Статті в журналах: "DNA Ligase 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.

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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.

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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.

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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.

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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.

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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.

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4

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.

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5

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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Howes, Timothy R. L., Annahita Sallmyr, Rhys Brooks, George E. Greco, Darin E. Jones, Yoshihiro Matsumoto, and Alan E. Tomkinson. "Erratum to “Structure-activity relationships among DNA ligase inhibitors; characterization of a selective uncompetitive DNA ligase I inhibitor” [DNA Repair 60C (2017) 29–39]." DNA Repair 61 (January 2018): 99. http://dx.doi.org/10.1016/j.dnarep.2017.12.001.

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10

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.

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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.

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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.

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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.

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

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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.

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GUL, Sheraz, Richard BROWN, Earl MAY, Marie MAZZULLA, Martin G. SMYTH, Colin BERRY, Andrew MORBY, and David J. POWELL. "Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay." Biochemical Journal 383, no. 3 (October 26, 2004): 551–59. http://dx.doi.org/10.1042/bj20040054.

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DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

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Chen, Xi, Shijun Zhong, Xiao Zhu, Barbara Dziegielewska, Tom Ellenberger, Gerald M. Wilson, Alexander D. MacKerell, and Alan E. Tomkinson. "Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair." Cancer Research 68, no. 9 (May 1, 2008): 3169–77. http://dx.doi.org/10.1158/0008-5472.can-07-6636.

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Buurman, Ed T., Valerie A. Laganas, Ce Feng Liu, and John I. Manchester. "Antimicrobial Activity of Adenine-Based Inhibitors of NAD+-Dependent DNA Ligase." ACS Medicinal Chemistry Letters 3, no. 8 (July 16, 2012): 663–67. http://dx.doi.org/10.1021/ml300169x.

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Hale, Michael R., Claire Brassington, Dan Carcanague, Kevin Embrey, Charles J. Eyermann, Robert A. Giacobbe, Lakshmaiah Gingipalli, et al. "From fragments to leads: novel bacterial NAD + -dependent DNA ligase inhibitors." Tetrahedron Letters 56, no. 23 (June 2015): 3108–12. http://dx.doi.org/10.1016/j.tetlet.2014.12.067.

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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.

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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.

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Howard, Steven, Nader Amin, Andrew B. Benowitz, Elisabetta Chiarparin, Haifeng Cui, Xiaodong Deng, Tom D. Heightman, et al. "Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase." ACS Medicinal Chemistry Letters 4, no. 12 (October 18, 2013): 1208–12. http://dx.doi.org/10.1021/ml4003277.

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Swift, Robert V., and Rommie E. Amaro. "Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms." Expert Opinion on Drug Discovery 4, no. 12 (November 24, 2009): 1281–94. http://dx.doi.org/10.1517/17460440903373617.

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Stokes, Suzanne S., Hoan Huynh, Madhusudhan Gowravaram, Robert Albert, Marta Cavero-Tomas, Brendan Chen, Jenna Harang, et al. "Discovery of bacterial NAD+-dependent DNA ligase inhibitors: Optimization of antibacterial activity." Bioorganic & Medicinal Chemistry Letters 21, no. 15 (August 2011): 4556–60. http://dx.doi.org/10.1016/j.bmcl.2011.05.128.

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Mandalapu, Dhanaraju, Deependra Kumar Singh, Sonal Gupta, Vishal M. Balaramnavar, Mohammad Shafiq, Dibyendu Banerjee, and Vishnu Lal Sharma. "Discovery of monocarbonyl curcumin hybrids as a novel class of human DNA ligase I inhibitors: in silico design, synthesis and biology." RSC Advances 6, no. 31 (2016): 26003–18. http://dx.doi.org/10.1039/c5ra25853g.

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A pharmacophore model identified a novel class of hLigI inhibitors to treat cancer. 36 compounds were synthesized and the identified inhibitor, compound 23 shown antiligase activity at IC₅₀ 24.9 μM by abolishing the interaction between hLigI and DNA.

