Mutual Prodrugs of 5-Fluorouracil: From a Classic Chemotherapeutic Agent to Novel Potential Anticancer Drugs

The development of potent antitumor agents with a low toxicological profile against healthy cells is still one of the greatest challenges facing medicinal chemistry. In this context, the “mutual prodrug” approach has emerged as a potential tool to overcome undesirable physicochemical features and mitigate the side effects of approved drugs. Among broad-spectrum chemotherapeutics available for clinical use today, 5-fluorouracil (5-FU) is one of the most representative, also included in the World Health Organization model list of essential medicines. Unfortunately, severe side effects and drug resistance phenomena are still the primary limits and drawbacks in its clinical use. This review describes the progress made over the last ten years in developing 5-FU-based mutual prodrugs to improve the therapeutic profile and achieve targeted delivery to cancer tissues.     

Synthesis of Isocryptolepine-Triazole Adducts and Evaluation of Their Cytotoxic Activity

Eighteen hybrid compounds between 8-bromo-2-fluoro-isocryptolepine ( 4 ) and 1,2,3-triazole were synthesized via azide rearrangement-annulation reaction. Compound 4 underwent regioselective N-propargylation and click reaction to form 8-bromo-2-fluoro-isocryptolepine-triazole hybrids 11 which were evaluated for cytotoxic activity. Compound 11 c containing 1-anisyltriazole was the most effective in inhibiting HepG2, HuCCA-1 and A549 cell lines (IC₅₀ values of 1.65–3.07 μM) while compounds 11 a (1-phenyltriazole), 11 j (1-para-CF₃-benzyltriazole) and 11 l (1-meta-Cl-benzyltriazole) were potent inhibitors of HuCCA-1, HepG2 and A549 cell lines, respectively. Moreover, 11 l showed the lowest cytotoxicity to normal human kidney cell line. Compounds 11 c and 11 l provided improvement of cytotoxic activity over 4 . Compounds 4 , 11 c and 11 l were selected to investigate their mechanisms of action. The results showed that 4 could induce G2/M cell cycle arrest and was involved in the upregulation of p53 and p21 proteins. However, the mechanisms of growth inhibition by 11 c and 11 l were associated with G0/G1 cell cycle arrest and mediated by induction of oxidative stress.     

Cover Feature: Superior Proton-Transfer Catalytic Promiscuity of Cytochrome c in Self-Organized Media (7/2021)

Evolutionarily elderly proteins commonly feature greater catalytic promiscuity. Cytochrome c is among the first set of proteins in evolution to have known prospects in electron transport and peroxidative properties. Here, we report that cyt c is also a proficient proton-transfer catalyst and enhances the Kemp elimination (KE; model reaction to show proton transfer catalytic property) by ∼750-fold on self-organized systems like micelles and vesicles. The self-organized systems mimic the mitochondrial environment in vitro for cyt c. Using an array of biophysical and biochemical mutational assays, both acid–base and redox mechanistic pathways have been explored. The histidine moiety close to hemin group (His18) is mainly responsible for proton abstraction to promote the concerted E2 pathway for KE catalysis when cyt c is in its oxidized form; this has also been confirmed by a H18A mutant of cyt c. However, the redox pathway is predominant under reducing conditions in the presence of dithiothreitol over the pH range 6–7.4. Interestingly, we found almost 750-fold enhanced KE catalysis by cyt c compared to aqueous buffer. Overall, in addition to providing mechanistic insights, the data reveal an unprecedented catalytic property of cyt c that could be of high importance in an evolutionary perspective considering its role in delineating the phylogenic tree and also towards generating programmable designer biocatalysts.     

A “Three-in-One” Multifunctional Probe for Bcl-2/Mcl-1 Profiling and Visualizing in Situ

Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously in situ. Herein, we report a multifunctional molecular probe (BnN₃-OPD-Alk) by a “three-in-one” molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN₃-OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both in vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.     

Preparation and Bioactivity of Iridium(III) Phenanthroline Complexes with Halide Ions and Pyridine Leaving Groups

A series of half-sandwich structural iridium(III) phenanthroline (Phen) complexes with halide ions (Cl⁻, Br⁻, I⁻) and pyridine leaving groups ([(η⁵-Cp^X)Ir(Phen)Z](PF₆)_n, Cp^x: electron-rich cyclopentadienyl group, Z: leaving group) have been prepared. Target complexes, especially the Cp^xbiph (biphenyl-substituted cyclopentadienyl)-based one, showed favourable anticancer activity against human lung cancer (A549) cells; the best one ( Ir8 ) was almost five times that of cisplatin under the same conditions. Compared with complexes involving halide ion leaving groups, the pyridine-based one did not display hydrolysis but effectively caused lysosomal damage, leading to accumulation in the cytosol, inducing an increase in the level of intracellular reactive oxygen species and apoptosis; this indicated an anticancer mechanism of oxidation. Additionally, these complexes could bind to serum albumin through a static quenching mechanism. The data highlight the potential value of half-sandwich iridium(III) phenanthroline complexes as anticancer drugs.     

An Autophagy-Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation

A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described. This compound was identified from cell-based high-content screening of an imidazole library. The results showed that Atz was internalized into lysosomes of cells where it induced lysosomal membrane permeabilization (LMP). This process generated nonfunctional autolysosomes, thereby inhibiting autophagy. In addition, Atz was found to promote LMP-mediated apoptosis. Specifically, LMP induced by Atz caused release of cathepsins from lysosomes into the cytosol. Cathepsins in the cytosol cleaved Bid to generate tBid, which subsequently activated Bax to induce mitochondrial outer membrane permeabilization (MOMP). This event led to cancer cell death via caspase activation. Overall, the findings suggest that Atz will serve as a new chemical probe in efforts aimed at gaining a better understanding of the autophagic process.     

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.     

Identification of the Catalytic Residues in the Cyclase Domain of the Class IV Lanthipeptide Synthetase SgbL

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into different classes based on their processing enzymes. The three-domain class IV lanthipeptide synthetases (LanL enzymes) consist of N-terminal lyase, central kinase, and C-terminal cyclase domains. While the catalytic residues of the kinase domains (mediating ATP-dependent Ser/Thr phosphorylations) and the lyase domains (carrying out subsequent phosphoserine/phosphothreonine (pSer/pThr) eliminations to yield dehydroalanine/dehydrobutyrine (Dha/Dhb) residues) have been characterized previously, such studies are missing for LanL cyclase domains. To close this gap of knowledge, this study reports on the identification and validation of the catalytic residues in the cyclase domain of the class IV lanthipeptide synthetase SgbL, which facilitate the nucleophilic attacks by Cys thiols on Dha/Dhb residues for the formation of β-thioether crosslinks.     

Efficient Linear dsDNA Tagging Using Deoxyuridine Excision**

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3’ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3’ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5’ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.     

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip - 3 retained the α-helical structure and bound to AurA with a K_D value of 13.7 μM. Bip - 3 and the adenosine-tethered peptide Bip - 3 - Adc provided IC₅₀ values of 103 μM and 7.7 μM, respectively, suggesting that Bip - 3 - Adc bivalently inhibited AurA. In addition, the selectivity of Bip - 3 - Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.