Potential Anticancer Drug Target Research Articles

<i>In silico</i> Pharmacokinetic, Molecular Docking and Molecular Dynamics Simulation Studies of Phytochemicals isolated from <i>Cascabela thevetia</i> as Potential Anticancer Agents

Cascabela thevetia generally recognized as yellow oleander, Lippold or lucky nut (in English) and Peeli Kaner (in Hindi), is Indigenous to Central America and Mexico and widely distributed in the Indian subcontinent as well, is a medicinally important herb that has long been employed to treat disorders like ulcers, scabies, haemorrhoids, and tumour dissolution. Seven Cardenolides (Compounds 1-7) isolated from fruits of Cascabela thevetia are selected as ligands for in silico pharmacokinetic (ADME properties) and drug-likeness studies via the Swiss ADME online server. Further, molecular docking studies were accomplished for the selected ligands via the Auto Dock Vina module of PyRx to explore the receptor-ligand interaction with β-tubulin (a potential target for anticancer drugs, PDB ID: 1SA0), to find out potential therapeutic ligands for cancer chemotherapy. From the studies performed it was found that most of the ligands (compounds) have the capability of good binding affinity with the receptor, β-tubulin, good drug-likeness and ADME properties among which compounds 3, 6 and 7 are the best drug candidates because they followed the Lipinski rule of 5 with 0 violation and also have good binding affinities (-8.3, -8.0 and -7.9 kcal/mol respectively) against the target protein. Hence, compounds 3, 6 and 7 can be developed as novel therapeutic agents in cancer chemotherapy. Further, a Molecular Dynamics (MD) simulation study of best-docked ligand 3 was performed to establish its stability and validate molecular docking results. Based on simulation results, it may be stated that ligand 3 was firmly attached to the receptor protein during MD simulation since none of the conformations of the receptor-ligand complex were unstable, and no folding or unfolding of the complex took place. Therefore, compound 3 can be considered a good inhibitor of β-tubulin after validation of various parameters of drug discovery.

Exploring the Effect of Fidgetin-Like 1 on Colorectal Cancer Through Tissue Chip and In Vitro Experiments

Fidgetin-like 1 (FIGNL1) is extensively overexpressed in a variety of cancers. It facilitates non‑small cell lung cancer tumor cell proliferation and hepatocellular carcinoma formation due to abnormal DNA repair. Clinically relevant data indicates that its high expression is linked with the poor prognosis of patients with renal clear-cell carcinoma, low-grade gliomas, and hepatocellular carcinoma. Nevertheless, the scope of FIGNL1’s involvement in cancer, particularly colorectal cancer (CRC), remains unclear. To investigate the function of FIGNL1 in CRC. Cell culture study. The TCGA database and immunohistochemistry analysis were employed to investigate FIGNL1 expression in CRC tissue. A cell viability assay was performed using the Cell Counting Kit-8. The cell migration and invasion were evaluated using the transwell assay. Small interfering RNA (siRNA) transfection was conducted to knockdown FIGNL1 expression. Infection with FIGNL1 overexpression lentivirus was performed to promote FIGNL1 overexpression. The STRING database was employed for predicting protein interaction. FIGNL1 was substantially upregulated in human CRC tissues and was associated with TNM stages and lymph node metastasis in patients. The inhibition of CRC cell proliferation, migration, and invasion in Caco-2 cells was achieved by silencing FIGNL1 using siRNA. Additional investigations suggested that FIGNL1 overexpression could promote CRC cell proliferation, migration, and invasion via P38 signaling pathway activation in Colo-205 cells. Subsequent experiments demonstrated that FIGNL1-mediated P38 phosphorylation was contingent upon SPIDR interaction. These results implied that FIGNL1 was a potential anticancer drug target, which also offered a novel strategy for future CRC treatment.

Effect of impaired autophagic flux on breast carcinogenesis through the enhanced LC3-p62-NRF2 feedback loop.