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22

Sun, Daekyu, and Rheanna Urrabaz. "Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I." Journal of Biochemical and Biophysical Methods 59, no. 1 (April 2004): 49–59. http://dx.doi.org/10.1016/s0165-022x(02)00071-4.

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23

Jahagirdar, Devashree, Shruti Purohit, and Nilesh K. Sharma. "Combinatorial Use of DNA Ligase Inhibitor L189 and Temozolomide Potentiates Cell Growth Arrest in HeLa." Current Cancer Therapy Reviews 15, no. 1 (February 22, 2019): 65–73. http://dx.doi.org/10.2174/1573394714666180216150332.

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Introduction: The issues of carcinoma drug resistance to alkylating agents such as Temozolomide (TMZ) are considered as a major concern in therapeutics. The potential ways to achieve better cancer cell growth arrest and cytotoxicity have been suggested including the combinatorial use of DNA repair protein inhibitors and genotoxic drug TMZ. Here, authors assess the ability of DNA ligase inhibitor (L189) to modulate TMZ mediated HeLa cell growth arrest and cytotoxicity. Materials and Methods: Here, authors have employed Trypan blue dye exclusion and propidium iodide (PI) using FACS to determine HeLa cell viability after exposure to TMZ with or without L189 inhibitor. Additionally, authors show the DNA ligase III protein level using ELISA and fluorescent microscopy to support the observed effects of combinatorial use of TMZ and L189. Results: In this paper, data indicate that the addition of L189 produced appreciable decrease in the growth of HeLa cells. However, combined treatment of L189 and TMZ showed enhanced TMZinduced HeLa growth arrest possibly in G2/M cell cycle phase without employing cell death mechanisms. Conclusions: These results underscore the combinatorial treatment using TMZ and L189 to bring desirable cancer cell growth arrest and future molecular study to dissect out the participating pathways.

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Zhong, Shijun, Xi Chen, Xiao Zhu, Barbara Dziegielewska, Kurtis E. Bachman, Tom Ellenberger, Jeff D. Ballin, Gerald M. Wilson, Alan E. Tomkinson, and Alexander D. MacKerell. "Identification and Validation of Human DNA Ligase Inhibitors Using Computer-Aided Drug Design." Journal of Medicinal Chemistry 51, no. 15 (August 2008): 4553–62. http://dx.doi.org/10.1021/jm8001668.

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25

Leckie, Gregor W., Dwight D. Erickson, Qizhi He, Ingrid E. Facey, Bor-Chian Lin, Jianli Cao, and Folim G. Halaka. "Method for Reduction of Inhibition in aMycobacterium tuberculosis-Specific Ligase Chain Reaction DNA Amplification Assay." Journal of Clinical Microbiology 36, no. 3 (1998): 764–67. http://dx.doi.org/10.1128/jcm.36.3.764-767.1998.

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The present study describes the identification of inhibitors of aMycobacterium tuberculosis-specific gap ligase chain reaction (LCR) DNA amplification assay as well as a method for their removal. A major contributor to inhibition was deduced to be a calcium phosphate precipitate, CaHPO4. The precipitate forms duringN-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH) decontamination, digestion, and concentration of respiratory specimens. The solubility product of CaHPO4 precipitate at pH 7.8, the pH at which gap LCR is optimized, indicates that the precipitate releases an amount of phosphate ions sufficient to inhibit amplification. A method for removal of the precipitate was identified. The precipitate is dissociated by exposing it to a mildly acidic (pH 4.1) buffer during the first of two centrifugation steps; the inhibitory phosphate ions are removed by the centrifugation steps. When 100 NALC-NaOH respiratory sediments were tested by gap LCR, none of the sediments were inhibitory when the acidic buffer was used while 24 samples were inhibitory when TE buffer, pH 7.8, was used. In another study, when the acidic buffer wash was applied to 1,440 NALC-NaOH respiratory sediments, only 10 sediments were found to be inhibitory. None of the inhibited sediments were culture positive for M. tuberculosis. This work demonstrates that when inhibition mechanisms are identified, relatively simple protocols can be used to obtain low inhibition rates and to allow the use of larger volume equivalents in amplification reactions.

