Potential Drug Targets Research Articles

Dual RNA-seq in filarial nematodes and Wolbachia endosymbionts using RNase H based ribosomal RNA depletion

Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium Wolbachia as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80–90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and Wolbachia from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from Brugia malayi containing the Wolbachia symbiont (wBm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5′-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of Wolbachia mRNA reads, resulting in a 16–1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the Brugia malayi depletion probes can remove rRNA from the filarial worm Dirofilaria immitis and the majority of rRNA from the more distantly related free living nematode Caenorhabditis elegans. These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library.

Potential drug targets for tumors identified through Mendelian randomization analysis.

According to the latest cancer research data, there are a significant number of new cancer cases and a substantial mortality rate each year. Although a substantial number of clinical patients are treated with existing cancer drugs each year, the efficacy is unsatisfactory. The incidence is still high and the effectiveness of most cancer drugs remains unsatisfactory. Therefore, we evaluated the human proteins for their causal relationship to for cancer risk and therefore also their potential as drug targets. We used summary tumors data from the FinnGen and cis protein quantitative trait loci (cis-pQTL) data from a genome-wide association study, and employed Mendelian randomization (MR) to explore the association between potential drug targets and nine tumors, including breast, colorectal, lung, liver, bladder, prostate, kidney, head and neck, pancreatic caners. Furthermore, we conducted MR analysis on external cohort. Moreover, Bidirectional MR, Steiger filtering, and colocalization were employed to validate the main results. The DrugBank database was used to discover potential drugs of tumors. Under the threshold of False discovery rate (FDR) < 0.05, results showed that S100A16 was protective protein and S100A14 was risk protein for human epidermal growth factor receptor 2-positive (HER-positive) breast cancer, phosphodiesterase 5A (PDE5A) was risk protein for colorectal cancer, and melanoma inhibitory activity (MIA) was protective protein for non-small cell lung carcinoma (NSCLC). And there was no reverse causal association between them. Colocalization analysis showed that S100A14 (PP.H4.abf = 0.920) and S100A16 (PP.H4.abf = 0.932) shared causal variation with HER-positive breast cancer, and PDE5A (PP.H4.abf = 0.857) shared causal variation with colorectal cancer (CRC). The MR results of all pQTL of PDE5A and MIA were consistent with main results. In addition, the MR results of MIA and external outcome cohort were consistent with main results. In this study, genetic predictions indicate that circulating S100 calcium binding protein A14 (S100A14) and S100 calcium binding protein A16 (S100A16) are associated with increase and decrease in the risk of HER-positive breast cancer, respectively. Circulating PDE5A is associated with increased risk of CRC, while circulating MIA is associated with decreased risk of NSCLC. These findings suggest that four proteins may serve as biomarkers for cancer prevention and as potential drug targets that could be expected for approval.

Abstract A013: Functional and clinical consequences of hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer

Abstract Recent whole-genome sequencing studies have unveiled intriguing hotspot mutations within non-coding genomic regions, yet their implications in cancer remain unknown. Here, we characterized the role of twin hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer: chr8: 29,952,919 G&amp;gt;A and chr8: 29,952,919 C&amp;gt;T. Either mutation is found in ~20% of bladder tumors, and the mutations do not typically co-occur. Through a comprehensive analysis integrating ChiP-seq, ATAC-seq, and Hi-C data from bladder cancer cell lines, we observed that these mutations co-localize with enhancer elements that interact with multiple nearby genes. In vitro dual luciferase reporter assays showed that both mutations are functional and likely affect gene expression in a genomic context. CRISPR-Cas9-mediated knocking out of the putative enhancer region identified LEPROTL1, DCTN6, and SARAF as target genes that are likely co-regulated, which was further supported by strong positive correlations of their mRNA expression in TCGA data. Isogenic cell clones generated by CRISPR-mediated base-editing showed that the mutations result in simultaneous upregulation of the three target genes, which were also expressed at higher levels in TCGA tumors harboring the non-coding mutations. Little is known about the function of LEPROTL1, DCTN6, and SARAF. Utilizing siRNA knockdown models complemented by live-cell imaging, viability assays, and cell cycle analysis, we found that they promote cell proliferation and cell cycle progression. Furthermore, our base-edited isogenic cell lines showed more rapid proliferation and cell cycle progression, as well as resistance to cisplatin. The mutations were also associated with shorter disease-specific survival in a TCGA cohort, and DCTN6 showed a negative association with overall survival. These results underscore the clinical significance of the non-coding mutations. Currently, we are assessing the effect of these mutations on tumor growth and cisplatin response in vivo using xenograft mouse models and evaluating the mechanisms by which LEPROTL1, DCTN6, and SARAF modulate cell cycle progression. Taken together, our data suggest that non-coding enhancer mutations near LEPROTL1 contribute to bladder carcinogenesis by increasing the expression of three co-regulated novel oncogenes, all of which were previously uncharacterized in bladder cancer. Since we found the mutations to be clinically significant, our study also raises the possibility of using the non-coding mutations and their target genes as clinical biomarkers and potential therapeutic drug targets. Citation Format: Kelly Butler, Bilal Lone, Alexandra Dobbins, Arup Chakraborty, Salah Boudajadi, Andrea Apolo, Rouf Banday. Functional and clinical consequences of hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A013.

Sestrin2 remedies neuroinflammatory response by inhibiting A1 astrocyte conversion via autophagy.

Most central nervous diseases are accompanied by astrocyte activation. Autophagy, an important pathway for cells to protect themselves and maintain homeostasis, is widely involved in regulation of astrocyte activation. Reactive astrocytes may play a protective or harmful role in different diseases due to different phenotypes of astrocytes. It is an urgent task to clarify the formation mechanisms of inflammatory astrocyte phenotype, A1 astrocytes. Sestrin2 is a highly conserved protein that can be induced under a variety of stress conditions as a potential protective role in oxidative damage process. However, whether Sestrin2 can affect autophagy and involve in A1 astrocyte conversion is still uncovered. In this study, we reported that Sestrin2 and autophagy were significantly induced in mouse hippocampus after multiple intraperitoneal injections of lipopolysaccharide, with the elevation of A1 astrocyte conversion and inflammatory mediators. Knockdown Sestrin2 in C8-D1A astrocytes promoted the levels of A1 astrocyte marker C3 mRNA and inflammatory factors, which was rescued by autophagy inducer rapamycin. Overexpression of Sestrin2 in C8-D1A astrocytes attenuated A1 astrocyte conversion and reduced inflammatory factor levels via abundant autophagy. Moreover, Sestrin2 overexpression improved mitochondrial structure and morphology. These results suggest that Sestrin2 can suppress neuroinflammation by inhibiting A1 astrocyte conversion via autophagy, which is a potential drug target for treating neuroinflammation.

Evaluating the effects of various ethanolic medicinal plant extracts on metastatic breast cancer proliferation, invasion, and expression of a novel potential drug target; CD82 metastatic suppressor protein, and on in vivo angiogenesis using the ex ovo yolk sac membrane (YSM) assay.

