Journal articles: 'Ligase I Inhibitors'

1

Alomari, Arqam, Robert Gowland, Callum Southwood, Jak Barrow, Zoe Bentley, Jashel Calvin-Nelson, Alice Kaminski, et al. "Identification of Novel Inhibitors of Escherichia coli DNA Ligase (LigA)." Molecules 26, no. 9 (April 25, 2021): 2508. http://dx.doi.org/10.3390/molecules26092508.

Full text

Abstract:

Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3′ hydroxyl and a 5′ phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11–2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.

APA, Harvard, Vancouver, ISO, and other styles

2

Ciarrocchi, Giovanni, Donald G. MacPhee, Les W. Deady, and Leann Tilley. "Specific Inhibition of the Eubacterial DNA Ligase by Arylamino Compounds." Antimicrobial Agents and Chemotherapy 43, no. 11 (November 1, 1999): 2766–72. http://dx.doi.org/10.1128/aac.43.11.2766.

Full text

Abstract:

ABSTRACT All known DNA ligases catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA via very similar mechanisms. The ligase family can, however, be divided into two classes: eubacterial ligases, which require NAD+ as a cofactor, and other ligases, from viruses, archaea, and eukaryotes, which use ATP. Drugs that discriminate between DNA ligases from different sources may have antieubacterial activity. We now report that a group of arylamino compounds, including some commonly used antimalarial and anti-inflammatory drugs and a novel series of bisquinoline compounds, are specific inhibitors of eubacterial DNA ligases. Members of this group of inhibitors have different heterocyclic ring systems with a common amino side chain in which the two nitrogens are separated by four carbon atoms. The potency, but not the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD+. The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a SalmonellaLig− strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parentalSalmonella strain.

APA, Harvard, Vancouver, ISO, and other styles

3

Lama, Rati, Samuel L. Galster, Chao Xu, Luke W. Davison, Sherry R. Chemler, and Xinjiang Wang. "Dual Targeting of MDM4 and FTH1 by MMRi71 for Induced Protein Degradation and p53-Independent Apoptosis in Leukemia Cells." Molecules 27, no. 22 (November 8, 2022): 7665. http://dx.doi.org/10.3390/molecules27227665.

Full text

Abstract:

MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored. Here, we describe the synthesis and characterization of quinolinol derivatives for the identification of analogs that are capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, the targeted degradation of MDM4 and FTH1 in cells, and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity that conventional MDM2-p53 disrupting inhibitors lack. This study provides a prototype structure for developing MDM4/FTH1 dual-targeting inhibitors as potential cancer therapeutics.

APA, Harvard, Vancouver, ISO, and other styles

4

Shapiro, Adam B., Ann E. Eakin, Grant K. Walkup, and Olga Rivin. "A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase." Journal of Biomolecular Screening 16, no. 5 (March 11, 2011): 486–93. http://dx.doi.org/10.1177/1087057111398295.

Full text

Abstract:

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

APA, Harvard, Vancouver, ISO, and other styles

5

Gorelik, Maryna, Stephen Orlicky, Maria A. Sartori, Xiaojing Tang, Edyta Marcon, Igor Kurinov, Jack F. Greenblatt, et al. "Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface." Proceedings of the National Academy of Sciences 113, no. 13 (March 14, 2016): 3527–32. http://dx.doi.org/10.1073/pnas.1519389113.

Full text

Abstract:

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

APA, Harvard, Vancouver, ISO, and other styles

6

Marblestone, Jeffrey G., K. G. Suresh Kumar, Michael J. Eddins, Craig A. Leach, David E. Sterner, Michael R. Mattern, and Benjamin Nicholson. "Novel Approach for Characterizing Ubiquitin E3 Ligase Function." Journal of Biomolecular Screening 15, no. 10 (September 23, 2010): 1220–28. http://dx.doi.org/10.1177/1087057110380456.

Full text

Abstract:

The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)–compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain’s inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform’s broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.

APA, Harvard, Vancouver, ISO, and other styles

7

Tobin, Lisa A., Aaron P. Rapoport, Ivana Gojo, Maria R. Baer, Alan E. Tomkinson, and Feyruz V. Rassool. "DNA Ligase III Alpha and (Poly-ADP) Ribose Polymerase (PARP1) Are Therapeutic Targets in Imatinib-Resistant (IR) Chronic Myeloid Leukemia (CML)." Blood 114, no. 22 (November 20, 2009): 853. http://dx.doi.org/10.1182/blood.v114.22.853.853.

Full text

Abstract:

Abstract Abstract 853 Therapy with the tyrosine kinase inhibitor imatinib, targeting the constitutively active BCR-ABL kinase has been remarkably successful in Philadelphia chromosome-positive (Ph+) CML, but resistance to tyrosine kinase inhibitors is a growing clinical problem, prompting the search for new therapeutic targets. BCR-ABL expression leads to increased reactive oxygen species (ROS), repair errors and genomic instability. We have previously shown that an error-prone alternative non-homologous end-joining (ALT NHEJ) pathway involving PARP1 and DNA ligase IIIa/XRCC1 is upregulated in Ph+ CML, providing a mechanism for the repair errors and genomic instability. To determine whether ALT NHEJ components may be novel therapeutic targets in IR CML, we characterized two IR cell lines (P210Mo7eIR, Baf3P210IR) for DSB repair abnormalities. Both IR cell lines demonstrate significantly higher levels of DSBs and NHEJ abnormalities (P<0.05) compared with their imatinib-sensitive (IS) counterparts. Notably, whereas steady state levels of the ALT NHEJ components DNA ligase IIIa and PARP1 are increased in IS P210Mo7e and Baf3P210 cells, compared with parental Mo7e and Baf3, the levels of these proteins are increased even further in the IR cells. Presence of increased DNA ligase IIIa and PARP1 levels in the IR cell lines suggests that these enzymes may be targets for therapy using the DNA ligase inhibitors that we have previously identified and PARP1 inhibitors, which have been used successfully in the treatment of cancers with DSB repair defects. Initial tests for cytotoxicity in BCR-ABL-positive cell lines and parental controls showed that the DNA ligase inhibitor L67, which specifically inhibits DNA ligase I and IIIα, is cytotoxic in BCR-ABL-positive cells and parental controls at concentrations of >10 μM, and that cytotoxicity is not influenced by BCR-ABL1 expression. Therefore, we examined the effect of a subtoxic concentration of L67 (0.3 μM) in the presence or absence of the PARP1 inhibitor Nu1025 (Calbiochem) at 50 μM in IR versus IS and parental cells. Combined treatment with L67 and Nu1025 significantly (p<0.001) reduces survival of IR cells compared with IS and parental controls, which were not significantly affected. To determine whether cells from CML patients that are resistant to imatinib are also sensitive to the combination of DNA ligase and PARP inhibitors, we next tested primary bone marrow mononuclear cells (BM MNC) from 6 CML patients with IR disease, compared with normal BM MNC. Cells from 3 of the 6 patients demonstrated a significant decrease in colony survival in response to the combination of DNA repair inhibitors, similar to the sensitivity demonstrated by the two IR cell lines studied. Interestingly, the patient demonstrating the highest sensitivity to the combination of DNA repair inhibitors had significantly increased levels of both DNA ligase IIIa and PARP1, whereas patients demonstrating less sensitivity had increased levels of either DNA ligase IIIa or PARP1, compared with normal BM MNC. Importantly, sensitivity to the DNA repair inhibitors is not correlated with mutations in BCR-ABL because the BCR-ABL mutation T315I that is found in Baf3P210IR cells when overexpressed in Baf3 cells has no effect on colony survival following drug treatment. Together, our results suggest that the process of acquiring IR may select for cells with high levels of PARP1 and DNA ligase IIIa and/or may upregulate ALT NHEJ pathways. Thus, patients with high levels of these proteins are likely to benefit from therapy using inhibitors of ALT NHEJ. Disclosures: No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

