Compound Exposure Research Articles

Discovery of HC-7366: An Orally Bioavailable and Efficacious GCN2 Kinase Activator.

A series of activators of GCN2 (general control nonderepressible 2) kinase have been developed, leading to HC-7366, which has entered the clinic as an antitumor therapy. Optimization resulted in improved permeability compared to that of the original indazole hinge binding scaffold, while maintaining potency at GCN2 and selectivity over PERK (protein kinase RNA-like endoplasmic reticulum kinase). The improved ADME properties of this series led to robust in vivo compound exposure in both rats and mice, allowing HC-7366 to be dosed in xenograft models, demonstrating that activation of the GCN2 pathway by this compound leads to tumor growth inhibition.

Associations of urinary volatile organic compounds with cardiovascular disease among the general adult population

ABSTRACT This study was to estimate the associations of volatile organic compounds (VOCs) exposure with the prevalence of total and specific cardiovascular disease (CVD) among the general adult population. This cross-sectional study analyzed 15 urinary VOC metabolites in the general population using the 2011–2016 National Health and Nutrition Examination Survey (n = 5,213). The weighted study population with 47.0 years median age, was primarily female (51.2%). The prevalence of total CVD in the overall population was 7.9%. The single-exposure analyzes of AAMA, ATCA, CEMA, CYMA, DHBMA, 3HPMA, and 3MHA +4MHA were significantly associated with increased prevalence of total CVD. Qgcomp regression consistently showed that urinary VOCs-mixed exposure was positively correlated with the prevalence of total and specific CVDs (chronic heart failure, angina, and stroke), and highlighted each VOCs metabolite weights and direction. The similar results were observed for the WQS regression using mixed analysis methods. In conclusion, exposure to VOCs increases CVD prevalence and advances the identification of risk factors for CVD for environmental study.

Abstract 4598: Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers

Abstract Homozygous deletions of the p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus, which is responsible for regulating the cell cycle, are commonly found in cancer and often involve the deletion of adjacent genes. One such adjacent gene is methylthioadenosine phosphorylase (MTAP), involved in metabolism, located on chromosome 9p21 in close proximity to the p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP is observed in approximately 80-90% of tumors with homozygous deletion of CDKN2A, representing 10-15% of all human tumors. These tumor types, including non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma, and mesothelioma, have a poor prognosis, highlighting the significant unmet medical need in this area. Deletion of MTAP leads to a significant accumulation of methylthioadenosine (MTA) in cells. MTA, at high concentrations, selectively inhibits the PRMT5 methyltransferase enzyme, competing with the substrate S-adenosylmethionine (SAM) required for methylation reactions. As a result, the overall level of symmetric arginine dimethylation throughout the proteome is reduced. This heightened sensitivity to modulation of methylosome activity makes cells with MTAP deletion more susceptible to therapeutic targeting of PRMT5. Hence, selective targeting of PRMT5 in cancers with homozygous MTAP deletion represents a promising strategy for specifically eliminating cancer cells with this genetic alteration. Ryvu has developed MTA-cooperative PRMT5 inhibitors characterized by good drug-like physicochemical properties and inhibition of methyltransferase activity with IC50 values in the low nanomolar range. A structure-based lead optimization delivered compounds coming from two independent series with high selective potency in MTAP-deleted cell lines and DMPK profiles allowing an oral administration. The antitumor activities were compared in vitro and in vivo to MRTX1719 and AMG193 in MTAP null tumors such as HCT116 MTAP KO, DoHH-2 and Lu99. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies. Taken together, these studies confirm that MTA cooperative PRMT5 inhibitors exert strong synthetic lethal phenotype in MTAP deleted cancers and offer an exciting therapeutic opportunity for a large patient population. Citation Format: Adam Radzimierski, Aneta Bobowska, Agata Stachowicz, Kamil Kuś, Kamila Kozłowska-Tomczyk, Agnieszka Ludwig-Słomczyńska, Paulina Podkalicka-Gołda, Aniela Gołas, Monika Żukowska, Pavlo Lebed, Przemysław Wyrębek, Daria Szukiel, Matylda Stefaniak, Oleksandr Popika, Julia Krzywik, Sujit Sasmal, Paulina Niedziejko-Ćwiertnia, Klara Korta-Piątek, Karolina Fijołkowska, Aleksandra Więckowska, Marta Olszak-Płachta, Swapnil Nipunge, Mateusz Świrski, Marek Wronowski, Henryk Pawlik, Quỳnh Vũ, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Igor Tomczyk, Ewelina Gabor-Worwa, Nilesh Gaud, Anna Kowal-Chwast, Dawid Gogola, Magdalena Miodek, Róża Starczak, Agata Dudek, Jacek Faber, Bożena Winnik, Sanja Novak-Ratajczak, Agnieszka Świrska, Karolina Gluza, Paweł Guzik, Katarzyna Banaszak, Nicolas Boutard, Grzegorz Ćwiertnia, Krzysztof Brzózka, Mateusz Nowak, Anna Bartosik, Didier Pez. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4598.