Breast cancer metastasis relies on cellular invasion and angiogenesis facilitated by the downregulation of metastatic suppressor proteins like Cluster of Differentiation 82 (CD82). Currently, no medicines target multiple systems to prevent metastatic progression through CD82 upregulation. This study screened for plant extracts displaying effects on cell proliferation, invasion, and CD82 expression in breast cancer cells, and in vivo angiogenesis, and further correlated between the biological activities and effect on CD82 expression. Seventeen ethanolic plant extracts were screened for their effect on cell proliferation (against MDA-MB-231 and MCF-7 breast cancer and Hek293 kidney cells), cell invasion and effect on CD82 expression in metastatic MDA-MB-231 cells. Selected extracts were further evaluated for in vivo anti-angiogenesis. Extracts displayed varying antiproliferative activity against the different cell lines, and those that showed selectivity indexes (SI) > 0.5 against MDA-MB-231 were selected for anti-invasion evaluation. Buddleja saligna Willd. (BS), Combretum apiculatum Sond. (CA), Foeniculum vulgare, Greyia radlkoferi, Gunnera perpensa and Persicaria senegalensis (Meisn.) Soják (PS) displayed 50% inhibitory concentration (IC50) values of 44.46 ± 3.46, 74.00 ± 4.48, 180.43 ± 4.51, 96.97 ± 2.29, 55.29 ± 9.88 and 243.60 ± 2.69µg/mL, respectively against MDA-MB-231, and compared to Hek293 showed SI of 0.9, 0.7, 1.4, 1.1, 2.2 and 0.5. Significant invasion inhibition was observed at both 20 and 40µg/mL for BS (94.10 ± 0.74 and 96.73 ± 0.95%) and CA (87.42 ± 6.54 and 98.24 ± 0.63%), whereas GR (14.91 ± 1.62 and 41 ± 1.78%) and PS (36.58 ± 0.54 and 51.51 ± 0.83%), only showed significant inhibition at 40µg/mL, and FV (< 5% inhibition) and GP (10 ± 1.03 and 22 ± 1.31%) did not show significant inhibition at both concentrations. Due to the significant anti-invasive activity of BS, CA and PS at 40µg/mL, these extracts were further evaluated for their potential to stimulate CD82. BS showed significant (p < 0.05) reduction in CD82 at 20 and 40µg/mL (13.2 ± 2.2% and 20.3 ± 1.5% decrease, respectively), whereas both CA and PS at 20µg/mL increased (p < 0.05) CD82 expression (16.4 ± 0.8% and 5.4 ± 0.6% increase, respectively), and at 40µg/mL significantly reduced CD82 expression (23.4 ± 3.1% and 11.2 ± 2.9% decrease, respectively). Using the yolk sac membrane assay, BS (59.52 ± 4.12 and 56.72 ± 3.13% newly formed vessels) and CA (83.33 ± 3.17 and 74.00 ± 2.12%) at both 20 and 40µg/egg showed significant (p < 0.001) angiogenesis inhibition, with BS showing statistical similar activity to the positive control, combretastatin A4 (10nmol/egg), whereas PS only displayed significant (p < 0.001) angiogenesis stimulation at 40µg/egg (120.81 ± 3.34% newly formed vessels). BS exhibits antiproliferative, anti-invasive, and anti-angiogenic activity despite inhibiting CD82, suggesting an alternative mode of action. CA at 20µg/mL shows moderate anti-invasive and anti-angiogenic potential by stimulating CD82, while at 40µg/mL it still displays these properties but inhibits CD82, suggesting an additional mode of action. PS, with the least antiproliferative activity, stimulates CD82 and inhibits angiogenesis at 20µg/mL but inhibits CD82 and increases angiogenesis at 40µg/mL, indicating CD82 targeting as a major mode of action. Future studies should explore breast cancer xenograft models to assess the extracts' impact on CD82 expression and angiogenesis in the tumor microenvironment, along with isolating bioactive compounds from the extracts.

Nlrp6 protects from corticosterone-induced NSPC ferroptosis by modulating RIG-1/MAVS-mediated mitophagy

Hippocampal neural stem/progenitor cells (NSPCs) are highly vulnerable to different stress stimuli, resulting in adult neurogenesis decline and eventual cognitive defects. Our previous study demonstrated that NOD-like receptor family pyrin domain-containing 6 (Nlrp6) highly expressed in NSPCs played a critical role in sustaining hippocampal neurogenesis to resist stress-induced depression, but the underlying mechnistms are still unclear. Here, we found that Nlrp6 depletion led to cognitive defects and hippocampal NSPC loss in mice. RNA-sequencing analysis of the primary NSPCs revealed that Nlrp6 deficiency altered gene expression profiles of mitochondrial energy generation and ferroptotic process. Upon siNlrp6 transfection, as well as corticosterone (CORT) exposure, downregulation of Nlrp6 suppressed retinoic acid-inducible gene I (RIG-1)/mitochondrial antiviral signaling proteins (MAVS)-mediated autophagy, but drove NSPC ferroptotic death. More interesting, short chain fatty acids (SCFAs) upregulated Nlrp6 expression and promoted RIG-1/MAVS-mediated mitophagy, preventing CORT-induced NSPC ferroptosis. Our study further demonstrates that Nlrp6 should be a sensor for RIG-1/MAVS-mediated mitophagy and play a critical role in maintain mitochondrial homeostasis of hippocampal NSPCs. These results suggests that Nlrp6 should be a potential drug target to combat neurodegenerative diseases relative with chronic stress.