8

TAN, Ghee T., Sangkook LEE, Ik-Soo LEE, Jingwen CHEN, Pete LEITNER, Jeffrey M. BESTERMAN, Douglas A. KINGHORN, and John M. PEZZUTO. "Natural-product inhibitors of human DNA ligase I." Biochemical Journal 314, no. 3 (March 15, 1996): 993–1000. http://dx.doi.org/10.1042/bj3140993.

Full text

Abstract:

Enzymic activity mediated by recombinant human DNA ligase I (hLI), in conjunction with tannin removal procedures, has been applied to a natural-product screen involving ~1000 plant extracts and various pure compounds. The primary hLI activity assay involved the measurement of the amount of radiolabelled phosphate in a synthetic nucleic acid hybrid that becomes resistant to alkaline phosphatase as a result of ligation. A bioactivity-guided fractionation scheme resulted in the isolation of ursolic [IC50 = 100 μg/ml (216 μM)] and oleanolic [IC50 = 100 μg/ml (216 μM)] acids from Tricalysia niamniamensis Hiern (Rubiaceae), which demonstrated similar DNA ligase inhibition profiles to other triterpenes such as aleuritolic acid. Protolichesterinic acid [IC50 = 6 μg/ml (20 μM)], swertifrancheside [IC50 = 8 μg/ml (11 μM)] and fulvoplumierin [IC50 = 87 μg/ml (357 μM)] represent three additional natural-product structural classes that inhibit hLI. Fagaronine chloride [IC50 = 10 μg/ml (27 μM)] and certain flavonoids are also among the pure natural products that were found to disrupt the activity of the enzyme, consistent with their nucleic acid intercalative properties. Further analyses revealed that some of the hLI-inhibitory compounds interfered with the initial adenylation step of the ligation reaction, indicating a direct interaction with the enzyme protein. However, in all cases, this enzyme–inhibitor interaction did not disrupt the DNA relaxation activity mediated by hLI. These results indicate that, although the same enzyme active site may be involved in both enzyme adenylation and DNA relaxation, inhibitors may exert allosteric effects by inducing conformational changes that disrupt only one of these activities. Studies with inhibitors are important for the assignment of specific cellular functions to these enzymes, as well as for their development into clinically useful antitumour agents.

APA, Harvard, Vancouver, ISO, and other styles

9

Goldenberg, Seth J., Jeffrey G. Marblestone, Michael R. Mattern, and Benjamin Nicholson. "Strategies for the identification of ubiquitin ligase inhibitors." Biochemical Society Transactions 38, no. 1 (January 19, 2010): 132–36. http://dx.doi.org/10.1042/bst0380132.

Full text

Abstract:

Dysregulation of the UPS (ubiquitin–proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; however, toxicity with this target remains high. E3s (Ub–protein ligases) represent an alternative attractive therapeutic target in the UPS. In this paper, we will discuss current platforms that report on E3 ligase activity and can detect E3 inhibitors, and underline the advantages and disadvantages of each approach.

APA, Harvard, Vancouver, ISO, and other styles

10

Mills, Scott D., Ann E. Eakin, Ed T. Buurman, Joseph V. Newman, Ning Gao, Hoan Huynh, Kenneth D. Johnson, et al. "Novel Bacterial NAD+-Dependent DNA Ligase Inhibitors with Broad-Spectrum Activity and Antibacterial EfficacyIn Vivo." Antimicrobial Agents and Chemotherapy 55, no. 3 (December 28, 2010): 1088–96. http://dx.doi.org/10.1128/aac.01181-10.

Full text

Abstract:

ABSTRACTDNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities ofEscherichia coli,Haemophilus influenzae,Mycoplasma pneumoniae,Streptococcus pneumoniae, andStaphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such asS. aureus,S. pneumoniae,Streptococcus pyogenes, andM. pneumoniae, as well as against Gram-negative pathogens, such asH. influenzaeandMoraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains withligAmutations.In vivoefficacy was demonstrated in a murineS. aureusthigh infection model and a murineS. pneumoniaelung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

APA, Harvard, Vancouver, ISO, and other styles

11

Howes, Timothy R. L., Annahita Sallmyr, Rhys Brooks, George E. Greco, Darin E. Jones, Yoshihiro Matsumoto, and Alan E. Tomkinson. "Structure-activity relationships among DNA ligase inhibitors: Characterization of a selective uncompetitive DNA ligase I inhibitor." DNA Repair 60 (December 2017): 29–39. http://dx.doi.org/10.1016/j.dnarep.2017.10.002.

Full text

APA, Harvard, Vancouver, ISO, and other styles

12

Tomkinson, Alan E., Tasmin Naila, and Seema Khattri Bhandari. "Altered DNA ligase activity in human disease." Mutagenesis 35, no. 1 (October 20, 2019): 51–60. http://dx.doi.org/10.1093/mutage/gez026.

Full text

Abstract:

Abstract The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

APA, Harvard, Vancouver, ISO, and other styles

13

Giardina, Sarah F., Elena Valdambrini, Michael Peel, Manny D. Bacolod, Mace L. Rothenberg, Richard B. Lanman, J. David Warren, and Francis Barany. "Cure-PROs: Next-generation targeted protein degraders." Journal of Clinical Oncology 41, no. 16_suppl (June 1, 2023): e15101-e15101. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e15101.