Abstract 5942: Discovery of WRN inhibitors as targeted therapy in the treatment of microsatellite unstable (MSI-H) tumors

Abstract The synthetic lethality resulting from the inhibition of the WRN helicase protein has been observed in tumors characterized by high levels of microsatellite instability (MSI-H). This instability stems from a deficiency in the DNA mismatch repair (MMR) mechanisms, leading to the accumulation of DNA damage. This phenomenon is notably prevalent in 10-30% of colorectal, gastric, endometrial, and ovarian cancers. Specifically, the inhibition of WRN helicase activity induces DNA damage leading to cell cycle arrest and apoptosis exclusively in MSI-H cell lines. This selectivity underscores the therapeutic potential of WRN inhibitors, as they demonstrate efficacy against MSI-H cells while remaining non-toxic to microsatellite stable (MSS) cell lines. Ryvu is developing a chemical series of potent, selective WRN helicase inhibitors. Structure-based optimization facilitated the rapid expansion and delivery of a compound library with novel intellectual property (IP), demonstrating target engagement in cells and selective potency over other RecQ family members. The correlation between the cellular effect and selectivity upon treatment with these ligands was confirmed in the clonogenic assay, specifically in responder, microsatellite instable cell lines only, thereby leaving a substantial safety margin (>100x) relative to non-responder, microsatellite stable lines. Target engagement has been confirmed by dose dependent DNA damage pathway activation concomitant with WRN degradation in in vitro assays. The pharmacokinetic properties of these compounds are favorable for progressing to in vivo studies. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies in xenograft MSI-H cancer models. These data provide pharmacological proof-of-concept for the synthetic lethal effect of our inhibitors and support WRN inhibition as a new oncological therapy. Citation Format: Wojciech Schönemann, Agnieszka Ludwig-Słomczyńska, Łukasz Dudek, Marcin Król, Marco Farinone, Urszula Kulesza, Wojciech Jasnosz, Izabela Mac, Karol Zuchowicz, Katarzyna Wierzbicka, Aleksandra Buchcic-Szychowska, Andrzej Gondela, Anna Zagórska, Oliwia Matuszewska, Marta Sowińska, Marcin Nowogródzki, Marianna Girardi, Szymon Paluch, Svitlana Sukhomlinova, Agata Chłopek, Iana Levenets, Kamila Kozłowska-Tomczyk, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Maciej Mikulski, Justyna Jabłońska, Magdalena Wiśniewska, Katarzyna Palus-Chramiec, Paulina Podkalicka-Gołda, Anita Janiga, Sylwia Sudoł-Tałaj, Magdalena Sieprawska-Lupa, Magdalena Miodek, Jacek Faber, Róża Starczak, Bozena Winnik, Agnieszka Świrska, Anna Kowal-Chwast, Dawid Gogola, Sanja Novak Ratajczak, Agata Dudek, Nilesh Gaud, Grzegorz Satała, Agata Stachowicz, Martin Swarbrick, Krzysztof Brzózka, Magdalena I. Zawadzka. Discovery of WRN inhibitors as targeted therapy in the treatment of microsatellite unstable (MSI-H) tumors [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 5942.

Abstract 2113: HMPL-506, a novel, highly potent and differentiated menin-MLL inhibitor for the treatment of MLL-rearrangedand NPM1mutant acute leukemia in preclinical models

