3K9me2), a transcriptionally repressive mark. Aberrantly expressed G9a/GLP has been implicated in numerous cancers, including prostate, lung, and leukemia. The oncogenic activity of G9a/GLP is attributed to both its catalytic and non-catalytic functions, such as forming co-activator complexes with p300, CARM1, GRIP1, and Mediator to positively regulate gene transcription. As such, current G9a/GLP enzymatic inhibitors display limited anti-cancer activity. As an alternative therapeutic strategy, PROTAC degraders harness the ubiquitin-proteasome system (UPS) to effectively eliminate oncoproteins and abrogate all catalytic and non-catalytic functions. Here, we present the first-in-class G9a/GLP PROTAC degrader, MS8709, which potently degrades G9a and GLP in a time-, concentration-, and UPS-dependent manner. Moreover, MS8709 induces superior cell growth inhibition compared to its parent inhibitor in prostate, lung, and leukemia cell lines. Overall, MS8709 is a useful chemical tool to investigate G9a/GLP biology and offers a novel approach for the treatment of G9a/GLP-dependent cancers. Citation Format: Julia Velez, Yulin Han, Kwang-Su Park, Md Kabir, Peiyi Yang, Yan Xiong, H. Umit Kaniskan, Jian Jin. Discovery of the first-in-class G9a/GLP PROTAC degrader [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 4511.

Multifunctional fluorescence probe for simultaneous detection of viscosity, polarity, and ONOO− and its bioimaging in vitro and vivo

Intracellular microenvironment (viscosity and polarity) and peroxynitrite ions (ONOO−) are involved in maintaining cell morphology, cell function, and signaling so that it is crucial to explore their level changes in vitro and vivo. In this work, we designed and synthesized a mitochondria-targeted fluorescence probe XBL for monitoring the dynamic changes of viscosity, polarity, and ONOO− based on TICT and ICT mechanism. The fluorescence spectra showed obvious changes for polarity at 500 nm as well as ONOO− and viscosity at 660 nm, respectively. The XBL can image simultaneously viscosity, polarity, and ONOO− in cells, and the results showed excess ONOO− leaded to the increase of viscosity in mitochondrial. The ferroptosis process was accompanied by increase of intracellular viscosity and ONOO− levels (or decrease of polarity), which allowed us to better understand the relevant physiological and pathological processes. The XBL can distinguish normal cells and cancerous cells by the fluorescence intensity changes in green and red channels, and image viscosity in inflamed mice. Thus, XBL can provided the chemical tool to understand the physiological and pathological mechanisms of disease by simultaneous detection of viscosity, polarity and ONOO−.

Yeast Bxi1/Ybh3 mediates conserved mitophagy and apoptosis in yeast and mammalian cells: convergence in Bcl-2 family.

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one.

Illumination of understudied ciliary kinases.

Cilia are cellular signaling hubs. Given that human kinases are central regulators of signaling, it is not surprising that kinases are key players in cilia biology. In fact, many kinases modulate ciliogenesis, which is the generation of cilia, and distinct ciliary pathways. Several of these kinases are understudied with few publications dedicated to the interrogation of their function. Recent efforts to develop chemical probes for members of the cyclin-dependent kinase like (CDKL), never in mitosis gene A (NIMA) related kinase (NEK), and tau tubulin kinase (TTBK) families either have delivered or are working toward delivery of high-quality chemical tools to characterize the roles that specific kinases play in ciliary processes. A better understanding of ciliary kinases may shed light on whether modulation of these targets will slow or halt disease onset or progression. For example, both understudied human kinases and some that are more well-studied play important ciliary roles in neurons and have been implicated in neurodevelopmental, neurodegenerative, and other neurological diseases. Similarly, subsets of human ciliary kinases are associated with cancer and oncological pathways. Finally, a group of genetic disorders characterized by defects in cilia called ciliopathies have associated gene mutations that impact kinase activity and function. This review highlights both progress related to the understanding of ciliary kinases as well as in chemical inhibitor development for a subset of these kinases. We emphasize known roles of ciliary kinases in diseases of the brain and malignancies and focus on a subset of poorly characterized kinases that regulate ciliary biology.