6 Background: Breast cancer affects more women worldwide than any other type of cancer. Although some genes have been identified as potential causes, the exact molecular mechanism of the disease is still unknown. In our study, we aimed to investigate the role of NRF2 and the autophagy markers p62 and LC3 in breast cancer cell lines that have different Tp53 status using a new compound called 6,6'dihydroxythiobinupharidine (DTBN). In our previous research, we have shown that DTBN modulates key cellular signal transduction pathways that are relevant to disease biology, including cancer. Furthermore, we analyzed online databases to determine the correlation between NRF2, p62, LC3, and Tp53 in breast cancer patients. Methods: To evaluate the effect of DTBN on NRF2, p62 and LC3 in different types of breast cancer cell lines with varying TP53 status, we conducted molecular studies by inhibiting NRF2 activity. We synthesized the correlation between NRF2, p62, LC3 and TP53 expression and clinical parameters by analyzing TCGA and GEO datasets. Results: Our findings suggest that DTBN induces cell death and autophagy in all breast cancer cell lines, regardless of their Tp53 status. However, they also led us to hypothesize that NRF2 may hinder the sensitivity of cancer cells to DTBN-induced cell death and autophagy. To test this hypothesis, we inhibited NRF2 activity using an NRF2 inhibitor called brusatol. The results of the study indicate that the inhibition of NRF2 significantly increased cell death and autophagy in DTBN-treated cells. We observed a decrease in the expression of the autophagy marker p62, while an increase in the expression of another autophagy marker LC3. These findings suggest that the interaction between NRF2, LC3 and p62, which is activated in response to DTBN treatment, may serve as a potential anticancer drug target. We have validated the positive correlation between NRF2 and the autophagic gene p62 and LC3, in different Tp53 status breast cancer cell lines by analyzing TCGA and GEO data. Conclusions: Our study shows that high levels of Nrf2, LC3 and p62 expression are significantly associated with increased breast cancer cell proliferation and migration. This implies that patients with breast cancer have lower overall survival and a higher rate of recurrence. Our findings highlight the role of DTBN-induced NRF2 and LC3 expression and reduced p62 expression in breast cancer cells with varying Tp53 status. These results may represent a paradigm for better understanding the cancer cell response to therapies and designing more efficient combined anticancer therapies targeting NRF2, LC3 p62, and Tp53.

Establishment of a prognostic signature of disulfidptosis-related lncRNAs for predicting survival and immune landscape in clear cell renal cell carcinoma

Abstract Objectives A novel cell death pathway, disulfidptosis, marked by intracellular disulfide build-up, is a recently identified form of cell death. This study developed a dependable model using disulfidptosis-associated lncRNAs to predict outcomes and immune interactions in clear cell renal cell carcinoma (ccRCC) patients. Methods Data from ccRCC patients, including genomic and clinicopathological details, were sourced from The Cancer Genome Atlas database. We employed the least absolute shrinkage and selection operator (LASSO) along with regression analyses to construct a prognostic model consisting of 12 disulfidptosis-related lncRNAs (DRLs). The model’s validity was tested using the RECA-EU and GSE29609 datasets. Results The prognostic model, incorporating 12 DRLs – LINC01671, DOCK9-DT, AL078581.2, SPINT1-AS1, ZNF503-AS1, AL391883.1, AC002070.1, AP001372.2, AC068338.3, AC026401.3, AL355835.1, and AL162377.1 – distinguished high-risk ccRCC patients with diminished survival rates in both the training and validation cohorts. Further analyses through Cox regression confirmed this risk model’s independent prognostic capability regarding overall survival (OS). Functional enrichment analysis indicated significant involvement of differentially expressed genes in immune response mediator production. A prognostic nomogram, integrating DRLs with clinical features, showed strong predictive accuracy as confirmed by receiver operating characteristic curves. Additionally, assessments of immune functionality and tumor mutation burden varied across risk categories in the tumor microenvironment, highlighting potential targets for anticancer drugs. Conclusions The findings suggest the DRLs signature is a potent prognostic indicator and may serve to forecast responses to immunotherapy in ccRCC patients.

Cinnamaldehyde hydrazone derivatives as potential cathepsin B inhibitors: parallel in-vitro investigation in liver and cerebrospinal fluid

The emergence of cathepsins as a potential target for anticancer drugs has led to extensive research in the development of their inhibitors. In the present study, we designed, synthesized, and characterized several cinnamaldehyde schiff bases employing diverse hydrazines, as potential cathepsin B inhibitors. The parallel studies on cathepsin B isolated from liver and cerebrospinal fluid unveiled the significance of the synthesized compounds as cathepsin B inhibitors at nanomolar concentrations. The compound, 7 exhibited the highest inhibition of 83.48 % and 82.96 % with an IC50 value of 0.06 nM and 0.09 nM for liver and cerebrospinal fluid respectively. The inhibitory potential of synthesized compounds has been extremely effective in comparison to previous reports. With the help of molecular docking studies using iGEMDOCK software, we found that the active site -CH2SH group is involved in the case of α-N-benzoyl-D, l-arginine-b-naphthylamide (BANA), curcumin 2, 3, 6, and 7. For toxicity prediction, ADMET studies were conducted and the synthesized compounds emerged to be non-toxic. The results obtained from the in vitro studies were supported with in silico studies. The synthesized cinnamaldehyde schiff bases can be considered promising drug candidates in conditions with elevated cathepsin B levels.