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Yi, Lanhua, and Xin Lü. "New Strategy on Antimicrobial-resistance: Inhibitors of DNA Replication Enzymes." Current Medicinal Chemistry 26, no. 10 (June 20, 2019): 1761–87. http://dx.doi.org/10.2174/0929867324666171106160326.

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Background:Antimicrobial resistance is found in all microorganisms and has become one of the biggest threats to global health. New antimicrobials with different action mechanisms are effective weapons to fight against antibiotic-resistance.Objective:This review aims to find potential drugs which can be further developed into clinic practice and provide clues for developing more effective antimicrobials.Methods:DNA replication universally exists in all living organisms and is a complicated process in which multiple enzymes are involved in. Enzymes in bacterial DNA replication of initiation and elongation phases bring abundant targets for antimicrobial development as they are conserved and indispensable. In this review, enzyme inhibitors of DNA helicase, DNA primase, topoisomerases, DNA polymerase and DNA ligase were discussed. Special attentions were paid to structures, activities and action modes of these enzyme inhibitors.Results:Among these enzymes, type II topoisomerase is the most validated target with abundant inhibitors. For type II topoisomerase inhibitors (excluding quinolones), NBTIs and benzimidazole urea derivatives are the most promising inhibitors because of their good antimicrobial activity and physicochemical properties. Simultaneously, DNA gyrase targeted drugs are particularly attractive in the treatment of tuberculosis as DNA gyrase is the sole type II topoisomerase in Mycobacterium tuberculosis. Relatively, exploitation of antimicrobial inhibitors of the other DNA replication enzymes are primeval, in which inhibitors of topo III are even blank so far.Conclusion:This review demonstrates that inhibitors of DNA replication enzymes are abundant, diverse and promising, many of which can be developed into antimicrobials to deal with antibioticresistance.

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Stokes, Suzanne S., Madhusudhan Gowravaram, Hoan Huynh, Min Lu, George B. Mullen, Brendan Chen, Robert Albert, et al. "Discovery of bacterial NAD+-dependent DNA ligase inhibitors: Improvements in clearance of adenosine series." Bioorganic & Medicinal Chemistry Letters 22, no. 1 (January 2012): 85–89. http://dx.doi.org/10.1016/j.bmcl.2011.11.071.

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Sashidhara, Koneni V., L. Ravithej Singh, Mohammad Shameem, Sarika Shakya, Anoop Kumar, Tulsankar Sachin Laxman, Shagun Krishna, Mohammad Imran Siddiqi, Rabi S. Bhatta, and Dibyendu Banerjee. "Design, synthesis and anticancer activity of dihydropyrimidinone–semicarbazone hybrids as potential human DNA ligase 1 inhibitors." MedChemComm 7, no. 12 (2016): 2349–63. http://dx.doi.org/10.1039/c6md00447d.

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29

Murphy-Benenato, Kerry E., Lakshmaiah Gingipalli, P. Ann Boriack-Sjodin, Gabriel Martinez-Botella, Dan Carcanague, Charles J. Eyermann, Madhu Gowravaram, et al. "Negishi cross-coupling enabled synthesis of novel NAD+-dependent DNA ligase inhibitors and SAR development." Bioorganic & Medicinal Chemistry Letters 25, no. 22 (November 2015): 5172–77. http://dx.doi.org/10.1016/j.bmcl.2015.09.075.

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Elder, Rhoderick H., and Daphne J. Osborne. "Function of DNA synthesis and DNA repair in the survival of embryos during early germination and in dormancy." Seed Science Research 3, no. 1 (March 1993): 43–53. http://dx.doi.org/10.1017/s0960258500001550.