Dual-Targeting Gold Nanoparticles: Simultaneous Decoration with Ligands for Co-Transporters SGLT-1 and B0AT1

Sodium–glucose co-transporter 1 (SGLT1) and sodium-dependent neutral amino acid transporter (B0AT1) are mainly expressed on the membrane of enterocytes, a type of epithelial cell found in the intestines. In addition to their physiological role in the absorption of nutrients, a protective role in the integrity of the intestinal barrier has been established. The natural ligands of SGLT1 (d-glucose) and of B0AT1 (l-glutamine) can trigger a protective anti-inflammatory effect on the intestinal epithelium. The literature suggests the activation of common intracellular pathways upon engagement of the two transporters, whose functional forms are composed of oligomers or clusters. Simultaneous activation of these two co-transporters could lead to a potential multitarget and synergistic anti-inflammatory effect. Therefore, nanoplatforms containing multiple copies of the ligands could represent chemical tools to study the potential simultaneous activation of the two co-transporters. For these reasons, in this study, a set of different gold nanoparticles decorated with derivatives of d-glucose and of l-glutamine were designed and prepared. In particular, the synthesis of suitable sulfur-ending functionalized ligand derivatives, including a C-glucoside derivative, their anchoring to gold nanoparticles and their physical–chemical characterization have been carried out. The obtained nanostructures could represent promising multifunctional platforms for further investigation of the existence of possible multitarget and synergistic effects toward the two co-transporters SGLT1 and B0AT1.

O‐glycosylation in viruses: A sweet tango

AbstractO‐glycosylation is an ancient yet underappreciated protein posttranslational modification, on which many bacteria and viruses heavily rely to perform critical biological functions involved in numerous infectious diseases or even cancer. But due to the innate complexity of O‐glycosylation, research techniques have been limited to study its exact role in viral attachment and entry, assembly and exit, spreading in the host cells, and the innate and adaptive immunity of the host. Recently, the advent of many newly developed methodologies (e.g., mass spectrometry, chemical biology tools, and molecular dynamics simulations) has renewed and rekindled the interest in viral‐related O‐glycosylation in both viral proteins and host cells, which is further fueled by the COVID‐19 pandemic. In this review, we summarize recent advances in viral‐related O‐glycosylation, with a particular emphasis on the mucin‐type O‐linked α‐N‐acetylgalactosamine (O‐GalNAc) on viral proteins and the intracellular O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) modifications on host proteins. We hope to provide valuable insights into the development of antiviral reagents or vaccines for better prevention or treatment of infectious diseases.

Abstract 2275 Integrating Chemical Tools to Interrogate Protein Methylation in Nuclei

Abstract 2275 Integrating Chemical Tools to Interrogate Protein Methylation in Nuclei

Development of Selective Pyrido[2,3-d]pyrimidin-7(8H)-one-Based Mammalian STE20-Like (MST3/4) Kinase Inhibitors.

Mammalian STE20-like (MST) kinases 1-4 play key roles in regulating the Hippo and autophagy pathways, and their dysregulation has been implicated in cancer development. In contrast to the well-studied MST1/2, the roles of MST3/4 are less clear, in part due to the lack of potent and selective inhibitors. Here, we re-evaluated literature compounds, and used structure-guided design to optimize the p21-activated kinase (PAK) inhibitor G-5555 (8) to selectively target MST3/4. These efforts resulted in the development of MR24 (24) and MR30 (27) with good kinome-wide selectivity and high cellular potency. The distinct cellular functions of closely related MST kinases can now be elucidated with subfamily-selective chemical tool compounds using a combination of the MST1/2 inhibitor PF-06447475 (2) and the two MST3/4 inhibitors developed. We found that MST3/4-selective inhibition caused a cell-cycle arrest in the G1 phase, whereas MST1/2 inhibition resulted in accumulation of cells in the G2/M phase.

OrthoID: profiling dynamic proteomes through time and space using mutually orthogonal chemical tools

Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report “OrthoID”, a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were—to the best of our knowledge—previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.

Discovery and Structure-Activity Relationships of Novel ssDAF-12 Receptor Modulators.

The nuclear receptor ssDAF-12 has been recognized as the key molecular player regulating the life cycle of the nematode parasite Strongyloides stercoralis. ssDAF-12 ligands permit the receptor to function as an on/off switch modulating infection, making it vulnerable to therapeutic intervention. In this study, we report the design and synthesis of a set of novel dafachronic acid derivatives, which were used to outline the first structure-activity relationship targeting the ssDAF-12 receptor and to unveil hidden properties shared by the molecular shape of steroidal ligands that are relevant to the receptor binding and modulation. Moreover, biological results led to the discovery of sulfonamide 3 as a submicromolar ssDAF-12 agonist endowed with a high receptor selectivity, no toxicity, and improved properties, as well as to the identification of unprecedented ssDAF-12 antagonists that can be exploited in the search for novel chemical tools and alternative therapeutic approaches for treating parasitism such as Strongyloidiasis.