Block DNA Replication Research Articles

Dual Inhibition of SYK and EGFR Overcomes Chemoresistance by Inhibiting CDC6 and Blocking DNA Replication.

Targeting multiple signaling pathways has been proposed as a strategy to overcome resistance to single-pathway inhibition in cancer therapy. A previous study in epithelial ovarian cancers identified hyperactivity of spleen tyrosine kinase (SYK) and epidermal growth factor receptor (EGFR), which mutually phosphorylate and activate each other. Given the potential for pharmacologic inhibition of both kinases with clinically available agents, this study aimed to assess the antitumor efficacy of both pharmacologic and genetic SYK and EGFR co-inhibition using a multifaceted approach to analyze the global phosphoproteome and chemoresistant ovarian cancer cell lines, patient-derived organoids, and xenograft models. Dual inhibition of SYK and EGFR in chemoresistant ovarian cancer cells elicited a highly synergistic antitumor effect. Notably, the combined inhibition strategy activated the DNA damage response, induced G1 cell cycle arrest, and promoted apoptosis. The phosphoproteomic analysis revealed that perturbation of SYK and EGFR signaling induced a significant reduction in both phosphorylated and total protein levels of cell division cycle 6 (CDC6), a crucial initiator of DNA replication. Together, this study offers preclinical evidence supporting dual inhibition of SYK and EGFR as a promising treatment for chemoresistant ovarian cancer that disrupts DNA synthesis by impairing formation of the prereplication complex. These findings warrant further clinical investigation to explore the potential of this combination therapy in overcoming drug resistance and improving patient outcomes.

HMGB3 and SUB1 Bind to and Facilitate the Repair of N2-Alkylguanine Lesions in DNA.

N2-Alkyl-2'-deoxyguanosine (N2-alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The N2-alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for N2-alkyl-dG lesions remain incompletely elucidated. By utilizing a photo-cross-linking coupled with mass spectrometry-based quantitative proteomic analysis, we identified a series of candidate N2-alkyl-dG-binding proteins. We found that two of these proteins, i.e., high-mobility group protein B3 (HMGB3) and SUB1, could bind directly to N2-nBu-dG-containing duplex DNA in vitro and promote the repair of this lesion in cultured human cells. In addition, HMGB3 and SUB1 protected cells against benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of N2-BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to trans-N2-BPDE-dG; the protein, however, does not distinguish cis-N2-BPDE-dG from unmodified dG. Consistently, genetic ablation of HMGB3 conferred diminished repair of trans-N2-BPDE-dG, but not its cis counterpart, whereas loss of SUB1 conferred attenuated repair of both diastereomers. Together, we identified proteins involved in the cellular sensing and repair of minor-groove N2-alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of N2-BPDE-dG.

FANCD2–FANCI surveys DNA and recognizes double- to single-stranded junctions

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2–FANCI (D2–I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion1. However, D2–I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation2–4. At present, it is unclear how DNA crosslinks are recognized and how D2–I functions in replication fork protection. Here, using single-molecule imaging, we show that D2–I is a sliding clamp that binds to and diffuses on double-stranded DNA. Notably, sliding D2–I stalls on encountering single-stranded–double-stranded (ss–ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions5. Using cryogenic electron microscopy, we determined structures of D2–I on DNA that show that stalled D2–I makes specific interactions with the ss–dsDNA junction that are distinct from those made by sliding D2–I. Thus, D2–I surveys dsDNA and, when it reaches an ssDNA gap, it specifically clamps onto ss–dsDNA junctions. Because ss–dsDNA junctions are found at stalled replication forks, D2–I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2–I in the recognition and protection of stalled replication forks in several DNA repair pathways.

Gentisic acid attenuates 5-fluorouracil-induced ovotoxicity in rats via modulating Nrf2 signalling: An experimental approach

5-Fluorouracil (5-FU) is the third most used chemotherapeutic in the world with its anticancer effect resulting from its potential to block DNA replication. Like other cytotoxic agents, 5-FU has side effects on healthy tissues, and the reproductive system is among the tissues most affected by these undesirable effects. Gentisic acid (GEA) is a secondary metabolite that is abundant in fruits, vegetables and spices and has antioxidant activity. This study was conducted to investigate the toxicity of 5-FU in rat ovarian tissue and to determine the therapeutic activity of GEA on ovotoxicity caused by 5-FU. The results showed that 5-FU caused histopathological findings by suppressing Nrf2 pathway and accordingly increasing oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis. However, GEA treatments after 5-FU application ameliorated 5-FU-induced ovotoxicity dose-dependently through activation of Nrf2 pathway. All these findings provided strong evidence supporting the hypothesis that GEA treatment may have therapeutic effects against 5-FU-induced ovarian damage. However, the beneficial effect of GEA use in eliminating ovarian damage in women after 5-FU chemotherapy should continue to be investigated with more detailed molecular studies.

Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

A Unique Hybridization Route to Access Hydrazylnaphthalimidols as Novel Structural Scaffolds of Multitargeting Broad-Spectrum Antifungal Candidates.

This study developed a class of novel structural antifungal hydrazylnaphthalimidols (HNs) with multitargeting broad-spectrum potential via multicomponent hybridization to confront increasingly severe fungal invasion. Some prepared HNs exhibited considerable antifungal potency; especially nitrofuryl HN 4a (MIC = 0.001 mM) exhibited a potent antifungal activity against Candida albicans, which is 13-fold higher than that of fluconazole. Furthermore, nitrofuryl HN 4a displayed low cytotoxicity, hemolysis and resistance, as well as a rapid fungicidal efficacy. Preliminary mechanistic investigations revealed that nitrofuryl HN 4a could inhibit lactate dehydrogenase to decrease metabolic activity and promote the accumulation of reactive oxygen species, leading to oxidative stress. Moreover, nitrofuryl HN 4a did not exhibit membrane-targeting ability; it could embed into DNA to block DNA replication but could not cleave DNA. These findings implied that HNs are promising as novel structural scaffolds of potential multitargeting broad-spectrum antifungal candidates for treating fungal infection.

Mutagenesis and Repair of γ-Radiation- and Radical-Induced Tandem DNA Lesions

Ionizing radiation induces many different types of DNA lesions. But one of its characteristics is to produce complex DNA damage, of which tandem DNA damage has received much attention, owing to its promise of distinctive biological properties. Oxidative stresses in response to inflammation in tissues and metal-catalyzed reactions that result in generation of radicals also form these DNA lesions. In this minireview, we have summarized the formation of the tandem lesions as well as the replication and repair studies carried out on them after site-specific synthesis. Many of these lesions are resistant to the traditional base excision repair, so that they can only be repaired by the nucleotide excision repair pathway. They also block DNA replication and, when lesion bypass occurs, it may be significantly error-prone. Some of these tandem DNA lesions may contribute to ageing, neurological diseases, and cancer.

The Metabolism, Detrimental Effects, and Signal Transduction Mechanism of Reactive Oxygen Species in Plants under Abiotic Stress

Plants are a major source of bio based materials, and their productivity is significantly impacted by environmental adversities with the continuous deterioration of the environment. The disruption of reactive oxygen species (ROS) production and scavenging balance is a common consequence of various abiotic stresses in plants, resulting in the excessive accumulation of ROS. However, it is crucial to acknowledging the dual role of ROS, acting both as harmful substances and signaling molecules. ROS can interact with membrane proteins, initiating a chain reaction of membrane lipid peroxidation. This process leads to a decrease in membrane lipid unsaturation and fluidity, as well as an increase in membrane permeability. Inhibited enzyme activity even lead to loss of protein function, caused DNA damage and mutations, blocked DNA replication and transcription, eventually leading to cell death. This can result in significant reductions in crop yield and biomass in bio based agricultural production. ROS also played vital role in signal molecule transduction in the process of plant resistance to stress, and worked in concert with other signal molecules to induce the stress resistance genes’ expression and enhance plant resistance. Hereto, scavenging mechanism for ROS in plants is of the essence to regulate the concentration of ROS, thus determining which role ROS play. The thermal dissipation of excess light energy effectively avoids ROS production, and the enzymatic and non-enzymatic antioxidant defense systems in plants work cooperatively to clear excessive ROS. This review provides a comprehensive summary of the production pathway, detrimental effects, scavenging mechanism and signal transduction of ROS in plants under abiotic stress.

CDC7 inhibition drives an inflammatory response and a p53-dependent senescent-like state in breast epithelial cells.

