Specific Inhibitor Research Articles

The Sirtuin 5 Inhibitor MC3482 Ameliorates Microglia‑induced Neuroinflammation Following Ischaemic Stroke by Upregulating the Succinylation Level of Annexin-A1.

In our previous study, we concluded that sirtuin 5 (SIRT5) was highly expressed in microglia following ischaemic stroke, which induced excessive neuroinflammation and neuronal injury. Therefore, SIRT5-targeting interventions should reduce neuroinflammation and protect against ischaemic brain injury. Here, we showed that treatment with a specific SIRT5 inhibitor, MC3482, alleviated microglia-induced neuroinflammation and improved long-term neurological function in a mouse model of stroke. The mice were administrated with either vehicle or 2mg/kg MC3482 daily for 7days via lateral ventricular injection following the onset of middle cerebral artery occlusion. The outcome was assessed by a panel of tests, including a neurological outcome score, declarative memory, sensorimotor tests, anxiety-like behavior and a series of inflammatory factors. We observed a significant reduction of infarct size and inflammatory factors, and the improvement of long-term neurological function in the early stages during ischaemic stroke when the mice were treated with MC3482. Mechanistically, the administration of MC3482 suppressed the desuccinylation of annexin-A1, thereby promoting its membrane recruitment and extracellular secretion, which in turn alleviated neuroinflammation during ischaemic stroke. Based on our findings, MC3482 offers promise as an anti-ischaemic stroke treatment that targets directly the disease's underlying factors.

Inhibition of ROS/caspase-3/GSDME-mediated pyroptosis alleviates high glucose-induced injury in AML-12 cells

Diabetic liver injury (DLI) is a chronic complication of the liver caused by diabetes, and its has become one of the main causes of nonalcoholic fatty liver disease (NAFLD). The gasdermin E (GSDME)-dependent pyroptosis signaling pathway is involved in various physiological and pathological processes; however, its role and mechanism in DLI are still unknown. This study was performed to investigate the role of GSDME-mediated pyroptosis in AML-12 cell injury induced by high glucose and to evaluate the therapeutic potential of caspase-3 inhibition for DLI. The results showed that high glucose activated apoptosis by regulating the apoptotic protein levels including Bax, Bcl-2, and enhanced cleavage of caspase-3 and PARP. Notably, some of the hepatocytes treated with high glucose became swollen, accompanied by GSDME-N generation, indicating that pyroptosis was further induced by active caspase-3. Moreover, the effects of high glucose on AML-12 cells could be partly reversed by a reactive oxygen scavenger (NAC) and caspase-3 specific inhibitor (Z-DEVD-FMK), which suggests high glucose induced GSDME-dependent pyroptosis in AML-12 cells through increasing ROS levels and activating caspase-3. In conclusion, our results show that high glucose can induce pyroptosis in AML-12 cells, at least in part, through the ROS/caspase-3/GSDME pathway，and inhibition of caspase-3 can ameliorate high glucose-induced hepatocyte injury, providing an important basis for clarifying the pathogenesis and treatment of DLI.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

Identification of a De Novo Peptide against Palmitoyl Acyltransferase 6 to Block Survivability and Infectivity of Leishmania donovani.

Palmitoylation is an essential post-translational modification in Leishmania donovani, catalyzed by enzymes called palmitoyl acyl transferases (PATs) and has an essential role in virulence. Due to the toxicity and promiscuity of known PAT inhibitors, identification of new molecules is needed. Herein, we identified a specific novel de novo peptide inhibitor, PS1, against the PAT6 Leishmania donovani palmitoyl acyl transferase (LdPAT6). To demonstrate specific inhibition of LdPAT6 by PS1, we employed a bacterial orthologue system and metabolic labeling-coupled click chemistry where both LdPAT6 and PS1 were coexpressed and displayed palmitoylation suppression. Furthermore, strong binding of the LdPAT6-DHHC domain with PS1 was observed through analysis using microscale thermophoresis, ELISA, and dot blot assay. PS1 specific to LdPAT6 showed significant growth inhibition in promastigotes and amastigotes by expressing low cytokines levels and invasion. This study reveals discovery of a novel de novo peptide against LdPAT6-DHHC which has potential to block survivability and infectivity of L. donovani.