Full text

Abstract:

e15101 Background: Many proteins, including transcription factors and scaffolding proteins, are not amenable to targeting by traditional small molecule inhibitors due to the lack of a well-defined binding pocket or active site. Proteolysis-Targeting Chimeras (PROTACs) are a new class of hetero-bifunctional molecules that bind both a target protein and an E3 ubiquitin ligase, bringing the two into proximity for appending ubiquitin, and subsequently marking the target protein for proteasomal degradation. Currently, thirteen PROTACs are in clinical trials for oncology indications. However, the clinical utility of PROTACs is challenged by their large size and long development timelines. Also, resistance mutations in the E3 ligase or transporter overexpression inevitably evolve. Thus, a new platform for small-molecule degraders that enables ultra-rapid drug development timelines, efficient cellular uptake, and can be developed to overcome innate and acquired drug resistance is needed. Methods: Coferons, developed in our laboratory, are small molecules that self-assemble upon binding to a target, where they form reversible covalent dimers through bio-orthogonal linker chemistries. We have combined features of the Coferon platform and PROTACs to generate CURE-PROs (Combinatorial Ubiquitination REal-time PROteolysis), consisting of one target protein ligand and one E3 ligase ligand that form reversible heterodimers that lead to targeted protein degradation within cells. By modifying known ligands for BRD4, and the E3 ubiquitin ligases Cereblon, VHL, and MDM2, with linkers able to reversibly join the BRD4 to the ligase ligands, we synthesized libraries of CURE-PRO monomers that can be combined to create thousands of CURE-PRO dimer combinations. We explored whether this platform could yield meaningful BRD4 degradation in vitro and in vivo. Results: Rapid combinatorial cell-based screening identified several BRD4-E3 ligase CURE-PRO combinations that induced greater than 50% BRD4 degradation, with the most promising CURE-PRO pairs achieving more than 95% protein degradation. Consistent with a PROTAC mechanism-of-action, successful CURE-PRO combinations confirmed significant protein degradation which was inhibited by proteasome inhibitors or competition with parent ligands. Significant BRD4 degradation was also observed in mice bearing bilateral human xenograft tumors, confirming CURE-PRO proof-of-mechanism in vivo. Conclusions: The combinatorial nature of our platform has the potential to significantly reduce synthesis time and effort to identify the optimal linker length and E3 ligase for each target protein. The CURE-PRO platform consists of expanding libraries of monomers for both additional oncoprotein targets as well as E3 ligases, which can be redeployed to shorten lead development timelines.

APA, Harvard, Vancouver, ISO, and other styles

14

Watanabe, Bunta, Hiroaki Kirikae, Takao Koeduka, Yoshinori Takeuchi, Tomoki Asai, Yoshiyuki Naito, Hideya Tokuoka, et al. "Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase inhibitors." Bioorganic & Medicinal Chemistry 26, no. 9 (May 2018): 2466–74. http://dx.doi.org/10.1016/j.bmc.2018.04.006.

Full text

APA, Harvard, Vancouver, ISO, and other styles

15

Baum, Ellen Z., Steven M. Crespo-Carbone, Darren Abbanat, Barbara Foleno, Amy Maden, Raul Goldschmidt, and Karen Bush. "Utility of Muropeptide Ligase for Identification of Inhibitors of the Cell Wall Biosynthesis Enzyme MurF." Antimicrobial Agents and Chemotherapy 50, no. 1 (January 2006): 230–36. http://dx.doi.org/10.1128/aac.50.1.230-236.2006.

Full text

Abstract:

ABSTRACT MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc-l-Ala-γ-d-Glu-meso-diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc-l-Ala-γ-d-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic l-Ala-γ-d-Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing l-Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 μM was identified.

APA, Harvard, Vancouver, ISO, and other styles

16

Kristan, Katja, Miha Kotnik, Marko Oblak, and Uroš Urleb. "New High-Throughput Fluorimetric Assay for Discovering Inhibitors of UDP-N-Acetylmuramyl-l-Alanine: d-Glutamate (MurD) Ligase." Journal of Biomolecular Screening 14, no. 4 (April 2009): 412–18. http://dx.doi.org/10.1177/1087057109332597.

Full text

Abstract:

A novel assay for monitoring the activity of the bacterial enzyme UDP- N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5′-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP Quest™ assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-alanine (UMA) and ATP that are in agreement with the data reported in the literature. A counterscreen assay was introduced to eliminate false positives, and some of the known MurD inhibitors have been retested to compare the data measured with different methods. Moreover, a focused library of around 1000 compounds was screened for the inhibition of MurD to assess the performance and robustness of the assay. Finally, a novel MurD inhibitor belonging to a new structural class, with an IC50 value of 105 µM, was discovered. ( Journal of Biomolecular Screening 2009:412-418)

APA, Harvard, Vancouver, ISO, and other styles

17

Pavlides, Savvas C., Kuang-Tzu Huang, Dylan A. Reid, Lily Wu, Stephanie V. Blank, Khushbakhat Mittal, Lankai Guo, et al. "Inhibitors of SCF-Skp2/Cks1 E3 Ligase Block Estrogen-Induced Growth Stimulation and Degradation of Nuclear p27kip1: Therapeutic Potential for Endometrial Cancer." Endocrinology 154, no. 11 (November 1, 2013): 4030–45. http://dx.doi.org/10.1210/en.2013-1757.

Full text

Abstract:

In many human cancers, the tumor suppressor, p27kip1 (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%–62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

APA, Harvard, Vancouver, ISO, and other styles

18

Powell, Jason A., Melissa R. Pitman, Julia R. Zebol, Paul A. B. Moretti, Heidi A. Neubauer, Lorena T. Davies, Alexander C. Lewis, et al. "Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1." Biochemical Journal 476, no. 21 (November 11, 2019): 3211–26. http://dx.doi.org/10.1042/bcj20190245.

Full text

Abstract:

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

APA, Harvard, Vancouver, ISO, and other styles

19

Wan, Yichao, Chunxing Yan, Han Gao, and Tingting Liu. "Small-molecule PROTACs: novel agents for cancer therapy." Future Medicinal Chemistry 12, no. 10 (May 2020): 915–38. http://dx.doi.org/10.4155/fmc-2019-0340.

Full text

Abstract:

Proteolysis-targeting chimera (PROTAC) is a new technology to selectively degrade target proteins via ubiquitin-proteasome system. PROTAC molecules (PROTACs) are a class of heterobifunctional molecules, which contain a ligand targeting the protein of interest, a ligand recruiting an E3 ligase and a linker connecting these two ligands. They provide several advantages over traditional inhibitors in potency, selectivity and drug resistance. Thus, many promising PROTACs have been developed in the recent two decades, especially small-molecule PROTACs. In this review, we briefly introduce the mechanism of PROTACs and focus on the progress of small-molecule PROTACs based on different E3 ligases. In addition, we also introduce the opportunities and challenges of small-molecule PROTACs for cancer therapy.

