A NRAS mRNA G-quadruplex structure-targeting small-molecule ligand reactivating DNA damage response in human cancer cells for combination therapy with clinical PI3K inhibitors.

The Neuroblastoma RAS (NRAS) oncogene homologue plays crucial roles in diverse cellular processes such as cell proliferation, survival, and differentiation. Several strategies have been developed to inhibit NRAS or its downstream effectors; however, there is no effective drug available to treat NRAS-driven cancers and thus new approaches are needed to be established. The mRNA sequence expressing NRAS containing several guanine(G)-rich regions may form quadruplex structures (G4s) and regulate NRAS translation. Therefore, targeting NRAS mRNA G4s to repress NRAS expression at translational level with ligands may be a feasible strategy against NRAS-driven cancers but it is underexplored. We reported herein a NRAS mRNA G4-targeting ligand, B3C, specifically localized in cytoplasm in HeLa cells. It effectively downregulates NRAS proteins, reactivates the DNA damage response (DDR), causes cell cycle arrest in G2/M phase, and induces apoptosis and senescence. Moreover, combination therapy with NARS mRNA G4-targeting ligands and clinical PI3K inhibitors for cancer cells inhibition treatment is unexplored, and we demonstrated that B3C combining with PI3Ki (pictilisib (GDC-0941)) showed potent antiproliferation activity against HeLa cells (IC50 = 1.03 µM (combined with 10 µM PI3Ki) and 0.42 µM (combined with 20 µM PI3Ki)) and exhibited strong synergistic effects in inhibiting cell proliferation. This study provides new insights into drug discovery against RAS-driven cancers using this conceptually new combination therapy strategy.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 µM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Discovery of a novel class of rosmarinic acid derivatives as antibacterial agents: Synthesis, structure-activity relationship and mechanism of action.

Twenty-seven rosmarinic acid derivatives were synthesized, among which compound RA-N8 exhibited the most potent antibacterial ability. The minimum inhibition concentration of RA-N8 against both S. aureus (ATCC 29213) and MRSA (ATCC BAA41 and ATCC 43300) was found to be 6 µg/mL, and RA-N8 killed E. coli (ATCC 25922) at 3 µg/mL in the presence of polymyxin B nonapeptide (PMBN) which increased the permeability of E. coli. RA-N8 exhibited a weak hemolytic effect at the minimum inhibitory concentration. SYTOX Green assay, SEM, and LIVE/DEAD fluorescence staining assay proved that the mode of action of RA-N8 is targeting bacterial cell membranes. Furthermore, no resistance in wildtype S. aureus developed after incubation with RA-N8 for 20 passages. Cytotoxicity studies further demonstrated that RA-N8 is non-toxic to the human normal cell line (HFF1). RA-N8 also exerted potent inhibitory ability against biofilm formation of S. aureus and even collapsed the shaped biofilm.

Structurally diverse G-quadruplexes as the noncanonical nucleic acid drug target for live cell imaging and antibacterial study.

The formation of G-quadruplex structures (G4s) in vitro from guanine (G)-rich nucleic acid sequences of DNA and RNA stabilized with monovalent cations, typically K+ and Na+, under physiological conditions, has been verified experimentally and some of them have high-resolution NMR or X-ray crystal structures; however, the biofunction of these special noncanonical secondary structures of nucleic acids has not been fully understood and their existence in vivo is still controversial at present. It is generally believed that the folding and unfolding of G4s in vivo is a transient process. Accumulating evidence has shown that G4s may play a role in the regulation of certain important cellular functions including telomere maintenance, replication, transcription and translation. Therefore, both DNA and RNA G4s of human cancer hallmark genes are recognized as the potential anticancer drug target for the investigation in cancer biology, chemical biology and drug discovery. The relationship between the sequence, structure and stability of G4s, the interaction of G4s with small molecules, and insights into the rational design of G4-selective binding ligands have been intensively studied over the decade. At present, some G4-ligands have achieved a new milestone and successfully entered the human clinical trials for anticancer therapy. Over the past few decades, numerous efforts have been devoted to anticancer therapy; however, G4s for molecular recognition and live cell imaging and for application as antibacterial agents and antibiofilms against antibiotic resistance have been obviously underexplored. The recent advances in G4-ligands in these areas are thus selected and discussed concentratedly in this article in order to shed light on the emerging role of G4s in chemical biology and therapeutic prospects against bacterial infections. In addition, the recently published molecular scaffolds for designing small ligands selectively targeting G4s in live cell imaging, bacterial biofilm imaging, and antibacterial studies are discussed. Furthermore, a number of underexplored G4-targets from the cytoplasmic membrane-associated DNA, the conserved promoter region of K. pneumoniae genomes, the RNA G4-sites in the transcriptome of E. coli and P. aeruginosa, and the mRNA G4-sites in the sequence for coding the vital bacterial FtsZ protein are highlighted to further explore in G4-drug development against human diseases.

