CD32 (FcγRIIB) expression is low on CD21low B cells from systemic sclerosis patients with digital ulcers, interstitial lung disease, and anti-topoisomerase I autoantibodies.

CD21low B cells have recently been found increased in SSc-associated digital ulcers (DUs) or interstitial lung disease (ILD). To further characterize CD21low B cells which encompass autoreactive cells, we analyzed their expression of the inhibitory CD32 receptor in SSc. Peripheral blood mononuclear cells from 27 patients with SSc and 15 age-and sex-matched healthy controls (HCs) were analyzed with multicolor flow cytometry. CD21low B cells were significantly increased in patients with DUs (51.3%) compared to HCs (28.1%) and in patients with ILD (53.1%) compared to HCs. CD21lowCD32low B cells were significantly increased in patients with DUs (23.8%) compared to HCs (4.4%), in patients with ILD (28.4%) compared to HCs, and in anti-topoisomerase I (+) patients (21.5%) compared to HCs and to anti-topoisomerase I (-) patients (2.4%). Autoreactive B cells recognizing Topoisomerase I were predominantly within CD32low cell fraction. Our study further supports the autoreactive status of CD21lowCD32low B cells in SSc patients.

TROPHY-U-01, a phase II open-label study of sacituzumab govitecan in patients with metastatic urothelial carcinoma progressing after platinum-based chemotherapy and checkpoint inhibitors: updated safety and efficacy outcomes.

BACKGROUND: Sacituzumab govitecan (SG) is a Trop-2-directed antibody-drug conjugate containing cytotoxic SN-38, the active metabolite of irinotecan. SG received accelerated US Food and Drug Administration approval for locally advanced (LA) or metastatic urothelial carcinoma (mUC) previously treated with platinum-based chemotherapy and a checkpoint inhibitor, based on cohort 1 of the TROPHY-U-01 study. Mutations in the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene are associated with increased adverse events (AEs) with irinotecan-based therapies. Whether UGT1A1 status could impact SG toxicity and efficacy remains unclear. PATIENTS AND METHODS: TROPHY-U-01 (NCT03547973) is a multicohort, open-label, phase II registrational study. Cohort 1 includes patients with LA or mUC who progressed after platinum- and checkpoint inhibitor-based therapies. SG was administered at 10 mg/kg intravenously on days 1 and 8 of 21-day cycles. The primary endpoint was objective response rate (ORR) per central review; secondary endpoints included progression-free survival, overall survival, and safety. Post hoc safety analyses were exploratory with descriptive statistics. Updated analyses include longer follow-up. RESULTS: Cohort 1 included 113 patients. At a median follow-up of 10.5 months, ORR was 28% (95% CI 20.2% to 37.6%). Median progression-free survival and overall survival were 5.4 months (95% CI 3.5-6.9 months) and 10.9 months (95% CI 8.9-13.8 months), respectively. Occurrence of grade ≥3 treatment-related AEs and treatment-related discontinuation were consistent with prior reports. UGT1A1 status was wildtype (∗1|∗1) in 40%, heterozygous (∗1|∗28) in 42%, homozygous (∗28|∗28) in 12%, and missing in 6% of patients. In patients with ∗1|∗1, ∗1|∗28, and ∗28|∗28 genotypes, any grade treatment-related AEs occurred in 93%, 94%, and 100% of patients, respectively, and were managed similarly regardless of UGT1A1 status. CONCLUSIONS: With longer follow-up, the ORR remains high in patients with heavily pretreated LA or mUC. Safety data were consistent with the known SG toxicity profile. AE incidence varied across UGT1A1 subgroups; however, discontinuation rates remained relatively low for all groups.

Activation of STING by the novel liposomal TLC388 enhances the therapeutic response to anti-PD-1 antibodies in combination with radiotherapy.

Current immune checkpoint inhibiters (ICIs) have contrasting clinical results in poorly immunogenic cancers such as microsatellite-stable colorectal cancer (MSS-CRC). Therefore, understanding and developing the combinational therapeutics for ICI-unresponsive cancers is critical. Here, we demonstrated that the novel topoisomerase I inhibitor TLC388 can reshape the tumor immune landscape, corroborating their antitumor effects combined with radiotherapy as well as immunotherapy. We found that TLC388 significantly triggered cytosolic single-stranded DNA (ssDNA) accumulation for STING activation, leading to type I interferons (IFN-Is) production for increased cancer immunogenicity to enhance antitumor immunity. TLC388-treated tumors were infiltrated by a vast number of dendritic cells, immune cells, and costimulatory molecules, contributing to the favorable antitumor immune response within the tumor microenvironment. The infiltration of cytotoxic T and NK cells were more profoundly existed within tumors in combination with radiotherapy and ICIs, leading to superior therapeutic efficacy in poorly immunogenic MSS-CRC. Taken together, these results showed that the novel topoisomerase I inhibitor TLC388 increased cancer immunogenicity by ssDNA/STING-mediated IFN-I production, enhancing antitumor immunity for better therapeutic efficacy in combination with radiotherapy and ICIs for poorly immunogenic cancer.

Two-Phase Electrosynthesis of Dihydroxycoumestans: Discovery of a New Scaffold for Topoisomerase I Poison.

Coumestan represents a biologically relevant structural motif distributed in a number of natural products, and the rapid construction of related derivatives as well as the characterization of targets would accelerate lead compound discovery in medicinal chemistry. In this work, a general and scalable approach to 8,9-dihydroxycoumestans via two-electrode constant current electrolysis was developed. The application of a two-phase (aqueous/organic) system plays a crucial role for success, protecting the sensitive o-benzoquinone intermediates from over-oxidation. Based on the structurally diverse products, a primary SAR study on coumestan scaffold was completed, and compound 3 r exhibited potent antiproliferative activities and a robust topoisomerase I (Top1) inhibitory activity. Further mechanism studies demonstrates that compound 3 r was a novel Top1 poison, which might open an avenue for the development of Top1-targeted antitumor agent.

Design, synthesis, and biological evaluation of novel benzo[6,7]indolo[3,4-c]isoquinolines as anticancer agents with topoisomerase I inhibition.

A novel series of benzo[6,7]indolo[3,4-c]isoquinolines 3a-3f was designed by scaffold hopping of topoisomerase I inhibitor benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs), which were developed by structural modification of the natural marine product lamellarin. The unconventional pentacycle was constructed by Bischler-Napieralski-type condensation of amide 11 and subsequent intramolecular Heck reaction. In vitro anticancer activity of the synthesized benzo[6,7]indolo[3,4-c]isoquinolines was evaluated on a panel of 39 human cancer cell lines (JFCR39). Among the compounds tested, N-(3-morpholinopropyl) derivative 3e showed the most potent antiproliferative activity, with a mean GI50 value of 39 nM. This compound inhibited topoisomerase I activity by stabilizing the enzyme-DNA complex.

Identification of FTY720 and COH29 as novel topoisomerase I catalytic inhibitors by experimental and computational studies.

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.

Spectroscopic, biochemical and computational studies of bioactive DNA minor groove binders targeting 5'-WGWWCW-3' motif.

Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5'-d(CGCACTAGTGCG)-3' and 5'-d(CGCAGTACTGCG)-3'. The ligands were carefully designed to target the DNA response element, 5'-WGWWCW-3', the binding site for several nuclear receptors. Basic 1D 1H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D 1H and 31P-{1H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents.

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.

Natural Immunomodulatory Agents as a Complementary Therapy for Poxviruses.