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AbstractDNA synthesis following the addition of water to excised embryos of non-dormant rye and to embryos of non-dormant and dormant genetic lines of Avena fatua has been examined. All the samples exhibit an early unscheduled DNA synthesis, have similar responses to DNA polymerase inhibitors and a similar increase in DNA ligase function for the first 24 h of imbibition, irrespective of whether they germinate or remain dormant. A β-polymerase-mediated DNA repair activity is indicated immediately upon imbibition with the stable incorporation of 3H-methyl-thymidine into high molecular weight DNA. Following γ-irradiation of dry or imbibed embryos, inhibitor results suggest the appearance of an additional α- or δ-polymerase activity in the ensuing repair. Abscisic acid permits the early unirradiated repair synthesis, but like aphidicolin, it inhibits replicative DNA synthesis and partly inhibits the post-irradiation incorporation of thymidine. DNA synthesis takes place continuously throughout dormancy of imbibed embryos: precursor incorporation into DNA cannot be chased in the short term and occurs in the absence of an S-phase or endoreduplication of nuclear DNA. It is proposed that dormant imbibed embryos maintain the integrity of the genome by a continuous but slow replacement of DNA steered by a non-amplifying and modified form of replicative DNA synthesis and that abscisic acid may play a determining role in this process.

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Gao, Shan-Shan, Hua Guan, Shuang Yan, Sai Hu, Man Song, Zong-Pei Guo, Da-Fei Xie, et al. "TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy." Science Advances 6, no. 28 (July 2020): eaba7822. http://dx.doi.org/10.1126/sciadv.aba7822.

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Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.

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Cerqueira, Sofia A., Min Tan, Shijun Li, Franceline Juillard, Colin E. McVey, Kenneth M. Kaye, and J. Pedro Simas. "Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation." Journal of Virology 90, no. 17 (June 15, 2016): 7667–83. http://dx.doi.org/10.1128/jvi.00813-16.

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ABSTRACTViruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesisin vivoremains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5SmLANA) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence.In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease.IMPORTANCEThe gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure, and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease.

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Hayes, Andrew J., Jiulia Satiaputra, Louise M. Sternicki, Ashleigh S. Paparella, Zikai Feng, Kwang J. Lee, Beatriz Blanco-Rodriguez, et al. "Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase." Antibiotics 9, no. 4 (April 6, 2020): 165. http://dx.doi.org/10.3390/antibiotics9040165.

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Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

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Tripathi, Rama P., Jyoti Pandey, Vandana Kukshal, Arya Ajay, Mridul Mishra, Divya Dube, Deepti Chopra, R. Dwivedi, Vinita Chaturvedi, and Ravishankar Ramachandran. "Synthesis, in silico screening and bioevaluation of dispiro-cycloalkanones as antitubercular and mycobacterial NAD+-dependent DNA ligase inhibitors." MedChemComm 2, no. 5 (2011): 378. http://dx.doi.org/10.1039/c0md00246a.

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Robert, Carine, Nidal E. Muvarak, Vu H. Duong, Maria R. Baer, Stephen B. Baylin, and Feyruz Rassool. "Demethylating Agents Reprogram Myelodysplastic Syndrome and Leukemia Cells, Sensitizing Them To Poly-(ADP)-Ribose Polymerase Inhibitors." Blood 122, no. 21 (November 15, 2013): 3778. http://dx.doi.org/10.1182/blood.v122.21.3778.3778.