NDM-1 Zn1-binding residue His116 plays critical roles in antibiotic hydrolysis.

Bacteria expressing NDM-1 have been labeled as superbugs because it confers upon them resistance to a broad range of ß-lactam antibiotics. The enzyme has a di­zinc active centre, with the Zn2 site extensively studied. The roles of active-site Zn1 ligand residues are, however, still not fully understood. We carried out structure-function studies using the mutants, H116A, H116N, and H116Q. Zinc content analysis showed that Zn1 binding was weakened by 40 to 60% in the H116 mutants. The enzymatic-activity studies showed that the lower hydrolysis rates were mainly caused by their weaker substrate binding. The catalytic efficiency (kcat/Km) of the mutants followed the order: WT > > H116Q (decreased by 4-20 fold) > H116A (decreased by 20-700 fold) ≥ H116N (decreased by 6-800 fold). The maximum effect was observed on H116N against penicillin G, whereas ampicillin was not hydrolyzed at all. The fold-increase of Km values, which informs the weakening of substrate binding, were: H116A by 5-45 fold; H116N by 6-100 fold; H116Q by 2-10 fold. Molecular dynamics simulations suggested that the Zn1 site mutations affected the positions of Zn2 and the bridging hydroxide, by 0.8 to 1.2 Å, with the largest changes of ~1.5 Å observed on Zn2 ligand C221. A native hydrogen bond between H118 and D236 was disrupted in the H116N and H116Q mutants, which led to increased flexibility of loop 10. Consequently, residue N233 was no longer maintained at an optimal position for substrate binding. H116 connected loop 7 across Zn1 to loop 10, thereby contributed to the overall integrity. This work revealed that the H116-Zn1 interaction plays a critical role in defining the substrate-binding site. From these results, it can be inferred that inhibition strategies targeting the zinc ions may be a new direction for drug development.

Rational structural modification of the isatin scaffold to develop new and potent antimicrobial agents targeting bacterial peptidoglycan glycosyltransferase.

A series of isatin derivatives bearing three different substituent groups at the N-1, C-3 and C-5 positions of the isatin scaffold were systematically designed and synthesized to study the structure-activity relationship of their inhibition of bacterial peptidoglycan glycosyltransferase (PGT) activity and antimicrobial susceptibility against S. aureus, E. coli and methicillin-resistant Staphylococcus aureus (MRSA (BAA41)) strains. The substituents at these sites are pointing towards three different directions from the isatin scaffold to interact with the amino acid residues in the binding pocket of PGT. Comparative studies of their structure-activity relationship allow us to gain better understanding of the direction of the substituents that contribute critical interactions leading to inhibition activity against the bacterial enzyme. Our results indicate that the modification of these sites is able to maximize the antimicrobial potency and inhibitory action against the bacterial enzyme. Two compounds show good antimicrobial potency (MIC = 3 µg mL-1 against S. aureus and MRSA; 12-24 µg mL-1 against E. coli). Results of the inhibition study against the bacterial enzyme (E. coli PBP 1b) reveal that some compounds are able to achieve excellent in vitro inhibitions of bacterial enzymatic activity (up to 100%). The best half maximal inhibitory concentration (IC50) observed among the new compounds is 8.9 µM.

N-Terminal selective modification of peptides and proteins using 2-ethynylbenzaldehydes.

Selective modification of the N-terminus of peptides and proteins is a promising strategy for single site modification methods. Here we report N-terminal selective modification of peptides and proteins by using 2-ethynylbenzaldehydes (2-EBA) for the production of well-defined bioconjugates. After reaction screening with a series of 2-EBA, excellent N-terminal selectivity is achieved by the reaction in slightly acidic phosphate-buffered saline using 2-EBA with electron-donating substituents. Selective modification of a library of peptides XSKFR (X = either one of 20 natural amino acids) by 2-ethynyl-4-hydroxy-5-methoxybenzaldehyde (2d) results in good-to-excellent N-terminal selectivity in peptides (up to >99:1). Lysozyme, ribonuclease A and a therapeutic recombinant Bacillus caldovelox arginase mutant (BCArg mutant) are N-terminally modified using alkyne- and fluorescein-linked 2-EBA. Alkyne-linked BCArg mutant is further modified by rhodamine azide via copper(I)-catalyzed [3 + 2] cycloaddition indicating that the reaction has high functional group compatibility. Moreover, the BCArg mutant modified by 2-ethynyl-5-methoxybenzaldehyde (2b) exhibits comparable activity in enzymatic and cytotoxic assays with the unmodified one.