APA, Harvard, Vancouver, ISO, and other styles

20

Pandey, Monica, Sujeet Kumar, Gunaseelan Goldsmith, Mrinal Srivastava, Santhini Elango, Mohammad Shameem, Dibyendu Bannerjee, Bibha Choudhary, Subhas S. Karki, and Sathees C. Raghavan. "Identification and characterization of novel ligase I inhibitors." Molecular Carcinogenesis 56, no. 2 (June 27, 2016): 550–66. http://dx.doi.org/10.1002/mc.22516.

Full text

APA, Harvard, Vancouver, ISO, and other styles

21

Watt, Jessica E., Gregory R. Hughes, Samuel Walpole, Serena Monaco, G. Richard Stephenson, Philip C. Bulman Page, Andrew M. Hemmings, Jesus Angulo, and Andrew Chantry. "Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors." Chemistry - A European Journal 24, no. 67 (November 6, 2018): 17677–80. http://dx.doi.org/10.1002/chem.201804169.

Full text

APA, Harvard, Vancouver, ISO, and other styles

22

Kuai, Jun, Yingzhi Bi, Yilin Qi, Deborah Conrady, Rajiv Govindaraj, Graham Hone, R. Aldrin Denny, Ken Carson, Geraldine Harriman, and Fang Wang. "864 Identification of a novel allosteric oral Cbl-b inhibitor that augmented T cell response and enhanced NK cell killing in vitro and in vivo." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A905. http://dx.doi.org/10.1136/jitc-2021-sitc2021.864.

Full text

Abstract:

BackgroundImmunotherapies aiming to boost anti-tumor cell responses in cancer patients has been proven successful by checkpoint inhibitors targeting PD1 or CTLA-4, but the majority of cancer patients do not garner durable benefit. Co-stimulation through the CD28 pathway is one potential approach to maximize the benefits of immunotherapies. The E3 ubiquitin ligase Cbl-b (casitas b-lineage lymphoma proto-oncogene b) has been established as a master negative regulator of T-cells and NK cells and plays an important role in immune suppression. Genetic ablation of Cbl-b or functional inactivation of its E3 ligase activity in mice resulted in CD8 T-cell-mediated rejection of primary tumors in several mouse models. Based on the overwhelming evidence supporting the role of Cbl-b in immune suppression, targeting Cbl-b with small molecule inhibitors is attractive for cancer immunotherapy.MethodsCbl-b is activated by tyrosine kinases and undergoes a large conformational change from closed inactive form to open active form. Historically, it had been difficult to identify inhibitors of Cbl-b. Through the utilization of our proprietary SpotFinder platform, a druggable phosphoregulatory pocket was identified in the inactive form of Cbl-b. Learnings from the platform allowed for the development of screening assays utilizing specifically designed protein constructs. Assays were developed to identify inhibitors that bind to the hotspot and lock Cbl-b in its inactive form.ResultsHere we report on a member of our lead series of inhibitors, a low nanomolar potent inhibitor identified via application of our SpotFinder platform. This inhibitor binds to the inactive form of Cbl-b, its binding mode in the identified hotspot confirmed by co-crystal structures. It inhibits the phosphorylation of Cbl-b by kinases, inhibits the E3 ligase activity of Cbl-b, promotes cytokine release and enhances T cell proliferation well as NK cell activation and killing. In vivo, our CBL-B inhibitors efficaciously augmented the T cell response in anti-CD3 treated mice.ConclusionsWe herein demonstrated the validation of our proprietary SpotFinder platform via the prediction and drugging of a regulatory hotspot on an important immune oncology target that has to date been very difficult to drug.

APA, Harvard, Vancouver, ISO, and other styles

23

Nicholson, B., Suresh Kumar, S. Agarwal, M. J. Eddins, J. G. Marblestone, J. Wu, M. P. Kodrasov, J. P. LaRocque, D. E. Sterner, and M. R. Mattern. "Discovery of Therapeutic Deubiquitylase Effector Molecules." Journal of Biomolecular Screening 19, no. 7 (March 14, 2014): 989–99. http://dx.doi.org/10.1177/1087057114527312.

Full text

Abstract:

The approval of proteasome inhibitors bortezomib and carfilzomib and the E3 ligase antagonist thalidomide and its analogs, lenalidomide and pomalidomide, validates the ubiquitin–proteasome pathway as a source of novel drugs for treating cancer and, potentially, a variety of devastating illnesses, including inflammation, cardiovascular disease, and neurodegenerative disease. All elements of this critical regulatory pathway—the proteasome itself, E3 ligases (which conjugate ubiquitin to target proteins), and deubiquitylating enzymes (which deconjugate ubiquitin, reversing ligase action)—are potential therapeutic targets, and all have been worked on extensively during the past decade. No deubiquitylase inhibitors or activators have yet progressed to clinical trial, however, despite compelling target validation and several years of high-throughput screening and preclinical development of hits by numerous pharmaceutical companies, biotechnology organizations, and academic groups. The appropriateness of deubiquitylases as therapeutic targets in many disease areas is reviewed, followed by evidence that selective inhibitors of these cysteine proteases can be discovered. Because the lack of progress in drug-discovery efforts with deubiquitylases suggests a need for improved discovery methodologies, currently available platforms and strategies are analyzed, and improved or completely novel, unrelated approaches are considered in terms of their likelihood of producing clinically viable effectors of deubiquitylases.

APA, Harvard, Vancouver, ISO, and other styles

24

Bjij, Imane, Pritika Ramharack, Shama Khan, Driss Cherqaoui, and Mahmoud Soliman. "Tracing Potential Covalent Inhibitors of an E3 Ubiquitin Ligase Through Target-Focused Modelling." Proceedings 22, no. 1 (November 14, 2019): 103. http://dx.doi.org/10.3390/proceedings2019022103.

Full text

Abstract:

The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the Nedd4-1 E3 Ubiquitin ligase is an enzyme that may be targeted either covalently, or non-covalently, there are few studies that demonstrate effective inhibitors of the enzyme. In this work, we aimed to identify covalent inhibitors of Nedd4-1. This task however, proved to be challenging due to the limited available electrophilic moieties in virtual libraries. We therefore opted to divide an existing covalent Nedd4-1 inhibitor in two parts: A non-covalent binding part and a pre-selected α, β-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on the active site binding investigations followed by validating the covalent conjugation. Thirty compounds were selected and covalently docked into the catalytic site of the Nedd4-1. Multiple filtrations were effected before selecting 5 hits that were later analysed by molecular dynamic simulations to check their stability and explore their binding landscape in complex with the protein. All in all, two inhibitors with optimum overall stability and more stabilising interactions were kept for eventual biological evaluation. Our improved pharmacophore model approach serves as a robust method that will illuminate the screening for novel covalent inhibitor in drug discovery.

