Bacterial Metabolism Affects the C. elegans Response to Cancer Chemotherapeutics.

The human microbiota greatly affects physiology and disease; however, the contribution of bacteria to the response to chemotherapeutic drugs remains poorly understood. Caenorhabditis elegans and its bacterial diet provide a powerful system to study host-bacteria interactions. Here, we use this system to study how bacteria affect the C. elegans response to chemotherapeutics. We find that different bacterial species can increase the response to one drug yet decrease the effect of another. We perform genetic screens in two bacterial species using three chemotherapeutic drugs: 5-fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FUDR), and camptothecin (CPT). We find numerous bacterial nucleotide metabolism genes that affect drug efficacy in C. elegans. Surprisingly, we find that 5-FU and FUDR act through bacterial ribonucleotide metabolism to elicit their cytotoxic effects in C. elegans rather than by thymineless death or DNA damage. Our study provides a blueprint for characterizing the role of bacteria in the host response to chemotherapeutics.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair.

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination.

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.

SLFN11 Blocks Stressed Replication Forks Independently of ATR.

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

Development of an ELISA with acidification treatment for an antibody conjugate incorporating Exatecans.

The successful development of Sacituzumab Govitecan and Trastuzumab Deruxtecan has made camptothecin derivatives one of the most popular payloads for antibody-drug conjugates (ADCs). Camptothecin and its derivatives all exist in a pH-dependent equilibrium between the carboxylate and lactone forms. Such transformation may lead to differences in the ratio of the two molecular forms in calibration standards and biological matrix (bio-matrix) samples, thereby leading to inaccurate conjugated antibody results. In this study, we reported an enzyme-linked immunosorbent assay (ELISA) free of the aforementioned influence for the detection of the Exatecans-conjugated antibody (conjugated SM001) in cynomolgus monkey serum. The assay was developed by first acidifying all samples with glacial acetic acid (HAc), then performing neutralization and thereafter capturing conjugated SM001 with anti-Exatecan monoclonal antibody (mAb) and detecting it with biotinylated Nectin4 (hNectin4-Bio) and horseradish peroxidase-labeled streptavidin (SA-HRP). Results showed that all tested performance parameters met the acceptance criteria. The conjugated SM001 concentrations obtained were in parallel to but slightly lower than total antibody (TAb) throughout the pharmacokinetic (PK) study, revealing that the assay strategy implemented for conjugated SM001 measurement worked well for the elimination of interference triggered by the heterogeneous existence of the lactone and carboxylate forms of Exatecan (lactone-Exatecan and carboxylate-Exatecan).

The Effect of Lipid Composition on the Liposomal Delivery of Camptothecin Developed by Active Click Loading.

In the present study, we investigated the role of lipid composition of camptothecin (CPT)-loaded liposomes (CPT-Lips) to adjust their residence time, drug distribution, and therefore the toxicities and antitumor activity. The CPT was loaded into liposomes using a click drug loading method, which utilized liposomes preloaded with GSH and then exposed to CPT-maleimide. The method produced CPT-Lips with a high encapsulation efficiency (>95%) and sustained drug release. It is shown that the residence times of CPT-Lips in the body were highly dependent on lipid compositions with an order of non-PEGylated liposomes of unsaturated lipids < non-PEGylated liposomes of saturated lipids < PEGylated liposomes of saturated lipids. Interestingly, the fast clearance of CPT-Lips resulted in significantly decreased toxicities but did not cause a significant decrease in their in vivo antitumor activity. These results suggested that the lipid composition could effectively adjust the residence time of CPT-Lips in the body and further optimize their therapeutic index, which would guide the development of a liposomal formulation of CPT.

Natural-source payloads used in the conjugated drugs architecture for cancer therapy: Recent advances and future directions.