Drugs that block DNA replication prevent cell proliferation, which may result in anticancer activity. The latter is dependent on the drug's mode of action as well as on cell type-dependent responses to treatment. The inhibition of Cell division cycle 7-related protein kinase (CDC7), a key regulator of DNA replication, decreases the efficiency of origin firing and hampers the restarting of paused replication forks. Here, we show that upon prolonged CDC7 inhibition, breast-derived MCF10A cells progressively withdraw from the cell cycle and enter a reversible senescent-like state. This is characterised by the rewiring of the transcriptional programme with the induction of cytokine and chemokine expression and correlates with the accumulation of Cyclic GMP-AMP synthase (cGAS)-positive micronuclei. Importantly, cell fate depends on Cellular tumour antigen p53 (p53) function as cells no longer enter senescence but are funnelled into apoptosis upon p53 knockout. This work uncovers key features of the secondary response to CDC7 inhibitors, which could aid the development of these compounds as anticancer drugs.

Abstract 7590: Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair

Abstract Colorectal Cancer (CRC) is the third most common cause of cancer mortality in the US. A major problem in CRC management is relapse and progression to metastatic disease leading to poor overall survival. The tumor suppressor p53 is mutated in most CRCs and often drives metastasis and therapy resistance. Current therapeutic options targeting p53 mutant cancers show poor efficacy and frequently exhibit high toxicity. To address this clinical problem, we developed a novel therapeutic strategy for selective targeting of p53 mutant cancers. The strategy combines a thymidine analogue (e.g., trifluorthymidine/TFT, a component of TAS102) and poly (ADP-ribose) polymerase inhibitor (PARPi). We observed that TAS102 does not block DNA replication, but rather prompts post-replicative base-excision DNA repair (BER), and PARPi increases double-strand DNA breaks (DSBs) in p53 mutant cancer cells. Normal p53 wild type (p53wt) cells are arrested in the G1 phase and repair DNA. Thus, the TAS102-PARPi combination selectively targets p53-mutant cancer cells, resulting in the accumulation of DSBs and cell death. This novel strategy was examined in p53wt and p53-mutant tumor cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. The drug combination was significantly more effective in inhibiting p53 mutant tumor growth and in prolonging survival compared to monotherapies. This strategy is currently being tested in a first-in-human dose-escalation Phase I study (NCT04511039) in patients with advanced CRC. Further investigation revealed that the TAS102-PARPi combination induced DNA damage response (DDR) and the G2 checkpoint, leading to G2 arrest in p53 mutant cancer cells. We observed that TAS102-PARPi induced phosphorylation of CDC2 (also known as CDK1) but did not increase phosphorylation of histone H3 (a marker of mitosis), therefore indicating activation of the G2 checkpoint. Furthermore, the roles of ATM and ATR kinases in the DDR and G2 responses were clarified using highly specific kinase inhibitors. The analysis of the DDR/G2 signaling response in isogenic p53wt and mutant CRC cell lines showed that inhibition of WEE1 kinase can selectively abrogate the G2 checkpoint in p53-mutant cells, resulting in marked DNA damage and cell death. Importantly, the blockade of WEE1 strongly synergized with the TAS102-PARPi combination, further enhancing its potency and efficacy against p53-mutant cancer cells. In summary, our research provides a foundation for a novel therapeutic strategy for p53 mutant cancers that may provide a paradigm shift in the treatment of these deadly cancers, increasing efficacy while minimizing toxic adverse effects. Citation Format: Mohammed M. Alruwaili, Natsumi Naranjo, Priyanka Rajan, Kyeong Beom Jo, Dae-Kyum Kim, Thomas Melendy, Robert Straubinger, Christos Fountzilas, Andrei Bakin. Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7590.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

Bacterial cell death to overcome drug resistance with multitargeting bis-naphthalimides as potent antibacterial agents against Enterococcus faecalis.

The increasing frequency of drug-resistant pathogens poses serious health issues to humans around the globe, leading to the development of new antibacterial agents to conquer drug resistance and bacterial infections. In view of this, we have synthesized a series of bis-naphthalimides to respond to awful drug resistance. Bioactivity assay and structure-activity relationship disclosed that compounds 5d and 5o exhibit potent antibacterial activity against E. faecalis, outperforming the marketed antibiotics. These drug candidates not only inhibit the biofilm formation of E. faecalis but also display rapid bactericidal properties, thus delaying the development of drug resistance within 20 passages. To explore the mechanism of antibacterial activity against E. faecalis, biofunctional examination was carried out which unveiled that 5d and 5o effectively disrupt bacterial cell membranes, causing the leakage of cytoplasmic contents and metabolic activity loss. Concurrently, 5d and 5o effectively intercalate with DNA to block DNA replication, causing the build-up of excessive reactive oxygen species and inhibiting the glutathione activity, ultimately leading to oxidative damage of E. faecalis and cell death. In addition, these compounds readily bind with HSA with a high binding constant, indicating that these drug candidates could be easily delivered to the target site. The above finding manifested that these newly synthesized bis-naphthalimides with multitargeting antibacterial properties offer a new prospect to overcome drug resistance.