APA, Harvard, Vancouver, ISO, and other styles

25

Gutierrez-Lugo, Maria-Teresa, Heather Baker, Joseph Shiloach, Helena Boshoff, and Carole A. Bewley. "Dequalinium, a New Inhibitor of Mycobacterium tuberculosis Mycothiol Ligase Identified by High-Throughput Screening." Journal of Biomolecular Screening 14, no. 6 (June 12, 2009): 643–52. http://dx.doi.org/10.1177/1087057109335743.

Full text

Abstract:

Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)—dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z′ ≥ 0.65). Fifteen hits (0.48%) were identified from the screen, and 2 hits were confirmed in a secondary assay that measures production of CGI. The structures of both hits contain N-substituted quinolinium moieties, and the more potent of the 2—namely, dequalinium chloride—inhibits MshC with an IC50 value of 24 ± 1 µM. Further studies showed dequalinium to be an ATP-competitive inhibitor of MshC, to bind MshC with a KD of 0.22 µM, and to inhibit the growth of M. tuberculosis under aerobic and anaerobic conditions with minimum inhibitory and anaerobic bactericidal concentrations of 1.2 and 0.3 µg/mL, respectively. The screening protocol described is robust and has enabled the identification of new MshC inhibitors. ( Journal of Biomolecular Screening 2009:643-652)

APA, Harvard, Vancouver, ISO, and other styles

26

Chu, Yu-Yi, Mei-Kuang Chen, Yongkun Wei, Heng-Huan Lee, Weiya Xia, Ying-Nai Wang, Clinton Yam, et al. "Targeting the ALK–CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex." Nature Cancer 3, no. 10 (October 17, 2022): 1211–27. http://dx.doi.org/10.1038/s43018-022-00438-2.

Full text

Abstract:

AbstractPoly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK–p-Tyr19-CDK9 expression.

APA, Harvard, Vancouver, ISO, and other styles

27

Lu, Jing, Yimin Qian, Kanak Raina, Martha Altieri, Hanqing Dong, Jing Wang, Xin Chen, et al. "BRD4 Degradation By Protacs Represents a More Effective Therapeutic Strategy Than BRD4 Inhibitors in DLBCL." Blood 126, no. 23 (December 3, 2015): 2050. http://dx.doi.org/10.1182/blood.v126.23.2050.2050.

Full text

Abstract:

Abstract Introduction: BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as an attractive target in multiple oncology indications, particularly hematological malignancies. As a crucial chromatin reader, BRD4 is frequently found to be preferentially localized at super-enhancer regions, which often reside upstream of important oncogenes, including c-Myc, Bcl-xL and Bcl-6. Thus BRD4 inhibitors have gained attention as promising therapeutics in malignancies driven by these key oncogenes, including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). However, we have recently found that BRD4 inhibitors lead to a compensatory accumulation of BRD4, which results in an incomplete inhibition of c-MYC and lack of apoptosis. These limitations could severely hamper the therapeutic potential of BRD4 inhibitors. To circumvent these limitations, we designed BRD4 degraders using our PROTAC (PROteolysis TArgeting Chimera) platform (Lu et al. Chem Biol. 22:755-763, 2015) and investigated the effects of BRD4 degradation in DLBCL in comparison with inhibitors. Methods and Results: We designed specific BRD4 PROTACs, which contain a BRD4 binding moiety connected to an E3 ubiquitin ligase-recruiting moiety. In all ABC-DLBCL, and GCB-DLBCL cell lines tested, BRD4 PROTACs lead to potent (<10 nM), fast (<4hrs) and robust (>90%) degradation of BRD4. This degradation is accompanied by more pronounced and longer-lasting suppression of downstream signaling events, such as c-MYC, than small molecule inhibitors (JQ1 and OTX015) treatment. We confirmed that BRD4 degradation is E3 ligase-mediated and proteasome-dependent as both excessive E3 ligase binding ligand and proteasome inhibitors (MG132 and carfilzomib) were able to prevent PROTAC-induced BRD4 degradation. Most importantly, BRD4 PROTACs were more effective in inhibiting DLBCL cell proliferation (measured by CellTiter-Glo® Luminescent Cell Viability Assay) and inducing apoptosis (evaluated by caspase3/7 assay and PARP cleavage) compared to BRD4 inhibitors. Conclusion: Hijacking E3 ubiquitin ligases, through PROTAC technology, is an innovative approach to achieve targeted, specific protein degradation. Such targeted degradation can provide important therapeutic advantages. Here, we demonstrated that BRD4 PROTACs lead to robust BRD4 downregulation, which results in better downstream signaling suppression, more pronounced inhibition of proliferation and induction of apoptosis compared to the effects of small molecule inhibitors. The results of on-going efficacy studies using a DLBCL tumor xenograft model will be presented at the meeting. Taken together, BRD4 degradation by PROTACs is a promising therapeutic strategy in DLBCL. Disclosures Lu: Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership. Raina:Arvinas, LLC: Employment, Equity Ownership. Altieri:Arvinas, LLC: Employment, Equity Ownership. Dong:Arvinas, LLC: Employment, Equity Ownership. Wang:Arvinas, LLC: Employment, Equity Ownership. Chen:Arvinas, LLC: Employment, Equity Ownership. Crew:Arvinas, LLC: Employment, Equity Ownership. Coleman:Arvinas, LLC: Employment, Equity Ownership. Crews:Arvinas, LLC: Employment, Equity Ownership. Winkler:Arvinas, LLC: Employment, Equity Ownership.

APA, Harvard, Vancouver, ISO, and other styles

28

Martinez-Iglesias, Olaia, Alba Casas-Pais, Raquel Castosa, Andrea Díaz-Díaz, Daniel Roca-Lema, Ángel Concha, Álvaro Cortés, Federico Gago, and Angélica Figueroa. "Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression." Cancers 12, no. 5 (May 23, 2020): 1340. http://dx.doi.org/10.3390/cancers12051340.

Full text

Abstract:

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression.

APA, Harvard, Vancouver, ISO, and other styles

29

Chen, Qing, Weilin Xie, Deborah J. Kuhn, Peter M. Voorhees, Antonia Lopez-Girona, Derek Mendy, Laura G. Corral, et al. "Targeting the p27 E3 ligase SCFSkp2 results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy." Blood 111, no. 9 (May 1, 2008): 4690–99. http://dx.doi.org/10.1182/blood-2007-09-112904.

Full text

Abstract:

Abstract Decreased p27Kip1 levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCFSkp2 E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27Kip1, agents inhibiting SCFSkp2 may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCFSkp2 ligase function in vitro, and induced specific accumulation of p21 and other SCFSkp2 substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCFSkp2 ligase, and induced G1/S cell-cycle arrest as well as SCFSkp2- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCFSkp2 inhibitors as a novel class of antitumor agents.