The immune cells have complex causal regulation effects on cancers

BackgroundThere was a large body of evidence linking immune cells to cancer risk. However, the causal relationship between immune cells, cancer, and what genes play an important role is unclear. MethodsIn this study, we performed comprehensive two-sample Mendelian randomization analysis (TSMR) to determine the causal relationship between immune cells and common cancers. We also performed Multimarker Analysis of Genomic Annotation (MAGMA) on immune cells causally associated with cancer to identify their relevant genes and used data summary-based MR (SMR) analysis to investigate the causal relationship between their gene expression, methylation, and cancer, and further used drug prediction and molecular docking to validate the medicinal value of the targets. Finally, reverse TSMR analysis was performed on cancer and immune cells to rule out reverse causality. ResultsAfter FDR correction (PFDR < 0.05), the results showed that 2 immune cells were associated with lung cancer risk, and 1 immune cell was significantly associated with pancreatic cancer risk. The expression of OSBPL10, CHD4, SMDT1, PHETA2, and NAGA was positively and causally related to the risk of lung cancer by SMR analysis and HEIDI test. We also found that increased expression of ANP32E decreased the risk of pancreatic cancer and that the methylation level of OSBPL10, CHD4, SULF2, CENPM, and CYP2D6 had a causal association with lung cancer. The methylation level of FCGR3A was causally associated with pancreatic cancer. The results of molecular docking indicated a strong affinity between the drugs and proteins that possessed existing structural information. ConclusionThis data-driven Mendelian randomization (MR) study demonstrates the causal role of immune cells in cancers. In addition, this study identifies candidate genes that may be potential anti-cancer drug targets.

Abstract LB171: First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer

Abstract METTL1 is an RNA methyltransferase that catalyzes N7-methylation on guanine at position 46 (m7G46) in a subset of transfer RNAs (tRNAs). These tRNAs are stabilized by m7G46, and this increases the translation of mRNAs containing the cognate codons. METTL1 is often over-expressed in cancer tissue, and its gene locus is frequently amplified in specific tumor types. Recent studies have shown that reducing METTL1 expression leads to tumor growth inhibition highlighting this enzyme as a potential novel target for anti-cancer drugs. We optimized small molecule inhibitors of METTL1 from a HTS hit using structure-guided medicinal chemistry. Representative compounds of two distinct chemical series exhibit potent in vitro METTL1 inhibition at low nanomolar concentrations while displaying exquisite selectivity over other RNA and protein methyltransferases. The effects of inhibiting METTL1 were measured by cellular mechanistic assays and modification-induced misincorporation tRNA sequencing (mim-tRNAseq). As expected, pharmacological inhibition led to decreased m7G46 methylation, lower levels of a subgroup of tRNAs and reduced cancer cell proliferation.The efficacy of METTL1 inhibition in a panel of cancer cell lines representing a wide range of solid and hematologic tumor types was determined. Cell proliferation was impaired in a subset of cell lines originating from various cancer types, including lymphoma, sarcoma, esophageal squamous cell carcinoma, glioblastoma, colorectal and lung adenocarcinoma. Importantly, viability was not affected in non-transformed cell lines, indicating a highly selective mechanism of action. The phenotypic consequences of METTL1 inhibition in selected cell lines were further investigated by flow cytometry and western blotting. Cell lines sensitive to METTL1 inhibition displayed reduced cell cycle progression and lowered expression of cell cycle regulators. RNA sequencing and proteome analyses identified molecular changes at both transcript and protein level. METTL1 inhibition led to induction of ATF4 expression and activation of the integrated stress response, as well as global changes to the translation machinery. Studies on cell-line derived xenograft or syngeneic in vivo mouse models were conducted to assess tumor growth inhibition after oral or intraperitoneal administration of the potent METTL1 small molecule inhibitor STM9005. Inhibition of METTL1 by this compound reduced tumor growth in both immune-deficient and immune-competent mouse models, effects accompanied by dose-dependent changes in pharmacodynamic biomarkers. Here, we describe the discovery of first-in-class inhibitors of METTL1 tRNA methyltransferase. To our knowledge, our data provide the first evidence that pharmacological inhibition of a tRNA methyltransferase reduces tumor growth in vivo. Citation Format: Alexandra Sapetschnig, Beth Thomas, Eliza Yankova, Harry Fischl, Aleksandra Azevedo, Sarah Bucknell, Richard Fosbeary, Sapphire Sawyer, Sian Evans, Carmen Livi, Byron Andrews, Jack Rogan, Natalie Webster, Matthew Fyfe, Konstantinos Tzelepis, Oliver Rausch. First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB171.

