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The 5-year relative survival rate for triple-negative breast cancer (TNBC) is 77%, which is notably lower than 90%, the overall survival rate for breast cancer. The primary systemic treatment for TNBC remains to be chemotherapy. However, patients frequently develop resistance to conventional chemotherapy, greatly compromising the anti-tumor effects of chemodrugs. Therefore, this study is aimed to enhance the effects of chemotherapy. The RNA-binding protein Hu antigen R (HuR) plays an important role in chemotherapy resistance. HuR post-transcriptionally regulates the stability of the target mRNA by binding to the U- or AU-rich elements (ARE) mainly in the 3’ untranslated region (UTR) of mRNA. In most cases, the binding stabilizes mRNA, thereby enhancing the translation of the encoded protein, many of which are implicated in multiple cancer hallmarks, including chemoresistance. The overexpression of HuR, and accumulated cytoplasmic expression, are reported to be related to chemoresistance in many types of cancer cells. We hypothesized that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of mRNA and thus reduce the translation of proteins contributing to chemoresistance. Previously, our lab reported a small molecule HuR inhibitor, KH-3, which potently inhibits HuR function by disrupting the HuR-mRNA interactions. To test our hypothesis, we utilized KH-3 as a tool compound to assess whether HuR inhibition enhances the efficacy of chemotherapy for TNBC cells. We generated a cell sub-line (231-TR) derived from the human TNBC cell line MDA-MB-231 with acquired resistance against docetaxel (TXT). Compared with the parental cell line, 231-TR exhibited similar sensitivity to KH-3 in the MTT-based cytotoxicity assay and the colony formation assay. The in vitro and in vivo combination of KH-3 and TXT synergized in inhibiting cell proliferation and tumor growth of multiple TNBC cell lines. Regarding mechanisms of action, the apoptosis pathway was downregulated and the Wnt signaling pathway was upregulated in 231-TR cells. KH-3 treatment downregulated β-Catenin, involved in promoting cell proliferation, in a time and dose-dependent manner. KH-3 was also identified to induce apoptosis cell death via inhibiting the anti-apoptotic protein BCL2. The cell cycle analysis revealed that KH-3 treatment caused the S phase accumulation. Therefore, the cell proliferation inhibition by KH-3 results from a combination of apoptosis and cell cycle arrest. Furtherly, KH-3 restored the effects of docetaxel in inducing apoptotic cell death in 231-TR cells. Together, this study provides a new strategy to overcome chemotherapy resistance in TNBC cells by functional inhibiting HuR. Citation Format: Lanjing Wei, Qi Zhang, Cuncong Zhong, Jeffrey Aubé, Danny R. Welch, Xiaoqing Wu, Liang Xu. Inhibition of RNA binding protein HuR function sensitizes the TNBC to chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-08.

Mucosal-associated invariant T (MAIT) cells are promising innate-like lymphocytes with potential for use in anti-tumor immunotherapy. Existing MAIT cell expansion protocols are associated with potentially decremental phenotypic changes, including increased frequency of CD4+ MAIT cells and higher inhibitory receptor expression. In this study, we compared the effect on expansion of human MAIT cells of a serum replacement, Physiologix XF SR (Phx), with traditional serum FBS for supplementing RPMI 1640 media. Using flow cytometry, we found that Phx supported a significantly higher proliferative capacity for MAIT cells and resulted in a lower frequency of CD4+ MAIT cells, which have been associated with reduced Th1 effector and cytolytic functions. We saw that culturing MAIT cells in Phx led to better survival of MAIT cells and lower frequency of PD-1+ MAIT cells than FBS-supplemented media. Functionally, we saw that Phx supplementation was associated with a higher frequency of IFN-γ+ MAIT cells after stimulation with Escherichia coli than FBS-supplemented RPMI. In conclusion, we show that MAIT cells cultured in Phx have higher proliferative capacity, lower expression of inhibitory receptors, and higher capacity to produce IFN-γ after E. coli stimulation than FBS-supplemented RPMI. This work shows that expanding MAIT cells with Phx compared with FBS-supplemented RPMI results in a more functionally desirable MAIT cell for future anti-tumor immunotherapy.

Huntington's disease is a progressive and lethal neurodegenerative disease caused by an increased CAG repeat mutation in exon 1 of the huntingtin gene (mutant huntingtin). Current drug treatments provide only limited symptomatic relief without impacting disease progression. Previous studies in our lab and others identified the abnormal binding of mutant huntingtin protein with calmodulin, a key regulator of calcium signaling. Disrupting the abnormal binding of mutant huntingtin to calmodulin reduces perturbations caused by mutant huntingtin in cell and mouse models of Huntington's disease and importantly normalizes receptor-stimulated calcium release. Using a series of high-throughput

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. SAR exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human Phase 1 clinical trial.

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF. In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 β-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

Suppl. Table 1. Origin of cancer cell lines used in this study. Suppl. Table 2. Quantitative HTS screen in PANC1 cells grown as spheroids. Results of full ten concentration drug-response curves of 1,912 individual agents and compounds in PANC1 spheroids. SID, Sample ID; CCLASS, Curve class; CCLASS2, Curve class (latest version); LAC50, log AC50 (M); HILL, Hill coefficient; ZERO, fitted response at minimum concentration; INF, Fitted response at maximum concentration; MAXR, Observed response at maximum concentration; DATA0 - DATA10, Responses; C0 - C10, Doses (in µM); auc, Area Under the Curve (via integration of the fit). This data is available via the PubChem database (AID 1259363). Suppl. Table 3 Top twenty hits of KiNativ profiling of PANC1 cells with at least {greater than or equal to}50 percent inhibition of ADP binding following treatment with 500 nM of inhibitor #1. Suppl. Table 4. In vitro activity of inhibitor #1 against a panel of over 400 kinases (DiscoveRx's KinomeScan) and in situ activity of inhibitor #1 in PANC1 spheroid cells (KiNative). Suppl. Table 5. Outcomes of both 6Ã-6 and 10Ã-10 combination drug screening of inhibitor #1 versus a collection of approved and investigational drugs.

Suppl. Fig. 1: Procedures for synthesis of NCGC00188382 (Inhibitor#1) Suppl. Fig. 2: qPCR array of 96 stem cell genes from PANC1 adherent and PANC1 spheroids reveal up-regulation of six stem cell genes in organotypic Panc1 spheres. Suppl. Fig. 3: Inhibitor #1 induces apoptosis in KLM-1 spheroids. Flow cytometry measuring fraction of cells with exposed extracellular phosphatidylserine via annexin V-FITC (in PI stained cells; y-axis). Suppl. Fig. 4: In vivo activity of inhibitor #1. Suppl. Fig. 5: Inhibitor #1 suppresses downstream signaling of aurora kinase B, and ephrin B2 receptor activation identified as targets within in situ kinome screen. Suppl. Fig. 6: Outcomes of combination drug screening of inhibitor #1 versus a collection of approved and investigational drugs. Suppl. Fig. 7: Confirmation of synergies derived from 10Ã-10 matrix screening with inhibitor #1 in PANC1 spheroids. Suppl. Fig. 8: A. Flow cytometry measuring DNA breaks and fragments after labeling of 3'-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-UTP) after 96-hour treatment with #1 in PANC1 spheroids and 2D monolayer cells.

Pancreatic cancer has poor prognosis and treatment outcomes due to its highly metastatic nature and resistance to current treatments. The RNA-binding protein (RBP) Hu-antigen R (HuR) is a central player in posttranscriptional regulation of cancer-related gene expression, and contributes to tumorigenesis, tumor growth, metastasis, and drug resistance. HuR has been suggested to regulate pancreatic cancer epithelial-to-mesenchymal transition (EMT), but the mechanism was not well understood. Here, we further elucidated the role HuR plays in pancreatic cancer cell EMT, and developed a novel inhibitor specifically interrupting HuR–RNA binding. The data showed that HuR binds to the 3′-UTR of the mRNA of the transcription factor Snail, resulting in stabilization of Snail mRNA and enhanced Snail protein expression, thus promoted EMT, metastasis, and formation of stem-like cancer cells (CSC) in pancreatic cancer cells. siRNA silencing or CRISPR/Cas9 gene deletion of HuR inhibited pancreatic cancer cell EMT, migration, invasion, and inhibited CSCs. HuR knockout cells had dampened tumorigenicity in immunocompromised mice. A novel compound KH-3 interrupted HuR–RNA binding, and KH-3 inhibited pancreatic cancer cell viability, EMT, migration/invasion in vitro. KH-3 showed HuR-dependent activity and inhibited HuR-positive tumor growth and metastasis in vivo.

Supplementary Data

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell–like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine–based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo. An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Suppl. Table 1. Origin of cancer cell lines used in this study. Suppl. Table 2. Quantitative HTS screen in PANC1 cells grown as spheroids. Results of full ten concentration drug-response curves of 1,912 individual agents and compounds in PANC1 spheroids. SID, Sample ID; CCLASS, Curve class; CCLASS2, Curve class (latest version); LAC50, log AC50 (M); HILL, Hill coefficient; ZERO, fitted response at minimum concentration; INF, Fitted response at maximum concentration; MAXR, Observed response at maximum concentration; DATA0 - DATA10, Responses; C0 - C10, Doses (in µM); auc, Area Under the Curve (via integration of the fit). This data is available via the PubChem database (AID 1259363). Suppl. Table 3 Top twenty hits of KiNativ profiling of PANC1 cells with at least {greater than or equal to}50 percent inhibition of ADP binding following treatment with 500 nM of inhibitor #1. Suppl. Table 4. In vitro activity of inhibitor #1 against a panel of over 400 kinases (DiscoveRx's KinomeScan) and in situ activity of inhibitor #1 in PANC1 spheroid cells (KiNative). Suppl. Table 5. Outcomes of both 6Ã-6 and 10Ã-10 combination drug screening of inhibitor #1 versus a collection of approved and investigational drugs.

RNA molecules can show high levels of cooperativity in their global folding and interactions with divalent ions. However, cooperativity at individual ligand-RNA interaction sites remains poorly understood. Here, we investigated the binding of thiamine and methylene diphosphonic acid (MDP, a soluble structural analogue of pyrophosphate) to the thiamine pyrophosphate riboswitch. These ligands each bind weakly at proximal subsites, with 10 μM and 1 mM affinities, respectively. The affinity of MDP moderately improves when thiamine or thiamine-like fragments are pre-bound to the RNA. Covalent linking of thiamine and MDP substantially increases riboswitch binding to a notable high affinity of 20 nM. Crystal structures and single-molecule correlated chemical probing revealed favorable induced fit effects upon binding of individual ligands and, unexpectedly, a substantial thermodynamically unfavorable RNA structural rearrangement upon binding of the linked thiamine-MDP ligand. Thus, linking of two ligands of modest affinity, accompanied by an unfavorable structural rearrangement, still yields a potent linked RNA-binding compound. Since complex ligands often bind riboswitches and other RNAs at proximal subsites, principles derived from this work inform and support fragment-linking strategies for identifying small molecules that interact with RNA specifically and with high affinity.

Homodimers are the most abundant type of enzyme in cells and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on the similar event at the second site, the most informative state is the asymmetric single bound (lig(1)) form, yet it tends to be elusive thermodynamically. Here we obtain milligram quantities of lig(1) of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu((I)-)catalyzed azide–alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins, and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig(1) intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but should also facilitate other structural applications where specific linkage of proteins is helpful.

Mucosal-associated invariant T (MAIT) cells are promising innate-like lymphocytes with potential for use in anti-tumor immunotherapy. Existing MAIT cell expansion protocols are associated with potentially decremental phenotypic changes, including increased frequency of CD4+ MAIT cells and higher inhibitory receptor expression. In this study, we compared the effect on expansion of human MAIT cells of a serum replacement, Physiologix XF SR (Phx), with traditional serum fetal bovine serum (FBS) for supplementing RPMI-1640 media. Using flow cytometry, we found that Phx supported a significantly higher proliferative capacity for MAIT cells and resulted in a lower frequency of CD4+ MAIT cells, which have been associated with reduced Th1 effector and cytolytic functions. We saw that culturing MAIT cells in Phx led to better survival of MAIT cells and lower frequency of PD-1+ MAIT cells compared to FBS-supplemented media. Functionally, we saw that Phx supplementation was associated with a higher frequency of IFN-γ+ MAIT cells after stimulation withE. colicompared to FBS-supplemented RPMI. In conclusion, we show that MAIT cells cultured in Phx have higher proliferative capacity, lower expression of inhibitory receptors, and have higher capacity to produce IFN-γ afterE. colistimulation, compared to FBS-supplemented RPMI. This work shows that expanding MAIT cells with Phx compared to FBS-supplemented RPMI result in a more functionally desirable MAIT cell for future anti-tumor immunotherapy.

Select triazole-based small molecules possess potent and selective kappa opioid receptor (KOR) agonism. Here, we designed twenty new analogs to investigate the structure–activity relationship effects for all three functional groups attached to the triazole core. We identified specific groups that are critical for KOR potency and further extended the range of moieties explored. These efforts revealed analogs with potency on par with our lead triazole probe molecule, Triazole 1.1, in addition to analogs possessing a spectrum of less potent KOR agonist activity.

Triple-negative breast cancer (TNBC) has a much lower 5-year relative survival rate (77%) than the overall breast cancer (91%). Chemotherapy remains the primary choice for the treatment of TNBC. However, patients often develop resistance to conventional chemotherapy after long-term exposure to the chemo-drugs, resulting in poorer prognosis and higher tumor reoccurrence compared to other subtypes of breast cancer. Therefore, understanding and overcoming drug resistance is critical for the successful treatment of TNBC. The Hu antigen R (HuR) or ELAVL1 (embryonic lethal, abnormal vision, Drosophila-like protein 1) plays an important role in chemotherapy resistance. The RNA-binding protein HuR is a posttranscriptional regulator, which can stabilize target mRNAs by binding to U- or AU-rich elements (ARE) mainly in 3’ untranslated region (UTR) of mRNAs and upregulate the translation of them. The encoded proteins are implicated in multiple cancer hallmarks, including chemoresistance. The overexpression of HuR, especially accumulated cytoplasmic expression, has been identified to be related to chemoresistance in many types of cancer. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNAs can accelerate the decay of mRNAs and thus reduce the translation of proteins responsible for chemoresistance.Recently, we reported a small molecule HuR inhibitor, KH-3, which potently inhibit HuR function by disrupting the HuR-mRNA interaction. KH-3 can effectively suppress the growth and invasion of TNBC cells in vitro and in vivo. In this study, we aim to verify that HuR is a target for overcoming chemoresistance and evaluate that KH-3 as a HuR functional inhibitor can enhance the efficacy of chemotherapy for TNBC cells. To determine whether HuR inhibition can overcome acquired chemotherapy resistance of TNBCs, we generated MDA-MB-231 sub-cell lines with acquired resistance against docetaxel or doxorubicin. Our results show that inhibition of HuR by KH-3 could synergize chemotherapy for TNBC in vitro and in vivo. More interestingly, KH-3 treatment could re-sensitize resistant TNBC cells to chemo-drugs, indicating that HuR inhibition can overcome acquired chemoresistance. In the study of mechanism of actions, several pathways and HuR direct target mRNAs are found to be involved in acquired docetaxel and doxorubicin resistance. The detailed molecular mechanisms of how KH-3 sensitizes TNBC to chemotherapy is currently under investigation. This study provides a new strategy to overcome chemotherapy resistance and improve the overall survival rate of patients with TNBC. Citation Format: Lanjing Wei, Qi Zhang, Gulhumay Gardashova, Cuncong Zhong, Jeffrey Aubé, Danny R. Welch, Xiaoqing Wu, Liang Xu. Targeting the RNA-binding protein HuR to overcome chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-20.

1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) is a polar, strongly hydrogen bond-donating solvent that has found numerous uses in organic synthesis due to its ability to stabilize ionic species, transfer protons, and engage in a range of other intermolecular interactions. The use of this solvent has exponentially increased in the past decade and has become a solvent of choice in some areas, such as C-H functionalization chemistry. In this review, following a brief history of HFIP in organic synthesis and an overview of its physical properties, literature examples of organic reactions using HFIP as a solvent or an additive are presented, emphasizing the effect of solvent of each reaction.

The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening and SHAPE-MaP RNA structure probing to discover small-molecule fragments that bind an RNA structure of interest. We identified fragments and cooperatively binding fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch with millimolar to micromolar affinities. We then used structure-activity relationship information to efficiently design a linked-fragment ligand, with no resemblance to the native ligand, with high ligand efficiency and druglikeness, that binds to the TPP thiM riboswitch with high nanomolar affinity and that modulates RNA conformation during cotranscriptional folding. Principles from this work are broadly applicable, leveraging cooperativity and multisite binding, for developing high-quality ligands for diverse RNA targets.

Pancreatic cancer has poor prognosis and treatment outcomes due to its highly metastatic nature and resistance to current treatments. The RNA-binding protein (RBP) Hu-antigen R (HuR) is a central player in posttranscriptional regulation of cancer-related gene expression, and contributes to tumorigenesis, tumor growth, metastasis, and drug resistance. HuR has been suggested to regulate pancreatic cancer epithelial-to-mesenchymal transition (EMT), but the mechanism was not well understood. Here, we further elucidated the role HuR plays in pancreatic cancer cell EMT, and developed a novel inhibitor specifically interrupting HuR–RNA binding. The data showed that HuR binds to the 3′-UTR of the mRNA of the transcription factor Snail, resulting in stabilization of Snail mRNA and enhanced Snail protein expression, thus promoted EMT, metastasis, and formation of stem-like cancer cells (CSC) in pancreatic cancer cells. siRNA silencing or CRISPR/Cas9 gene deletion of HuR inhibited pancreatic cancer cell EMT, migration, invasion, and inhibited CSCs. HuR knockout cells had dampened tumorigenicity in immunocompromised mice. A novel compound KH-3 interrupted HuR–RNA binding, and KH-3 inhibited pancreatic cancer cell viability, EMT, migration/invasion in vitro. KH-3 showed HuR-dependent activity and inhibited HuR-positive tumor growth and metastasis in vivo.

To identify novel D3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a β-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 (20), potently promotes D3R-mediated β-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.

Focused modification of a sulfonamide-based kappa opioid receptor (KOR) antagonist series previously reported by this laboratory was investigated. A total of 32 analogues were prepared to explore linker replacement, constraint manipulation, and aryl group or amine substitution. All analogues were assayed for KOR antagonist activity, and the initial lead compound was assessed for

The 5-year relative survival rate for triple-negative breast cancer (TNBC) is 77%, which is notably lower than 90%, the overall survival rate for breast cancer. The primary systemic treatment for TNBC remains to be chemotherapy. However, patients frequently develop resistance to conventional chemotherapy, greatly compromising the anti-tumor effects of chemodrugs. Therefore, this study is aimed to enhance the effects of chemotherapy. The RNA-binding protein Hu antigen R (HuR) plays an important role in chemotherapy resistance. HuR post-transcriptionally regulates the stability of the target mRNA by binding to the U- or AU-rich elements (ARE) mainly in the 3’ untranslated region (UTR) of mRNA. In most cases, the binding stabilizes mRNA, thereby enhancing the translation of the encoded protein, many of which are implicated in multiple cancer hallmarks, including chemoresistance. The overexpression of HuR, and accumulated cytoplasmic expression, are reported to be related to chemoresistance in many types of cancer cells. We hypothesized that inhibition of HuR function by disrupting its interaction with mRNA can accelerate the decay of mRNA and thus reduce the translation of proteins contributing to chemoresistance.Previously, our lab reported a small molecule HuR inhibitor, KH-3, which potently inhibits HuR function by disrupting the HuR-mRNA interactions. To test our hypothesis, we utilized KH-3 as a tool compound to assess whether HuR inhibition enhances the efficacy of chemotherapy for TNBC cells. We generated a cell sub-line (231-TR) derived from the human TNBC cell line MDA-MB-231 with acquired resistance against docetaxel (TXT). Compared with the parental cell line, 231-TR exhibited similar sensitivity to KH-3 in the MTT-based cytotoxicity assay and the colony formation assay. The in vitro and in vivo combination of KH-3 and TXT synergized in inhibiting cell proliferation and tumor growth of multiple TNBC cell lines. Regarding mechanisms of action, the apoptosis pathway was downregulated and the Wnt signaling pathway was upregulated in 231-TR cells. KH-3 treatment downregulated β-Catenin, involved in promoting cell proliferation, in a time and dose-dependent manner. KH-3 was also identified to induce apoptosis cell death via inhibiting the anti-apoptotic protein BCL2. The cell cycle analysis revealed that KH-3 treatment caused the S phase accumulation. Therefore, the cell proliferation inhibition by KH-3 results from a combination of apoptosis and cell cycle arrest. Furtherly, KH-3 restored the effects of docetaxel in inducing apoptotic cell death in 231-TR cells. Together, this study provides a new strategy to overcome chemotherapy resistance in TNBC cells by functional inhibiting HuR. Citation Format: Lanjing Wei, Qi Zhang, Cuncong Zhong, Jeffrey Aubé, Danny Welch, Xiaoqing Wu, Liang Xu. Functional inhibition of the RNA-binding protein HuR sensitizes triple-negative breast cancer to chemotherapy [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 1753.

Cytochromes P450 17A1 (CYP7A1) and 21A2 (CYP21A2) catalyze key reactions in the production of steroid hormones, including mineralocorticoids, glucocorticoids, and androgens. With the ultimate goal of designing probes that are selectively metabolized to each of these steroid types, fluorinated derivatives of the endogenous substrates, pregnenolone and progesterone, were prepared to study the effects on CYP17A1 and CYP21A2 activity. In the functional assays, the hydroxylase reactions catalyzed by each of these enzymes were blocked when fluorine was introduced at the site of metabolism ( positions 17 and 21 of the steroid core, respectively). CYP17A1, furthermore, performed the 17,20-lyase reaction on substrates with a fluorine installed at the 21-position. Importantly, none of the substitutions examined herein prevented compound entry into the active sites of either CYP17A1 or CYP21A2 as demonstrated by spectral binding assays. Taken together, the results suggest that fluorine might be used to redirect the metabolic pathways of pregnenolone and progesterone to specific types of steroids.

Mu opioid receptor (MOR)-targeting analgesics are efficacious pain treatments, but notorious for their abuse potential. In preclinical animal models, coadministration of traditional kappa opioid receptor (KOR)-targeting agonists with MOR-targeting analgesics can decrease reward and potentiate analgesia. However, traditional KOR-targeting agonists are well known for inducing antitherapeutic side effects (psychotomimesis, depression, anxiety, dysphoria). Recent data suggest that some functionally selective, or biased, KOR-targeting agonists might retain the therapeutic effects of KOR activation without inducing undesirable side effects. Nalfurafine, used safely in Japan since 2009 for uremic pruritus, is one such functionally selective KOR-targeting agonist. Here, we quantify the bias of nalfurafine and several other KOR agonists relative to an unbiased reference standard (U50,488) and show that nalfurafine and EOM-salvinorin-B demonstrate marked G protein-signaling bias. While nalfurafine (0.015 mg/kg) and EOM-salvinorin-B (1 mg/kg) produced spinal antinociception equivalent to 5 mg/kg U50,488, only nalfurafine significantly enhanced the supraspinal analgesic effect of 5 mg/kg morphine. In addition, 0.015 mg/kg nalfurafine did not produce significant conditioned place aversion, yet retained the ability to reduce morphine-induced conditioned place preference in C57BL/6J mice. Nalfurafine and EOM-salvinorin-B each produced robust inhibition of both spontaneous and morphine-stimulated locomotor behavior, suggesting a persistence of sedative effects when coadministered with morphine. Taken together, these findings suggest that nalfurafine produces analgesic augmentation, while also reducing opioid-induced reward with less risk of dysphoria. Thus, adjuvant administration of G protein-biased KOR agonists like nalfurafine may be beneficial in enhancing the therapeutic potential of MOR-targeting analgesics, such as morphine.

Loss-of-function mutations in genes encoding TET DNA dioxygenase occur frequently in hematopoietic malignancy, but rarely in solid tumors, which instead commonly have reduced activity. The impact of decreased TET activity in solid tumors is not known. Here we show that TET2 mediates the IFN-γ/JAK/STAT signaling pathway to control chemokine and PD-L1 expression, lymphocyte infiltration, and cancer immunity. IFN-γ stimulated STAT1 to bind TET2 and recruit TET2 to hydroxymethylate chemokine and PD-L1 genes. Reduced TET activity was associated with decreased Th1-type chemokines and tumor-infiltrating lymphocytes and the progression of human colon cancer. Deletion of Tet2 in murine melanoma and colon tumor cells reduced chemokine expression and tumor-infiltrating lymphocytes, enabling tumors to evade antitumor immunity and to resist anti–PD-L1 therapy. Conversely, stimulating TET activity by systematic injection of its cofactor ascorbate/vitamin C increased chemokines and tumor-infiltrating lymphocytes, leading to enhanced antitumor immunity and anti–PD-L1 efficacy and extended lifespan of tumor-bearing mice. These results suggest an IFN-γ/JAK/STAT/TET signaling pathway that mediates tumor response to anti–PD-L1/PD-1 therapy and is frequently disrupted in solid tumors. Our findings also suggest TET activity as a biomarker for predicting the efficacy of and patient response to anti–PD-1/PD-L1 therapy, and stimulation of TET activity as an adjuvant immunotherapy of solid tumors.