APA, Harvard, Vancouver, ISO, and other styles

30

Bjij, Imane, Pritika Ramharack, Shama Khan, Driss Cherqaoui, and Mahmoud E. S. Soliman. "Tracing Potential Covalent Inhibitors of an E3 Ubiquitin Ligase through Target-Focused Modelling." Molecules 24, no. 17 (August 28, 2019): 3125. http://dx.doi.org/10.3390/molecules24173125.

Full text

Abstract:

The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the enzyme may be targeted both covalently and non-covalently, minimal studies provide effective inhibitors against it. Recently, research has focused on covalent inhibitors based on their characteristic, highly-selective warheads and ability to prevent drug resistance. This prompted us to screen for new covalent inhibitors of Nedd4-1 using a combination of computational approaches. However, this task proved challenging due to the limited number of electrophilic moieties available in virtual libraries. Therefore, we opted to divide an existing covalent Nedd4-1 inhibitor into two parts: a non-covalent binding group and a pre-selected α, β-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on molecular interactions at the binding site. The pharmacophore was then subjected to virtual screening to identify structurally similar hit compounds. Multiple filtrations were implemented prior to selecting four hits, which were validated with a covalent conjugation and later assessed by molecular dynamic simulations. The results showed that, of the four hit molecules, Zinc00937975 exhibited advantageous molecular groups, allowing for favourable interactions with one of the characteristic cysteine residues. Predictive pharmacokinetic analysis further justified the compound as a potential lead molecule, prompting its recommendation for confirmatory biological evaluation. Our inhouse, refined, pharmacophore model approach serves as a robust method that will encourage screening for novel covalent inhibitors in drug discovery.

APA, Harvard, Vancouver, ISO, and other styles

31

Sallmyr, Annahita, Lisa Tobin, Alan E. Tomkinson, and Feyruz V. Rassool. "Inhibiting Alternative Non Homologus Endjoining (NHEJ) Pathways: Therapeutic Targets in Chronic Myeloid Leukemia (CML)." Blood 112, no. 11 (November 16, 2008): 1088. http://dx.doi.org/10.1182/blood.v112.11.1088.1088.

Full text

Abstract:

Abstract BCR-ABL fusion tyrosine kinase in chronic myeloid leukemia (CML), induces high levels of ROS that generate DNA double strand breaks (DSBs). We previously showed that CML cells repair DSBs by aberrant non homologous end-joining (NHEJ) that is characterized by large DNA deletions. The generation of DNA deletions represents a mechanism by which genomic alterations may be acquired in the progression of chronic phase CML to blast crisis. Recently, we demonstrated that a “back-up” or alternative NHEJ pathway is involved in aberrant repair of DSBs in CML. Proteins in this pathway include, DNA ligase IIIα, XRCC1 and poly(-ADP) ribose polymerase (PARP). We have identified that NHEJ proteins, DNA ligase IIIα and WRN are overexpressed in CML. This increased expression appears to be dependent on the presence of BCR-ABL. “Knockdown” of these proteins leads to an accumulation of unrepaired DSBs, demonstrating their essential involvement in DSB repair in CML cells. The goal of the current study is to evaluate the effect of inhibiting “back-up” DNA repair proteins in proliferation and apoptosis of BCR-ABL-positive CML compared with standard Imatinib therapy. To evaluate whether “back-up” repair proteins may be therapeutic targets, we used siRNA down-regulation and small molecule inhibition of DNA ligase IIIα and PARP in BCR-ABL positive cell lines. Importantly, we have recently identified small molecule inhibitors of DNA Ligases by computer aided drug design (CADD). Inhibition of DNA ligases and PARP result in a significant increase in apoptosis of CML cells (K562, Kasumi 4, MEG01 and KU812 and P210 MO7e), comparable with the cell death observed with imatinib treatment. Importantly, CML cell lines resistant to imatinib treatment demonstrate similar apoptotic levels in response to “back-up” repair protein inhibition. These inhibitors are in the process of being tested in CML xenografts and mouse models for therapeutic efficacy in vivo. Our data suggest that the survival of CML cells is at least in part maintained by repair of DSBs using “Back-up” NHEJ. The main proteins involved in this pathway, which include DNA ligase IIIα, XRCCI, DNA Ligase I, PARP and WRN have the potential to be novel therapeutic targets in CML patients that have acquired resistance to imatinib.

APA, Harvard, Vancouver, ISO, and other styles

32

Landré, Vivien, Barak Rotblat, Sonia Melino, Francesca Bernassola, and Gerry Melino. "Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities." Oncotarget 5, no. 18 (September 3, 2014): 7988–8013. http://dx.doi.org/10.18632/oncotarget.2431.

Full text

APA, Harvard, Vancouver, ISO, and other styles

33

Parsons, William H., Arthur A. Patchett, Herbert G. Bull, William R. Schoen, David Taub, Jacqueline Davidson, Patricia L. Combs, James P. Springer, and Hans Gadebusch. "Phosphinic acid inhibitors of D-alanyl-D-alanine ligase." Journal of Medicinal Chemistry 31, no. 9 (September 1988): 1772–78. http://dx.doi.org/10.1021/jm00117a017.

Full text

APA, Harvard, Vancouver, ISO, and other styles

34

van der Meer, Laurens T., Jurgen A. F. Marteijn, Theo M. de Witte, Joop H. Jansen, and Bert A. van der Reijden. "Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1." Blood 108, no. 11 (November 16, 2006): 1173. http://dx.doi.org/10.1182/blood.v108.11.1173.1173.

Full text

Abstract:

Abstract The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover. Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.

APA, Harvard, Vancouver, ISO, and other styles

35

Tsukahara, Fujiko, and Yoshiro Maru. "Bag1 directly routes immature BCR-ABL for proteasomal degradation." Blood 116, no. 18 (November 4, 2010): 3582–92. http://dx.doi.org/10.1182/blood-2009-10-249623.

Full text

Abstract:

Abstract Degradation of BCR-ABL oncoproteins by heat shock protein 90 (Hsp90) inhibitors in chronic myelogenous leukemia is expected to overcome resistance to ABL tyrosine kinase inhibitors. However, the precise mechanisms still remain to be uncovered. We found that while c-Cbl E3 ligase induced ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, another E3 ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) degraded immature BCR-ABL proteins and efficiently suppressed BCR-ABL–dependent leukemic growth. Interestingly, Bag1 (Bcl-2-associated athanogene-1), a nucleotide exchange factor for Hsc70, directly bound BCR-ABL with a high affinity, which was enhanced by CHIP and Hsp90 inhibitors, inhibited by imatinib and competed with Hsc70. Bag1 knockdown abrogated Hsp90 inhibitor-induced BCR-ABL degradation. Bag1 induced binding of immature BCR-ABL to proteasome. Expression of Bag1 induced BCR-ABL degradation and growth suppression in Ba/F3 cells when Hsc70 was knocked down with or without CHIP induction. CHIP appears to sort newly synthesized Hsp90-unchaperoned BCR-ABL to the proteasome not only by inhibiting Hsc70 and thereby promoting Bag1 to bind BCR-ABL, but also by ubiquitinating BCR-ABL. Bag1 may direct CHIP/Hsc70-regulated protein triage decisions on BCR-ABL immediately after translation to the degradation pathway.