Lycopene promoted M2 macrophage polarization via inhibition of NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to Escherichia coli infection in J744A.1 cells.

Escherichia coli (E. coli) can induce severe clinical bovine mastitis, which is to blame for large losses experienced by dairy farms. Macrophage polarization into various states is in response to pathogen infections. Lycopene, a naturally occurring hydrocarbon carotenoid, relieved inflammation by controlling M1/M2 status of macrophages. Thus, we wanted to explore the effect of lycopene on polarization states of macrophages in E. coli-induced mastitis. Macrophages were cultivated with lycopene for 24, before E. coli inoculation for 6h. Lycopene (0.5μmol/L) significantly enhanced cell viabilities and significantly reduced lactic dehydrogenase (LDH) levels in macrophages, whereas 2 and 3μmol/L lycopene significantly enhanced LDH activities. Lycopene treatment significantly reduced the increase in LDH release, iNOS, CD86, TNF-α, IL-1β and phosphatase and tensin homolog (PTEN) expressions in E. coli group. 0.5μmol/L lycopene significantly increased E. coli-induced downregulation of CD206, arginase I (ARG1), indoleamine 2,3-dioxygenase (IDO), chitinase 3-like 3 (YM1), PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, jumonji domain-containing protein-3 (JMJD3) and interferon regulatory factor 4 (IRF4) levels. Moreover, Ginkgolic acid C17:1 (a specific PTEN inhibitor), 740YPDGFR (a specific PI3K activator), SC79 (a specific AKT activator) or CHPG sodium salt (a specific NF-κB activator) significantly decreased CD206, AGR1, IDO and YM1 expressions in lycopene and E. coli-treated macrophages. Therefore, lycopene increased M2 macrophages via inhibiting NOTCH1-PI3K-mTOR-NF-κB-JMJD3-IRF4 pathway in response to E. coli infection in macrophages. These results contribute to revealing the pathogenesis of E. coli-caused bovine mastitis, providing the new angle of the prevention and management of mastitis.

A CARM1 Inhibitor Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

CARM1, belonging to the protein arginine methyltransferase (PRMT) family, is intricately associated with the progression of cancer and is viewed as a promising target for both cancer diagnosis and therapy. However, the number of specific and potent CARM1 inhibitors is limited. We herein discovered a CARM1 inhibitor, iCARM1, that showed better specificity and activity toward CARM1 compared to the known CARM1 inhibitors, EZM2302 and TP-064. Similar to CARM1 knockdown, iCARM1 suppressed the expression of oncogenic estrogen/ERα-target genes, whereas activated type I interferon (IFN) and IFN-induced genes (ISGs) in breast cancer cells. Consequently, iCARM1 potently suppressed breast cancer cell growth both in vitro and in vivo. The combination of iCARM1 with either endocrine therapy drugs or etoposide demonstrated synergistic effects in inhibiting the growth of breast tumors. In summary, targeting CARM1 by iCARM1 effectively suppresses breast tumor growth, offering a promising therapeutic approach for managing breast cancers in clinical settings.

Gastrodin regulates the expression of renin-angiotensin system-SIRT3 and proinflammatory mediators in reactive astrocytes via activated microglia.

Gastrodin, an anti-inflammatory herbal agent, is known to suppress microglia activation. Here, we investigated whether it would exert a similar effect in reactive astrocytes and whether it might act through the renin-angiotensin system (RAS) and sirtuin 3 (SIRT3). Angiotensinogen (ATO), angiotensin-converting enzyme (ACE), angiotensin II type 1 (AT1) and type 2 (AT2) receptor and SIRT3 expression was detected in TNC-1 astrocytes treated with BV-2 microglia conditioned medium (CM) with or without gastrodin and lipopolysaccharide (LPS) pre-treatment by RT-PCR, immunofluorescence and western blotting analysis. Expression of C3 (A1 astrocyte marker), S100A10 (A2 astrocyte marker), proinflammatory cytokines and neurotrophic factors was then evaluated. The results showed a significant increase of ATO, ACE, AT1, SIRT3, C3, proinflammatory cytokines and neurotrophic factors expression in TNC-1 astrocytes incubated in CM + LPS when compared with cells incubated in the CM, but AT2 and S100A10 expression was reduced. TNC-1 astrocytes responded vigorously to BV-2 CM treated with gastrodin + LPS as compared with the control. This was evident by the decreased expression of the abovementioned protein markers, except for AT2 and S100A10. Interestingly, SIRT3, IGF-1 and BDNF expression was enhanced, suggesting that gastrodin inhibited the expression of RAS and proinflammatory mediators but promoted the expression of neurotrophic factors. And gastrodin regulated the phenotypic changes of astrocytes through AT1. Additionally, azilsartan (a specific inhibitor of AT1) inhibited the expression of C3 and S100A10, which remained unaffected in gastrodin and azilsartan combination treatment. These findings provide evidence that gastrodin may have a therapeutic effect via regulating RAS-SIRT3.