Poxviruses target innate immunity mediators such as tumor necrosis factors, interleukins, interferons, complement, and chemokines. It also targets adaptive immunity such as CD4+ T cells, CD4+ T cells, and B cells. Emerging of the recent epidemic of monkeypox virus (MPXV), a zoonotic disease native to Central and Western Africa, besides the lack of permitted treatments for poxviruses infections, encouraged researchers to identify effective inhibitors to help in preventing and treating poxviruses infections. Natural bioactive components, particularly polyphenolics, are promising for creating powerful antioxidants, anti-inflammatory, immune-stimulating, and antiviral agents. As a result, they are potentially effective therapies for preventing and treating viral diseases, such as infections caused by poxviruses including the recent pandemic MPXV. Polyphenolics: rosmarinic acid, caffeic acid, resveratrol, quercitrin, myricitrin, gingerol, gallotannin, and propolis-benzofuran A, as well as isoquinoline alkaloids: galanthamine and thalimonine represent prospective antiviral agents against MPXV, they can inhibit MPXV and other poxviruses via targeting different viral elements including DNA Topoisomerase I (TOP1), Thymidine Kinase (TK), serine/threonine protein kinase (Ser/Thr kinase), and protein A48R. The bioactive extracts of different traditional plants including Guiera senegalensis, Larrea tridentata, Sarracenia purpurea, Kalanchoe pinnata (Lam.) Pers., Zingiber officinale Roscoe, Quercus infectoria, Rhus chinensis, Prunella vulgaris L., Salvia rosmarinus, and Origanum vulgare also can inhibit the growth of different poxviruses including MPXV, vaccinia virus (VACV), variola virus, buffalopox virus, fowlpox virus, and cowpox virus. There is an urgent need for additional molecular studies to identify and confirm the anti-poxviruses properties of various natural bioactive components, especially those that showed potent antiviral activity against other viruses.

Inhibition of DNA Topoisomerase Ι by Flavonoids and Polyacetylenes Isolated from Bidens pilosa L.

Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2-19) and nine polyacetylenes (21-29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 µM, respectively. The structure-activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.

Molecular Dynamics Investigations of Human DNA-Topoisomerase I Interacting with Novel Dewar Valence Photo-Adducts: Insights into Inhibitory Activity.

Chronic exposure to ultraviolet (UV) radiation is known to induce the formation of DNA photo-adducts, including cyclobutane pyrimidine dimers (CPDs) and Dewar valence derivatives (DVs). While CPDs usually occur at higher frequency than DVs, recent studies have shown that the latter display superior selectivity and significant stability in interaction with the human DNA/topoisomerase 1 complex (TOP1). With the aim to deeply investigate the mechanism of interaction of DVs with TOP1, we report here four all-atom molecular dynamic simulations spanning one microsecond. These simulations are focused on the stability and conformational changes of two DNA/TOP1-DV complexes in solution, the data being compared with the biomimetic thymine dimer counterparts. Results from root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) analyses unequivocally confirmed increased stability of the DNA/TOP1-DV complexes throughout the simulation duration. Detailed interaction analyses, uncovering the presence of salt bridges, hydrogen bonds, water-mediated interactions, and hydrophobic interactions, as well as pinpointing the non-covalent interactions within the complexes, enabled the identification of specific TOP1 residues involved in the interactions over time and suggested a potential TOP1 inhibition mechanism in action.

QSAR prediction, synthesis, anticancer evaluation, and mechanistic investigations of novel sophoridine derivatives as topoisomerase I inhibitors.

Sophoridine, which is derived from the Leguminous plant Sophora alopecuroides L., has certain pharmacological activity as a new anticancer drug. Herein, a series of novel N-substituted sophoridine derivatives was designed, synthesized and evaluated with anticancer activity. Through QSAR prediction models, it was discovered that the introduction of a benzene ring as a main pharmacophore and reintroduced into a benzene in para position on the phenyl ring in the novel sophoridine derivatives improved the anticancer activity effectively. In vitro, 28 novel compounds were evaluated for anticancer activity against four human tumor cell lines (A549, CNE-2, HepG-2, and HEC-1-B). In particular, Compound 26 exhibited remarkable inhibitory effects, with an IC50 value of 15.6 µM against HepG-2 cells, surpassing cis-Dichlorodiamineplatinum (II). Molecular docking studies verified that the derivatives exhibit stronger binding affinity with DNA topoisomerase I compared to sophoridine. In addition, 26 demonstrated significant inhibition of DNA Topoisomerase I and could arrest cells in G0/G1 phase. This study provides valuable insights into the design and synthesis of N-substituted sophoridine derivatives with anticancer activity.

Unveiling the mechanism of hesperidin-induced LdTopI-mediated cell death pathway in protozoan parasite Leishmania donovani.

Unicellular protozoan parasite Leishmania donovani is the causative agent for visceral leishmaniasis (VL) or Kala-azar, a neglected fatal parasitic disease. The conventional treatment of VL consists of therapeutic agents having several shortcomings such as toxicity, high cost, efficacy variance and increased drug resistance. Therefore, there is a desperate need to develop an effective treatment against the parasite. Previous reports suggested that flavonoids can inhibit the enzyme Leishmania donovani DNA topoisomerase I (LdTopILS). Therefore, for the first time in this present study, we divulge HSP (one of the natural sources of flavonoids), as a potent natural antileishmanial compound with efficacy in BALB/c mice at 20 mg/kg of body weight, inhibits LdTopILS at 97 % of its activity at 160 µM in preincubation condition (competitively). It binds with free enzyme and does not allow it to bind with the substrate DNA. Moreover, HSP does not stabilize DNA-topoisomerase I cleavable complex. Thus, HSP acts a catalytic topoisomerase I inhibitor, which inhibits complete activity by binding with Lys269 and Thr411 of large subunit of the enzyme. On the other hand, HSP induces the topo I-mediated programmed cell death process by the formation of cellular reactive oxygen species, resulting in depolarization of mitochondrial membrane potential, followed by fragmentation of nuclear DNA. Therefore, the present study illuminates a natural flavonoid that in future might be a promising lead for the treatment of VL.

Design, synthesis, and biological evaluation of harmine derivatives as topoisomerase I inhibitors for cancer treatment.

A series of ß-carboline derivatives were designed and synthesized by introducing the chalcone moiety into the harmine. The synthesized derivatives were evaluated their anti-proliferative activities against six human cancer cell lines (MCF-7, MDA-MB-231, HepG2, HT29, A549, and PC-3) and one normal cell line (L02). Among them, compound G11 exhibited the potent anti-proliferative activity against MCF-7 cell line, with an IC50 value of 0.34 µM. Further biological studies revealed that compound G11 inhibited colony formation of MCF-7 cells, suppressed MCF-7 cell migration by downregulating migration-associated protein MMP-2. In addition, it could induce apoptosis of MCF-7 cells by downregulating Bcl-2 and upregulating Cleaved-PARP, Bax, and phosphorylated Bim proteins. Furthermore, compound G11 can act as a Topo I inhibitor, affecting DNA synthesis and transcription, thereby inhibiting cancer cell proliferation. Moreover, compound G11 inhibited tumor growth in 4T1 syngeneic transplant mice with an inhibition rate of 43.19 % at a dose of 10 mg/kg, and 63.87 % at 20 mg/kg, without causing significant toxicity to the mice or their organs, achieving the goal of reduced toxicity and increased efficacy. All these results indicate of G11 has enormous potential as an anti-tumor agent and merits further investigation.

Dimeric Bis-Benzimidazole-Pyrroles DB2Py(n) - AT-Site-Specific Ligands: Synthesis, Physicochemical Analysis, and Biological Activity.

Its broad spectrum of biological activity makes benzimidazole a fundamental pharmacophore in pharmaceutics. The paper describes newly synthesized AT-specific fluorescent bis-benzimidazole molecules DB2Py(n) that contain a pyrrolcarboxamide fragment of the antibiotic drug netropsin. Physico-chemical methods using absorption, fluorescence, and circular dichroism spectra have shown the ability of bis-benzimidazole- pyrroles to form complexes with DNA. The new DB2Py(n) series have turned out to be more toxic to human tumor lines and less vulnerable to non-tumor cell lines. Bis-benzimidazole-pyrroles penetrated the cell nucleus, affected the cell-cycle synthesis (S) phase, and inhibited eukaryotic topoisomerase I in a cellfree model at low concentrations. A real-time tumor cell proliferation test confirmed the molecule's enhanced toxic properties upon dimerization. Preliminary cytotoxicity data for the bis-benzimidazole-pyrroles tested in a cell model with a MDR phenotype showed that monomeric compounds can overcome MDR, while dimerization weakens this ability to its intermediate values as compared to doxorubicin. In this respect, the newly synthesized cytotoxic structures seem promising for further, in-depth study of their properties and action mechanism in relation to human tumor cells, as well as for designing new AT-specific ligands.

Levels of anti-topoisomerase I antibody correlated with short onset of cardiopulmonary involvement in Thai systemic sclerosis patients.

Anti-topoisomerase-I antibody (ATA) is associated with disease severity and internal organ involvement in patients with systemic sclerosis (SSc). The correlation between ATA levels and the clinical course of SSc is unclear. We aimed to determine the correlation between ATA level and survival time and the onset of internal organ fibrosis in SSc patients. This historical cohort study was conducted in adult SSc patients with quantitative tests of ATA between January 2019 and December 2022. Patients with overlap syndrome and no quantitative ATA test were excluded. According to the sample size calculation, and 10% compensated for missing data, a total of 153 patients were needed. The respective mean age on the study date and median ATA level was 59.9 ± 11.3 years and 370 U/mL (range 195-652). Most cases (107 cases; 69.9%) were the diffuse cutaneous SSc subset. According to a multivariable analysis, the ATA titer had a negative correlation with the onset of cardiac involvement (Rho - 0.47, p = 0.01), and had a positive correlation with skin thickness progression (Rho 0.39, p = 0.04). Eleven cases exhibited ATA levels < 7 U/mL and outlier ATA levels were excluded, 142 cases were included in the sensitivity analysis, and multivariable analysis showed the correlation between early onset of ILD and cardiac involvement (Rho - 0.43, p = 0.03 and Rho - 0.51, p = 0.01, respectively). The ATA level was correlated with neither the survival time nor the onset of renal crisis in both analyses. High ATA levels were correlated with a short onset of ILD and cardiac involvement and the presence of extensive skin tightness. Quantitative tests of ATA could serve as an effective tool for identifying patients at risk of an unfavorable prognosis.

Exploratory clinical subgroup clustering in systemic sclerosis: Results from the Indian Progressive Systemic Sclerosis Registry.

Objective: To conduct an exploratory cluster analysis of systemic sclerosis patients from the baseline data of the Indian systemic sclerosis registry. Methods: Patients satisfying American College of Rheumatology-European League Against Rheumatism classification criteria for systemic sclerosis were included. The clusters formed using clinical and immunological parameters were compared. Results: Of the 564 systemic sclerosis registry participants, 404 patients were included. We derived four clusters of which three were anti-topoisomerase I predominant and one was anti-centromere antibody 2 dominant. Cluster 1 (n-82 (20.3%)) had diffuse cutaneous systemic sclerosis patients with the most severe skin disease, anti-topoisomerase I positivity, males, younger age of onset and high prevalence of musculoskeletal, vasculopathic and gastrointestinal features. Cluster 2 (n-141 (34.9%)) was also diffuse cutaneous systemic sclerosis and anti-topoisomerase I predominant but with less severe skin phenotype than cluster 1 and a lesser prevalence of musculoskeletal, vasculopathic and gastrointestinal features. Cluster 3 (n-119 (29.5%)) had limited cutaneous systemic sclerosis patients with anti-topoisomerase I positivity along with other antibodies. The proximal muscle weakness was higher and digital pitting scars were lower, while other organ involvement was similar between clusters 2 and 3. Cluster 4 (n-62 (15.30%)) was the least severe group with limited cutaneous systemic sclerosis and anti-centromere antibody predominance. Age of onset was higher with low musculoskeletal disease and a higher presence of upper gastrointestinal features. The prevalence of interstitial lung disease was similar in the three anti-topoisomerase I predominant clusters. Conclusion: With exploratory cluster analysis, we confirmed the possibility of subclassification of systemic sclerosis along a spectrum based on clinical and immunological characteristics. We also corroborated the presence of anti-topoisomerase I in limited cutaneous systemic sclerosis and the association of interstitial lung disease with anti-topoisomerase I.

Camptothecin structure simplification elaborated new imidazo[2,1-b]quinazoline derivative as a human topoisomerase I inhibitor with efficacy against bone cancer cells and colon adenocarcinoma.

Camptothecin is a pentacyclic natural alkaloid that inhibits the hTop1 enzyme involved in DNA transcription and cancer cell growth. Camptothecin structure pitfalls prompted us to design new congeners using a structure simplification strategy to reduce the ring extension number from pentacyclic to tetracyclic while maintaining potential stacking of the new compounds with the DNA base pairs at the Top1-mediated cleavage complex and aqueous solubility, as well as minimizing compound-liver toxicity. The principal axis of this study was the verification of hTop1 inhibiting activity as a possible mechanism of action and the elaboration of new simplified inhibitors with improved pharmacodynamic and pharmacokinetic profiling using three structure panels (A-C) of (isoquinolinoimidazoquinazoline), (imidazoquinazoline), and (imidazoisoquinoline), respectively. DNA relaxation assay identified five compounds as hTop1 inhibitors belonging to the imidazoisoquinolines 3a,b, the imidazoquinazolines 12, and the isoquinolinoimidazoquinazolines 7a,b. In an MTT cytotoxicity assay against different cancer cell lines, compound 12 was the most potent against HOS bone cancer cells (IC50 = 1.47 µM). At the same time, the other inhibitors had no detectable activity against any cancer cell type. Compound (12) demonstrated great penetrating power in the HOS cancer cells' 3D-multicellular tumor spheroid model. Bioinformatics research of the hTop1 gene revealed that the TP53 cell proliferative gene is in the network of hTop1. The finding is confirmed empirically using the gene expression assay that proved the increase in p53 expression. The impact of structure simplification on compound 12 profile, characterized by the absence of acute oral liver toxicity when compared to Doxorubicin as a standard inhibitor, the lethal dose measured on Swiss Albino female mice and reported at LD50 = 250 mg/kg, and therapeutic significance in reducing colon adenocarcinoma tumor volume by 75.36 % after five weeks of treatment with compound 12. The molecular docking solutions of the active CPT-based derivative 12 and the inactive congener 14 into the active site of hTop1 and the activity cliffing of such MMP directed us to recommend the addition of HBD and HBA variables to compound 12 imidazoquinazoline core scaffold to enhance the potency via hydrogen bond formation with the major groove amino acids (Asp533, Lys532) as well as maintaining the hydrogen bond with the minor groove amino acid Arg364.

Methods to Quantitatively Measure Topological Changes Induced by DNA-Binding Proteins In Vivo and In Vitro.

Agarose gel electrophoresis in the presence of chloroquine (an intercalating agent) can be used to resolve and characterize the population of topoisomers present in supercoiled plasmid DNA. Here, we describe how chloroquine gel electrophoresis can capture changes in the topoisomer distribution of plasmid DNA that bears a recognition site for a given protein, if that plasmid is isolated from cells producing the protein of interest. We also describe two complementary in vitro assays, which can be used to capture transient changes in DNA supercoiling caused when the purified protein of interest engages its recognition site. These are the topoisomerase I-mediated relaxation assay (TMRA) and the ligase-mediated supercoiling assay (LMSA). Together, these in vivo and in vitro methods allow the capture and measurement of changes in DNA topology that are triggered by DNA-binding proteins, especially those that multimerize on or spread along DNA.

TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.