APA, Harvard, Vancouver, ISO, and other styles

36

Wilson, Brice A. P., Donna Voeller, Emily A. Smith, Antony Wamiru, Ekaterina I. Goncharova, Gang Liu, Stanley Lipkowitz, and Barry R. O’Keefe. "In Vitro Ubiquitination Platform Identifies Methyl Ellipticiniums as Ubiquitin Ligase Inhibitors." SLAS DISCOVERY: Advancing the Science of Drug Discovery 26, no. 7 (April 21, 2021): 870–84. http://dx.doi.org/10.1177/24725552211000675.

Full text

Abstract:

The transfer of the small protein ubiquitin to a target protein is an intricately orchestrated process called ubiquitination that results in modulation of protein function or stability. Proper regulation of ubiquitination is essential, and dysregulation of this process is implicated in several human diseases. An example of a ubiquitination cascade that is a central signaling node in important disease-associated pathways is that of CBLB [a human homolog of a viral oncogene Casitas B-lineage lymphoma (CBL) from the Cas NS-1 murine retrovirus], a RING finger ubiquitin ligase (E3) whose substrates include a number of important cell-signaling kinases. These include kinases important in immune function that act in the T cell receptor and costimulatory pathways, the Tyro/Axl/MerTK (TAM) receptor family in natural killer (NK) cells, as well as growth factor receptor kinases like epidermal growth factor receptor (EGFR). Loss of CBLB has been shown to increase innate and adaptive antitumor immunity. This suggests that small-molecule modulation of CBLB E3 activity could enhance antitumor immunity in patients. To explore the hypothesis that enzymatic inhibition of E3s may result in modulation of disease-related signaling pathways, we established a high-throughput screen of >70,000 chemical entities for inhibition of CBLB activity. Although CBLB was chosen as a proof-of-principle target for inhibitor discovery, we demonstrate that our assay is generalizable to monitoring the activity of other ubiquitin ligases. We further extended our observed in vitro inhibition with additional cell-based models of CBLB activity. From these studies, we demonstrate that a class of natural product–based alkaloids, known as methyl ellipticiniums (MEs), is capable of inhibiting ubiquitin ligases intracellularly.

APA, Harvard, Vancouver, ISO, and other styles

37

Rana, Sandeep, Jayapal Reddy Mallareddy, Sarbjit Singh, Lidia Boghean, and Amarnath Natarajan. "Inhibitors, PROTACs and Molecular Glues as Diverse Therapeutic Modalities to Target Cyclin-Dependent Kinase." Cancers 13, no. 21 (November 2, 2021): 5506. http://dx.doi.org/10.3390/cancers13215506.

Full text

Abstract:

The cyclin-dependent kinase (CDK) family of proteins play prominent roles in transcription, mRNA processing, and cell cycle regulation, making them attractive cancer targets. Palbociclib was the first FDA-approved CDK inhibitor that non-selectively targets the ATP binding sites of CDK4 and CDK6. In this review, we will briefly inventory CDK inhibitors that are either part of over 30 active clinical trials or recruiting patients. The lack of selectivity among CDKs and dose-limiting toxicities are major challenges associated with the development of CDK inhibitors. Proteolysis Targeting Chimeras (PROTACs) and Molecular Glues have emerged as alternative therapeutic modalities to target proteins. PROTACs and Molecular glues utilize the cellular protein degradation machinery to destroy the target protein. PROTACs are heterobifunctional molecules that form a ternary complex with the target protein and E3-ligase by making two distinct small molecule–protein interactions. On the other hand, Molecular glues function by converting the target protein into a “neo-substrate” for an E3 ligase. Unlike small molecule inhibitors, preclinical studies with CDK targeted PROTACs have exhibited improved CDK selectivity. Moreover, the efficacy of PROTACs and molecular glues are not tied to the dose of these molecular entities but to the formation of the ternary complex. Here, we provide an overview of PROTACs and molecular glues that modulate CDK function as emerging therapeutic modalities.

APA, Harvard, Vancouver, ISO, and other styles

38

Casaletto, Jessica B., Leta K. Nutt, Qiju Wu, Jonathan D. Moore, Laurence D. Etkin, Peter K. Jackson, Tim Hunt, and Sally Kornbluth. "Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase." Journal of Cell Biology 169, no. 1 (April 11, 2005): 61–71. http://dx.doi.org/10.1083/jcb.200411056.

Full text

Abstract:

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

APA, Harvard, Vancouver, ISO, and other styles

39

Montecucco, A., M. Lestingi, G. Pedrali-Noy, S. Spadari, and G. Ciarrocchi. "Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation." Biochemical Journal 271, no. 1 (October 1, 1990): 265–68. http://dx.doi.org/10.1042/bj2710265.

Full text

Abstract:

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

APA, Harvard, Vancouver, ISO, and other styles

40

Chen, Wei-Yi, Jui-Hsia Weng, Chen-Che Huang, and Bon-chu Chung. "Histone Deacetylase Inhibitors Reduce Steroidogenesis through SCF-Mediated Ubiquitination and Degradation of Steroidogenic Factor 1 (NR5A1)." Molecular and Cellular Biology 27, no. 20 (August 20, 2007): 7284–90. http://dx.doi.org/10.1128/mcb.00476-07.

Full text

Abstract:

ABSTRACT Histone deacetylase (HDAC) inhibitors such as trichostatin A and valproic acid modulate transcription of many genes by inhibiting the activities of HDACs, resulting in the remodeling of chromatin. Yet this effect is not universal for all genes. Here we show that HDAC inhibitors suppressed the expression of steroidogenic gene CYP11A1 and decreased steroid secretion by increasing the ubiquitination and degradation of SF-1, a factor important for the transcription of all steroidogenic genes. This was accompanied by increased expression of Ube2D1 and SKP1A, an E2 ubiquitin conjugase and a subunit of the E3 ubiquitin ligase in the Skp1/Cul1/F-box protein (SCF) family, respectively. Reducing SKP1A expression with small interfering RNA resulted in recovery of SF-1 levels, demonstrating that the activity of SCF E3 ubiquitin ligase is required for the SF-1 degradation induced by HDAC inhibitors. Overexpression of exogenous SF-1 restored steroidogenic activities even in the presence of HDAC inhibitors. Thus, increased SF-1 degradation is the cause of the reduction in steroidogenesis caused by HDAC inhibitors. The increased SKP1A expression and SCF-mediated protein degradation could be the mechanism underlying the mode of action of HDAC inhibitors.