Drug conjugates are obtained from tumor-located vectors connected to cytotoxic agents via linkers, which are designed to deliver hyper-toxic payloads directly to targeted cancer cells. These drug conjugates include antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), small molecule-drug conjugates (SMDCs), nucleic acid aptamer-drug conjugates (ApDCs), and virus-like drug conjugate (VDCs), which show great therapeutic value in the clinic. Drug conjugates consist of a targeting carrier, a linker, and a payload. Payloads are key therapy components. Cytotoxic molecules and their derivatives derived from natural products are commonly used in the payload portion of conjugates. The ideal payload should have sufficient toxicity, stability, coupling sites, and the ability to be released under specific conditions to kill tumor cells. Microtubule protein inhibitors, DNA damage agents, and RNA inhibitors are common cytotoxic molecules. Among these conjugates, cytotoxic molecules of natural origin are summarized based on their mechanism of action, conformational relationships, and the discovery of new derivatives. This paper also mentions some cytotoxic molecules that have the potential to be payloads. It also summarizes the latest technologies and novel conjugates developed in recent years to overcome the shortcomings of ADCs, PDCs, SMDCs, ApDCs, and VDCs. In addition, this paper summarizes the clinical trials conducted on conjugates of these cytotoxic molecules over the last five years. It provides a reference for designing and developing safer and more efficient conjugates.

Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases.

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 µg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC-MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 µM) and MCF7 (3.2 µM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.

Camptothecin bioprocessing from Aspergillus terreus, an endophyte of Catharanthus roseus: antiproliferative activity, topoisomerase inhibition and cell cycle analysis.

Attenuation of camptothecin (CPT) productivity by fungi with preservation and subculturing is the challenge that halts fungi to be an industrial platform of CPT production. Thus, screening for novel endophytic fungal isolates with metabolic stability for CPT production was the objective. Catharanthus roseus is one of the medicinal plants with diverse bioactive metabolites that could have a plethora of novel endophytes with unique metabolites. Among the endophytes of C. roseus, Aspergillus terreus EFBL-NV OR131583.1 had the most CPT producing potency (90.2 µg/l), the chemical identity of the putative CPT was verified by HPLC, FT-IR, NMR and LC-MS/MS. The putative A. terreus CPT had the same molecular mass (349 m/z), and molecular fragmentation patterns of the authentic one, as revealed from the MS/MS analyses. The purified CPT had a strong activity against MCF7 (5.27 µM) and UO-31 (2.2 µM), with a potential inhibition to Topo II (IC50 value 0.52 nM) than Topo 1 (IC50 value 6.9 nM). The CPT displayed a high wound healing activity to UO-31 cells, stopping their metastasis, matrix formation and cell immigration. The purified CPT had a potential inducing activity to the cellular apoptosis of UO-31 by ~ 17 folds, as well as, arresting their cellular division at the S-phase, compared to the control cells. Upon Plackett-Burman design, the yield of CPT by A. terreus was increased by ~ 2.6 folds, compared to control. The yield of CPT by A. terreus was sequentially suppressed with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by 3rd month and 5th generation. However, the productivity of the attenuated A. terreus culture was completely restored by adding 1% surface sterilized leaves of C. roseus, and the CPT yield was increased over-the-first culture by ~ 3.2 folds (315.2 µg/l). The restoring of CPT productivity of A. terreus in response to indigenous microbiome of C. roseus, ensures the A. terreus-microbiome interactions, releasing a chemical signal that triggers the CPT productivity of A. terreus. This is the first reports exploring the potency of A. terreus, endophyte of C. roseus" to be a platform for industrial production of CPT, with an affordable sustainability with addition of C. roseus microbiome.

Design and synthesis of TH19P01-Camptothecin based hybrid peptides inducing effective anticancer responses on sortilin positive cancer cells.

Recently, the sortilin receptor (SORT1) was found to be preferentially over-expressed on the surface of many cancer cells, which makes SORT1 a novel anticancer target. The SORT1 binding proprietary peptide TH19P01 could achieve the SORT1-mediated cancer cell binding and subsequent internalization. Inspired by the peptide-drug conjugate (PDC) strategy, the TH19P01-camptothecin (CPT) conjugates were designed, efficiently synthesized, and evaluated for their anticancer potential in this study. The water solubility, in vitro anticancer activity, time-kill kinetics, cellular uptake, anti-migration activity, and hemolysis effects were systematically estimated. Besides, in order to monitor the release of CPT from conjugates in real-time, the CPT/Dnp-based "turn on" hybrid peptide was designed, which indicted that CPT could be sustainably released from the hybrid peptide in both human serum and cancer cellular environments. Strikingly, compared with free CPT, the water solubility, cellular uptake, and selectivity towards cancer cells of hybrid peptide LYJ-2 have all been significantly enhanced. Moreover, unlike free CPT or TH19P01, LYJ-2 exhibited selective anti-proliferative and anti-migration effects against SORT1-positive MDA-MB-231 cells. Collectively, this study not only established efficient strategies to improve the solubility and anticancer potential of chemotherapeutic agent CPT, but also provided important references for the future development of TH19P01 based PDCs targeting SORT1.

Identification of 7-aminourea or 7-aminothiourea derivatives of camptothecin as selective topoisomerase I inhibitors with anti-colorectal cancer activities.

Colorectal cancer (CRC) remains one of the most prevalent malignant tumors of the digestive system, yet the availability of safe and effective chemotherapeutic agents for clinical use remains limited. Camptothecin (CPT) and its derivatives, though approved for cancer treatment, have encountered significant challenges in clinical application due to their low bioavailability and high systemic toxicity. Strategic modification at the 7-position of CPT enables the development of novel CPT derivatives with high activity. In the present study, a series of compounds incorporating aminoureas, amino thioureas, and acylamino thioureas as substituents at the 7-position were screened. These compounds were subsequently evaluated for their cytotoxicity against the human gastric cancer (GC) cell line AGS and the CRC cell line HCT116. Two derivatives, XSJ05 (IC50 = 0.006 ± 0.003 µM) and XSJ07 (IC50 = 0.013 ± 0.003 µM), exhibited remarkably effective anti-CRC activity, being better than TPT. In addition, they have a better safety profile. In vitro mechanistic studies revealed that XSJ05 and XSJ07 exerted their inhibitory effects on CRC cell proliferation by suppressing the activity of topoisomerase I (Topo I). This suppression triggers DNA double-strand breaks, leads to DNA damage and subsequently causes CRC cells to arrest in the G2/M phase. Ultimately, the cells undergo apoptosis. Collectively, these findings indicate that XSJ05 and XSJ07 possess superior activity coupled with favorable safety profiles, suggesting their potential as lead compounds for the development of CRC therapeutics.

Biological impact and therapeutic potential of a novel camptothecin derivative (FLQY2) in pancreatic cancer through inactivation of the PDK1/AKT/mTOR pathway.

BACKGROUND: Camptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest. AIM OF THE STUDY: This study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs. METHODS: The cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2's antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model. RESULTS: FLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects. CONCLUSION: The study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer.

Biotechnological approaches for the production of camptothecin.

Camptothecin (CPT), an indole alkaloid popular for its anticancer property, is considered the third most promising drug after taxol and famous alkaloids from Vinca for the treatment of cancer in humans. Camptothecin was first identified in Camptotheca acuminata followed by several other plant species and endophytic fungi. Increased harvesting driven by rising global demand is depleting the availability of elite plant genotypes, such as Camptotheca acuminata and Nothapodytes nimmoniana, crucial for producing alkaloids used in treating diseases like cancer. Conservation of these genotypes for the future is imperative. Therefore, research on different plant tissue culture techniques such as cell suspension culture, hairy roots, adventitious root culture, elicitation strategies, and endophytic fungi has been adopted for the production of CPT to meet the increasing demand without affecting the source plant's existence. Currently, another strategy to increase camptothecin yield by genetic manipulation is underway. The present review discusses the plants and endophytes that are employed for camptothecin production and throws light on the plant tissue culture techniques for the regeneration of plants, callus culture, and selection of cell lines for the highest camptothecin production. The review further explains the simple, accurate, and cost-effective extraction and quantification methods. There is enormous potential for the sustainable production of CPT which could be met by culturing of suitable endophytes or plant cell or organ culture in a bioreactor scale production. Also, different gene editing tools provide opportunities for engineering the biosynthetic pathway of CPT, and the overall CPT production can be improved . KEY POINTS: • Camptothecin is a naturally occurring alkaloid with potent anticancer properties, primarily known for its ability to inhibit DNA topoisomerase I. • Plants and endophytes offer a potential approach for camptothecin production. • Biotechnology approaches like plant tissue culture techniques enhanced camptothecin production.

Impact of camptothecin exposures on the development and larval midgut metabolomic profiles of Spodoptera frugiperda.

Spodoptera frugiperda is an economic agricultural pest that has invaded many countries around the world and caused huge losses in grain production. Camptothecin (CPT) is one of the botanical compounds with insecticidal activity and has the potential for pest control. However, the effects of CPT on development and metabolism of S. frugiperda remain unknown. In this study, we have investigated the adverse effects of 1.0 and 5.0 mg/kg CPT exposures on the growth and development of S. frugiperda. Our results found that 1.0 and 5.0 mg/kg CPT treatments altered the parameters of the life cycle, including inducing larval mortality, altering the weight of larvae, pupae, and adults, the larval duration, and decreasing the pupation rate and emergence rate. In addition, comparative metabolomics analysis was performed in the larval midgut of S. frugiperda to explore the toxicity mechanism of CPT. A total of 261 and 348 differential metabolites were identified in the groups with 1.0 and 5.0 mg/kg CPT treatments, respectively. Further analysis found that pantothenate and CoA biosynthesis, sulfur relay system, selenocompound metabolism, and fatty acid biosynthesis pathways were significantly altered by 5.0 mg/kg CPT exposure. Our results provided new insight into the toxicological mechanisms of CPT against S. frugiperda and laid the foundation for the field application of CPT in pest control.

Development of Chitosan-Coated Graphene Oxide and Iron Oxide Nanocomposites for Targeted Delivery of Camptothecin to Liver Cancer Cells.

Innovative drug delivery platforms for selective, regulated, and sustained release of anticancer drugs are crucial in cancer treatment. This study presents nanoparticles developed from chitosan (CS), graphene oxide (GO), and magnetite (Fe3O4), and their nanocomposites, to enhance the loading and release efficiency of camptothecin (CPT). Nanostructures were characterized using imaging microscopy, FT-IR, and X-ray diffraction, with an average crystallite size of 5.5 nm. Camptothecin binding proportions were 70% for CS, 81% for CS@Fe3O4, 58% for CS@GO, and 74% for CS@GO/Fe3O4. At pH 5.0, CPT release ratios were 87%, 80%, 88%, and 90%, respectively, and at pH 7.4, 84%, 72%, 89%, and 87%. Cytotoxicity was assessed using the MTT assay against HepG2 and SMMC-7721 cancer cells. CPT-CS@GO/Fe3O4 exhibited the highest survival at 5 µM and 12.5 µM concentrations, indicating it as the most effective nanocarrier for camptothecin delivery. The study demonstrates CS@GO/Fe3O4's potential as a superior drug delivery system.

Optimisation of enzyme-assisted extraction of camptothecin from Nothapodytes nimmoniana and its characterisation.

INTRODUCTION: Efficient extraction of camptothecin (CPT), an anticancer agent from the commercial source Nothapodytes nimmoniana (J. Graham) Mabb in India, is of paramount importance. CPT is present in the highest concentration in the stem portion, and the stem can be readily harvested without uprooting the plant. The fluorescence microscopy mapping of the bark matrix for CPT revealed its presence in a free form within both the outer (epidermal and cortical tissues) and inner (xylem and phloem tissues) sections. The bark matrix primarily consists of cellulose, hemicellulose, and lignin, rendering it woody, rigid, and resistant to efficient solvent penetration for CPT extraction. We proposed a hypothesis that subjecting it to disruption through treatment with hydrolytic enzymes like cellulase and xylanase could enhance solvent diffusion, thereby enabling a swift and effective extraction of CPT. OBJECTIVE: The present study was aimed at enzyme-assisted extraction, using cellulase and xylanase for hydrolytic disruption of the cells to readily access CPT from the stem of the plant N. nimmoniana (J. Graham) Mabb. METHODOLOGY: The hydrolytic cell disruption of ground powder from the stem bark was studied using cellulase and xylanase enzymes. The enzymatically pretreated stem bark powder was subsequently recovered by filtration, dried, and subjected to extraction with methanol to isolate CPT. This process was optimised through a Box-Behnken design, employing a one-factor-at-a-time approach to assess parameters such as enzyme concentration (2-10% w/w), pH (3-7), incubation time (6-24 h), and solid-to-solvent ratio (1:30-1:70 g/mL). CPT was characterised using proton nuclear magnetic resonance (1H-NMR) and Fourier transform infrared (FTIR) spectra, and a high-performance liquid chromatography (HPLC) method was developed for quantification. RESULTS: The cellulase and xylanase treatment resulted in the highest yields of 0.285% w/w and 0.343% w/w, with efficiencies of 67% and 81%, respectively, achieved in a significantly shorter time compared to the untreated material, which yielded 0.18% with an efficiency of only 42%. Extraction by utilising the predicted optimised process parameters, a nearly two-fold increase in the yield, was observed for xylanase, with incubation and solvent extraction times set at 16 and 2 h, respectively. Scanning electron microscopy (SEM) images of the spent material indicated perforations attributed to enzymatic action, suggesting that this could be a primary factor contributing to the enhanced extraction. CONCLUSION: Enzyme-mediated hydrolytic cell disruption could be a potential approach for efficient and rapid isolation of CPT from the bark of N. nimmoniana.

Facile synthesis and cytotoxicity of substituted uracil-1'(N)-acetic acid and 4-pyridone-1'(N)-acetic acid esters of 20(S)-camptothecins.

A series of novel substituted uracil-1'(N)-acetic acid esters (5-9) and 4-pyridone-1'(N)-acetic acid esters (10-11) of 20(S)-camptothecins (CPTs) have been synthesized by the acylation method. All of these new esters were assayed for in vitro cytotoxicity against five human cancer cell lines A549, Bel7402, BGC-823, HCT-8 and A2780. The in vitro bioassay results showed that all the synthesized compounds 5-11 had cytotoxities that were higher than TPT and comparable to CPT on these five tumor cell lines, some of them even showed comparable or superior cytotoxic activity to CPT. The in vitro data exhibited the cytotoxicity of the ester depended on that of its parent compound. The ester 5, 6, 8, 10, 11 even possessed the cytotoxity activity comparable to or even a little better than CPT on A549, HCT-8 and A2780. The compound 11 had the same level of cytoxity on Bel7402 as that of CPT. Here the synthesis and the in vitro antitumor evaluation of a series of novel 20-O-linked substituted uracil-1'(N)-acetic acid and 4-pyridone-1'(N)-acetic acid esters derivatives of CPTs are reported.

γH2A/γH2AX Mediates DNA Damage-Specific Control of Checkpoint Signaling in Saccharomyces cerevisiae.

DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In Saccharomyces cerevisiae, phosphorylation of histone H2A (γH2A, equivalent to γH2AX in mammals) is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that γH2A negatively regulates the G2/M checkpoint in response to the genotoxin camptothecin, which is a DNA topoisomerase I poison. γH2A also suppresses DDC signaling induced by the DNA alkylating agent methyl methanesulfonate. These results differ from prior findings, which demonstrate positive or no roles of γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, which suggest that γH2A has DNA damage-specific effects on DDC signaling. We also find evidence supporting the notion that γH2A regulates DDC signaling by mediating the competitive recruitment of the DDC mediator Rad9 and the DNA repair factor Rtt107 to DNA lesions. We propose that γH2A/γH2AX serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.

Recent Advances in the Synthesis of Rosettacin.

Camptothecin and its analogues show important antitumor activity and have been used in clinical studies. However, hydrolysis of lactone in the E ring seriously attenuates the antitumor activity. To change this situation, aromathecin alkaloids are investigated in order to replace camptothecins. Potential antitumor activity has obtained more and more attention from organic and pharmaceutical chemists. As a member of the aromathecin alkaloids, rosettacin has been synthesized via different methods. This review summarizes recent advances in the synthesis of rosettacin.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa.

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C-10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring-functionalized congeners (e.g., C-9, C-10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa, via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 (NtCPT9H) which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin, as well as two methyltransferases (NtOMT1/2, converting 9-hydroxycamptothecin to 9-methoxycamptothecin), and a uridine diphosphate-glycosyltransferase (NtUGT5, decorating 9-hydroxycamptothecin to 9-ß-D-glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of NtCPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C-9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.