Identifying potential drug targets for varicose veins through integration of GWAS and eQTL summary data

BackgroundVaricose veins (VV) are a common chronic venous disease that is influenced by multiple factors. It affects the quality of life of patients and imposes a huge economic burden on the healthcare system. This study aimed to use integrated analysis methods, including Mendelian randomization analysis, to identify potential pathogenic genes and drug targets for VV treatment.MethodsThis study conducted Summary-data-based Mendelian Randomization (SMR) analysis and colocalization analysis on data collected from genome-wide association studies and cis-expression quantitative trait loci databases. Only genes with PP.H4 &amp;gt; 0.7 in colocalization were chosen from the significant SMR results. After the above analysis, we screened 12 genes and performed Mendelian Randomization (MR) analysis on them. After sensitivity analysis, we identified four genes with potential causal relationships with VV. Finally, we used transcriptome-wide association studies and The Drug-Gene Interaction Database data to identify and screen the remaining genes and identified four drug targets for the treatment of VV.ResultsWe identified four genes significantly associated with VV, namely, KRTAP5-AS1 [Odds ratio (OR) = 1.08, 95% Confidence interval (CI): 1.05–1.11, p = 1.42e-10] and PLEKHA5 (OR = 1.13, 95% CI: 1.06–1.20, p = 6.90e-5), CBWD1 (OR = 1.05, 95% CI: 1.01–1.11, p = 1.42e-2) and CRIM1 (OR = 0.87, 95% CI: 0.81–0.95, p = 3.67e-3). Increased expression of three genes, namely, KRTAP5-AS1, PLEKHA5, and CBWD1, was associated with increased risk of the disease, and increased expression of CRIM1 was associated with decreased risk of the disease. These four genes could be targeted for VV therapy.ConclusionWe identified four potential causal proteins for varicose veins with MR. A comprehensive analysis indicated that KRTAP5-AS1, PLEKHA5, CBWD1, and CRIM1 might be potential drug targets for varicose veins.

MSphere of Influence: Retreading old ground-the beauty in multi-omic data sets.

Ethan L. Morgan works on human papillomaviruses (HPVs), with a specific interest in identifying how HPV induces tumor formation. In this mSphere of Influence article, he reflects on how three papers influenced him. "Comprehensive genomic characterization of head and neck squamous cell carcinomas" (The Cancer Genome Atlas Network, Nature 517:576-582, 2015, https://doi.org/10.1038/nature14129) and "Integrated genomic and molecular characterization of cervical cancer" (The Cancer Genome Atlas Network, Nature 543: 378-384, 2017, https://doi.org/10.1038/nature21386) showed him the power behind comprehensive multi-omic analyses to understand disease biology, while "Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response" (J. Sitz et al., Proc Natl Acad Sci U S A 116:19552-19562, 2019, https://doi.org/10.1073/pnas.1906102116) reinforced how this can be used to undercover potential new drug targets in HPV-associated disease.

Identifying Key Regulatory Genes in Drug Resistance Acquisition: Modeling Pseudotime Trajectories of Breast Cancer Single-Cell Transcriptome

Single-cell RNA-sequencing (scRNA-seq) technology has provided significant insights into cancer drug resistance at the single-cell level. However, understanding dynamic cell transitions at the molecular systems level remains limited, requiring a systems biology approach. We present an approach that combines mathematical modeling with a pseudotime analysis using time-series scRNA-seq data obtained from the breast cancer cell line MCF-7 treated with tamoxifen. Our single-cell analysis identified five distinct subpopulations, including tamoxifen-sensitive and -resistant groups. Using a single-gene mathematical model, we discovered approximately 560–680 genes out of 6000 exhibiting multistable expression states in each subpopulation, including key estrogen-receptor-positive breast cancer cell survival genes, such as RPS6KB1. A bifurcation analysis elucidated their regulatory mechanisms, and we mapped these genes into a molecular network associated with cell survival and metastasis-related pathways. Our modeling approach comprehensively identifies key regulatory genes for drug resistance acquisition, enhancing our understanding of potential drug targets in breast cancer.