APA, Harvard, Vancouver, ISO, and other styles

41

Triola, Gemma, Stefan Wetzel, Bernhard Ellinger, Marcus A. Koch, Katja Hübel, Daniel Rauh, and Herbert Waldmann. "ATP competitive inhibitors of d-alanine–d-alanine ligase based on protein kinase inhibitor scaffolds." Bioorganic & Medicinal Chemistry 17, no. 3 (February 2009): 1079–87. http://dx.doi.org/10.1016/j.bmc.2008.02.046.

Full text

APA, Harvard, Vancouver, ISO, and other styles

42

Shrivastava, Nidhi, Jeetendra K. Nag, Jyoti Pandey, Rama Pati Tripathi, Priyanka Shah, Mohammad Imran Siddiqi, and Shailja Misra-Bhattacharya. "Homology Modeling of NAD+-Dependent DNA Ligase of the Wolbachia Endosymbiont of Brugia malayi and Its Drug Target Potential Using Dispiro-Cycloalkanones." Antimicrobial Agents and Chemotherapy 59, no. 7 (April 6, 2015): 3736–47. http://dx.doi.org/10.1128/aac.03449-14.

Full text

Abstract:

ABSTRACTLymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiontWolbachia. Depletion ofWolbachiaproduces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD+-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD+-dependent DNA ligase of theWolbachiasymbiont ofBrugia malayi(wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD+cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages ofB. malayiin vitro, and the most active compounds were further monitoredin vivoin jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effectsin vitroon the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 μM), followed by compound 5 (IC50, 2.3 μM) and compound 1 (IC50, 2.9 μM). These compounds also exhibited the same adverse effect on adult worms and microfilariaein vivo(P< 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P< 0.05).wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

APA, Harvard, Vancouver, ISO, and other styles

43

Ashraf, Zaman, Aamer Saeed, and Humaira Nadeem. "Design, synthesis and docking studies of some novel isocoumarin analogues as antimicrobial agents." RSC Adv. 4, no. 96 (2014): 53842–53. http://dx.doi.org/10.1039/c4ra07223e.

Full text

APA, Harvard, Vancouver, ISO, and other styles

44

Kovač, Andreja, Vita Majce, Roman Lenaršič, Sergeja Bombek, Julieanne M. Bostock, Ian Chopra, Slovenko Polanc, and Stanislav Gobec. "Diazenedicarboxamides as inhibitors of d-alanine-d-alanine ligase (Ddl)." Bioorganic & Medicinal Chemistry Letters 17, no. 7 (April 2007): 2047–54. http://dx.doi.org/10.1016/j.bmcl.2007.01.015.

Full text

APA, Harvard, Vancouver, ISO, and other styles

45

Duckworth, Benjamin P., Todd W. Geders, Divya Tiwari, Helena I. Boshoff, Paul A. Sibbald, Clifton E. Barry, Dirk Schnappinger, Barry C. Finzel, and Courtney C. Aldrich. "Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium tuberculosis." Chemistry & Biology 18, no. 11 (November 2011): 1432–41. http://dx.doi.org/10.1016/j.chembiol.2011.08.013.

Full text

APA, Harvard, Vancouver, ISO, and other styles

46

Lee, Junglim, Deanne W. Sammond, Zeno Fiorini, Jonel P. Saludes, Michael G. Resch, Bing Hao, Wei Wang, Hang Yin, and Xuedong Liu. "Computationally Designed Peptide Inhibitors of the Ubiquitin E3 Ligase SCFFbx4." ChemBioChem 14, no. 4 (February 11, 2013): 445–51. http://dx.doi.org/10.1002/cbic.201200777.

Full text

APA, Harvard, Vancouver, ISO, and other styles

47

Bruning, John B., Ana C. Murillo, Ofelia Chacon, Raúl G. Barletta, and James C. Sacchettini. "Structure of theMycobacterium tuberculosisd-Alanine:d-Alanine Ligase, a Target of the Antituberculosis Drug d-Cycloserine." Antimicrobial Agents and Chemotherapy 55, no. 1 (October 18, 2010): 291–301. http://dx.doi.org/10.1128/aac.00558-10.

Full text

Abstract:

ABSTRACTd-Alanine:d-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of twod-alanine (d-Ala) molecules to form thed-alanyl:d-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development.d-Cycloserine (DCS), an analog ofd-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure ofMycobacterium tuberculosisDdl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. TheM. tuberculosisapo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide andd-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP andd-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed ford-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC50) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

APA, Harvard, Vancouver, ISO, and other styles

48

Ando, Kiyohiro, Yusuke Suenaga, and Takehiko Kamijo. "DNA Ligase 4 Contributes to Cell Proliferation against DNA-PK Inhibition in MYCN-Amplified Neuroblastoma IMR32 Cells." International Journal of Molecular Sciences 24, no. 10 (May 19, 2023): 9012. http://dx.doi.org/10.3390/ijms24109012.

Full text

Abstract:

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.

APA, Harvard, Vancouver, ISO, and other styles

49

Sicari, Daria, Janine Weber, Elena Maspero, and Simona Polo. "The NEDD4 ubiquitin E3 ligase: a snapshot view of its functional activity and regulation." Biochemical Society Transactions 50, no. 1 (February 7, 2022): 473–85. http://dx.doi.org/10.1042/bst20210731.

Full text

Abstract:

Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mechanisms underlying ubiquitination is of central importance. Being responsible for chain specificity and substrate recognition, E3 ligases are the selective elements of the ubiquitination process. In this review, we discuss different cellular pathways regulated by one of the first identified E3 ligase, NEDD4, focusing on its pathophysiological role, its known targets and modulators. In addition, we highlight small molecule inhibitors that act on NEDD4 and discuss new strategies to effectively target this E3 enzyme.

APA, Harvard, Vancouver, ISO, and other styles

50

Rajalingam, Krishnaraj, and Ivan Dikic. "Inhibitors of apoptosis catch ubiquitin." Biochemical Journal 417, no. 1 (December 12, 2008): e1-e3. http://dx.doi.org/10.1042/bj20082215.

Full text

Abstract:

IAP (inhibitor of apoptosis) proteins are a class of anti-apoptotic regulators characterized by the presence of BIR (baculoviral IAP repeat) domains. Some of the IAPs also possess a RING (really interesting new gene) domain with E3 ubiquitin ligase activity. In this issue of the Biochemical Journal, Blankenship et al. unveil the presence of an UBA (ubiquitin-associated domain) in several IAPs. UBAs in c-IAPs (cellular IAPs) bind to monoubiquitin and ubiquitin chains and are implicated in degradation of c-IAPs by promoting their interaction with proteasomes as well as in regulation of TNF-α (tumour necrosis factor-α)-induced apoptosis. These novel observations establish IAPs as ubiquitin-interacting proteins and opens up new lines of investigation.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'Ligase I Inhibitors'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles