Ligase Inhibitors Research Articles

AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.

Strategic advancement of E3 ubiquitin ligase in the management of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) presents a significant global health challenge due to its high incidence, poor prognosis, and limited treatment options. As a pivotal regulator of protein stability, E3 ubiquitin ligase plays a crucial role in tumorigenesis and development. This review provides an overview of the latest research on the involvement of E3 ubiquitin ligase in hepatocellular carcinoma and elucidates its significance in hepatocellular carcinoma cell proliferation, invasion, and evasion from immune surveillance. Special attention is given to the functions of RING, HECT, and RBR E3 ubiquitin ligases and their association with hepatocellular carcinoma progression. By dissecting the molecular mechanisms and regulatory networks governed by E3 ubiquitin ligase, several potential therapeutic strategies are proposed: including the development of specific inhibitors targeting E3 ligases; augmentation of their tumor suppressor activity through drug or gene therapy; utilization of E3 ubiquitin ligase to modulate immune checkpoint proteins for improved efficacy of immunotherapy; combination strategies integrating traditional therapies with E3 ubiquitin ligase inhibitors; as well as biomarker development based on E3 ubiquitin ligase activity. Furthermore, this review discusses the prospect of overcoming drug resistance in hepatocellular carcinoma treatment through these novel approaches. Overall, this review establishes a theoretical foundation and offers fresh insights into harnessing the potential of E3 ubiquitin ligase for treating hepatocellular carcinoma while highlighting future research directions that pave the way for clinical translation studies and new drug discoveries.

YF550-C1, an E3 ligase inhibitor of CBL-B, to demonstrate T cell and NK cell activation and anti-tumor activities in syngeneic and PDX tumor model.

e14570 Background: Casitas B-lineage lymphoma b (CBL-B) is one of ligase in E3 ubiquitin family, which mainly expressed in matured hematopoietic cells, including T and NK cells. Study showed that CBL-B deficiency upregulated interleukin-2 and interferon-gamma secretion during anti-CD3 and anti-CD28 stimulated TCR activation, even without co-stimulator anti-CD28[3]. Except T cells, CBL-B plays important role in NK cells as well. CBL-B was upregulated during NK cell activation. While downregulation of CBL-B enhanced the cytotoxicity of primary human NK cells against hematological cancer cell lines. In vivo studies proved that CBL-B deficiency protected mice from tumor growth by inducing antitumor immunity. Thus, CBL-B shows a promising target for cancer therapy. Methods: We developed a small molecule YF550-C1 that targeted CBL-B, and evaluated the activation on T cell and NK cell function, and the therapeutical effect on syngeneic tumor models. The T cell activation was indicated by levels of IL-2 and IFN-γ from CBL-B knockout or WT CD8+ T cells since stimulated with anti-CD3 or anti-CD3/28 for 48 hours. The resistance of PGE2-induced immunosuppressive was evaluated by measuring IL-2 and IFN-γ since human T cells treated with PGE2 and followed by series diluted YF550-C1 or comparable inhibitor cpd23. OVA specific antigen stimulated CD8+ T activation assay was conducted by coculture OT-1 CD8+ T cells and WT DC cells with 50:1 for 72 hours in the present of 5ug/ml OVA peptide and series diluted YF550-C1. The NK cell cytotoxicity was evaluated by co-culture human NK cells and K562-luc cells with 1.25:1 ratio in the present of 1000nM or 333nM YF550-C1, which indicated by luminance. Syngeneic mouse model that inoculated with different types of murine cancer cell lines was used to evaluate tumor growth. Results: CBL-B deficiency suppressed tumor growth in mouse tumor models and enhanced cytokines release in T cells upon TCR engagement. CBL-B inhibitor, YF550-C1, alleviated PGE2-mediated immunosuppression of human T cell activation. CBL-B inhibitor, YF550-C1, enhanced OVA-specific antigen stimulated CD8+ T cell activation. CBL-B inhibitor, YF550-C1, enhanced cytotoxicity of human NK cell against K562-luc cells. CBL-B inhibitor, YF550-C1, or in combinate with anti-PD-L1 suppressed tumor growth in syngeneic tumor models. Single-agent of CBL-B inhibitor YF550-C1 treatment was sufficient to suppress PD1/PD-L1 resistant NSCLC patient-derived tumor growth. Conclusions: These studies provided insight into the activity of the inhibitor of CBL-B, demonstrating that YF550-C1 displays excellently T cell activation, T cell immunosuppression alleviation, NK cell activation and antitumor activity in multiple preclinical tumor models. Our data showed that YF550-C1 is a promising candidate for cancer treatment as monotherapy or in combination with PD-L1 blockade.

Impact of DNA ligase inhibition on the nick sealing of polβ nucleotide insertion products at the downstream steps of base excision repair pathway.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps. We previously demonstrated that a functional interplay between polβ and BER ligases is critical for efficient repair, and polβ mismatch or oxidized nucleotide insertions confound final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polβ+/+ cells more than polβ-/- null cells. We showed less efficient ligation after polβ nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polβ inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polβ, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

Functional analysis of the ube3a response in Japanese flounder (Paralichthys olivaceus) to CSBV infection

Ube3a is a member of the E3 ubiquitin ligase HECTc family, and its role has been established in neurodevelopmental disorders. However, studies on its role in Japanese flounder are scarce. Thus, in this study, the ube3a of Japanese flounder was cloned, and its role in conferring resistance against Chinook salmon bafnivirus (CSBV) was analyzed. Japanese flounder ube3a encoded a protein containing 834 amino acids. Interestingly, its homology with the Atlantic halibut was determined to be 94%. In addition, there were differential expressions of ube3a in different tissues of Japanese flounder, with the highest expression level observed in the fin, followed by the gills and skin (P ≤ 0.05). Subcellular localization analysis revealed that Ube3a is a cytoplasmic protein. We established an in vitro CSBV infection model using Japanese flounder gill cell line (FG). After ube3a overexpression, the viral load was significantly lower than that of the control group (P ≤ 0.05). Contrastingly, after incubation of FG cells with an E3 ubiquitin ligase inhibitor, the viral load was significantly higher than in the control group (P ≤ 0.01). Then, the expression levels of nf-κb, traf3, and tnf-α after incubation with an E3 ubiquitin ligase inhibitor were examined. The results demonstrated that ube3a may exerted a significant antiviral effect in Japanese flounder via the ubiquitination pathway.

Selective inhibition of DNA ligase IV provides additional efficacy to the treatment of anaplastic thyroid cancer.

Although the incidence of anaplastic thyroid carcinoma (ATC) is low (2.5% of thyroid cancer cases), this cancer has a very poor prognosis (survival rates < 5 months) and accounts for 14-39% of deaths. Conventional therapies based on surgery in combination with radiotherapy or chemotherapy showed limited effectiveness primarily due to the robust and protective DNA damage response in thyroid cancer cells. We used single-cell transcriptomic data from patients with different subtypes of thyroid cancer to study expression of genes involved in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Then, we investigated the mechanisms of DNA damage and repair in anaplastic (C643 and Hth74) and papillary (TPC-1) thyroid cancer cell lines. The effect of caffeine (inhibitor of ATM and ATR) and UCN-01 (CHK1 inhibitor) was evaluated in cell cycle progression of thyroid cancer cells after γ-radiation or doxorubicin treatment. The DNA damage response was monitored after staining of phosphorylated γ-H2AX and 53BP1. Reporter plasmids were used to determine the efficacy of double-strand DNA breaks (DSBs) repair by HR and NHEJ in thyroid cancer cells. We evaluated the combination of selective inhibition of the DNA ligase IV by SCR7 and doxorubicin on cellular apoptosis and tumor growth in xenograft murine models of anaplastic thyroid cancer. Single-cell RNA-Seq showed that NHEJ- and HR-related genes are expressed in ATC and PTC patients. We showed that ATC cells undergo mitosis in the presence of unrepaired DNA damage caused by γ-radiation and doxorubicin treatment. To proliferate and survive, these cells efficiently repair DNA lesions using homologous recombination (HR) and non-homologous end joining (NHEJ). The combination of SCR7 with doxorubicin, significantly increased apoptosis and impaired ATC tumor growth in a xenograft mouse model compared to doxorubicin monotherapy. This study shows the therapeutic value of the combination of a DNA ligase IV inhibitor and DNA-damaging agents (doxorubicin and/or γ-radiation) for the treatment of anaplastic thyroid cancer.

In Silico Drug Repurposing Studies for the Discovery of Novel Salicyl-AMP Ligase (MbtA)Inhibitors.