IKS03, a Novel CD19-Targeted Antibody Drug Conjugate, Induces Target Dependent In Vivo Cell Killing of B-Cell Lymphoma Xenografts By DNA Crosslinking

CD19 is widely expressed in B-cell non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). IKS03 is a CD19-targeting antibody drug conjugate (ADC) with a novel linker payload design for tumor-selective payload release. Site-specific conjugation using farnesyltransferase-catalysed ConjuALL technology to a DNA crosslinking agent yields a homogenous ADC with drug-to-antibody ratio (DAR) of 2. To minimize systemic release of the highly potent payload in human plasma, IKS03 incorporates a linker specifically cleavable by the lysosomal enzyme beta-glucuronidase. In addition, the payload is a pro-drug with a protecting moiety cleavable by the same enzyme. Therefore, following CD19-dependent binding and uptake, IKS03 requires intracellular lysosomal processing in the target cell to both release and fully activate the free pyrrolobenzodiazepine (PBD) dimer payload which induces DNA crosslinking, blocks DNA replication and ultimately leads to tumor selective cell death. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. IKS03 was highly active against Farage and OCI-LY10 DLBCL xenografts as well as Granta-519 MCL xenografts in SCID mice with durable tumor regressions observed following treatment with a single dose of 0.1 to 0.3 mg/kg. Likewise, in a Ramos lymphoma model a single IKS03 dose of 0.3 mg/kg significantly inhibited tumor growth. IKS03 demonstrated greater in vivo efficacy than the clinical benchmark ADCs polatuzumab vedotin and loncastuximab tesirine approved for the treatment of patients with B-cell non-Hodgkin lymphomas. PBD cytotoxic agents have been described to induce DNA crosslinking leading to DNA damage. To demonstrate that IKS03 in vivo activity is the result of targeted payload release, Ramos xenografts were established in immunodeficient mice and treated with up to 1 mg/kg of IKS03 in comparison with a non-targeted ADC utilizing the same linker payload format. Tumors were excised at 1 day and 3 days post-treatment and assessed for the presence of phospho-H2AX positive foci, a marker of DNA damage, by immunohistochemistry. IKS03 treatment increased both frequency and staining intensity of nuclear phospho-H2AX foci in Ramos xenografts in a dose- and time-dependent manner. In contrast, the non-targeted control ADC did not result in any changes as compared to a vehicle control. This confirms the specific induction of DNA damage in CD19 positive lymphomas as a pharmacodynamic response to IKS03 treatment in vivo. In conclusion, IKS03 is highly effective in an array of preclinical MCL and DLBCL xenograft models at doses that are well tolerated. In vivo, IKS03 treatment increased the level of phospho-H2AX indicative of irreversible DNA damage in a target-dependent manner. This is consistent with the proposed mechanism of action of CD19-mediated delivery of a PBD payload specifically to B-cell tumors. Clinical investigation in patients with advanced B-Cell Non-Hodgkin Lymphomas is being initiated (NCT05365659).

Unraveling Novel Mechanisms for Targeting the Intra-S-Phase Checkpoint in p53-Mutated Acute Myeloid Leukemia

