Improved camptothecin encapsulation and efficacy by environmentally sensitive block copolymer/chitosan/fucoidan nanoparticles for targeting lung cancer cells.

In this study, thermo-sensitive poly(N-isopropyl acrylamide) (PNP) was polymerized with pH-sensitive poly(acrylic acid) (PAA) to prepare a PAA-b-PNP block copolymer. Above its cloud point, the block copolymer self-assembled into nanoparticles (NPs), encapsulating the anticancer drug camptothecin (CPT) in situ. Chitosan (CS) and fucoidan (Fu) further modified these NPs, forming Fu-CPT-NPs to enhance biocompatibility, drug encapsulation efficiency (EE), and loading content (LC), crucially facilitating P-selectin targeting of lung cancer cells through a pulmonary drug delivery system. The EE and LC reached 82 % and 3.5 %, respectively. According to transmission electron microscope observation, these Fu-CPT-NPs had uniform spherical shapes with an average diameter of ca. 250 nm. They could maintain their stability in a pH range of 5.0-6.8. In vitro experimental results revealed that the Fu-CPT-NPs exhibited good biocompatibility and had anticancer activity after encapsulating CPT. It could deliver CPT to cancer cells by targeting P-selectin, effectively increasing cell uptake and inducing cell apoptosis. Animal study results showed that the Fu-CPT-NPs inhibited lung tumor growth by increasing tumor cell apoptosis without causing significant tissue damage related to generating reactive oxygen species in lung cancer cells. This system can effectively improve drug-delivery efficiency and treatment effects and has great potential for treating lung cancer.

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.

Polymeric micelles paving the Way: Recent breakthroughs in camptothecin delivery for enhanced chemotherapy.

Camptothecin, a natural alkaloid, was first isolated from the bark and stem of the Camptotheca acuminate tree in China. It, along with its analogs, has demonstrated potent anti-cancer activity in preclinical studies, particularly against solid tumors such as lung, breast, ovarian, and colon cancer. Despite its promising anti-cancer activity, the application of camptothecin is limited due to its poor solubility, toxicity, and limited biodistribution. Nanotechnology-based drug delivery systems have been used to overcome limited bioavailability and ensure greater biodistribution after administration. Additionally, various drug delivery systems, particularly polymeric micelles, have been investigated to enhance the solubility, stability, and efficacy of camptothecin. Polymeric micelles offer a promising approach for the delivery of camptothecin. Polymeric micelles possess a core-shell structure, with a typical hydrophobic core, which exhibits a high capacity to incorporate hydrophobic drugs. The structure of polymeric micelles can be engineered to have a high drug loading capacity, thereby enabling them to carry a large amount of hydrophobic drug within their core. The shell portion of polymeric micelles is composed of hydrophilic polymers Furthermore, the hydrophilic segment of polymeric micelles plays an important role in protecting against the reticuloendothelial system (RES). This review provides a discussion on recent research and developments in the delivery of camptothecin using polymeric micelles for the treatment of cancers.

Camptothecin-loaded mesoporous silica nanoparticles functionalized with CpG oligodeoxynucleotide as a new approach for skin cancer treatment.

The therapeutic efficacy of camptothecin (CPT), a potent antitumor alkaloid, is hindered by its hydrophobic nature and instability, limiting its clinical use in treating cutaneous squamous cell carcinoma (SCC). This study introduces a novel nano drug delivery system (NDDS) utilizing functionalized mesoporous silica nanoparticles (FMSNs) for efficient CPT delivery. The FMSNs were loaded with CPT and subsequently coated with chitosan (CS) for enhanced stability and bioadhesion. Importantly, CpG oligodeoxynucleotide (CpG ODN) was attached onto the CS-coated FMSNs to leverage the immunostimulatory properties of CpG ODN, augmenting the chemotherapy's efficacy. The final formulation FMSN-CPT-CS-CpG displayed an average size of 241 nm and PDI of 0.316 with an encapsulation efficiency of 95 %. Comprehensive in vitro and in vivo analyses, including B16F10 cells and DMBA/TPA-induced SCC murine model, demonstrated that the FMSN-CPT-CS-CpG formulation significantly enhanced cytotoxicity against B16F10 cells and induced complete regression in 40 % of the in vivo subjects, surpassing the efficacy of standard CPT and FMSN-CPT treatments. This study highlights the potential of combining chemotherapeutic and immunotherapeutic agents in an NDDS for targeted, efficient skin cancer treatment.

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.

Targeted delivery of SN38 to breast cancer using amphiphilic diblock copolymers PHPMA-b-PBAEM as micellar carriers with AS1411 aptamer.

Breast cancer treatment can be challenging, but a targeted drug delivery system (DDS) has the potential to make it more effective and reduce side effects. This study presents a novel nanotherapeutic targeted DDS developed through the self-assembly of an amphiphilic di-block copolymer to deliver the chemotherapy drug SN38 specifically to breast cancer cells. The vehicle was constructed from the PHPMA-b-PEAMA diblock copolymer synthesized via RAFT polymerization. A single emulsion method was then used to encapsulate SN38 within nanoparticles (NPs) formed from the PHPMA-b-PEAMA copolymer. The AS1411 DNA aptamer was covalently bonded to the surface of the micellar NPs, producing a targeted DDS. Molecular dynamics (MD) simulation studies were also performed on the di block polymeric system, demonstrating that SN38 interacted well with the di block. The in vitro results demonstrated that AS1411- decorated SN38-loaded HPMA NPs were highly toxic to breast cancer cells while having a minimal effect on non-cancerous cells. Remarkably, in vivo studies elucidated the ability of the targeted DDS to enhance the antitumor effect of SN38, suppressing tumor growth and improving survival rates compared to free SN38.

Molecularly imprinted beads based on modified cellulose hydrogel:A novel solid phase extraction filler for specific adsorption of camptothecin.

Traditional solid phase extraction (SPE) suffers from a lack of specific adsorption. To overcome this problem, a combination of adsorption method and molecular imprinting technology by polydopamine modification was proposed to realize specific recognition of target compounds in SPE, which is of great significance to improve the separation efficiency of SPE. Cellulose hydrogel beads were prepared by dual cross-linking curing method and modified with polydopamine to make them hydrophilic and biocompatible. Subsequently, cellulose hydrogel-based molecularly imprinted beads (MIBs) were synthesized by surface molecular imprinting technology and used as novel column fillers in SPE to achieve efficient adsorption (34.16 mg·g-1) with specific selectivity towards camptothecin (CPT) in 120 min. The simulation and NMR analysis revealed that recognition mechanism of MIBs involved hydrogen bond interactions and Van der Waals effect. The MIBs were successful used in separating CPT from Camptotheca acuminata fruits, exhibiting impressive adsorption capacity (1.19 mg·g-1) and efficient recovery of CPT (81.54 %). Thus, an environmentally friendly column filler for SPE was developed, offering a promising avenue for utilizing cellulose-based materials in the selective separation of natural products.

Exploring the influence of organ differentiation on biosynthesis and accumulation of camptothecin in Ophiorrhiza rugosa var. decumbens.

Genus Ophiorrhiza has recently emerged as one of the promising sources of Camptothecin (CPT), an antitumour monoterpene indole alkaloid. It possesses CPT in its every part and has a relatively short life span. To determine whether differentiation plays any role in the synthesis and/or accumulation of CPT, the concentration of CPT was analyzed across various tissues of Ophiorrhiza rugosa var. decumbens obtained through both direct as well as indirect modes of regeneration. The results revealed that the plants obtained from both types of regeneration showed similar levels of CPT. It was also observed that with differentiation, the accumulation of CPT increases, as the callus, being an undifferentiated mass of cells, had only traces of CPT. In contrast, the completely differentiated in-vitro plant obtained from it showed a significantly higher percentage of CPT in shoots (0.22% dry weight) and roots (0.247% dw). The CPT when analyzed after hardening, varied among different organs of the plant. It was also observed that the inflorescence accumulated the highest concentration of CPT (0.348% dw) once the flowering began, accompanied by a decrease in remaining organs. This decrease may result from CPT being mobilized to the inflorescence as a chemical defense mechanism. These findings allowed us to determine the ideal plant harvesting age for CPT extraction. The findings could be used to decide the right stage of plant harvest, which is just before the onset of blooming. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03999-4.

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.

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.

Isolation, anticancer potency, and camptothecin-producing ability of endophytic fungi isolated from Ixora chinensis.