Revisiting methotrexate and phototrexate Zinc15 library-based derivatives using deep learning in-silico drug design approach.

Introduction: Cancer is the second most prevalent cause of mortality in the world, despite the availability of several medications for cancer treatment. Therefore, the cancer research community emphasized on computational techniques to speed up the discovery of novel anticancer drugs. Methods: In the current study, QSAR-based virtual screening was performed on the Zinc15 compound library (271 derivatives of methotrexate (MTX) and phototrexate (PTX)) to predict their inhibitory activity against dihydrofolate reductase (DHFR), a potential anticancer drug target. The deep learning-based ADMET parameters were employed to generate a 2D QSAR model using the multiple linear regression (MPL) methods with Leave-one-out cross-validated (LOO-CV) Q2 and correlation coefficient R2 values as high as 0.77 and 0.81, respectively. Results: From the QSAR model and virtual screening analysis, the top hits (09, 27, 41, 68, 74, 85, 99, 180) exhibited pIC50 ranging from 5.85 to 7.20 with a minimum binding score of -11.6 to -11.0 kcal/mol and were subjected to further investigation. The ADMET attributes using the message-passing neural network (MPNN) model demonstrated the potential of selected hits as an oral medication based on lipophilic profile Log P (0.19-2.69) and bioavailability (76.30% to 78.46%). The clinical toxicity score was 31.24% to 35.30%, with the least toxicity score (8.30%) observed with compound 180. The DFT calculations were carried out to determine the stability, physicochemical parameters and chemical reactivity of selected compounds. The docking results were further validated by 100ns molecular dynamic simulation analysis. Conclusion: The promising lead compounds found endorsed compared to standard reference drugs MTX and PTX that are best for anticancer activity and can lead to novel therapies after experimental validations. Furthermore, it is suggested to unveil the inhibitory potential of identified hits via in-vitro and in-vivo approaches.

Molecular modeling of novel 2-aminopyridine derivatives as potential JAK2 inhibitors: a rational strategy for promising anticancer agents

Janus kinase 2(JAK2) is a potential target for anticancer drugs in the treatment of numerous myeloproliferative diseases due to its central role in the JAK/STAT signaling cascade. In this study, the binding behavior of 2 amino-pyridine derivatives as JAK2 inhibitors was investigated by using multifaceted strategies including 3D-QSAR, molecular docking, Fingerprint analysis, MD simulations, and MM-PBSA calculations. A credible COMFA (q 2 = 0.606 and r 2 = 0.919) and COMSIA (q 2 = 0.641 and r 2 = 0.992) model was developed, where the internal and external validation revealed that the obtained 3D-QSAR models could be capable of predicting bioactivities of JAK2 inhibitors. The structural criteria provided by the contour maps of model were used to computationally develop more potent 100 new JAK2 inhibitors. Docking studies were conducted on the model data set and newly developed compounds (in-house library) to demonstrate their binding mechanism and highlight the key interacting residues within JAK2 active site. The selected docked complexes underwent MD simulation (100 ns), which contributed in the further study of the binding interactions. Binding free energy analyses (MMGB/PBSA) revealed that key residues such as Glu930, Leu932 (hinge region), Asp939 (solvent accessible region), Arg980, Asn981and Asp994 (catalytic site) have a significantly facilitate ligand-protein interactions through H-bonding and van der Waals interactions. The preliminary in-silico ADMET evaluation revealed encouraging results for all the modeled and in-house library compounds. The findings of this research have the potential to offer valuable recommendations for the advancement of novel, potent, and efficacious JAK2 inhibitors. Overall, this work has successfully employed a wide range of computer-based methodologies to understand the interaction dynamics between 2-amino-pyridine derivatives and the JAK2 enzyme, which is a crucial target in myeloproliferative disorders. Communicated by Ramaswamy H. Sarma

19F Nuclear Magnetic Resonance Fingerprinting Technique for Identifying and Quantifying G-Quadruplex Topology in Human Telomeric Overhangs.

G-quadruplexes (G4s) are noncanonical nucleic acid secondary structures with diverse topological features and biological roles. Human telomeric (Htelo) overhangs consisting of TTAGGG repeats can fold into G4s that adopt different topologies under physiological conditions. These G4s are potential targets for anticancer drugs. Despite intensive research, the existence and topology of G4s at Htelo overhangs in vivo are still unclear because there is no method to distinguish and quantify the topology of Htelo overhangs with native lengths that can form more than three tandem G4s in living cells. Herein, we present a novel 19F chemical shift fingerprinting technique to identify and quantify the topology of the Htelo overhangs up to five G-quadruplexes (G4s) and 120 nucleotides long both in vitro and in living cells. Our results show that longer overhang sequences tend to form stable G4s at the 5'- and 3'-ends, while the interior G4s are dynamic and "sliding" along the sequence, with TTA or 1-3 TTAGGG repeats as a linker. Each G4 in the longer overhang is conformationally heterogeneous, but the predominant ones are hybrid-2, two- or three-tetrad antiparallel, and hybrid-1 at the 5'-terminal, interior, and 3'-terminal, respectively. Additionally, we observed a distinct behavior of different lengths of telomeric sequences in living cells, suggesting that the overhang length and protein accessibility are related to its function. This technique provides a powerful tool for quickly identifying the folding topology and relative population of long Htelo overhangs, which may provide valuable insights into telomere functionality and be beneficial for structure-based anticancer drug development targeting G4s.

Regulation of Eukaryotic Translation Initiation Factor 4E as a Potential Anticancer Strategy.

Eukaryotic translation initiation factors (eIFs) are highly expressed in cancer cells, especially eIF4E, the central regulatory node driving cancer cell growth and a potential target for anticancer drugs. eIF4E-targeting strategies primarily focus on inhibiting eIF4E synthesis, interfering with eIF4E/eIF4G interactions, and targeting eIF4E phosphorylation and peptide inhibitors. Although some small-molecule inhibitors are in clinical trials, no eIF4E inhibitors are available for clinical use. We provide an overview of the regulatory mechanisms of eIF4E and summarize the progress in developing and discovering eIF4E inhibitor strategies. We propose that interference with eIF4E/eIF4G interactions will provide a new perspective for the design of eIF4E inhibitors and may be a preferred strategy.

Discovery of a pyrrole-pyridinimidazole derivative as novel SIRT6 inhibitor for sensitizing pancreatic cancer to gemcitabine

Pancreatic cancer is a highly aggressive cancer, and is primarily treated with gemcitabine, with increasing resistance. SIRT6 as a member of sirtuin family plays important roles in lifespan and diverse diseases, such as cancer, diabetes, inflammation and neurodegenerative diseases. Considering the role of SIRT6 in the cytoprotective effect, it might be a potential anticancer drug target, and is associated with resistance to anticancer therapy. However, very few SIRT6 inhibitors have been reported. Here, we reported the discovery of a pyrrole-pyridinimidazole derivative, 8a, as a new non-competitive SIRT6 inhibitor, and studied its roles and mechanisms in the antitumor activity and sensitization of pancreatic cancer to gemcitabine. Firstly, we found a potent SIRT6 inhibitor compound 8a by virtual screening and identified by molecular and cellular SIRT6 activity assays. 8a could effectively inhibit SIRT6 deacetylation activity with IC50 values of 7.46 ± 0.79 μM in FLUOR DE LYS assay, and 8a significantly increased the acetylation levels of H3 in cells. Then, we found that 8a could inhibit the cell proliferation and induce cell apoptosis in pancreatic cancer cells. We further demonstrate that 8a sensitize pancreatic cancer cells to gemcitabine via reversing the activation of PI3K/AKT/mTOR and ERK signaling pathways induced by gemcitabine and blocking the DNA damage repair pathway. Moreover, combination of 8a and gemcitabine induces cooperative antitumor activity in pancreatic cancer xenograft model in vivo. Overall, we demonstrate that 8a, a novel SIRT6 inhibitor, could be a promising potential drug candidate for pancreatic cancer treatment.

Adamantaniline Derivatives Target ATP5B to Inhibit Translation of Hypoxia Inducible Factor-1α.

Hypoxia inducible factor-1α (HIF-1α) plays a critical role in cellular adaptation to hypoxia and it is a potential therapeutic target for anti-cancer drugs. Applying high-throughput screening, here it is found that HI-101, a small molecule containing an adamantaniline moiety, effectively reduces HIF-1α protein expression. With the compound as a hit, a probe (HI-102) is developed for target identification by affinity-based protein profiling. The catalytic β subunit of mitochondrial FO F1 -ATP synthase, ATP5B, is identified as the binding protein of HI-derivatives. Mechanistically, HI-101 promotes the binding of HIF-1α mRNA to ATP5B, thus inhibiting HIF-1α translation and the following transcriptional activity. Further modifications of HI-101 lead to HI-104, a compound with good pharmacokinetic properties, exhibiting antitumor activity in MHCC97-L mice xenograft model, and HI-105, the most potent compound with an IC50 of 26nm. The findings provide a new strategy for further developing HIF-1α inhibitors by translational inhibition through ATP5B.

Towardα-1,3/4 fucosyltransferases targeted drug discovery: In silico uncovering of promising natural inhibitors of fucosyltransferase 6.

Sialyl Lewis X (sLex ) antigen is a fucosylated cell-surface glycan that is normally involved in cell-cell interactions. The enhanced expression of sLex on cell surface glycans, which is attributed to the upregulation of fucosyltransferase 6 (FUT6), has been implicated in facilitating metastasis in human colorectal, lung, prostate, and oral cancers. The role that the upregulated FUT6 plays in the progression of tumor to malignancy, with reduced survival rates, makes it a potential target for anticancer drugs. Unfortunately, the lack of experimental structures for FUT6 has hampered the design and development of its inhibitors. In this study, we used in silico techniques to identify potential FUT6 inhibitors. We first modeled the three-dimensional structure of human FUT6 using AlphaFold. Then, we screened the natural compound libraries from the COCONUT database to sort out potential natural products (NPs) with best affinity towardthe FUT6 model. As a result of these simulations, we identified three NPs for which we predicted binding affinities and interaction patterns quite similar to those we calculated for two experimentally tested FUT6 inhibitors, that is, fucose mimetic-1 and a GDP-triazole derived compound. We also performed molecular dynamics (MD) simulations for the FUT6 complexes with identified NPs, to investigate their stability. Analysis of the MD simulations showed that the identified NPs establish stable contacts with FUT6 under dynamics conditions. On these grounds, the three screened compounds appear as promising natural alternatives to experimentally tested FUT6 synthetic inhibitors, with expected comparable binding affinity. This envisages good prospects for future experimental validation towardFUT6 inhibition.

Abstract 4631: Multiplex IHC assay to explore regional heterogeneity of FAP and inflammatory cell density in localized prostatic adenocarcinoma

Abstract Cancer-associated fibroblasts (CAFs) expressing FAP promote tumor growth, invasion and immune suppression. FAP is a molecular imaging target in cancer and its protease activity is a potential anti-cancer drug target. While FAP expression was associated with biochemical recurrence after prostatectomy using tissue microarrays (TMAs)(DOI:10.1158/2767-9764.CRC-21-0183), its expression has not been comprehensively correlated with morphological features of disease aggressiveness beyond grade. 48 cases of localized prostatic adenocarcinoma were used for a multiplex immunohistochemical assay that included FAP, CD45, CD163, p63 and Keratin 8. Each of these markers were individually optimized by single-plex staining and were then performed as an iterative IHC assay (PMID:35188986) and quantification was performed using the HALO image analysis platform (Indica Labs). We developed a second multiplex assay including FAP, vimentin, smooth muscle actin, desmin, CD31, S100, keratin 8 and p63. FAP IHC staining was analytically validated using a series of positive and negative cell lines. In normal appearing (non-inflamed) prostate regions, FAP expression was negative. By contrast, upregulation of FAP was observed in the stromal compartment in regions of chronic inflammation and some proliferative inflammatory atrophy (PIA) lesions. In tumor regions, only 1 case was totally negative for FAP. Except for 2 cases, in which there was focal weak tumor cell staining, all FAP expression was present in the tumor stromal compartment (TSC). The vast majority of FAP expression was associated with Vim+/Des- fibroblasts, which at times were also positive for SMA (Vim+/Des-/SMA+), indicating they are myofibroblasts. There was very infrequent FAP expression associated with M2 macrophages, endothelial cells, nerves and vimentin-negative smooth muscle cells, indicating that the majority of FAP expression occurs in fibroblasts. Preliminary quantitative data using visual estimations of the fraction of TSC expressing FAP (range, 0-80%, mean=35.8%, median=30%, SD=23.7) was not associated with the overall GS/grade group, but was associated with non-organ confined disease. Furthermore, there was a trend towards increased FAP expression in cases showing intraductal carcinoma and large cribriform formation. Ongoing studies are in process to obtain quantitative data by image analysis and to correlate FAP expression with the presence of overall inflammatory cell density and the density of M2 macrophages. Additionally, we will characterize the cases in terms of molecular features including TMPRSS2-ERG status and PTEN genomic status. Future use of these panels will facilitate studies of associations of FAP with clinical outcome, MRI and PET imaging, response to specific treatments and to help characterize FAP as a predictor of response to emerging FAP inhibitors and FAP-targeted prodrugs. Citation Format: Fernanda Caramella Pereira, Alan K. Meeker, Kenneth Pienta, Qizhi Zheng, Tracy Jones, Srinivasan Yegnasubramanian, Jessica L. Hicks, Sujayita Roy, Angelo M. De Marzo, W. Nathaniel Brennen. Multiplex IHC assay to explore regional heterogeneity of FAP and inflammatory cell density in localized prostatic adenocarcinoma. [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 4631.

High-Throughput Exonuclease Assay Based on the Fluorescent Base Analogue 2-Aminopurine.

Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair, and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited. Here, we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3' and 5' DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the Mycobacterium tuberculosis PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high-throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance.

Inhibition of secretory leukocyte protease inhibitor (SLPI) promotes the PUMA-mediated apoptosis and chemosensitivity to cisplatin in colorectal cancer cells

BackgroundAberrant expression of Secretory Leukocyte Protease Inhibitor (SLPI) has been associated with human cancer growth and its suppression was identified as a potential target for anti-cancer drugs, particularly in colorectal cancer. However, the underlying mechanism by which SLPI affected the development of drug resistance in CRC remains unclear.ObjectiveThis study investigated the role of SLPI in the p53-up-regulated modulator of apoptosis (PUMA)-mediated CRC cells’ apoptosis and their chemosensitivity to Cisplatin.MethodsA series of qRT-PCR and western blot analyses were performed to characterize the expressions of SLPI, PUMA, and Akt in CRC lines. Tunel, transwell, and CCK-8 analyses were monitored to define the impacts of the siRNA-mediated knockdown of SLPI on CRC cell development. Furthermore, in vivo development of CRC was evaluated in nude mice infected with siSLPI or Cisplatin alone or both, and Ki67 and caspase-3 immunohistochemistry assay was monitored on multiple tissue microarray from the same cohort.ResultsOur results showed that SLPI inhibition strongly promoted the expressions of the pro-apoptotic protein PUMA, cleaved-caspase3 and Bax and reduced the cell viability of HT29 and HT116 cell lines in vitro. In addition, siSLPI knockdown effectively suppressed both Akt and FoxO3 proteins and improved the sensitivity to cisplatin chemotherapy. Xenograft tumor assay revealed a lowered growth in mice treated with Cisplatin, while combined treatment of siSLPI achieved more significant anticancer effects than Cisplatin alone.ConclusionsTaken together, these findings demonstrated that suppression of SLPI might repress the growth of human colorectal cancer cells both in vitro and in vivo. These results suggested SLPI as a novel resistance factor to Cisplatin, and a combination of Cisplatin and SLPI inhibitor be beneficial for colorectal cancer therapy.

Identification of Lignan Compounds as New 6-Phosphogluconate Dehydrogenase Inhibitors for Lung Cancer.

Targeting pentose phosphate pathway (PPP) enzymes has emerged as a promising strategy to combat cancer. 6-Phosphogluconate dehydrogenase (6-PGD), the third critical enzyme of the PPP, catalyzes oxidative decarboxylation of 6-phosphogluconate (6-PG) to produce ribulose-5-phosphate (Ru-5-P) and CO2. Overexpression of 6-PGD has been reported in multiple cancers and is recognized as a potential anticancer drug target. The current study is focused on the utilization of indispensable virtual screening tools for structure-based drug discovery. During the study, 17,000 natural compounds were screened against the 3-phosphoglycerate (3-PG) binding site of 6-PGD through a molecular operating environment (MOE), which revealed 115 inhibitors with higher selectivity and binding affinity. Out of the 115 best-fit compounds within the 6-PGD binding cavity, 15 compounds were selected and optimized through stringent in silico ADMET assessment models that justified the desirable pharmacokinetic, pharmacodynamic and physicochemical profiles of 5 ligands. Further protein−ligand stability assessment through molecular dynamics (MD) simulation illustrated three potential hits, secoisolariciresinol, syringaresinol and cleomiscosin A, with stable confirmation. Moreover, 6-PGD inhibitor validation was performed by an in vitro enzymatic assay using human erythrocytes purified 6-PGD protein and A549 cell lysate protein. The results of the in vitro assays supported the in silico findings. In order to gain insight into the anticancer activity of the aforementioned compounds, they were subjected to CLC-Pred, an in silico cytotoxicity browsing tool, which proved their anticancer activity against several cancer cell lines at Pa > 0.5. Additionally, a confirmation for in silico cytotoxicity was made by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for commercially available hits syringaresinol and cleomiscosin A against lung cancer (A549) cells. The results demonstrated that syringaresinol has an IC50 value of 36.9 μg/mL, while cleomiscosin A has an IC50 value of 133 μg/mL. After MTT, flow cytometry analysis confirmed that compounds induced apoptosis in A549 cells in a dose-dependent manner. This study suggested that the respective lignan compounds can serve as lead candidates for lung cancer therapy via 6-PGD inhibition. Furthermore, in vivo experiments need to be conducted to confirm their efficacy.

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples.

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, protein amounts, or other relevant molecules. These methods have shown to be substantial alternatives to PCR or ELISA for clinical and research applications. Moreover, the detection of protein amount (by Western blot or immunohistochemistry) is often insufficient to provide information for cancer diagnosis, whereas the measurement of enzyme activity represents a valuable biomarker. Measurement of enzyme activity also allows for the diagnosis and potential treatment of pathogen-borne diseases. In all eukaryotes, topoisomerases are the key DNA-binding enzymes involved in the control of the DNA topological state during important cellular processes and are among the important biomarkers for cancer prognosis and treatment. Over the years, topoisomerases have been substantially investigated as a potential target of antiparasitic and anticancer drugs with libraries of natural and synthetic small-molecule compounds that are investigated every year. Here, the rolling circle amplification method, termed rolling circle enhanced enzyme activity detection (REEAD) assay that allows for the quantitative measurement of topoisomerase 1 (TOP1) activity in a simple, fast, and gel-free manner is presented.By cleaving and ligating a specially designed DNA substrate, TOP1 converts a DNA oligonucleotide into a closed circle, which becomes the template for rolling circle amplification, yielding ~103 tandem repeat rolling circle products. Depending on the nucleotide incorporation during the amplification, there is the possibility of different readout methods, from fluorescence to chemiluminescence to colorimetric. As each TOP1-mediated cleavage-ligation generates one closed DNA circle, the assay is highly sensitive and directly quantitative.

P326 DNA replication initiator proteins stabilize the kinetochore in the human fungal pathogen Cryptococcus neoformans

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesDNA replication licensing ensures maintenance of the ploidy state by limiting DNA replication once per cell cycle. The mini-chromosome maintenance (MCM) complex is an evolutionarily conserved DNA helicase in eukaryotes that aids this process. Upon activation in the S phase by several protein kinases, the MCM complex unwinds the double-stranded DNA and moves away from DNA replication origins preventing re-initiation of replication in the remainder of the cell cycle. Deregulation of MCM function can lead to malignant transformation of proliferating cells as indicated by their upregulated expression in human cancer and pre-cancerous cells. MCMs, therefore, serve as important biomarkers and potential targets for anti-cancer drugs. This study aims to decipher the effect of deregulated expression of the MCM complex on the cell cycle events of the human fungal pathogen, Cryptococcus neoformans, which is more similar to metazoans than other budding yeast. Aneuploidy-mediated drug resistance is a common mechanism in many major human fungal pathogens including Cryptococcus and Candida.MethodsWe generated conditional mutants of individual subunits of the MCM complex in C. neoformans and assayed the impact of their altered expression on cell cycle events. We also tested the alternative role of MCM subunits in the assembly of the kinetochore, a vital component of the chromosome segregation machinery.ResultsOur screen with MCM conditional mutants identified two in vivo functional subcomplexes that comprise the MCM complex in C. neoformans. Although upregulated expression of either Mcm2 or Mcm3 does not affect cell cycle progression, overexpression of either Mcm6 or Mcm7 led to the accumulation of cells in the large bud stage with nuclear segregation defects. This work provides evidence for the first time for a mitotic role of pre-replication complex proteins, MCM 2-7 complex in Cryptococcus. Depletion of Mcm2 led to arrest of cells in the S and G2/M stages of the cell cycle with defects in nuclear segregation. Localization and expression of several kinetochore proteins upon depletion of Mcm2 established that Mcm2 is vital for kinetochore assembly/integrity. Although the centromeric histone H3 variant, CENP-A, remains largely clustered, the outer kinetochore did not mature indicating that Mcm2 alone or as part of the MCM complex plays a role in kinetochore assembly/integrity and thereby in chromosome segregation.ConclusionA conserved eukaryotic DNA helicase, MCM 2-7 complex, has an unexplored non-canonical role in kinetochore assembly/integrity and chromosome segregation.