Tp53 mutated acute myeloid leukemia (AML) is a highly challenging subtype to treat due to its aggressive nature and resistance to standard therapies. Even in cases where initial responses to conventional treatments occur, residual minor clonal populations harboring p53 mutations place patients at a higher risk of relapse and lower overall survival rates. Thus, innovative treatment strategies are essential to tackle the challenges of p53 mutated AML and enhance patient outcomes. Although it plays a substantial role in G1/S and G2/M checkpoints, p53's significance in the intra-S-phase checkpoint is limited. This checkpoint is vital in detecting replication defects and its disruption leads to catastrophic genome instability, massive DNA breakage, and cell death. We endeavored to target this pathway by employing a non-ATP cell cycle inhibitor, LBS-007, that blocks the kinase activity of CDC7 that is responsible for phosphorylating MCM2 prior to DNA replication initiation. Thus, we hypothesized that inhibition of CDC7 can disrupt DNA synthesis, leading to genomic instability and potentially p53-independent cell death. Initially, we explored LBS-007 as a single agent in a p53 deleted (17p-) AML cell line and two AML lines with hot-spot p53 mutation (R175H or R248Q). LBS-007 showed significant effectiveness in all three cases with altered p53 status and with GI 50 values comparable to those observed in wild-type p53 AML cells (62 nM, 40 nM, and 59 nM versus 133 nM). To further enhance its therapeutic impact, we investigated combinations with other DNA replication inhibitors. When used sequentially with LBS-007, hydroxyurea provided a robust response yielding GI 50's between 1 and 3 nM for LBS-007, effectively blocking DNA replication origins (LBS-007) and inducing fork stalls (HU). Surprisingly, the combination of LBS-007 and an ATR inhibitor, Ceralasertib, had a paradoxical effect, suggesting the effectiveness of LBS-007 requires intra-S-phase checkpoint activation. To determine the underlying molecular mechanisms, we conducted RPPA analyses on p53 defective AML cells treated with LBS-007. We observed significant upregulation of apoptotic proteins and pathways when used as a single agent (Figure 1A). However, in combination with hydroxyurea, activation of DNA damage sensors and intra-S-phase checkpoint proteins were overrepresented (Figure 1B). The observed effectiveness of LBS-007 in driving robust p53-independent apoptosis and cell cycle checkpoints indicates its potential as a powerful new agent to treat p53-mutated AML. To discover synergistic combinations that enhance the efficacy of LBS-007, we utilized venetoclax, a BCL2 inhibitor that facilitates apoptosis. The inclusion of venetoclax significantly increased the efficacy of LBS-007, resulting in combinatorial GI 50's below 10 nM in both p53 mutated and p53 deleted AML. Most impressively, the combination of LBS-007 and venetoclax was exponentially enhanced when used in conjunction with ceralasertib (4.8 pM). Collectively, these results provide compelling evidence that combining CDC7 inhibition and venetoclax with a potential ATR inhibitor will have a profound impact on treatment of p53 altered AML (Figure 2). We are now evaluating the efficacy of these combinations using xenograft transplant models. To this end, cohorts of mice transplanted with AML cell lines harboring either wild-type or mutated p53 will be treated with strategies identified through our in vitro and RPPA analyses. Further studies will address the cooperation between mutations common to AML and p53 alterations using defined PDX models. Our results identify a unique and potentially efficacious strategy to treat one of the most difficult to treat subtypes of AML. These findings will provide the rationale for potential clinical trials using CDC7 inhibition in p53 mutated or R/R AML.

Novel Fluoroquinolones with Possible Antibacterial Activity in Gram-Negative Resistant Pathogens: In Silico Drug Discovery.

Antibiotic resistance is a global threat to public health, and the search for new antibacterial therapies is a current research priority. The aim of this in silico study was to test nine new fluoroquinolones previously designed with potential leishmanicidal activity against Campylobacter jejuni, Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Salmonella typhi, all of which are considered by the World Health Organization to resistant pathogens of global concern, through molecular docking and molecular dynamics (MD) simulations using wild-type (WT) and mutant-type (MT) DNA gyrases as biological targets. Our results showed that compound 9FQ had the best binding energy with the active site of E. coli in both molecular docking and molecular dynamics simulations. Compound 9FQ interacted with residues of quinolone resistance-determining region (QRDR) in GyrA and GyrB chains, which are important to enzyme activity and through which it could block DNA replication. In addition to compound 9FQ, compound 1FQ also showed a good affinity for DNA gyrase. Thus, these newly designed molecules could have antibacterial activity against Gram-negative microorganisms. These findings represent a promising starting point for further investigation through in vitro assays, which can validate the hypothesis and potentially facilitate the development of novel antibiotic drugs.

Liquid Chromatography-Mass Spectrometry Screening of Cyclophosphamide DNA Damage In Vitro and in Patients Undergoing Chemotherapy Treatment.

DNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and often severe side effects in patients undergoing alkylating-drug based therapies. The development of biomarkers to identify patients who effectively respond with tolerable toxicities vs patients who develop serious side effects is needed. Cyclophosphamide (CPA) is a commonly used chemotherapeutic drug and lacks biomarkers to evaluate its therapeutic effect and toxicity. Upon administration, CPA is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity, respectively. Previous studies have explored the detection of the major DNA adduct of CPA, the interstrand DNA-DNA cross-link G-NOR-G, finding differences in the cross-link amount between Fanconi Anemia and non-Fanconi Anemia patients undergoing chemotherapy treatment. In this study, we take advantage of our DNA adductomic approach to comprehensively profile CPA's and its metabolites' reactions with DNA in vitro and in patients undergoing CPA-based chemotherapy. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with CPA. Moreover, acrolein-derived DNA adducts were quantified in patient samples. The results suggest that CPA-DNA damage is very complex, and an evaluation of DNA adduct profiles is necessary when evaluating the relationship between CPA-DNA damage and patient outcome.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.

A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-(2-carbamoyl-2-hydroxyethyl)formamidopyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-dfG, a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis (TLS) polymerases (Polη, Polι, Polκ, and Polζ), and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-knockout cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.

226-LB: Repurposing a Cancer Drug, AZD6738 (ATRi), for the Prevention of Type 1 Diabetes

Type 1 diabetes (T1D) is a chronic, incurable autoimmune disorder caused by the destruction of pancreatic β cells by diabetogenic T cells, leading to insulin deficiency and hyperglycemia. It affects 3 million Americans and 80 new people are diagnosed daily. Despite treatment with insulin, patients have a significantly reduced life expectancy. This study proposes a new approach to prevent T1D by specifically targeting diabetogenic T cells undergoing the Warburg effect, a metabolic process similar to cancer cells, with a cancer drug approved by the FDA. The use of FDA-approved drugs accelerates the development of new therapies as these agents have already undergone initial efficacy and toxicity tests in humans. One promising drug for this purpose is AZD6738 (ATRi), a cancer drug that is currently in clinical trials. ATRi is a small molecule inhibitor that targets ATR, a central DNA damage response kinase, whose inhibition blocks DNA replication. We demonstrate that ATRi treatment significantly delays T1D onset in an adoptive transfer mouse model by reducing the survival of diabetogenic CD4+ T cells by inhibiting their proliferation. Furthermore, an FDA-approved glycolysis inhibitor PFK158 potentiates inhibition of diabetogenic CD4+ T cells by ATRi. These agents target different properties of the Warburg effect. While ATRi inhibits rapid proliferation, PFK158 blocks glycolysis. Combination therapy allows for increased efficacy while reducing potential toxicity by allowing the use of lower doses of each agent. These data show promise in the rapid establishment of novel T1D prevention methods by repurposing FDA-approved cancer drugs. Disclosure N. Sugitani: None. H. R. Mason: None. S. Roy: None. J. D. Piganelli: None. Funding National Institutes of Health (R01DK132583); JDRF (2-SRA2020-910-S-B)

Novel Thiazolylketenyl Quinazolinones as Potential Anti-MRSA Agents and Allosteric Modulator for PBP2a.

Bacterial infections caused by methicillin-resistant Staphylococcus aureus have seriously threatened public health. There is an urgent need to propose an existing regimen to overcome multidrug resistance of MRSA. A unique class of novel anti-MRSA thiazolylketenyl quinazolinones (TQs) and their analogs were developed. Some synthesized compounds showed good bacteriostatic potency. Especially TQ 4 was found to exhibit excellent inhibition against MRSA with a low MIC of 0.5 μg/mL, which was 8-fold more effective than norfloxacin. The combination of TQ 4 with cefdinir showed stronger antibacterial potency. Further investigation revealed that TQ 4, with low hemolytic toxicity and low drug resistance, was not only able to inhibit biofilm formation but also could reduce MRSA metabolic activity and showed good drug-likeness. Mechanistic explorations revealed that TQ 4 could cause leakage of proteins by disrupting membrane integrity and block DNA replication by intercalated DNA. Furthermore, the synergistic antibacterial effect with cefdinir might be attributed to TQ 4 with the ability to induce PBP2a allosteric regulation of MRSA and further trigger the opening of the active site to promote the binding of cefdinir to the active site, thus inhibiting the expression of PBP2a, thereby overcoming MRSA resistance and significantly enhancing the anti-MRSA activity of cefdinir. A new strategy provided by these findings was that TQ 4, possessing both excellent anti-MRSA activity and allosteric effect of PBP2a, merited further development as a novel class of antibacterial agents to overcome increasingly severe MRSA infections.