Camptothecin (CPT) is an important alkaloid used for anticancer treatment. It is mainly produced by two endangered and overharvested Camptotheca acuminata and Nothapodytes nimmoniana plants. Endophytic fungi are promising alternative sources for CPT production. In the present study, fungi residing within explants of Ixora chinensis were isolated and their CPT-producing capability of their endophytes was verified via thin-layer chromatography, high-performance liquid chromatography, liquid chromatography/high resolution mass spectrometry, and nuclear magnetic resonance analyses and compared with standards. In addition, MTT and sulforhodamine B assays were selected to test the anticancer effect. The endophytic fungi collection of 62 isolates were assigned to 11 genera, with four common genera (Diaporthe, Phyllosticta, Colletotrichum, and Phomopsis) and seven less common genera (Penicillium, Botryosphaeria, Fusarium, Pestalotiopsis, Aspergillus, and Didymella). Moreover, the anticancer activity of extracts was assessed against human lung carcinoma (A549). Among eight potential extracts, only Penicillium sp. I3R2 was found to be a source of CPT, while the remaining seven extracts have not been discovered potential secondary compounds. Thus, other prominent endophytic fungi might be potential candidates of phytochemicals with anticancer properties.

Engineering H2O2 Self-Supplying Platform for Xdynamic Therapies via Ru-Cu Peroxide Nanocarrier: Tumor Microenvironment-Mediated Synergistic Therapy.

Of the most common, hypoxia, overexpressed glutathione (GSH), and insufficient H2O2 concentration in the tumor microenvironment (TME) are the main barriers to the advancment of reactive oxygen species (ROS) mediated Xdynamic therapies (X = photo, chemodynamic, chemo). Maximizing Fenton catalytic efficiency is crucial in chemodynamic therapy (CDT), yet endogenous H2O2 levels are not sufficient to attain better anticancer efficacy. Specifically, there is a need to amplify Fenton reactivity within tumors, leveraging the unique attributes of the TME. Herein, for the first time, we design RuxCu1-xO2-Ce6/CPT (RCpCCPT) anticancer nanoagent for TME-mediated synergistic therapy based on heterogeneous Ru-Cu peroxide nanodots (RuxCu1-xO2 NDs) and chlorine e6 (Ce6), loaded with ROS-responsive thioketal (TK) linked-camptothecin (CPT). The Ru-Cu peroxide NDs (RCp NDs, x = 0.50) possess the highest oxygen vacancy (OV) density, which grants them the potential to form massive Lewis's acid sites for peroxide adsorption, while the dispersibility and targetability of the NDs were improved via surface modification using hyaluronic acid (HA). In TME, RCpCCPT degrades, releasing H2O2, Ru2+/3+, and Cu+/2+ ions, which cooperatively facilitate hydroxyl radical (•OH) formation and deactivate antioxidant GSH enzymes through a cocatalytic loop, resulting in excellent tumor therapeutic efficacy. Furthermore, when combined with laser treatment, RCpCCPT produces singlet oxygen (1O2) for PDT, which induces cell apoptosis at tumor sites. Following ROS generation, the TK linkage is disrupted, releasing up to 92% of the CPT within 48 h. In vitro investigations showed that laser-treated RCpCCPT caused 81.5% cell death from PDT/CDT and chemotherapy (CT). RCpCCPT in cancer cells produces red-blue emission in images of cells taking them in, which allows for fluorescence image-guided Xdynamic treatment. The overall results show that RCp NDs and RCpCCPT are more biocompatible and have excellent Xdynamic therapeutic effectiveness in vitro and in vivo.

Pold4 subunit of replicative polymerase δ promotes fork slowing at broken templates.

Single-strand breaks (SSBs) are the most frequent type of lesion, and replication across such lesions leads to double-strand breaks (DSBs). DSBs that arise during replication are repaired by homologous recombination (HR) and are suppressed by fork reversal. Poly[ADP-ribose] polymerase I (PARP1) and the proofreading exonuclease activity of replicative polymerase ε (Polε) are required for fork reversal when leading strand replication encounters SSBs. However, the mechanism underlying fork reversal at the SSB during lagging-strand replication remains elusive. We here demonstrate that the Pold4 subunit of replicative polymerase δ (Polδ) plays a role in promoting fork reversal during lagging strand replication on a broken template. POLD4-/- cells exhibited heightened sensitivity to camptothecin (CPT) but not to other DNA-damaging agents compared to wild-type cells. This selective CPT sensitivity in POLD4-/- cells suggests that Pold4 suppresses DSBs during replication, as CPT induces significant SSBs during replication, which subsequently lead to DSBs. To explore the functional interactions among Pold4, Polε exonuclease, and PARP1 in DSB suppression, we generated PARP1-/-, POLD4-/-, Polε exonuclease-deficient POLE1exo-/-, PARP1-/-/POLD4-/-, and POLD4-/-/POLE1exo-/- cells. These epistasis analyses showed that Pold4 is involved in the PARP1-Polε exonuclease-mediated fork reversal following CPT treatment. These results suggest that Pold4 aids in fork reversal when lagging strand replication stalls on a broken template. In conclusion, the Pold4 subunit of Polδ has roles in the PARP1-Polε exonuclease-mediated fork reversal, contributing to the suppression of DSBs.