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Abstract Several lines of evidence suggest that genomic instability in myeloid malignancies is promoted by increased endogenous DNA damage and error-prone repair that lead to disease progression and resistance to therapy. We recently reported increased levels of Poly-(ADP)-ribose polymerase (PARP) and DNA Ligase IIIα as well as increased activity of a highly error-prone pathway for repair of DNA double-strand breaks in myeloid leukemias. Importantly, these leukemia cells are sensitive to inhibitors of DNA Ligase IIIα and PARP, suggesting their dependence on these factors for survival. Parallel studies have shown that transient exposure to DNA demethylating agents at low nM concentrations reprograms cancer cells, altering heritable gene expression patterns in key cellular pathways, including DNA repair pathways, suggesting that pre-treatment of leukemia cells with demethylating agents may further sensitize them to PARP inhibitors. Thus, established cell lines from acute myeloid leukemia (AML; MV411, KASUMI-1), myelodysplastic syndrome transformed to AML (MDS; P39) and bone marrow mononuclear cells obtained from AML patient samples (N=7) were exposed to non-cytotoxic doses of DNA methyltransferase inhibitors (DNMTis, decitabine, DAC), followed by four days without drug treatment and subsequent treatment with low doses of PARP inhibitors (PARPis; ABT888, or BMN673) alone or in combination with DNMTis. Clonogenicity, apoptosis, DNA repair efficiency, and activity and expression level of DNA repair and DNA methyltransferase proteins were then studied. In all the cell lines tested, treatment with DAC (5-10nM) followed by ABT888 (500nM) induced a significant decrease in colony survival compared to control or single treatment. The use of a more potent PARPi, BMN673 (0.1-10nM), confirmed that treatment with DNMTis followed by PARPis induces a robust inhibition of AML and MDS cell line colony forming capacity. Interestingly, the same schedule treatment of decitabine followed by PARPis significantly decreases the clonogenic capacity in 4 out of 7 (57%) of bone marrow mononuclear cells from AML patient tested so far, suggesting that DNMTis and PARPis sequential treatment could be a valuable therapeutic option for AML and MDS patient. We next initiated studies to elucidate the mechanism by which DAC may sensitize myeloid malignancies to PARPis. As expected, DAC treatment alone was sufficient to decrease DNMT1 expression levels and increase caspase 3 cleavage in AML cell lines, compared to control treated cells. But surprisingly, DAC treatment alone also induced a decrease in PARP protein expression, with a further decrease in cells treated with DAC followed by PARPis, suggesting that both methylation and DNA repair signaling alter PARP1 steady-state levels. Moreover, preliminary results show that the presence of PARP on chromatin is decreased with DAC treatment and further decreased following PARPis. In conclusion, our results suggest that DNMTis reprogram cells, sensitizing them to PARP inhibition in AML/MDS patient and cell line models, paving the way for testing the therapeutic potential of sequential treatment with these agents in clinical trials. We are exploring one hypothesis that decreased levels of PARP on chromatin following DAC treatment may lead to more effective trapping of PARP1 at sites of DNA damage by PARPis, leading to abrogation of DNA repair. Understanding how these proteins interact may explain the mechanisms underlying the sensitization of epigenetically reprogrammed cells to PARPis, and may define the molecular subsets of AML patients that may respond to this novel therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

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Jo, Ukhyun, Yasuhisa Murai, Sirisha Chakka, Lu Chen, Ken Cheng, Junko Murai, Liton Kumar Saha, Lisa M. Miller Jenkins, and Yves Pommier. "SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors." Proceedings of the National Academy of Sciences 118, no. 6 (February 3, 2021): e2015654118. http://dx.doi.org/10.1073/pnas.2015654118.

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Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4CDT2 ubiquitin ligase.

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Keefe, Anthony D., Jeremy S. Disch, Jennifer Duffy, Esther C. Lee, Diana Gikunju, Betty Chan, Benjamin D. Levin, et al. "Abstract 5346: Discovery of new targeted protein degraders using DNA-encoded chemistry." Cancer Research 83, no. 7_Supplement (April 4, 2023): 5346. http://dx.doi.org/10.1158/1538-7445.am2023-5346.

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Abstract Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplars exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant that rapidly generated bispecific nanomolar degraders of ERα, with PROTACs inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization. Citation Format: Anthony D. Keefe, Jeremy S. Disch, Jennifer Duffy, Esther C. Lee, Diana Gikunju, Betty Chan, Benjamin D. Levin, Michael I. Monteiro, Sarah A. Talcott, Anthony Lau, Fei Zhou, Anton Kozhushnyan, Neil E. Westlund, Patrick B. Mullins, Yan Yu, Moritz von Rechenberg, Junyi Zhang, Yelena Arnautova, Yanbin Liu, Ying Zhang, Andrew J. McRiner, Anna Kohlmann, Matthew A. Clark, John W. Cuozzo, Christelle Huguet, Shilpi Arora. Discovery of new targeted protein degraders using DNA-encoded chemistry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5346.