Inhibition of QDPR synergistically modulates intracellular tetrahydrobiopterin profiles in cooperation with methotrexate

Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis in monoaminergic neurons, phenylalanine metabolism in hepatocytes, and nitric oxide synthesis in endothelial and immune cells. BH4 is consumed as a cofactor or is readily oxidized by autooxidation. Quinonoid dihydropteridine reductase (QDPR) is an enzyme that reduces quinonoid dihydrobiopterin (qBH2) back to BH4, and we have previously demonstrated the significance of QDPR in maintaining BH4 in vivo using Qdpr-KO mice. In addition to the levels of BH4 in the cells, the ratios of oxidized to reduced forms of BH4 are supposed to be important for regulating nitric oxide synthase (NOS) via the so-called uncoupling of NOS. However, previous studies were limited due to the absence of specific and high-affinity inhibitors against QDPR. Here, we performed a high-throughput screening for a QDPR inhibitor and identified Compound 9b with an IC50 of 0.72 μM. To understand the inhibition mechanism, we performed kinetic analyses and molecular dynamics simulations. Treatment with 9b combined with methotrexate (MTX), an inhibitor of another BH4-reducing enzyme, dihydrofolate reductase (DHFR), significantly oxidized intracellular redox states in HepG2, Jurkat, SH-SY5Y, and PC12D cells. Collectively, these findings suggest that 9b may enhance the anticancer and anti-autoimmune effects of MTX.

Comparison of the CYP3A selective inhibitors CYP3cide, clobetasol, and azamulin for their potential to distinguish CYP3A7 activity in the presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ~50% of the total CYP content in fetal and neonatal livers and is the predominant CYP involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-S (sulfate). The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low, but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We utilized three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and CYP inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that CP was not an MDI of CYP3A5, but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7 and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. Significance Statement These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect probe selection may have on CYP3A P450 inhibition studies.

Abstract PO2-23-12: Innovative Metastatic Breast Cancer Therapy, CBT300, Reverses Drug and Immune Resistance