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).

Polysarcosine as PEG Alternative for Enhanced Camptothecin-Induced Cancer Immunogenic Cell Death.

Nanomedicine-enhanced immunogenic cell death (ICD) has attracted considerable attention for its great potential in cancer treatment. Even though polyethylene glycol (PEG) is widely recognized as the gold standard for surface modification of nanomedicines, some shortcomings associated with this PEGylation, such as hindered cell endocytosis and accelerated blood clearance phenomenon, have been revealed in recent years. Notably, polysarcosine (PSar) as a highly biocompatible polymer can be finely synthesized by mild ring-opening polymerization (ROP) of sarcosine N-carboxyanhydrides (Sar-NCAs) and exhibit great potential as an alternative to PEG. In this article, PSar-b-polycamptothecin block copolymers are synthesized by sequential ROP of camptothecin-based NCAs (CPT-NCAs) and Sar-NCAs. Then, the detailed and systematic comparison between PEGylation and PSarylation against the 4T1 tumor model indicates that PSar decoration can facilitate the cell endocytosis, greatly enhancing the ICD effects and antitumor efficacy. Therefore, it is believed that this well-developed PSarylation technique will achieve effective and precise cancer treatment in the near future.

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.

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.

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.

Neutrophil elastase as a versatile cleavage enzyme for activation of αvß3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment.

Introduction: The development of bioconjugates for the targeted delivery of anticancer agents is gaining momentum after recent success of antibody drug conjugates (ADCs) in the clinic. Smaller format conjugates may have several advantages including better tumor penetration; however, cellular uptake and trafficking may be substantially different from ADCs. To fully leverage the potential of small molecule drug conjugates (SMDCs) with potent binding molecules mediating tumor homing, novel linker chemistries susceptible for efficient extracellular activation and payload release in the tumor microenvironment (TME) need to be explored. Methods: We designed a novel class of SMDCs, which target αvß3 integrins for tumor homing and are cleaved by neutrophil elastase (NE), a serine protease active in the TME. A peptidomimetic αvß3 ligand was attached via optimized linkers composed of substrate peptide sequences of NE connected to different functional groups of various payload classes, such as camptothecins, monomethyl auristatin E, kinesin spindle protein inhibitors (KSPi) and cyclin-dependent kinase 9 inhibitors (CDK-9i). Results: NE-mediated cleavage was found compatible with the diverse linker attachments via hindered ester bonds, amide bonds and sulfoximide bonds. Efficient and traceless release of the respective payloads was demonstrated in biochemical assays. The newly designed SMDCs were highly stable in buffer as well as in rat and human plasma. Cytotoxicity of the SMDCs in cancer cell lines was clearly dependent on NE. IC50 values were in the nanomolar or sub-nanomolar range across several cancer cell lines reaching similar potencies as compared to the respective payloads only in the presence of NE. In vivo pharmacokinetics evaluating SMDC and free payload exposures in rat and particularly the robust efficacy with good tolerability in triple negative breast and small cell lung cancer murine models demonstrate the utility of this approach for selective delivery of payloads to the tumor. Discussion: These results highlight the broad scope of potential payloads and suitable conjugation chemistries paving the way for future SMDCs harnessing the safety features of targeted delivery approaches in combination with NE cleavage in the TME.

Screening for cold tolerance genes in C. elegans, whose expressions are affected by anticancer drugs camptothecin and leptomycin B.

Temperature is a vital environmental factor affecting organisms' survival as they determine the mechanisms to tolerate rapid temperature changes. We demonstrate an experimental system for screening chemicals that affect cold tolerance in Caenorhabditis elegans. The anticancer drugs leptomycin B and camptothecin were among the 4000 chemicals that were screened as those affecting cold tolerance. Genes whose expression was affected by leptomycin B or camptothecin under cold stimuli were investigated by transcriptome analysis. Abnormal cold tolerance was detected in several mutants possessing genes that were rendered defective and whose expression altered after exposure to either leptomycin B or camptothecin. The genetic epistasis analysis revealed that leptomycin B or camptothecin may increase cold tolerance by affecting a pathway upstream of the insulin receptor DAF-2 that regulates cold tolerance in the intestine. Our experimental system combining drug and cold tolerance could be used for a comprehensive screening of genes that control cold tolerance at a low cost and in a short time period.