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Cabrera, Raninga, Khanna та Freire. "GSK3-β Stimulates Claspin Degradation via β-TrCP Ubiquitin Ligase and Alters Cancer Cell Survival". Cancers 11, № 8 (29 липня 2019): 1073. http://dx.doi.org/10.3390/cancers11081073.

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: Claspin is essential for activating the DNA damage checkpoint effector kinase Chk1, a target in oncotherapy. Claspin functions are tightly correlated to Claspin protein stability, regulated by ubiquitin-dependent proteasomal degradation. Here we identify Glycogen Synthase Kinase 3-β (GSK3-β) as a new regulator of Claspin stability. Interestingly, as Chk1, GSK3-β is a therapeutic target in cancer. GSK3-β inhibition or knockdown stabilizes Claspin, whereas a GSK3-β constitutively active form reduces Claspin protein levels by ubiquitination and proteasome-mediated degradation. Our results also suggest that GSK3-β modulates the interaction of Claspin with β-TrCP, a critical E3 ubiquitin ligase that regulates Claspin stability. Importantly, GSK3-β knock down increases Chk1 activation in response to DNA damage in a Claspin-dependent manner. Therefore, Chk1 activation could be a pro-survival mechanism that becomes activated upon GSK3-β inhibition. Importantly, treating triple negative breast cancer cell lines with Chk1 or GSK3-β inhibitors alone or in combination, demonstrates that Chk1/GSK3-β double inhibition restrains cell growth and triggers more apoptosis compared to individual treatments, thereby revealing novel possibilities for a combination therapy for cancer.

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Gu, Wenxin, Tiansheng Wang, Francois Maltais, Brian Ledford, Joseph Kennedy, Yunyi Wei, Christian H. Gross, et al. "Design, synthesis and biological evaluation of potent NAD+-dependent DNA ligase inhibitors as potential antibacterial agents. Part I: Aminoalkoxypyrimidine carboxamides." Bioorganic & Medicinal Chemistry Letters 22, no. 11 (June 2012): 3693–98. http://dx.doi.org/10.1016/j.bmcl.2012.04.037.

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von Klitzing, Christine, Florian Bassermann, Stephan W. Morris, Christian Peschel, and Justus Duyster. "A Role for NIPA in DNA Damage Response." Blood 104, no. 11 (November 16, 2004): 1265. http://dx.doi.org/10.1182/blood.v104.11.1265.1265.

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Abstract The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis. Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways. In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

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Wergeland, Line, Kevin B. Spurgers, Eystein Oveland, Torill Høiby, Manel Cascallo, Tsuyoshi Honda, James B. Lorens, Guillermina Lozano, Timothy J. McDonnell, and Bjorn T. Gjertsen. "Bcl-2 Protects against p53-Induced Apoptosis through Hdm2." Blood 112, no. 11 (November 16, 2008): 5333. http://dx.doi.org/10.1182/blood.v112.11.5333.5333.

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Abstract Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.

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Pettersson, Susanne, Michael Kelleher, Emmanuelle Pion, Maura Wallace, and Kathryn L. Ball. "Role of Mdm2 acid domain interactions in recognition and ubiquitination of the transcription factor IRF-2." Biochemical Journal 418, no. 3 (February 25, 2009): 575–85. http://dx.doi.org/10.1042/bj20082087.