Abstract For many patients with metastatic breast cancer (MBC), their disease is treatable but incurable. Although the rate of death from MBC has significantly decreased over the last 10 years due to early detection and new treatment options, MBC still accounts for over 40,000 deaths each year in the US that has been consistent for the past 30 years. Recently, it is estimated that 90% of those deaths were due to drug resistant recurrent disease. Nearly 45% of MBC deaths can be attributed to a subpopulation of patients with metastatic triple negative breast cancer (mTNBC). Despite MBC’s initial sensitivity to chemotherapy, it often recurs at greater than 40% in stage I-III patients and greater than 80% in stage IV patients which have a dismal 5-year survival of 12%. African American women have a 40% greater incidence of death from MBC despite a 4% lower risk of diagnosis. Unfortunately, current therapies do little to reduce drug resistance and death for recurrent drug resistant MBC. Currently, the only therapies approved by the FDA for recurrent TNBC patients are Keytruda plus chemotherapy and an Antibody Drug Conjugate (ADC) called Sacituzumab Govitecan. Keytruda (anti-PD-L1) is an immune checkpoint inhibitor(ICI) given with chemotherapy (nab-paclitaxel, paclitaxel, or gemcitabine plus carboplatin). The second recently approved therapy, Sacituzumab Govitecan (SG), is composed of an antibody to human trophoblast cell-surface antigen 2 (Trpo-2) coupled to SN-38 (topoisomerase I inhibitor). Although these results are very encouraging, the 80% of TNBC patients that either do not have high positive PD-L1 tumors, or don’t respond to these therapies and become drug resistant suggests that most patients with TNBC will have recurrent disease with little or no hope for survival. For contrast, our novel MBC therapy, CBT300, targets cell surface GRP78 that has been found in over 95% of MBC and in 93% of TNBC tumors. We can now show that inhibition of cell surface GRP78 can a) induce apoptosis of drug resistant TNBC cells in vitro and in vivo, b) eliminate drug resistance showing synergistic effects with chemotherapy in vitro and in vivo, c) decrease amount of chemotherapy in combination with CBT300 and d) reduce immune suppression. Recent publications show that GRP78 is found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, tumor cell surface GRP78 (csGRP78) is important for many aspects of MBC development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. Recently, it has been shown that increased cell surface GRP78 expression in TNBC patients was significantly associated with later stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival. In studies to help understand how cell surface GRP78 causes MBC progression and drug resistance, we discovered a novel GRP78 binding transmembrane protein on TNBC cells called Receptor Tyrosine Kinase Orphan Receptor-1 (ROR1). Using the GRP78 binding domain from ROR1 and a human Fc IgG1 domain, we created a biologic fusion protein that is a potent and cell surface specific GRP78 inhibitor, CBT300. We now show that CBT300’s elimination of cell surface GRP78 destabilizes and removes oncofetal proteins ROR1, Cripto-1, and checkpoint protein PD-L1 from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. Since ROR1, Cripto-1 and PD-L1 are three of the major redundant pathways for tumor drug resistance, immune suppression and stem cell formation, the ability to inhibit all these pathways with CBT300, a single safe and very efficacious therapy, is very innovative and could be a significant advance for the treatment of MBC. Citation Format: Elizabeth Stolarik, Jorryn Zelek, Mariah Thigpen, Anita Davidson, Donald Davidson. Innovative Metastatic Breast Cancer Therapy, CBT300, Reverses Drug and Immune Resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-23-12.

Abstract PO2-04-09: Combination of the PLK1 Inhibitor Onvansertib and the PI3Kα Inhibitor Alpelisib Overcomes Palbociclib Resistance in PIK3CA-mutated HR+ Breast Cancer

Abstract In the 1st line setting of hormone receptor-positive HER2 negative (HR+/HER2-) metastatic breast cancer, cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors (palbociclib, abemaciclib and ribociclib) are typically used in combination with endocrine therapy. Though initially effective, acquired resistance to either CDK4/6 inhibitors or endocrine therapy eventually leads to disease relapse in most patients. Activating mutations in PIK3CA, the gene encoding alpha catalytic subunit of phosphatidylinositol-4,5-bisphosphat 3-kinase (PI3K) are found in approximately 40% of HR+ breast cancer patients and have been implicated in mediating resistance to CDK4/6 inhibitors. The PI3Kα inhibitor alpelisib is currently approved for PI3KCA-mutant HR+ breast cancer after progression on CDK4/6 inhibitors. However, drug-associated resistance mechanisms may limit its clinical benefit. Synergistic combination therapies have the potential to improve efficacy and overcome resistance mechanisms. Polo-like kinase 1 (PLK1) is a serine-threonine-protein kinase, key cell cycle regulator that controls G2/M entry and mitotic progression, and that has been shown to mediate resistance to palbociclib in HR+ breast cancer. Onvansertib is an oral and highly specific PLK1 inhibitor currently on clinical development in solid tumors and hematological malignancies. Here we evaluated the potential of onvansertib to increase the activity of alpelisib in PIK3CA-mutant HR+ breast cancer preclinical models. The combination of onvansertib and alpelisib synergistically inhibited cell viability and/or colony formation in three PI3KCA-mutant HR+ breast cancer cell lines, MCF-7 (PIK3CA E545K), EFM-19 (PIK3CA H1047L), and T-47D (PIK3CA 1047R). A significant increase in apoptosis was observed in cells treated with the combination compared to either agent alone. We next tested the combination of onvansertib and alpelisib in patient-derived xenograft (PDX) models. For this purpose, palbociclib-resistant PIK3CA-mutant HR+ breast cancer PDXs were established from primary breast tumor (PDX HBCx-86, PIK3CA E545K) or from metastatic bone biopsies of patients who had progressed on endocrine therapy plus palbociclib (HBCx-180, PIK3CA H1047R) or on PI3Kα inhibitor (HBCx-134palboR31, PIK3CA H1047R). PDX tumors were grafted subcutaneously in nude mice and mice were treated with vehicle, onvansertib (45 mg/kg, oral, 5 days a week), alpelisib (25mg/kg, oral, 5 days a week) or the combination. The combination was well tolerated and showed superior anti-tumor activity than the single agents in the 3 PDX models. In the HBCx-86 PDX, although none of the single agents showed activity, the combination induced potent tumor growth inhibition. In the metastasis-derived PDX HBCx-134palboR31, the combination treatment induced pronounced tumor regression in 62% of mice (5/8), with complete response in 25% (2/8), while mice treated with the single agents showed tumor progression. Finally, in the HBCx-180 PDX, established from a patient with primary resistance to palbociclib, the efficacy of the combination was greater and more durable, with significant survival advantage, compared to the monotherapies. Collectively, our preclinical findings suggest that co-targeting PLK1 and PI3Kα with onvansertib and alpelisib respectively, may constitute a promising therapeutic combination strategy for patients with PIK3CA-mutant HR+ breast cancer failing to respond to first-line standard of care therapies. Citation Format: Sreeja Sreekumar, Pierre Painsec, Davis Klein, Dalia Gonzalez, Laura Sourd, Elodie Montaudon, Tod Smeal, Elisabetta Marangoni, Maya Ridinger. Combination of the PLK1 Inhibitor Onvansertib and the PI3Kα Inhibitor Alpelisib Overcomes Palbociclib Resistance in PIK3CA-mutated HR+ Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-09.

Abstract PO3-25-08: Artificial Intelligence-based screening of small molecule inhibitors of phosphoenolpyruvate carboxykinase 2 (PCK2), a novel cancer therapeutic target

Abstract Background: Metabolic rewiring is a hallmark of cancer that is often mediated by alterations in metabolic isozyme expression. Isozymes that are overexpressed in cancer and catalyze rate limiting steps in key metabolic processes are potential therapeutic targets. We previously identified phosphoenolpyruvate carboxykinase 2 (PCK2) as cancer dominant isoform, required for maintaining high metabolic activity and proliferation of lung, prostate, and breast cancer (BC) cells in vitro and in vivo. No PCK2 specific small molecule inhibitors exist. Methods: mRNA expression levels were assessed in the TCGA data. PCK2 MISSION shRNAs (Millipore) were used to knock-down expression of PCK2 in BC cells. Alterations in metabolic flux caused by PCK2 down-regulation were examined via 13C-labeling and mass spectrometry using a SCIEX 5500 QTRAP and SelexION instrument for mobility separation of metabolites. The AtomNetTM (Atomwise Inc.) deep convolutional neural network structure-based drug design software was used to identify candidate small molecule PCK2 inhibitors which were tested in PCK1 and PCK2 in vitro enzyme activity assays. The lead compound was assessed for drug-like properties by QickPropV4.0.001w, and its growth inhibitory activity was tested in MDA-MB-321 and BT-549 cells. Results: Analysis of mRNA levels in TNBC samples revealed a significant decrease in PCK1 expression compared to normal tissues, while PCK2 expression remained elevated or even increased. Quantification of mRNA levels in TNBC cell lines confirmed the higher expression of PCK2 compared to PCK1. To assess functional significance of PCK2 expression, we downregulated PCK2 using shRNA constructs in TNBC cell lines. Western blot analysis confirmed efficient knockdown of PCK2 protein without affecting PCK1 expression. Clonogenic assays demonstrated that PCK2-depleted BT-549 and MDA-MB-231 cells exhibited impaired proliferation, with reduced colony number and size compared to control cells. Metabolic analysis of PCK2-depleted TNBC cells demonstrated decreased levels of glycolytic and TCA intermediates and reduced flux through pyruvate carboxylase, indicating altered metabolic pathways upon PCK2 depletion. To identify potential inhibitors of PCK2, virtual screening was conducted using compound structures. We used a PCK2 protein structure homology model and designated the 3-mercaptopicolinic acid-binding site of the PCK1 structure as the site of interest to perform virtual drug screening of 7 million diverse compounds. The first screen yielded 86 structures to be tested in vitro, of which 5 demonstrated > 16% PCK2 inhibition. The top hit (IC50=2.4µM) was used to refine a second round of in silico screen that yielded 87 second-round candidates that were assessed for PCK2 inhibition in vitro. 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate emerged as the lead compound. QickProp predicted favorable absorption, distribution, metabolism, and excretion properties, but relatively low cell permeability (apparent Caco-2 and MDCK2 cell permeabilities 18 nm/sec and 8 nm/sec, respectively). The specificity of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was tested in PCK1 and PCK2 enzymatic assays and revealed IC50 of 500nM on PCK1 and an IC50 of 3 nM on PCK2. We next tested the cell growth inhibitory effect of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on parental MDA-MB-231 and BT-549 cells and their PCK2 knockdown clones. Cell growth and viability significantly decreased in the MDA-MD-231 and BT-549 parental cells (the IC50 for MDA-MB-231 was 173 μM) and in shRNA non-targeting control cells, but not in the PCK2 depleted clones, demonstrating PCK2-dependent inhibitory effect. Conclusion: We identified 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate as a high affinity selective PCK2 enzyme inhibitor. This compound also has significant growth inhibitory activity in BC cell lines in vitro and represents a novel therapeutic lead compound. Citation Format: Alejandro Rios Hoyo, Naing Lin Shan, Hao-Kuen Lin, Gerson Espinoza Campos, Mostafa Ahmed, Sheila Umlauf, Rebecca Cardone, Yulia V. Surovtseva, Vignesh Gunasekharan, Lajos pusztai. Artificial Intelligence-based screening of small molecule inhibitors of phosphoenolpyruvate carboxykinase 2 (PCK2), a novel cancer therapeutic target [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-08.

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

Filamentation and inhibition of prokaryotic CTP synthase with ligands

AbstractCytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo‐electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6‐diazo‐5‐oxo‐ l‐norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament‐forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent‐accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

Abstract ED09-01: Emerging approaches to targeting pathways using precision medicine in breast cancer

Abstract The past few decades have yielded significant strides forward in the pursuit to extend survival for patients with breast cancer. Much of this success is directly attributed to breakthroughs in the development of targeted therapies intersecting advances in genomic medicine. Despite these achievements, challenges remain, and targeted therapeutic development is still evolving rapidly to offset them. The focus of this educational session is centered around reviewing the latest in targeted therapeutic advancements and precision medicine. Recent advances have centered on novel selective estrogen receptor degraders (SERDs) for advanced ESR1-mutated or endocrine-resistant disease. Antibody-drug conjugates (ADCs) continued to make gains, targeting HER2-positive/low advanced cancers as well as an expanded indications for TROP2-targeted ADCs. New ADCs are also on the horizon leveraging new cell surface targets. Emerging are forward-looking cell-surface methodologies that harness bispecific antibodies and T-cell engagers. Various strategies combining established targeted immunotherapies such as PD-1 and PD-L1 with additional therapies continue. Attempts to target the frequently mutated PI3K pathway have yielded some success, but clinical exploration continues using new approaches that utilize mutation specific inhibitors. Second generation PARP inhibitors have emerged recently and are being explored in a variety of indications with promising early results. Finally, understanding treatment resistance in particular with the use of longitudinal liquid biopsies is fueling further target discovery. Altogether, development in precision medicine is advancing rapidly with advanced genomics, novel targets, and cutting-edge drug modalities. Citation Format: M. Radovich. Emerging approaches to targeting pathways using precision medicine in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED09-01.

TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway.

