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Cell Chemical Biology May 2022We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I,...
We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I, is a precursor of the anticancer duocarmycin family that preserves the class's sequence-specific DNA binding but lacks its signature DNA alkylating cyclopropyl warhead. While less active than duocarmycin, PDE-I retains comparable antimalarial potency to chloroquine. Importantly, PDE-I is >1,000-fold less toxic to human cell lines than duocarmycin, with mitigated impacts on eukaryotic chromosome stability. PDE-I treatment induces severe defects in parasite nuclear segregation leading to impaired daughter cell formation during schizogony. Time-of-addition studies implicate parasite DNA metabolism as the target of PDE-I, with defects observed in DNA replication and chromosome integrity. We find the effect of duocarmycin and PDE-I on parasites is phenotypically indistinguishable, indicating that the DNA binding specificity of duocarmycins is sufficient and the genotoxic cyclopropyl warhead is dispensable for the parasite-specific selectivity of this compound class.
Topics: Animals; Antimalarials; Biological Products; DNA; Duocarmycins; Folic Acid Antagonists; Humans; Malaria; Parasites
PubMed: 34710358
DOI: 10.1016/j.chembiol.2021.10.005 -
Scientific Reports Oct 2021A wide range of diseases have been shown to be influenced by the accumulation of senescent cells, from fibrosis to diabetes, cancer, Alzheimer's and other age-related...
A wide range of diseases have been shown to be influenced by the accumulation of senescent cells, from fibrosis to diabetes, cancer, Alzheimer's and other age-related pathologies. Consistent with this, clearance of senescent cells can prolong healthspan and lifespan in in vivo models. This provided a rationale for developing a new class of drugs, called senolytics, designed to selectively eliminate senescent cells in human tissues. The senolytics tested so far lack specificity and have significant off-target effects, suggesting that a targeted approach could be more clinically relevant. Here, we propose to use an extracellular epitope of B2M, a recently identified membrane marker of senescence, as a target for the specific delivery of toxic drugs into senescent cells. We show that an antibody-drug conjugate (ADC) against B2M clears senescent cells by releasing duocarmycin into them, while an isotype control ADC was not toxic for these cells. This effect was dependent on p53 expression and therefore more evident in stress-induced senescence. Non-senescent cells were not affected by either antibody, confirming the specificity of the treatment. Our results provide a proof-of-principle assessment of a novel approach for the specific elimination of senescent cells using a second generation targeted senolytic against proteins of their surfaceome, which could have clinical applications in pathological ageing and associated diseases.
Topics: Cell Line; Cellular Senescence; Duocarmycins; Gene Expression Regulation; Humans; Immunoconjugates; Senotherapeutics; Tumor Suppressor Protein p53; beta 2-Microglobulin
PubMed: 34645909
DOI: 10.1038/s41598-021-99852-2 -
Drug Metabolism and Disposition: the... Jan 2022Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of...
Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the isomer of the indole-based ICT2700 over the stereoisomer. Thus the ICT2700 configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.
Topics: Antineoplastic Agents, Phytogenic; Biomarkers; Cell Line, Tumor; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Drug Design; Duocarmycins; Humans; Prodrugs; Stereoisomerism
PubMed: 34607808
DOI: 10.1124/dmd.121.000642 -
Scientific Reports Sep 2021Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the...
Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cohort Studies; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P450 Family 2; Female; Head and Neck Neoplasms; Heterocyclic Compounds, 3-Ring; Humans; Indoles; Mice; Prodrugs; Pyrroles; Xenograft Model Antitumor Assays
PubMed: 34556703
DOI: 10.1038/s41598-021-98217-z -
MAbs 2021This study describes the characterization of conjugation sites for a random, lysine conjugated 2-iminothiolane (2-IT) based antibody-drug-conjugate synthesized from an...
This study describes the characterization of conjugation sites for a random, lysine conjugated 2-iminothiolane (2-IT) based antibody-drug-conjugate synthesized from an IgG1 antibody and a duocarmycin analog-based payload-linker. Of the 80 putative lysine sites, 78 were found to be conjugated via tryptic peptide mapping and LC-HRMS. Surprisingly, seven cysteine-linked conjugated peptides were also detected resulting from the conjugation of cysteine residues derived from the four inter-chain disulfide bonds during the reaction. This unexpected finding could be attributed to the free thiols of the 2-IT thiolated antibody intermediates and/or the 4-mercaptobutanamide by-product resulting from the hydrolysis of 2-IT. These free thiols could cause the four inter-chain disulfide bonds of the antibody to scramble via intra- or inter-molecular attack. The presence of only pair of non-reactive (unconjugated) lysine residues, along with the four intact intra-chain disulfide bonds, is attributed to their poor accessibility, which is consistent with solvent accessibility modeling analysis. We also discovered a major by-product derived from the hydrolysis of the amidine moiety of the -terminus conjugate. In contrast, the amidine moiety in lysine-linked conjugates appeared stable. Based on our results, we propose plausible formation mechanisms of cysteine-linked conjugates and the hydrolysis of the -terminus conjugate, which provide scientific insights that are beneficial to process development and drug quality control.
Topics: Cysteine; Drug Discovery; Duocarmycins; Humans; Immunoconjugates; Immunoglobulin G; Lysine
PubMed: 34486490
DOI: 10.1080/19420862.2021.1974150 -
Cancer Chemotherapy and Pharmacology Oct 2021Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by...
PURPOSE
Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue.
METHODS
Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively.
RESULTS
NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug.
CONCLUSIONS
The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.
Topics: Bystander Effect; Cell Hypoxia; Chromatography, Liquid; Coculture Techniques; Escherichia coli Proteins; HCT116 Cells; Humans; Indoles; Models, Biological; NADPH-Ferrihemoprotein Reductase; Nitroreductases; Prodrugs; Spheroids, Cellular; Tandem Mass Spectrometry
PubMed: 34245333
DOI: 10.1007/s00280-021-04320-3 -
Drug Discovery Today Aug 2021Duocarmycins are a class of DNA minor-groove-binding alkylating molecules. For the past decade, various duocarmycin analogues have been used as payloads in the... (Review)
Review
Duocarmycins are a class of DNA minor-groove-binding alkylating molecules. For the past decade, various duocarmycin analogues have been used as payloads in the development of antibody-drug conjugates (ADCs). Currently, more than 15 duocarmycin-based ADCs have been studied preclinically, and some of them such as SYD985 have been granted Fast-Track Designation status. Nevertheless, progress in duocarmycin-based ADCs also faces challenges, with setbacks including the termination of BMS-936561/MDX-1203. In this review, we discuss issues associated with the efficacy, pharmacokinetic profile, and toxicological activity of these biotherapeutics. Furthermore, we summarize the latest advances in duocarmycin-based ADCs that have different target specificities and linker chemistries. Evidence from preclinical and clinical studies has indicated that duocarmycin-based ADCs are promising biotherapeutics for oncological application in the future.
Topics: Animals; Antineoplastic Agents; Drug Development; Drug Evaluation, Preclinical; Duocarmycins; Humans; Immunoconjugates; Neoplasms
PubMed: 34224904
DOI: 10.1016/j.drudis.2021.06.012 -
Molecules (Basel, Switzerland) Jun 2021Triple-negative breast cancer (TNBC) is a heterogeneous subtype of tumors that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein....
Triple-negative breast cancer (TNBC) is a heterogeneous subtype of tumors that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. The mainstay of treatment remains chemotherapy, but the therapeutic outcome remains inadequate. This paper investigates the potential of a duocarmycin derivative, tafuramycin A (TFA), as a new and more effective chemotherapy agent in TNBC treatment. To this extent, we optimized the chemical synthesis of TFA, and we encapsulated TFA in a micellar system to reduce side effects and increase tumor accumulation. In vitro and in vivo studies suggest that both TFA and SMA-TFA possess high anticancer effects in TNBC models. Finally, the encapsulation of TFA offered a preferential avenue to tumor accumulation by increasing its concentration at the tumor tissues by around four times in comparison with the free drug. Overall, the results provide a new potential strategy useful for TNBC treatment.
Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Female; Humans; Indole Alkaloids; Maleates; Mice; Mice, Inbred BALB C; Micelles; Nanoparticles; Polystyrenes; Triple Negative Breast Neoplasms
PubMed: 34207832
DOI: 10.3390/molecules26123532 -
Frontiers in Pharmacology 2021Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For...
Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise 'off-switch' in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications.
PubMed: 34163368
DOI: 10.3389/fphar.2021.701456 -
Bioorganic & Medicinal Chemistry Jun 2021The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and...
The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Duocarmycins; Humans; Indoles; Molecular Structure; Structure-Activity Relationship
PubMed: 33932713
DOI: 10.1016/j.bmc.2021.116167