Tuberculosis (TB) continues to pose a global health challenge, exacerbated by the rise of drug-resistant strains. The development of new TB therapies is an arduous and time-consuming process. To expedite the discovery of effective treatments, computational structure-based drug repurposing has emerged as a promising strategy. From this perspective, conditionally essential targets present a valuable opportunity, and the mycobactin biosynthesis pathway stands out as a prime example highlighting the intricate response of Mycobacterium tuberculosis (Mtb) to changes in iron availability. This study focuses on the repurposing and revival of FDA-approved drugs (library) as potential inhibitors of MbtA, a crucial enzyme in mycobactin biosynthesis in Mtb conserved among all species of mycobacteria. The literature suggests this pathway to be associated with drug efflux pumps, which potentially contribute to drug resistance. This makes it a potential target for antitubercular drug discovery. Herein, we utilized cheminformatics and structure-based drug repurposing approaches, viz., molecular docking, dynamics, and PCA analysis, to decode the intermolecular interactions and binding affinity of the FDA-reported molecules against MbtA. Virtual screening revealed ten molecules with significant binding affinities and interactions with MbtA. These drugs, originally designed for different therapeutic indications (four antiviral, three anticancer, one CYP450 inhibitor, one ACE inhibitor, and one leukotriene antagonist), were repurposed as potential MbtA inhibitors. Furthermore, our study explores the binding modes and interactions between these drugs and MbtA, shedding light on the structural basis of their inhibitory potential. Principal component analysis highlighted significant motions in MbtA-bound ligands, emphasizing the stability of the top protein-ligand complexes (PLCs). This computational approach provides a swift and cost-effective method for identifying new MbtA inhibitors, which can subsequently undergo validation through experimental assays. This streamlined process is facilitated by the fact that these compounds are already FDA-approved and have established safety and efficacy profiles. This study has the potential to lay the groundwork for addressing the urgent global health challenge at hand, specifically in the context of combating antimicrobial resistance (AMR) and tuberculosis (TB).

Protective effects of compound M01 on retinal ganglion cells in experimental anterior ischemic optic neuropathy by inhibiting TXNIP/NLRP3 inflammasome pathway

Apoptotic death of retinal ganglion cells (RGCs) is a common pathologic feature in different types of optic neuropathy, including ischemic optic neuropathy and glaucoma, ultimately leading to irreversible visual function loss. Potent and effective protection against RGC death is determinative in developing a successful treatment for these optic neuropathies. This study evaluated the neuroprotective effect of a HECT domain-E3 ubiquitin ligase inhibitor, M01, on retinal ganglion cells after ischemic injury. Experimental anterior ischemic optic neuropathy (AION) was induced by photothrombotic occlusion of microvessels supplying optic nerve in rats. M01 was administered (100 mg/Kg and 200 mg/Kg) subcutaneously for three consecutive days after AION induction. Administration of M01 (100 mg/Kg) significantly increased RGC survival and preserved visual function after AION induction. The number of TUNEL-positive cells and ED1-positive cells was significantly decreased, and optic disc edema was reduced considerably after ischemic infarction with M01 treatment. Moreover, M01 effectively ameliorated optic nerve demyelination and enhanced M2 microglial polarization after AION induction. M01 enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2); subsequently, downregulated Thioredoxin interacting protein (TXNIP) expression, inhibited NLR family pyrin domain containing 3 (NLRP3) activation, and further decreased inflammatory factors, interleukin (IL)-1β and IL-6 in the retina after ischemic injury. These findings suggested that M01 has therapeutic potential by modulating Nrf2 and TXNIP/NLRP3 inflammasome pathways in the retina and optic nerve ischemic damage-related diseases.

MurF Ligase Inhibitors: An Overview of Antibacterial Activity

Abstract: ATP dependent MurC-F ligases are essential for the biosynthesis of peptidoglycan, an essential bacterial cell wall component that is required for bacterial cell survival. Last, in the series, MurF catalyzes the ATP-dependent addition of D-Ala-D-Ala dipeptide to UDP-N-acetylmuramyl-tripeptide to form the UDP-N-acetylmuramy-pentapeptide monomeric precursor of peptidoglycan. Owing to its critical essentiality in peptidoglycan biosynthesis and absence in eukaryotic counterparts, MurF is considered a promising target for the design and development of potent antibacterial agents. Several MurF inhibitors have been designed and evaluated for their MurF inhibitory and antibacterial activity. These include aminoalkylphosphinates, sulfonamides, diarylquinolones, hydroxylamines, phosphorylated hydroxylamines, thiazolylaminopyrimidines, 2,4,6-trisubstituted 1,3,5-triazines, etc. However, most of the inhibitors developed till date lack potent antibacterial activity against both Gram-positive and Gram-negative bacteria. In the present review, an updated status of MurF ligase inhibitors is presented that may provide a useful source for the design of novel MurF inhibitors with potent and broad-spectrum antibacterial activity.

Structure-guided design and synthesis of ATP-competitive N-acyl-substituted sulfamide d-alanine-d-alanine ligase inhibitors

d-Alanine-d-alanine ligase (Ddl) catalyses the ATP-dependent formation of d-Ala-d-Ala, a critical component in bacterial cell wall biosynthesis and is a validated target for new antimicrobial agents. Here, we describe the structure-guided design, synthesis, and evaluation of ATP-competitive N-acyl-substituted sulfamides 27–36, 42, 46, 47 as inhibitors of Staphylococcus aureus Ddl (SaDdl). A crystal structure of SaDdl complexed with ATP and d-Ala-d-Ala (PDB: 7U9K) identified ATP-mimetic 8 as an initial scaffold for further inhibitor design. Evaluation of 8 in SaDdl enzyme inhibition assays revealed the ability to reduce enzyme activity to 72 ± 8 % (IC50 = 1.6 mM). The sulfamide linker of 8 was extended with 2-(4-methoxyphenyl)ethanol to give 29, to investigate further interactions with the d-Ala pocket of SaDdl, as predicted by molecular docking. This compound reduced enzyme activity to 89 ± 1 %, with replacement of the 4-methoxyphenyl group in 29 with alternative phenyl substituents (27, 28, 31–33, 35, 36) failing to significantly improve on this (80–89 % remaining enzyme activity). Exchanging these phenyl substituents with selected heterocycles (42, 46, 47) did improve activity, with the most active compound (42) reducing SaDdl activity to 70 ± 1 % (IC50 = 1.7 mM), which compares favourably to the FDA-approved inhibitor d-cycloserine (DCS) (IC50 = 0.1 mM). To the best of our knowledge, this is the first reported study of bisubstrate SaDdl inhibitors.

Comparative functional and structural analysis of Pseudomonas aeruginosa d-alanine-d-alanine ligase isoforms as prospective antibiotic targets.

Pseudomonas aeruginosa is a major human pathogen in the healthcare setting. The emergence of multi-drug-resistant and extensive drug-resistant P. aeruginosa is of great concern, and clearly indicates that new alternatives to current first-line antibiotics are required in the future. Inhibition of d-alanine-d-alanine production presents as a promising avenue as it is a key component in the essential process of cell wall biosynthesis. In P. aeruginosa, d-alanine-d-alanine production is facilitated by two isoforms, d-alanine-d-alanine ligase A (PaDdlA) and d-alanine-d-alanine ligase B (PaDdlA), but neither enzyme has been individually characterised to date. Here, we present the functional and structural characterisation of PaDdlA and PaDdlB, and assess their potential as antibiotic targets. This was achieved using a combination of in vitro enzyme-activity assays and X-ray crystallography. The former revealed that both isoforms effectively catalyse d-alanine-d-alanine production with near identical efficiency, and that this is effectively disrupted by the model d-alanine-d-alanine ligase inhibitor, d-cycloserine. Next, each isoform was co-crystallised with ATP and either d-alanine-d-alanine or d-cycloserine, allowing direct comparison of the key structural features. Both isoforms possess the same structural architecture and share a high level of conservation within the active site. Although residues forming the d-alanine pocket are completely conserved, the ATP-binding pocket possesses several amino acid substitutions resulting in a differing chemical environment around the ATP adenine base. Together, these findings support that the discovery of dual PaDdlA/PaDdlB competitive inhibitors is a viable approach for developing new antibiotics against P. aeruginosa.

Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses

The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.

Design and Synthesis of Inhibitors of the E3 Ligase SMAD Specific E3 Ubiquitin Protein Ligase 1 as a Treatment for Lung Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a devastating rare disease, which despite currently available treatments, still represents a high unmet medical need. Specific E3 ubiquitin protein ligase 1 (SMURF1) is a HECT E3 ligase that ubiquitinates key signaling molecules from the TGFβ/BMP pathways, which are of great relevance in the pathophysiology of PAH. Herein, the design and synthesis of novel potent small-molecule SMURF1 ligase inhibitors are described. Lead molecule 38 has demonstrated good oral pharmacokinetics in rats and significant efficacy in a rodent model of pulmonary hypertension.

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.

Differential role for rapid proteostasis in Rho GTPase-mediated control of quiescent endothelial integrity

Endothelial monolayer permeability is regulated by actin dynamics and vesicular traffic. Recently, ubiquitination was also implicated in the integrity of quiescent endothelium, as it differentially controls the localization and stability of adhesion and signaling proteins. However, the more general effect of fast protein turnover on endothelial integrity is not clear. Here, we found that inhibition of E1 ubiquitin ligases induces a rapid, reversible loss of integrity in quiescent, primary human endothelial monolayers, accompanied by increased F-actin stress fibers and the formation of intercellular gaps. Concomitantly, total protein and activity of the actin-regulating GTPase RhoB, but not its close homolog RhoA, increase ∼10-fold in 5 to 8h. We determined that the depletion of RhoB, but not of RhoA, the inhibition of actin contractility, and the inhibition of protein synthesis all significantly rescue the loss of cell-cell contact induced by E1 ligase inhibition. Collectively, our data suggest that in quiescent human endothelial cells, the continuous and fast turnover of short-lived proteins that negatively regulate cell-cell contact is essential to preserve monolayer integrity.

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster.

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Decoding the Role of MDM2 as a Potential Ubiquitin E3 Ligase and Identifying the Therapeutic Efficiency of Alkaloids against MDM2 in Combating Glioblastoma.

Glioblastomas (GBMs) represent the most aggressive form of brain tumor arising from the malignant transformation of astrocytes. Despite various advancements, treatment options remain limited to chemotherapy and radiotherapy followed by surgery giving an overall survival of 14-15 months. These therapies are somewhere restricted in giving a better survival and cure. There is a need for new therapeutics that could potentially target GBM based on molecular pathways and pathology. Here, ubiquitin E3 ligases can be used as targets as they bind a wide array of substrates and therefore can be attractive targets for new inhibitors. Through this study, we have tried to sort various ubiquitin E3 ligases based on their expression, pathways to which these ligases are associated, and mutational frequencies, and then we tried to screen potent inhibitors against the most favorable E3 ligase as very few studies are available concerning inhibition of E3 ligase in GBM. Our study found MDM2 to be the most ideal E3 ligase and further we tried to target MDM2 against various compounds under the alkaloid class. Molecular Docking and MD simulations combined with ADMET properties and BBB scores revealed that only evodiamine and sanguinarine were effective in inhibiting MDM2. We also tried to give a proposed mechanism of how these inhibitors mediate the p53 signaling in GBM. Therefore, the new scaffolds predicted by the computational approach could help in designing promising therapeutic agents targeting MDM2 in glioblastoma.

Abstract 1693: Effect of selective inhibitor of E3 ligase, SMER3 on muscle atrophy: potential implication in targeting cancer associated cachexia

Abstract 1693: Effect of selective inhibitor of E3 ligase, SMER3 on muscle atrophy: potential implication in targeting cancer associated cachexia

Peptidomics-based identification of an antimicrobial peptide derived from goat milk fermented by Lactobacillus rhamnosus (C25).

Antimicrobial peptides (AMPs) are emerging as promising novel drug applicants. In the present study, goat milk was fermented using Lactobacillus rhamnosus C25 to generate bioactive peptides (BAPs). The peptide fractions generated were separated using ultrafiltration membranes with molecular weight cut-offs of 3, 5, and 10kDa, and their antimicrobial activity toward Gram-positive and Gram-negative bacteria was investigated. Isolated AMPs were characterized using RP-HPLC and identified by LC-MS/MS. A total of 569 sequences of peptides were identified by mass spectrometry. Out of the 569, 36 were predicted as AMPs, 21 were predicted as cationic, and out of 21, 6 AMPs were helical peptides. In silico analysis indicated that the majority of peptides were antimicrobial and cationic in nature, an important factor for peptide interaction with the negative charge membrane of bacteria. The results showed that the peptides of <5kDa exhibited maximum antibacterial activity against E. faecalis, E. coli, and S. typhi. Further, molecular docking was used to evaluate the potent MurD ligase inhibitors. On the basis of ligand binding energy, six predicted AMPs were selected and then analyzed by AutoDock tools. Among the six AMPs, peptides IGHFKLIFSLLRV (-7.5kcal/mol) and KSFCPAPVAPPPPT (-7.6kcal/mol), were predicted as a high-potent antimicrobial. Based on these findings, in silico investigations reveal that proteins of goat milk are a potential source of AMPs. This is for the first time that the antimicrobial peptides produced by Lactobacillus rhamnosus (C25) fermentation of goat milk have been identified via LC-MS/MS and predicted as AMPs, cationic charges, helical structure in nature, and potent MurD ligase inhibitors. These peptides can be synthesized and improved for use as antimicrobial agents. PRACTICAL APPLICATIONS: Goat milk is considered a high-quality source of milk protein. According to this study, goat milk protein is a potential source of AMPs, Fermentation can yield goat milk-derived peptides with a broad antibacterial activity spectrum at a low cost. The approach described here could be beneficial in that the significant AMPs can be synthesized and used in the pharmaceutical and food industries.

A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase.

RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small-molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12, that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure-activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12, which has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promoting proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.