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Mdm2 (murine double minute 2)-mediated ubiquitination of the p53 tumour suppressor requires interaction of the ligase at two distinct binding sites that form general multiprotein-docking sites for the p53 protein. The first Mdm2-binding site resides in the transactivation domain of p53 and is an allosteric effector site for Mdm2-mediated p53 ubiquitination; the second site requires the acid domain of Mdm2 to recognize a ‘ubiquitination signal’ within p53's DNA-binding core. In order to expand on fundamental requirements for a protein to function as an Mdm2 substrate and the role of the acid domain in recognition, we have carried out a bioinformatics search for open reading frames that have homology with the Mdm2-docking sites in p53. IRF-2 [IFN (interferon) regulatory factor-2], an IFN-regulated transcription factor, has been identified as an Mdm2-binding protein and substrate requiring interactions with both the hydrophobic pocket and the acid domain of Mdm2. Mutation of either of the two Mdm2-binding sites on IRF-2 can attenuate substrate ubiquitination, confirming the requirement of a dual-site substrate interaction mechanism. Ligands that bind to the hydrophobic pocket are not sufficient to inhibit Mdm2 E3-ligase activity. Rather, acid domain-binding ligands act as E3-ligase inhibitors, lending additional support to the idea that the acid domain of Mdm2 is key to understanding its mechanism of action. The ability of Mdm2 and IRF-2 to form a complex in cells complements the biochemical assays and together establishes a novel substrate with which to develop insights into E3-ubiquitin ligase–substrate interactions in vitro and in cells.

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43

Rowley, Roy, and Jun Zhang. "Caffeine-Mediated Override of Checkpoint Controls: A Requirement for rhp6 (Schizosaccharomyces pombe)." Genetics 152, no. 1 (May 1, 1999): 61–71. http://dx.doi.org/10.1093/genetics/152.1.61.

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Abstract Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are arrested or delayed in interphase through the action of checkpoint controls. If the arrested cell is exposed to caffeine, relatively normal cell cycle progression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viability. To address the mechanism of caffeine’s action on cell progression, fission yeast mutants that take up caffeine but are not sensitized to hydroxyurea (HU) by caffeine were selected. Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene. Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal checkpoint responses to HU, radiation, or inactivation of DNA ligase. However, the addition of caffeine does not override the associated cell cycle blocks. Both point and deletion mutations show synthetic lethality at room temperature with temperature-sensitive mutations in cyclin B (cdc13-117) or the phosphatase cdc25 (cdc25-22). These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, promotes entry to mitosis in response to caffeine treatment.

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44

Nichols, Gena J., Jerome Schaack, and David A. Ornelles. "Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication." Journal of Virology 83, no. 12 (March 25, 2009): 5987–98. http://dx.doi.org/10.1128/jvi.00091-09.

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ABSTRACT Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.

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45

Surivet, Jean-Philippe, Roland Lange, Christian Hubschwerlen, Wolfgang Keck, Jean-Luc Specklin, Daniel Ritz, Daniel Bur, et al. "Structure-guided design, synthesis and biological evaluation of novel DNA ligase inhibitors with in vitro and in vivo anti-staphylococcal activity." Bioorganic & Medicinal Chemistry Letters 22, no. 21 (November 2012): 6705–11. http://dx.doi.org/10.1016/j.bmcl.2012.08.094.

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46

R, Sri Dharani, Ranjitha R, Sripathi R, Ali Muhammad K S, and Ravi S. "DOCKING STUDIES IN TARGET PROTEINS INVOLVED IN ANTIBACTERIAL ACTION MECHANISMS: ALKALOIDS ISOLATED FROM SCUTELLARIA GENUS." Asian Journal of Pharmaceutical and Clinical Research 9, no. 5 (September 1, 2016): 121. http://dx.doi.org/10.22159/ajpcr.2016.v9i5.12693.

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ABSTRACTObjective: In the present work, docking study was performed for 22 selected alkaloids isolated from the genus Scutellaria to evaluate their affinityto bacterial proteins that are known targets for many antibiotics with a different mechanism of action: Inhibitors of cell wall synthesis, inhibitors ofnucleic acids synthesis and antimetabolites.Methods: Molecular docking study was carried out using AutoDock 4.2 version and the visualization result using Chimera 1.10 and DiscoveryStudio 4.5.Result: Among the 22 alkaloids studied, with the DNA gyrase protein 1KZN and a dihydropteroate synthase enzyme 3TYE, the compoundscutebarbatine E showed a docking score of −8.5 and −8.7 Kcal/mol, respectively, involving with hydrophilic and hydrophobic interactions. Withrespect to MurD ligase involved in cell wall synthesis 1UAG and 2X5O, the compound 6,7,nicotinyl scutebarbatine G fared well with a dockingscore of −10.1 and −10.2 Kcal/mol, respectively. Scutebarbatine G performed well with respect to 3UDI with binding scores of −9.3 K cal/mol.Conclusion: Overall, it seems that for the selected alkaloids from the genus Scutellaria, the main mechanism of the action is the inhibition of cell wallsynthesis.Keywords: Scutebarbatine, Alkaloids, Molecular docking, Antimicrobial studies.