Abstract Background: Mixed-lineage leukemia (MLL, or KMT2A) gene rearrangements (MLL-r) occur in 5%-10% acute leukemias and are associated with poor prognosis. Nucleophosmin 1 mutations (NPM1m) are the most common genetic alterations in acute myeloid leukemia (AML). Multiple studies have illuminated that those leukemogenesis are dependent on the interaction of menin-MLL, which controls downstream gene expressions associated with cell proliferation and differentiation, e.g. HOXA9 and CD11b. Herein, we introduce HMPL-506, a novel and highly differentiated small molecule compound targeting menin-MLL interaction. Methods and Results: Biochemical assay revealed that HMPL-506 potently blocked menin-MLL binding with IC50 of 1.0 nM. In cell lines carrying MLL-r or NPM1m, HMPL-506 substantially down-regulated menin-MLL target genes transcription of MEIS1 and HOXA9 and upregulated the differentiation marker of CD11b by RT-PCR assay, consequently, HMPL-506 attenuated tumor cell growth, with GI50 ranging from 3.0 to 12.1 nM in MLL-r cell lines (MV-4-11, MOML-13 and RS4;11) and 22.4 nM in NPM1m cell line (OCI-AML-3) in CellTiter-Glo cell viability assay. Notably, compared with the other 5 menin inhibitors in clinical stage, HMPL-506 showed the strongest inhibitory potency in MLL-r and NPM1m cell line models. In vivo studies demonstrated the dose- and exposure- dependent target regulation of MEIS1 and anti-tumor efficacy following HMPL-506 treatment in multiple tumor xenograft models. For instance, in MV-4-11 subcutaneous model, once daily oral administration of HMPL-506 at 5, 10 and 25 mg/kg for 4 days in BALB/c nude mice induced dose-dependent inhibition of MEIS1 gene expression, positively correlating with compound exposures in tumor tissues; continuous dosing of HMPL-506 at 5 mg/kg for 28 days reduced the tumor growth by 86%, which was comparable to the efficacy induced by SNDX-5613 at 50 mg/kg. Treatment of HMPL-506 at 10 mg/kg and 25 mg/kg resulted in tumor shrinkage in all treated animals, with tumor regression rates of 72% and 100%, respectively. Furthermore, HMPL-506 synergistically improved anti-tumor effect of azacytidine, venetoclax and gilteritinib against MLL-r leukemias both in vitro and in vivo. HMPL-506 displayed favorable PK profiles and high selectivity among multiple kinases, methyltransferases and safety related targets. More importantly, the IC50 of HMPL-506 on hERG patch clamp was over 60 µM, indicating low risk of QTc prolongation in human. Conclusion: HMPL-506 is a novel and highly differentiated menin-MLL inhibitor with robust anti-tumor activities, favorable ADME properties and low risk of cardiac toxicity, warranting it further clinical development for treatment of MLL-r and NPM1m acute leukemias. HMPL-506 IND is expected to be cleared in H1 2024. Citation Format: Min Cheng, Liang Ge, Zhihu Gao, Zeyu Zhong, An Jiang, Wei Zhang, Jia Hu, Shuwen Jiang, Na Li, Na Yang, Jian Wang, Yang Sai, Weiguo Qing, Yongxin Ren, Weiguo Su. HMPL-506, a novel, highly potent and differentiated menin-MLL inhibitor for the treatment of MLL-rearrangedand NPM1mutant acute leukemia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2113.

Abstract 2472: Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models

Abstract Background: ENPP1, a nucleotide pyrophosphatase and phosphodiesterase, has emerged as an important modulator of anti-tumor immune activation, as it regulates the concentrations of cGAMP and ATP in the TME. ENPP1 expression in tumors is common and correlates with a cold tumor phenotype. Its loss has been reported to suppress metastasis, restore immune cell infiltration into the tumor and potentiate the response to immune checkpoint blockade, while, conversely, the overexpression of ENPP1 promoted metastasis and rendered tumors resistant to checkpoint blockade. RBS2418 was designed as a potent and selective inhibitor of ENPP1 and is in clinical development. Methods: ENPP1 enzyme inhibition was measured in vitro using cGAMP and ATP substrates. Effects of pH and serum and selectivity against related enzymes were evaluated. In vivo efficacy was assessed in Hepa1-6, and GL261-luc syngeneic liver and glioblastoma mouse models. Compound exposures were determined in tumor and blood samples. Cures were confirmed by re-challenge of cured animals with the same cancer cells. Results: RBS2418 inhibited hydrolysis of cGAMP and ATP in vitro with similar potency (Ki =0.14 and 0.13 nM). Inhibition was also similar in human serum, consistent with low binding affinity to human serum albumin. In humans, oral dosing at doses of 100 mg could inhibit ENPP1 enzyme activity during the full dosing interval. Dosing of RBS2418 was performed in mice to achieve trough concentrations of RBS2418 in tumors and plasma exceeding ENPP1 EC90 levels for the complete dosing interval. In the Hepa1-6 model treatment with RBS2418 for either 2 or 10 days resulted in significant reduction of tumor growth and prolongation of survival as compared to vehicle control. There was no significant difference between the RBS2418 groups. Complete tumor regression was observed in 44% of treated mice across the two treatment groups. These mice were resistant to rechallenge with Hepa1-6 tumor cells. In the GL261-luc model treatment with RBS2418 showed significant reduction of tumor burden and prolongation of survival as compared to vehicle control. The mice that achieved complete tumor regression were resistant to rechallenge with GL261-luc tumor cells. Conclusions: RBS2418 inhibited ATP and cGAMP hydrolysis by ENPP1 with similar high potency. The pharmacology profile was optimized to achieve low serum protein binding and oral bioavailability in humans. RBS2418 treatment was well tolerated in mice under conditions where tumor and plasma levels exceeding ENPP1 serum EC90 levels were achieved. In the syngeneic mouse models Hepa1-6 and GL261-luc significant tumor growth reductions, complete tumor regression and cures were observed. Resistance to cancer cell rechallenge was consistent with treatment-induced anti-tumor immunity. Citation Format: Ningwu Huang, Junjun Cheng, Ron Hawley, Klaus Klumpp. Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2472.

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

Abstract BRM (SMARCA2) and BRG1 (SMARCA4) are highly homologous ATPases that are members of the BAF (also known as the mSWI/SNF) chromatin remodeling complex. BRM and BRG1 are mutually exclusive enzymatic subunits of BAF complexes, and functional genomic screens have shown a synthetic lethal relationship between the two genes. BRG1 is frequently mutated in cancer, including in approximately 10% of non-small cell lung carcinomas. Selective inhibition of BRM is a mechanism by which BRG1-mutated cancer cell growth would be affected by losing BRM function, while normal tissues should be spared as they still express functional BRG1. Given that the ATPase domains of BRM and BRG1 are 92% identical, the identification of selective enzymatic inhibitors of BRM has been challenging. Here, we report the discovery of novel, highly potent compounds that selectively inhibit BRM over BRG1 as seen in cell-based transcription and proliferation assays, that also possess excellent oral pharmacokinetics. Our lead compounds inhibit BRM in a cell-based reporter assay with an unbound IC50 of 1 nM and are greater than 20-fold selective over BRG1. They are also selective against other helicases and do not show any significant activity in off-target screening panels. Dosing of mice carrying BRG1-mutant xenografts causes target gene modulation that is correlated with compound exposure. Treatment of A549 and RERF-LC-AI xenografts with lead compounds led to tumor growth inhibition of 87% and 96%, respectively, at well-tolerated doses, and significant tumor growth inhibition was also achieved in multiple other BRG1-mutant models. The results suggest that our compounds have first-in-class potential as potent and selective BRM ATPase inhibitors for the treatment of BRG1-mutated cancers, and we are planning to file an IND in 2024. Citation Format: Janice Y. Lee, Nathan Brooks, Brandon Antonakos, Bryan Perria, Candace Langan, Bonita D. Jones, Robert Stephen Flack, Zhifang Li, David Terry, Ross Wallace, Robert Bondi, Gereint Sis, Ronee Baracani, Maralee McVean, Gabrielle Kolakowski, Dayo Osimboni, Kevin Wilson. Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3230.

Abstract 2097: ABT co-dose accelerated target validation with a fit-for-purpose HPK1 inhibitor tool

Abstract Target validation can be challenging when mouse efficacy studies require achieving a high therapeutic exposure of a tool compound, ideally with a low peak-to-trough (P/T) ratio to avoid Cmax-induced toxicity. Herein, we describe the daily co-administration of the CYP inhibitor aminobenzotriazole (ABT) as a well-tolerated PK enhancer for medium-term efficacy studies in mice, enabling faster decision-making in the early space with imperfect tools. Genetic studies in mice have suggested that HPK1 inhibition would lead to enhanced effector T-cell function, decreased tumor growth and enhanced αPD-L1 efficacy. However, it remains unknown whether immune activation by pharmacological inhibition of HPK1 will recapitulate the genetic deletions. We developed a fit for purpose tool compound with picomolar HPK1 potency, excellent kinome selectivity (98% >100x, ATP-Km corrected), dose-dependent inhibition of pSLP76, sustained IL2 elevation in PBMCs, and IL6/TNFa elevation in dendritic cells. The mouse PK for this tool was inadequate, having a low exposure (AUC 16 uM-hr at 100 mg/kg po) and a high P/T ratio of 13 that could lead to an erosion of kinome selectivity in vivo; complicating efficacy interpretation and limiting overall tolerability. However, when co-dosed with ABT (50 mg/kg po 2 h prior to the HPK1 inhibitor), improved exposure of our tool was achieved (AUC 63 uM-hr at 100 mg/kg po), 90% inhibition of pSLP76 was maintained at C(trough), and the P/T ratio was reduced to 3 to avoid Cmax-induced toxicity. The inhibitor was well-tolerated co-dosed with ABT for three weeks in mice, enabling studies sooner to probe HPK1 pharmacological inhibition in cancer immunotherapy as a monotherapy as well as in combination with a mouse anti-PD-1 antibody. Citation Format: Joey L. Methot, Joanna L. Chen, Elsie Yu, Ryan D. Otte, Haiyan Xu, Zangwei Xu, Brian Lacey, Jason Laskey, Robbie Mcleod, Dustin Smith, Alexander Pasternak. ABT co-dose accelerated target validation with a fit-for-purpose HPK1 inhibitor tool [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 2097.

Abstract 520: circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition

Abstract DHX9 is a multifunctional DEAH-box ATP-independent RNA helicase which has been reported to play important roles in replication, transcription, translation, RNA splicing and RNA processing which contribute to DHX9’s role in maintenance of genomic stability. DHX9 can bind specifically to inverted repeat Alu elements, preventing back-splicing events that lead to circular RNA and exon skipping of the linear transcript. Upon DHX9 inhibition, a robust induction of Alu-mediated circular RNA is observed. For example, circBRIP1 is an Alu-mediated circular RNA that is robustly elevated upon DHX9 loss, with no observed change to levels of linear BRIP1. Here we describe data demonstrating circBRIP1 as a DHX9 specific target engagement pharmacodynamic (PD) biomarker and its potential utility as a non-invasive clinical PD biomarker. We have previously demonstrated that DHX9 inhibition by novel small molecule inhibitors is efficacious in microsatellite instable (MSI) colorectal cancer (CRC) with defective mismatch repair (dMMR), both in vitro and in vivo. DHX9 inhibition in the HCT116 dMMR CRC cell line results in a robust dose dependent induction of the Alu-mediated circular RNA, circBRIP1, as early as 6 hours after compound treatment. The development of lead series DHX9 inhibitors exhibited a positive correlation between circBRIP1 EC50 and DHX9 biochemical activity, as well as proliferation inhibition in a dMMR CRC cell line. However, dose dependent induction of circBRIP1 following DHX9 inhibition occurs with similar potencies across DHX9i sensitive and insensitive CRC cell lines. This suggests that elevation of circBRIP1 is a unique proximal target engagement PD biomarker of DHX9 inhibition, but not a predictor of DHX9 sensitivity. Importantly, induction of circBRIP1 is observed intratumorally in human xenograft in vivo efficacy studies in mice. Induction in each individual tumor correlates with observed tool compound exposure, providing evidence of a predictable PK/PD relationship. We also observe dose dependent circBRIP1 induction in mouse peripheral blood mononuclear cells (PBMC). We have extended these studies to primary human whole blood, where we see robust dose dependent induction of circBRIP1 after ex vivo treatment with DHX9 inhibitor for 24 hours. Altogether, these studies show evidence for the utility of circBRIP1 as a non-invasive target engagement biomarker for DHX9 inhibitors in the clinic. Citation Format: David Brennan, Jennifer Castro, Matthew H. Daniels, Monique Laidlaw, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition [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 520.

Mechanism-Based Macrocyclic Inhibitors of Serine Proteases.

Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery.

Association of volatile organic compound exposure with metabolic syndrome and its components: a nationwide cross-sectional study

Background Metabolic syndrome (MetS) is a health issue consisting of multiple metabolic abnormalities. The impact of exposure to volatile organic compounds (VOCs) on MetS and its components remains uncertain. This study aimed to assess the associations of individual urinary metabolites of VOC (mVOCs) and mVOC mixtures with MetS and its components among the general adult population in the United States.Methods A total of 5345 participants with eligible data were filtered from the 2011–2020 cycles of the National Health and Nutrition Examination Survey. Multivariate logistic regression models were applied to assess the associations of individual mVOCs with MetS and its components. The least absolute shrinkage and selection operator (LASSO) regression models were constructed to identify more relevant mVOCs. The weight quantile sum regression model was applied to further explore the links between mVOC co-exposure and MetS and its components.ResultsThe results indicated positive associations between multiple mVOCs and MetS, including CEMA, DHBMA, and HMPMA. CEMA was found to be positively correlated with all components of MetS. HMPMA was associated with elevated triglyceride (TG), reduced high-density lipoprotein, and fasting blood glucose (FBG) impairment; 3HPMA was associated with an elevated risk of high TG and FBG impairment; and DHBMA had positive associations with elevated TG and high blood pressure. The co-exposure of LASSO-selected mVOCs was associated with an increased risk of elevated TG, high blood pressure, and FBG impairment.ConclusionPositive associations of certain individual urinary mVOCs and mVOC mixtures with MetS and its components were observed by utilizing multiple statistical models and large-scale national data. These findings may serve as the theoretical basis for future experimental and mechanistic studies and have important implications for public health.

Chloroform associated with bone mineral density and bone mineral content in adults: A population-based cross-sectional research.

Bone mineral density is an important indicator of osteoporosis, and its variation with volatile organic compounds exposure has rarely been studied. However, the relationship between chloroform (an essential volatile organic compounds component) and bone mineral density remains unclear. Consequently, we aimed to explore the relationship between chloroform alone and bone mineral density or bone mineral content. Herein, 2,553 individuals aged 18 and above from the National Health and Nutrition Examination Surveys (NHANES) in 2009-2010, 2013-2014, and 2017-2020, were included. We employed two independent t-tests and multi-linear regression models to statistically assess the relationship between chloroform exposure and BMD/BMC in the spine and femoral area. A "V"-shaped correlation between chloroform exposure and bone mineral density or bone mineral content (BMD/BMC) was observed in the unadjusted model, particularly in the Ward's triangle and femoral neck as a whole. A negative correlation was specifically observed for the Ward's triangle BMD/BMC and L4 BMD/BMC. On the other hand, in the adjusted model, a dominantly negative correlation between the L4 BMC and chloroform exposure was observed over a range of exposure levels. The subgroup analysis revealed a negative correlation between chloroform concentrations and BMC in the femur and spine, especially in women and the 65-80 age population. Our study revealed a "V" shaped correlation between chloroform and BMD/BMC of the femur and spine in U.S. adults. This finding highlights the fact that prolonged exposure to chloroform may cause the changes in BMD/BMC.

Association of the blood levels of specific volatile organic compounds with nonfatal cardio-cerebrovascular events in US adults

BackgroundCardio-cerebrovascular diseases constitute a major global public health burden. Volatile organic compounds (VOCs) exposure has become progressively severe, endangering human health and becoming one of the main concerns in environmental pollution. The associations of VOCs exposure with nonfatal cardio-cerebrovascular events have not been identified in observational study with a large sample size, so we aim to examine the association in US adult population.MethodsAdults aged > 18 years with complete data regarding selected blood levels of VOCs (including benzene, ethylbenzene, o-xylene, and m-/p-xylene) and nonfatal cardio-cerebrovascular events were included in the analysis (n = 3,968, National Health and Nutrition Examination Survey, NHANES, 2013–2018 survey cycle). Participants were classified into low- and high-exposure based on whether above selected VOCs low limit detect concentration or median value. Weighted multivariate logistic analyses and subgroup analyses were used to detect the association between selected VOCs exposure and nonfatal cardio-cerebrovascular events in US adults.ResultsWeighted multivariate logistic analyses showed that the high-VOCs exposure group had an increased risk of nonfatal cardio-cerebrovascular events compared with the low-VOCs exposure group; the adjusted odds ratios (OR) and 95% confidence intervals (CI) of nonfatal cardio-cerebrovascular events for the high-VOCs exposure group were 1.41 (0.91, 2.19), 1.37 (0.96, 1.95), 1.32 (0.96, 1.82), and 1.17 (0.82, 1.67) for benzene, ethylbenzene, o-xylene, and m-/p-xylene, respectively, which was not significant assuming statistical significance at a 0.05 significance level (95% CI) for a two-tailed test. Lastly, we found high-VOCs exposure was associated with increased incidence of nonfatal cardio-cerebrovascular events in both daily smokers an non-daily smokers (p-interaction > 0.01), but the association was not statistically significant in non-daily smokers.ConclusionsThis study found that VOCs (benzene, ethylbenzene, o-xylene, and m-/p-xylene) exposure was associated with increased incidence of nonfatal cardio-cerebrovascular events in US adults, and the results need to be confirmed by larger cohort studies.

(206) Novel Observations about Epididymal Cysts Found in a Cohort of Michigan Men

Abstract Introduction Epididymal cysts (EC) are an incidental finding on scrotal ultrasounds (SUS), with an anecdotal prevalence from 5-20% with peaks at age 2 and in puberty. A recent survey at a children’s hospital in Michigan found that up to 35% of boys 15 or older had ECs. While adult men often demonstrate EC, comparatively few papers characterize the prevalence of ECs in adults. It is presumed that ECs most commonly develop during their 20s-40s but no clear prevalence by age has been established in the general adult population. Objective This present research will investigate within the same geographic region the prevalence of ECs in adult men. Methods A retrospective review was conducted for men 18-years-of-age and older who underwent a Michigan hospital-based SUS between 2010 – 2015. Patients without EC and EC patients were compared using two-sample t test, associations between age and presence of ECs were evaluated by chi-square test, and associations between age and TTV were evaluated by one-way ANOVA and Pearson’s correlation coefficient. Results 357 out of 801 men (44.6%) were found to have sonographic evidence of ECs. When separated into age groups, the prevalence of ECs significantly increased (p=0.041) with increasing age. In men with only ECs, their TTV did not significantly decrease with age (r = 0.06, p=0.684), unlike men without EC (r = 0.18, p=0.028). Conclusions This geographic cohort of men have the highest prevalence of ECs reported in the global literature. Two novel observations are that ECs frequency increases with age and TTV is preserved with age in men with ECs. TTV correlates significantly with FSH, LH, testosterone, and sperm health – further investigation should examine how preserved TTV may correlate with these values. Future studies should also examine the link between endocrine disrupting compound exposure and development of ECs. Disclosure No.

Exploring spatial heterogeneity in synergistic effects of compound climate hazards: Extreme heat and wildfire smoke on cardiorespiratory hospitalizations in California.

Extreme heat and wildfire smoke events are increasingly co-occurring in the context of climate change, especially in California. Extreme heat and wildfire smoke may have synergistic effects on population health that vary over space. We leveraged high-resolution satellite and monitoring data to quantify spatially varying compound exposures to extreme heat and wildfire smoke in California (2006-2019) at ZIP Code Tabulation Area (ZCTA) level. We found synergistic effects between extreme heat and wildfire smoke on daily cardiorespiratory hospitalizations at the state level. We also found spatial heterogeneity in such synergistic effects across ZCTAs. Communities with lower education attainment, lower health insurance coverage, lower income, lower proportion of automobile ownership, lower tree canopy coverage, higher population density, and higher proportions of racial/ethnic minorities experienced higher synergistic effects. This study highlights the need to incorporate compound hazards and environmental justice considerations into evidence-based policy development to protect populations from increasingly prevalent compound hazards.

Exposure characteristics and health risk assessment of 97 typical chemical pollutants in human serum

Rapid industrial and agricultural developments in China have led to the wide use and discharge of chemical products and pesticides, resulting in extensive residues in environmental media. These residues can enter the human body through various pathways, leading to high exposure risks and health hazards. Because the human body is exposed to a variety of chemical pollutants, accurately quantifying the exposure levels of these pollutants in the human body and evaluating their health risks are of great importance. In this study, the serum concentrations of 97 typical chemical pollutants of 60 adults in central China were simultaneously determined using solid-phase extraction coupled with gas chromatography-tandem mass spectrometry (SPE-GC-MS/MS). In this method, 200 μL of a serum sample was mixed with 10 μL of an isotope-labeled internal standard solution. The sample was vortexed and refrigerated overnight at 4 ℃. Each sample was then deproteinized by the addition of 200 μL of 15% formic acid aqueous solution and vortexed. The serum sample was loaded into a preconditioned Oasis® PRiME HLB SPE cartridge and rinsed with 3 mL of methanol-water (6∶1, v/v). The SPE cartridge was subsequently vacuumed. The analytes were eluted with 3 mL of dichloromethane followed by 3 mL of n-hexane. The eluent was concentrated to near dryness under a gentle nitrogen stream and reconstituted with 100 μL of acetone. The samples were determined by GC-MS/MS and separated on a DB-5MS capillary column (30 m×0.25 mm×0.25 μm) with temperature programming. The column temperature was maintained at 70 ℃ for 2 min, increased at a rate of 25 ℃/min to 150 ℃, increased at a rate of 3 ℃/min to 200 ℃, and then held for 2 min. Finally, the column temperature was increased at a rate of 8 ℃/min to 300 ℃ and maintained at this temperature for 8 min. The samples were detected in multiple-reaction monitoring (MRM) mode and quantitatively analyzed using the internal standard method. Multiple linear regression models were used to analyze the effects of demographic characteristics, lifestyle habits, and diet on the concentrations of the chemical pollutants in the serum samples, and known biomonitoring equivalents (BEs) and human biomonitoring (HBM) values were combined to compute hazard quotients (HQs) and hazard indices (HIs) and evaluate the health risks of single and cumulative exposures to the chemical pollutants. The results showed that the main pollutants detected in human serum were organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). The detection rates of eight pollutants, including hexachlorobenzene (HCB) (100%), pentachlorophenol (PCP) (100%), p,p'-dichlorodiphenylene (p,p'-DDE) (100%), PCB-138 (100%), PCB-153 (98.3%), β-hexachlorocyclohexane (β-HCH) (91.7%), fluorene (Flu) (85.0%), and anthracene (Ant) (75.0%), were greater than 70%. The serum levels of β-HCH were higher in females than in males, and age was positively correlated with exposure to p,p'-DDE, PCB-138, PCB-153, and β-HCH. Increased exposure levels to p,p'-DDE and β-HCH may be associated with a high frequency of meat intake, whereas increased exposure level to PCP may be associated with a high frequency of vegetable intake. The serum HQ of PCP was greater than 1 in 6.7% of the samples, and no risk was observed for HCB and p,p'-DDE exposure in the study population. Approximately 28.3% of the study subjects had HI values greater than 1. Overall, the general adult population in this region is widely exposed to a wide range of chemical pollutants, and gender, age, and diet are likely to be the main factors influencing the concentration of chemical pollutants. The health risk of single and compound exposures to chemical pollutants should not be ignored.

Pesticides in small volume plasma samples: Method development and application to smallmouth bass (Micropterus dolomieu) from the Chesapeake Bay watershed, USA

Nontarget organisms are exposed to pesticides following applications in agricultural and urban settings, potentially resulting in deleterious effects. Direct measurements of pesticides in biological tissues may aid in characterizing exposure, accumulation, and potential toxicity versus analyses in environmental media alone (e.g., water, soil, and air). Plasma represents a nonlethal sampling medium that can be used to assess recent exposures to contaminants. Herein, a method was developed to test the extraction of 210 pesticides and their transformation products in small volume plasma samples (100 μL). Plasma samples were protein precipitated with 0.5 % formic acid in acetonitrile added to the sample (ratio of 3.5:1). Pass-through solid phase extraction was used for sample matrix and lipid removal and samples were analyzed by liquid chromatography and gas chromatography with tandem mass spectrometry. Recoveries of 70.0–129.8 % were achieved for 182 pesticides and degradates across the low (25 ng mL−1), medium (100 ng mL−1), and high (250 ng mL−1) spike levels. Method detection levels ranged 0.4–13.0 ng mL−1. Following development, the method was applied to smallmouth bass (Micropterus dolomieu) plasma samples (n = 10) collected from adults in the Chesapeake Bay watershed. Individual plasma samples resulted in four to seven analytes detected with summed concentrations ranging 16.4–95.0 ng mL−1. Biological multiresidue pesticide methods help elucidate recent exposures of bioactive compounds to nontarget organisms.

Insight into the adsorption behaviors and bioaccessibility of three altered microplastics through three types of advanced oxidation processes

Advanced oxidation processes (AOPs) can significantly alter the structural properties, environmental behaviors and human exposure level of microplastics in aquatic environments. Three typical microplastics (Polyethylene (PE), polypropylene (PP), and polystyrene (PS)) and three AOPs (Heat-K2S2O8 (PDS), UV-H2O2, UV-peracetic acid (PAA)) were adopted to simulate the process when microplastics exposed to the sewage disposal system. 2-Nitrofluorene (2-NFlu) adsorption experiments found the equilibrium time decreased to 24 hours and the capacity increased up to 610 μg g−1, which means the adsorption efficiency has been greatly improved. The fitting results indicate the adsorption mechanism shifted from the partition dominant on pristine microplastic to the physical adsorption (pore filling) dominant. The alteration of specific surface area (21 to 152 m2 g−1), pore volume (0.003 to 0.148 cm3 g−1) and the particle size (123 to 16 μm) of microplastics after AOPs are implying the improvement for pore filling. Besides, the investigation of bioaccessibility is more complex, AOPs alter microplastic with more oxygen-containing functional groups and lower hydrophobicity detected by XPS and water contact angle, those modifications have increased the sorption concentration, especially in the human intestinal tract. Therefore, this indicates the actual exposure of organic compounds loaded in microplastic may be higher than in the pristine microplastic. This study can help to assess the human health risk of microplastic pollution in actual environments.

Caffeic acid phenethyl ester mediates apoptosis in serum-starved HT29 colon cancer cells through modulation of heat shock proteins and MAPK pathways.

Colorectal cancer (CRC) is among the most prevalent gastrointestinal cancers of epithelial origin worldwide, with over 2 million cases detected every year. Emerging evidence suggests a significant increase in the levels of inflammatory and stress-related markers in patients with CRC, indicating that oxidative stress and lipid peroxidation may influence signalling cascades involved in the progression of the disease. However, the precise molecular and cellular basis underlying CRC and their modulations during bioactive compound exposure have not yet been deciphered. This study examines the effect of caffeic acid phenethyl ester (CAPE), a natural bioactive compound, in HT29 CRC cells grown under serum-supplemented and serum-deprived conditions. We found that CAPE inhibited cell cycle progression in the G2/M phase and induced apoptosis. Migration assay confirmed that CAPE repressed cancer invasiveness. Protein localisation by immunofluorescence microscopy and protein expression by western blot analysis reveal increased expressions of key inflammatory signalling mediators such as p38α, Jun N-terminal kinase and extracellular signal-regulated kinase (ERK) proteins. Molecular docking data demonstrates that CAPE shows a higher docking score of -5.35 versus -4.59 to known p38 inhibitor SB203580 as well as a docking score of -4.17 versus -3.86 to known ERK1/2 inhibitor AZD0364. Co-immunoprecipitation data reveals that CAPE treatment effectively downregulates heat shock protein (HSP) expression in both sera-supplemented and limited conditions through its interaction with mitogen-activated protein kinase 14 (MAPK14). These results suggest that stress induction via serum starvation in HT29 CRC cells leads to the induction of apoptosis and co-ordinated activation of MAPK-HSP pathways. Molecular docking studies support that CAPE could serve as an effective inhibitor to target p38 and MAPK compared to their currently known inhibitors.

Synthesis of 1-(4-chlorobenzoyl)-4-(dimethylamino) pyridin-1-ium chloride and determination of cytotoxicity and apoptosis in human colon cancer cells

BackgroundIn this experiment, we synthesized a new compound, 1-(4-chlorobenzoyl)-4-(dimethylamino) pyridin-1-ium chloride (SM-9), and examined its toxicity and anticancer activity in human colon cancer (HCT-116) cells. MethodsWe uncovered the underlying mechanisms of cell toxicity and apoptosis due to SM-9 compound exposure in HCT-116 cells by using the MTT assay and protein profiling array and measuring gene expression levels using reverse transcription-polymerase chain reaction. ResultsOur data show that the SM-9 compound activated the caspase-3, caspase-8, p21, p27, and p53 proteins involved in the apoptosis of HCT-116 cells, thereby inducing cytotoxicity, the formation of reactive oxygen species, and apoptosis. The results of this study show that the SM-9 compound has advantageous qualities and must be taken as an anticancer medication. We conclude that the SM-9 compound exerted an anticancer effect by inducing the apoptotic pathways in human colon cancer (HCT-116) cells.