Hypersensitive blood vessels in Clarkson disease.

Idiopathic systemic capillary leak syndrome (ISCLS) is a rare, recurrent condition with dramatically increased blood vessel permeability and, therefore, induction of systemic edema, which may lead to organ damage and death. In this issue of the JCI, Ablooglu et al. showed that ISCLS vessels were hypersensitive to agents known to increase vascular permeability, using human biopsies, cell culture, and mouse models. Several endothelium-specific proteins that regulate endothelial junctions were dysregulated and thereby compromised the vascular barrier. These findings suggest that endothelium-intrinsic dysregulation underlies hyperpermeability and implicate the cytoplasmic serine/threonine protein phosphatase 2A (PP2A) as a potential drug target for the treatment of ISCLS.

Exploring optimal drug targets through subtractive proteomics analysis and pangenomic insights for tailored drug design in tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, ranks among the top causes of global human mortality, as reported by the World Health Organization’s 2022 TB report. The prevalence of M. tuberculosis strains that are multiple and extensive-drug resistant represents a significant barrier to TB eradication. Fortunately, having many completely sequenced M. tuberculosis genomes available has made it possible to investigate the species pangenome, conduct a pan-phylogenetic investigation, and find potential new drug targets. The 442 complete genome dataset was used to estimate the pangenome of M. tuberculosis. This study involved phylogenomic classification and in-depth analyses. Sequential filters were applied to the conserved core genome containing 2754 proteins. These filters assessed non-human homology, virulence, essentiality, physiochemical properties, and pathway analysis. Through these intensive filtering approaches, promising broad-spectrum therapeutic targets were identified. These targets were docked with FDA-approved compounds readily available on the ZINC database. Selected highly ranked ligands with inhibitory potential include dihydroergotamine and abiraterone acetate. The effectiveness of the ligands has been supported by molecular dynamics simulation of the ligand–protein complexes, instilling optimism that the identified lead compounds may serve as a robust basis for the development of safe and efficient drugs for TB treatment, subject to further lead optimization and subsequent experimental validation.

Synthesis, Biological and Molecular Docking Studies of Thiazole-Thiadiazole derivatives as potential Anti-Tuberculosis Agents.

Tuberculosis remains a global health threat, with increasing infection rates and mortality despite existing anti-TB drugs. The present work focuses on the research findings regarding the development and evaluation of thiadiazole-linked thiazole derivatives as potential anti-tuberculosis agents. We present the synthesis data and confirm the compound structures using spectroscopic techniques. The current study reports twelve thiazole-thiadiazole compounds (5 a-5 l) for their anti-tuberculosis and related bioactivities. This paper emphasizes compounds 5 g, 5 i, and 5 l, which exhibited promising MIC values, leading to further in silico and interaction analysis. Pharmacophore mapping data included in the present analysis identified tubercular ThyX as potential drug targets. The compounds were evaluated for anti-tubercular activity using standard methods, revealing significant MIC values, particularly compound 5 l, with the best MIC value of 7.1285 μg/ml. Compounds 5 g and 5 i also demonstrated moderate to good MIC values against M. tuberculosis (H37Ra). Structural inspection of the docked poses revealed interactions such as hydrogen bonds, halogen bonds, and interactions containing Pi electron cloud, shedding light on conserved interactions with residues like Arg 95, Cys 43, His 69, and Arg 87 from the tubercular ThyX enzyme.

Uncovering novel drug targets for bipolar disorder: a Mendelian randomization analysis of brain, cerebrospinal fluid, and plasma proteomes.

There is a clear demand for innovative therapeutics for bipolar disorder (BD). We integrated the largest BD genome-wide association study (GWAS) dataset (NCase = 41 917, NControl = 371 549) with protein quantitative trait loci from brain, cerebrospinal fluid, and plasma. Using a range of integrative analyses, including Mendelian randomization (MR), Steiger filter analysis, Bayesian colocalization, and phenome-wide MR analysis, we prioritized novel drug targets for BD. Additionally, we incorporated data from the UK Biobank (NCase = 1064, NControl = 365 476) and the FinnGen study (NCase = 7006, NControl = 329 192) for robust biological validation. Through MR analysis, we found that in the brain, downregulation of DNM3, MCTP1, ABCB8 and elevation of DFNA5 and PDF were risk factors for BD. In cerebrospinal fluid, increased BD risk was associated with increased levels of FRZB, AGRP, and IL36A and decreased CTSF and LRP8. Plasma analysis revealed that decreased LMAN2L, CX3CL1, PI3, NCAM1, and TIMP4 correlated with increased BD risk, but ITIH1 did not. All these proteins passed Steiger filtering, and Bayesian colocalization confirmed that 12 proteins were colocalized with BD. Phenome-wide MR analysis revealed no significant side effects for potential drug targets, except for LRP8. External validation further underscored the concordance between the primary and validation cohorts, confirming MCTP1, DNM3, PDF, CTSF, AGRP, FRZB, LMAN2L, NCAM1, and TIMP4 are intriguing targets for BD. Our study identified druggable proteins for BD, including MCTP1, DNM3, and PDF in the brain; CTSF, AGRP, and FRZB in cerebrospinal fluid; and LMAN2L, NCAM1, and TIMP4 in plasma, delineating promising avenues to development of novel therapies.

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.

The complex regulation of Slo1 potassium channels from a structural perspective.

Fast and regulated potassium efflux by Slo1 channels is crucial in many tissues in animals including neurons, the kidney and smooth muscle. During the last decade, structures have revealed many details about the gating mechanism and regulation of these large and complex molecular machines. This review summarizes these findings and the current knowledge about the intricate regulation of these important channels. Slo1 integrates sensing of the membrane potential via a voltage-sensor domain that undergoes subtle but significant structural rearrangements with a calcium-induced expansion of parts of the intracellular gating ring. Together, these two signals synergistically lead to changes in the conformation and chemical nature of the pore domain, allowing potassium ions to be translocated. In many native tissues, Slo1 channels are assembled with at least three classes of auxiliary subunits that change the gating kinetics or allow the channel to open also in absence of one of the two signals. Finally, Slo1 is inhibited, activated or deregulated by natural toxins and synthetic compounds, underlining the importance of the channel for the organism and as a potential target for drugs and other molecules.

Bioinformatics analysis of PANoptosis regulators in the diagnosis and subtyping of steroid-induced osteonecrosis of the femoral head.

In this study, we aimed to investigate the involvement of PANoptosis, a form of regulated cell death, in the development of steroid-induced osteonecrosis of the femoral head (SONFH). The underlying pathogenesis of PANoptosis in SONFH remains unclear. To address this, we employed bioinformatics approaches to analyze the key genes associated with PANoptosis. Our analysis was based on the GSE123568 dataset, allowing us to investigate both the expression profiles of PANoptosis-related genes (PRGs) and the immune profiles in SONFHallowing us to investigate the expression profiles of PRGs as well as the immune profiles in SONFH. We conducted cluster classification based on PRGs and assessed immune cell infiltration. Additionally, we used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific hub genes. Furthermore, we developed an optimal machine learning model to identify the key predictive genes responsible for SONFH progression. We also constructed a nomogram model with high predictive accuracy for assessing risk factors in SONFH patients, and validated the model using external data (area under the curve; AUC = 1.000). Furthermore, we identified potential drug targets for SONFH through the Coremine medical database. Using the optimal machine learning model, we found that 2 PRGs, CASP1 and MLKL, were significantly correlated with the key predictive genes and exhibited higher expression levels in SONFH. Our analysis revealed the existence of 2 distinct PANoptosis molecular subtypes (C1 and C2) within SONFH. Importantly, we observed significant variations in the distribution of immune cells across these subtypes, with C2 displaying higher levels of immune cell infiltration. Gene set variation analysis indicated that C2 was closely associated with multiple immune responses. In conclusion, our study sheds light on the intricate relationship between PANoptosis and SONFH. We successfully developed a risk predictive model for SONFH patients and different SONFH subtypes. These findings enhance our understanding of the pathogenesis of SONFH and offer potential insights into therapeutic strategies.

Abstract PO3-24-09: May ESR1 mutant breast cancer cells influence fibroblast phenotype?

Abstract For almost 50 years, it has been postulated that carcinogenesis relies on the intrinsic genetic abnormalities of tumor cells, but in the last decades, it has been reported that their functional interactions with the tumor microenvironment (TME) might affect therapy response and contribute to the disease progression. Based on these observations, the aim of the present study was to investigate the functional interactions between one of the essential components of TME, the fibroblasts, and breast cancer (BC) cells, expressing mutations in the hormone-binding domain (HBD) of the gene encoding for the estrogen receptor alpha (ESR1). Thus, we employed estrogen receptor-positive MCF-7 BC cells CRISPR engineered to express the Y537S-HBD mutation. Y537S is the most commonly detected mutation harbored in HBD-ESR1 and is a major contributor to endocrine therapy resistance and poor clinical outcome in BC. We collected conditioned media (CM) from BC cells and fibroblasts isolated from primary human tissues (normal fibroblasts, NFs, and cancer-associated fibroblasts, CAFs) in order to assess in vitro co-culture systems that can mimic the complex in vivo TME. It was interesting to observe that CAFs displayed a significant increase in their proliferation and migration when exposed to CM of mutant BC cells with respect to the parental ones. These latter findings fit with the immunofluorescence analysis showing a more evident formation of actin stress fibers. However, proteomic analysis demonstrated, in the same circumstances, more significant changes in NFs than in CAFs. Among the up-regulated proteins revealed by NFs upon Y537S-CM exposure, most of them overlap the up-regulated proteins featuring the intrinsic phenotype of CAFs, mainly involved in the cellular ultrastructural organization (i.e. cytoskeleton, focal adhesion, vesicle, and organelle) as evidenced by Metacore functional analysis. On the other side, we demonstrated that mutant clones showed more aggressive behavior in terms of proliferation, growth, migration, and invasion, compared to the parental counterpart upon exposure to the CM-derived from NFs and CAFs. This led us to further investigate the intrinsic properties of the mutated clones through their genomic and proteomic profiles that evidenced, by using Metacore software, an enrichment of insulin-like growth factor 1 receptor (IGF1R) pathway. The relevance of the latter datum was confirmed by the higher content of the IGF-1 ligand found in the secretome of mutant cells. Thus, the short autocrine loop maintaining IGF1/IGF1R signaling activation in mutant clones might reasonably play an important role in mediating the autocrine and paracrine effects between mutant cells and fibroblasts. For instance, the pharmacological blockade of IGF1R signaling interfered with the reciprocal interactions between fibroblasts and BC cells that sustained tumor growth and progression as evidenced in both “in vitro” and “in vivo” experiments. In conclusion, our study addressed the IGF1R pathway as a potential druggable target helpful to disconnect mutant breast cancer cell-fibroblast crosstalk that potentiates breast tumor growth and progression. Citation Format: Luca Gelsomino, Amanda Caruso, Rocco Malivindi, Adel Elisabetta Leonetti, Emine Tasan, Salvatore Panza, Giuseppina Daniela Naimo, Ines Barone, Cinzia Giordano, Daniela Bonofiglio, Loredana Mauro, Guowei Gu, Suzanne Fuqua, Stefania Catalano, Sebastiano Andò. May ESR1 mutant breast cancer cells influence fibroblast phenotype? [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 PO3-24-09.

Artemisinin derivatives as potential drug candidates against Mycobacterium tuberculosis: insights from molecular docking, MD simulations, PCA, MM/GBSA and ADMET analysis

ABSTRACT Tuberculosis, one of the most ancient and formidable infectious diseases, primarily arises from the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Phosphoenolpyruvate carboxykinase (Pck), a potential drug target, is essential for the growth of Mtb that involves in the pathway of gluconeogenesis at the centre of phosphoenolpyruvate-pyruvate-oxaloacetate node. This study aims to understand drug-like properties of the compounds derived from artemisinin, and to investigate their inhibitory roles against Mtb. Molecular docking was performed on a set of 56 artemisinin compounds to identify their binding efficacy to the Mtb protein target. Subsequently, the selected top three complexes were subjected to molecular dynamics simulations. By the trajectory analysis, root mean square deviation and radius of gyration indicated the compactness of the systems without much fluctuation. The principal component analysis revealed that the complexes were less dynamic and energetically more favourable, and Gibbs energy landscape revealed the favourable energetic transitions between conformations. The artemisinin dimer primary alcohol holding good bioavailability scores appears highly stable in complex with Pck (MM/GBSA of −37.67 kcal/mol) that stands as a potential inhibitor of the Mtb target. However, further preclinical experiments and investigations are necessary to evaluate the intrinsic properties and to confirm effectiveness of the drug candidate.

Natural products and synthetic analogs as selective orphan nuclear receptor 4A (NR4A) modulators.

Although endogenous ligands for the orphan nuclear receptor 4A1 (NR4A1, Nur77), NR4A2 (Nurr1), and NR4A3 (Nor-1) have not been identified, several natural products and synthetic analogs bind NR4A members. These studies are becoming increasingly important since members of the NR4A subfamily of 3 receptors are potential drug targets for treating cancer and non-cancer endpoints and particularly those conditions associated with inflammatory diseases. Ligands that bind NR4A1, NR4A2, and NR4A3 including Cytosporone B, celastrol, bis-indole derived (CDIM) compounds, tryptophan/indolic, metabolites, prostaglandins, resveratrol, piperlongumine, fatty acids, flavonoids, alkaloids, peptides, and drug families including statins and antimalarial drugs. The structural diversity of NR4A ligands and their overlapping and unique effects on NR4A1, NR4A2, and NR4A3 suggest that NR4A ligands are selective NR4A modulators (SNR4AMs) that exhibit tissue-, structure-, and response-specific activities. The SNR4AM activities of NR4A ligands are exemplified among the Cytosporone B analogs where n-pentyl-2-[3,5-dihydroxy-2-(nonanoyl)]phenyl acetate (PDNPA) binds NR4A1, NR4A2 and NR4A3 but activates only NR4A1 and exhibits significant functional differences with other Cytosporone B analogs. The number of potential clinical applications of agents targeting NR4A is increasing and this should spur future development of SNR4AMs as therapeutics that act through NR4A1, NR4A2 and NR4A3.

Circulating Proteins and IgA Nephropathy.

The therapeutic options for IgA nephropathy are rapidly evolving, but early diagnosis and targeted treatment remain challenging. We aimed to identify circulating plasma proteins associated with IgA nephropathy by proteome-wide mendelian randomization studies across multiple ancestry populations. In this study, we applied Mendelian randomization and colocalization analyses to estimate the putative causal effects of 2615 proteins on IgA nephropathy in Europeans and 235 proteins in East Asians. Following two-stage network Mendelian randomization, multi-trait colocalization analysis and protein-altering variant annotation were performed to strengthen the reliability of the results. A protein-protein interaction network was constructed to investigate the interactions between the identified proteins and the targets of existing medications. Putative causal effects of 184 and 13 protein-disease pairs in European and East Asian ancestries were identified, respectively. Two protein-disease pairs showed shared causal effects across them (CFHR1 and FCRL2). Supported by the evidence from colocalization analysis, potential therapeutic targets were prioritized and four drug-repurposing opportunities were suggested. The protein-protein interaction network further provided strong evidence for existing medications and pathways that are known to be therapeutically important. Our study identified a number of circulating proteins associated with IgA nephropathy and prioritized several potential drug targets that require further investigation.