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47

Yu, Duonan, Martin Carroll, and Andrei Thomas-Tikhonenko. "p53 status dictates responses of B lymphomas to monotherapy with proteasome inhibitors." Blood 109, no. 11 (June 1, 2007): 4936–43. http://dx.doi.org/10.1182/blood-2006-10-050294.

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Abstract The proapoptotic function of p53 is thought to underlie most anticancer modalities and is also activated in response to oncogenic insults, such as overexpression of the Myc oncoprotein. Here we generated tractable B lymphomas using retroviral transduction of the MYC oncogene into hematopoietic cells with 2 knock-in alleles encoding a fusion between p53 and 4-hydroxytamoxifen (4OHT) receptor (p53ERTAM). In these polyclonal tumors, Myc is the only oncogenic lesion, and p53ERTAM status can be rapidly toggled between “off” and “on” with 4OHT, provided that the Trp53 promoter has been independently activated. Although 4OHT can trigger widespread apoptosis and overt tumor regression even in the absence of DNA-damaging agents, in tumors with high levels of Mdm2 these responses are blunted. However, cotreatment with proteasome inhibitors fully restores therapeutic effects in vivo. Similarly, human Burkitt lymphomas with wild-type p53 and overexpression of Hdm2 are highly sensitive to proteasome inhibitors, unless p53 levels are reduced using the HPV-E6 ubiquitin ligase. Therefore, proteasome inhibitors could be highly effective as a monotherapy against Myc-induced lymphomas, with no need for adjuvant chemotherapy or radiation therapy. On the other hand, their efficacy is crucially dependent on the wild-type p53 status of the tumor, placing important restrictions on patient selection.

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Lambert, Carsten, Tatjana Döring та Reinhild Prange. "Hepatitis B Virus Maturation Is Sensitive to Functional Inhibition of ESCRT-III, Vps4, and γ2-Adaptin". Journal of Virology 81, № 17 (6 червня 2007): 9050–60. http://dx.doi.org/10.1128/jvi.00479-07.

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ABSTRACT Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor γ2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role γ2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess γ2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed γ2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of γ2-adaptin.

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Deng, Liang, Peihong Dai, Anthony Ciro, Donald F. Smee, Hakim Djaballah, and Stewart Shuman. "Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly." Journal of Virology 81, no. 24 (October 10, 2007): 13392–402. http://dx.doi.org/10.1128/jvi.00770-07.

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ABSTRACT The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.

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Weber, Elisabeth, Ina Rothenaigner, Stefanie Brandner, Kamyar Hadian, and Kenji Schorpp. "A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein–Protein Interaction Inhibitors." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 3 (December 13, 2016): 316–23. http://dx.doi.org/10.1177/1087057116681408.

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The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer. Here, we present a novel high-throughput screening (HTS)–compatible assay based on the AlphaScreen technology to identify inhibitors of the RNF8-Ubc13 protein–protein interaction, along with a follow-up strategy for subsequent validation. We have adapted the AlphaScreen assay to a 384-well format and demonstrate its reliability, reproducibility, and suitability for automated HTS campaigns. In addition, we have established a biochemical orthogonal homogeneous time-resolved fluorescence (HTRF) assay in HTS format and a cellular microscopy-based assay allowing verification of the primary hits. This strategy will be useful for drug screening programs aimed at RNF8-Ubc13 modulation.

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