Intracerebral hemorrhage (ICH) often triggers oxidative stress through reactive oxygen species (ROS). Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in regulating oxidative stress and inflammation across various diseases. 5Z-7-Oxozeaenol (OZ), a specific inhibitor of TAK1, has exhibited therapeutic effects in various conditions. However, the impact of OZ following ICH and its underlying molecular mechanisms remain elusive. This study aimed to explore the possible role of OZ in ICH and its underlying mechanisms by inhibiting oxidative stress-mediated pyroptosis. Adult male Sprague-Dawley rats were subjected to an ICH model, followed by treatment with OZ. Neurobehavioral function, blood-brain barrier integrity, neuronal pyroptosis, and oxidative stress markers were assessed using various techniques including behavioral tests, immunofluorescence staining, western blotting, transmission electron microscopy, and biochemical assays. Our study revealed that OZ administration significantly inhibited phosphorylated TAK1 expression post-ICH. Furthermore, TAK1 blockade by OZ attenuated blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative damage while enhancing neurobehavioral function. Mechanistically, OZ administration markedly reduced ROS production and oxidative stress by facilitating nuclear factor-erythroid 2-related factor 2 (NRF2) nuclear translocation. This was accompanied by a subsequent suppression of the NOD-like receptor protein 3 (NLRP3) activation-mediated inflammatory cascade and neuronal pyroptosis. Our findings highlight that OZ alleviates brain injury and oxidative stress-mediated pyroptosis via the NRF2 pathway. Inhibition of TAK1 emerges as a promising approach for managing ICH.

A Novel Role for the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Delta Isoform in Hepatocellular Proliferation

The phosphatidylinositol-4,5-bisphosphate 3-kinase delta isoform (Pik3cd), usually considered immune-specific, was unexpectedly identified as a gene potentially related to either regeneration and/or differentiation in animals lacking hepatocellular Integrin Linked Kinase (ILK). Since a specific inhibitor (Idelalisib, or CAL101) for the catalytic subunit encoded by Pik3cd (p110δ) has reported hepatotoxicity when used for treating Chronic Lymphocytic Leukemia and other lymphomas, we aimed to elucidate if there is a role for p110δ in normal liver function. To determine the effect on normal liver regeneration, partial hepatectomy (PHx) was performed using mice in which p110δ was first inhibited using CAL101. Inhibition led to over a 50% decrease in proliferating hepatocytes in the first two days after PHx. This difference correlated with phosphorylation changes in the HGF and EGF receptors (MET and EGFR, respectively) and NF-κB signaling. Ingenuity Pathway Analyses implicated C/EBPβ, HGF and the EGFR heterodimeric partner, ERBB2, as three of the top 20 regulators downstream of p110δ signaling as their pathways were suppressed in the presence of CAL101 at one day post-PHx. A regulatory role for p110δ signaling in mouse and rat hepatocytes through MET and EGFR was further verified using hepatocyte primary cultures, in the presence or absence of CAL101. Combined, this data supports a role for p110δ as a downstream regulator of normal hepatocytes when stimulated to proliferate.

FGF21 overexpression alleviates VSMC senescence in diabetic mice by modulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway.

Diabetes accelerates vascular senescence, which is the basis for atherosclerosis and stiffness. The activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and oxidative stress are closely associated with progressive senescence in vascular smooth muscle cells (VSMCs). The vascular protective effect of FGF21 has gradually gained increasing attention, but its role in diabetes-induced vascular senescence needs further investigation. In this study, diabetic mice and primary VSMCs are transfected with an FGF21 activation plasmid and treated with a peroxisome proliferator-activated receptor γ (PPARγ) agonist (rosiglitazone), an NLRP3 inhibitor (MCC950), and a spleen tyrosine kinase (SYK)-specific inhibitor, R406, to detect senescence-associated markers. We find that FGF21 overexpression significantly restores the level of catalase (CAT), vascular relaxation, inhibits the intensity of ROSgreen fluorescence and p21 immunofluorescence, and reduces the area of SA-β-gal staining and collagen deposition in the aortas of diabetic mice. FGF21 overexpression restores CAT, inhibits the expression of p21, and limits the area of SA-β-gal staining in VSMCs under high glucose conditions. Mechanistically, FGF21 inhibits SYK phosphorylation, the production of the NLRP3 dimer, the expression of NLRP3, and the colocalization of NLRP3 with PYCARD (ASC), as well as NLRP3 with caspase-1, to reverse the cleavage of PPARγ, preserve CAT levels, suppress ROSgreen density, and reduce the expression of p21 in VSMCs under high glucose conditions. Our results suggest that FGF21 alleviates vascular senescence by regulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway in diabetic mice.

Inhibition of 12/15-lipoxygenase reduces orthodontically induced root resorption in rats

ABSTRACT Objectives To investigate whether the inhibition of 12/15-lipoxygenase (12/15-LOX), one of the core enzymes of the arachidonic acid cascade, suppresses orthodontically induced root resorption (OIRR), and examine the involvement of the hyaline degeneration of periodontal ligament cells and odontoclast differentiation. Materials and Methods The left maxillary first molars of 10-week-old male Wistar rats were moved mesially for 14 days using a closed-coil spring (25 cN) inserted between the first molar and incisor. The rats were intraperitoneally administered with a 12/15-LOX specific inhibitor (ML-351; 0.05 mmol/kg) daily in the experimental group or vehicle (dimethyl sulfoxide) in the control group. Tooth movement was measured using microcomputed tomography on day 14. The appearance of OIRR, hyaline degeneration, osteoclasts, and odontoclasts was evaluated via histological analysis. Immunohistochemical staining for receptor-activated NF-kB ligand (RANKL) and osteoprotegerin was performed. Results OIRR observed on day 14 in the control group was strongly suppressed by ML-351 treatment. Hyaline degeneration observed on the compression side on day 3 and the appearance of osteoclasts and odontoclasts on days 3 and 14 were significantly suppressed by ML-351. RANKL expression on day 3 was significantly suppressed by ML-351. These key processes in OIRR were substantially suppressed by ML-351 treatment. Conclusions Inhibition of 12/15-LOX reduced OIRR by suppressing hyaline degeneration and subsequent odontoclast differentiation.

EphB2 deficiency in hepatic stellate cells mitigates fibrogenesis in metabolic dysfunction-associated steatohepatitis by attenuating the TGFβ/SMAD signaling

Background &amp; Aims: The receptor tyrosine kinase EphB2 is now emerging as a critical regulator of inflammation and fibrogenesis in metabolic dysfunction-associated steatohepatitis (MASH) though the underlying mechanism remain unclear. We aimed to investigate the role or mechanism by which EphB2 contributes in the progression of MASH fibrogenesis. Hypothesis: We hypothesized that EphB2 in hepatic stellate cells (HSCs) promotes MASH-liver fibrosis Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for EphB2 expression. Human liver biopsies obtained from patients with MASH were used to assess EphB2 expression by immunohistochemistry. Primary human HSCs were stimulated with TGFβ with or without TGFβR/SMAD inhibitors (SB525334, CS208, and SIS3 at 1μM) and EphB2 expression assessed by qPCR, western blot and immunofluorescence staining. EphB2 was silenced in primary human HSCs using siRNA and the resulting HSCs assess for TGBβ-induced HSC-myofibroblasts transition. MASH models were developed in hepatocyte-specific EphB2 knockout and HSCs EphB2 knockout and wild-type mice fed a high fat diet for 26 weeks or a choline-deficient amino-acid defined (CDAA) high fat diet for 10 weeks. Statistical analysis was performed using GraphPad Prism and a p-value &lt; 0.05 was considered significant. Results: The expression of EphB2 was 3-fold significantly upregulated in liver specimens from patients with advanced MASH and correlated with disease severity. EphB2 was modestly expressed in hepatocyte, but strongly induced in HSCs in human and mouse MASH. Hepatocyte-specific EphB2 deficiency does not inhibit the development of steatohepatitis and fibrosis in mice fed the CDAA-diet. TGFβ/SMAD signaling promotes an 8-fold increase of EphB2 expression in human HSCs while siRNA silencing of EphB2 in human HSCs was suffcient to abolished TGFβ-mediated HSCs activation. Interestingly, HSCs-specific EphB2 knockout mice developed 2.5 to 6-fold less fibrosis than wild-type controls in response to high fat diets feeding suggesting that EphB2 expression on HSCs is required for the development of fibrogenesis in MASH. Deletion of EphB2 in activated HSCs after fibrosis was established in mice was suffcient to mitigate MASH fibrosis. Conclusions: EphB2 is upregulated in human and mouse MASH. Upregulation of EphB2 in human HSCs is mediated via the TGFβ/SMAD signaling. HSCs expressing EphB2 regulate the development of MASH-fibrosis. Specific EphB2 inhibitors could potentially mitigates fibrogenesis in MASH, opening up an avenue for the treatment of MASH. Funding sources: NIDDK, NIAMS, APS postdoctoral fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.