Discovery of Thioether-Cyclized Macrocyclic Covalent Inhibitors by mRNA Display.

Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS). Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date. This study outlines the workflow for integrating chemical functionalization─installation of a covalent warhead─with mRNA display and showcases its application in targeted covalent ligand discovery.

Gas-phase solvent-free complexation of phenolic acids in cyclodextrins: A mass spectrometric study.

Phenolic acids are phytochemicals commonly existing in plants that are also beneficial to the human body after consumption. As additives in the food industry, phenolic acids typically need stabilization by encapsulation. We have been studying cyclodextrins as potential encapsulating agents, elucidating the structure and energetics of the complexes they form with phenolic acids. To highlight the role of the solvent in these interactions and to investigate the possible component ratios available to these complexes, we first carried out gas-phase studies monitored through mass spectrometry that allowed us to work in solvent-free conditions. In addition to detecting the products of these complexation equilibria qualitatively, we were also able to find conditions to quantitative determine the relative binding affinities associated with these processes. As well as on the unusual complex stoichiometry observed in the gas phase for a series of phenolic acids with α-CD, ß-CD, and γ-CD. We propose an explanation of these uncommon stoichiometries through solvation effects involving the cyclodextrin host as the solvating species. We also report on the observed trends in the measured relative affinities of these interactions.

Development of Prodrug-Payloads for Targeted Therapeutic Applications of Platinum-Acridine Anticancer Agents.

A synthetic platform has been developed that provides access to platinum(IV) prodrugs of highly cytotoxic platinum-acridine anticancer agents and allows them to be incorporated into conjugation-ready prodrug-payloads (PPLs). The PPLs can be conveniently assembled in highly efficient microscale reactions utilizing strain-promoted azide-alkyne cycloaddition chemistry. Model reactions were performed to study the stability of the PPLs in buffers and media and to assess their compatibility with cysteine-maleimide Michael addition chemistry. Amide coupling was a successful strategy to generate a conjugate containing integrin-targeted cyclo[RGDfK] peptide. Reactions with ascorbate were performed to mimic the reductive activation of the PPLs and the latter conjugate, and a cyanine (Cy5) fluorophore-labeled PPL was used to probe the reduction of platinum(IV) in cancer cells by confocal microscopy. The PPL concept introduced here should be evaluated for treating solid tumors with PAs using cancer-targeting vehicles, such as antibody-drug conjugates.

Transcriptome-Wide Studies of RNA-Targeted Small Molecules Provide a Simple and Selective r(CUG)exp Degrader in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG)exp, harbored in the 3' untranslated region (3' UTR) of dystrophia myotonica protein kinase (DMPK) mRNA and drives disease through a gain-of-function mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG)expin vitro, affording perimidin-2-amine diazirine (1) that bound to r(CUG)exp. The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1. Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG)exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites.

Associations between prenatal exposure to polycyclic aromatic hydrocarbons and thyroid hormones in umbilical cord blood.

We explored the association between maternal urinary polycyclic aromatic hydrocarbon (PAH) metabolites and thyroid hormones in umbilical cord blood in 120 pairs of pregnant women and newborns. Maternal urinary PAH metabolites were measured using high-performance liquid chromatography with tandem mass spectrometry. Thyroid hormones were measured using a flow fluorescence assay. The dose-response relationship between PAH metabolites and thyroid hormones was analyzed using the generalized linear model and restricted cubic spline model. Results showed that Æ©OH PAHs in maternal urine had a negative effect on triiodothyronine (T3). Associations between maternal urinary PAH metabolites and thyroid hormones in umbilical cord blood plasma were observed. Prenatal exposure to PAHs could affect neonatal thyroid hormones, thereby disrupting neonatal thyroid function.

DNA Adduct Detection after Post-Labeling Technique with PCR Amplification (DNA-ADAPT-qPCR) Identifies the Pre-ribosomal RNA Gene as a Direct Target of Platinum-Acridine Anticancer Agents.

To study the DNA damage caused by a potent platinum-acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click chemistry-enabled, azide-modified derivative (APA) was developed. The method involves biotinylation, affinity capture, and bead-based enrichment of APA-modified genomic DNA. The key steps of the assay were validated and optimized in model duplexes, including full-length plasmids, restriction fragments, and a DNA ladder. Native DNA treated with APA and subsequently subjected to post-labeling with a biotin affinity tag was enzymatically digested and fragments were analyzed by in-line LC-MS and MS/MS. The monofunctional-intercalative adducts formed by APA in 5'-pyrimidine/guanine sequences in double-stranded DNA were quantitatively biotinylated by strain-promoted 1,3-dipolar cycloaddition chemistry. When applied to DNA extracted from A549 lung cancer cells, the assay in combination with qPCR amplification demonstrates that platinum-acridines form adducts in the gene sequences encoding pre-ribosomal RNA, a potential pharmacological target of these agents.

A membrane transporter determines the spectrum of activity of a potent platinum-acridine hybrid anticancer agent.

Cytotoxic drugs that are mechanistically distinct from current chemotherapies are attractive components of personalized combination regimens for combatting aggressive forms of cancer. To gain insight into the cellular mechanism of a potent platinum-acridine anticancer agent (compound 1), a correlation analysis of NCI-60 compound screening results and gene expression profiles was performed. A plasma membrane transporter, the solute carrier (SLC) human multidrug and toxin extrusion protein 1 (hMATE1, SLC47A1), emerged as the dominant predictor of cancer cell chemosensitivity to the hybrid agent (Pearson correlation analysis, p < 10-5) across a wide range of tissues of origin. The crucial role of hMATE1 was validated in lung adenocarcinoma cells (A549), which expresses high levels of the membrane transporter, using transporter inhibition assays and transient knockdown of the SLC47A1 gene, in conjunction with quantification of intracellular accumulation of compound 1 and cell viability screening. Preliminary data also show that HCT-116 colon cancer cells, in which hMATE1 is epigenetically repressed, can be sensitized to compound 1 by priming the cells with the drugs EPZ-6438 (tazemetostat) and EED226. Collectively, these results suggest that hMATE1 may have applications as a pan-cancer molecular marker to identify and target tumors that are likely to respond to platinum-acridines.

Discovery of a Chiral DNA-Targeted Platinum-Acridine Agent with Potent Enantioselective Anticancer Activity.

A structure-activity relationship study was performed for a set of rigidified platinum-acridine anticancer agents containing linkers derived from chiral pyrrolidine and piperidine scaffolds. Screening a library of microscale reactions and selected resynthesized compounds in non-small-cell lung cancer (NSCLC) cells showed that cytotoxicities varied by more than three orders of magnitude. A potent hit compound was discovered containing a (R)-N-(piperidin-3-yl) linker (P2-6R), which killed NCI-H460 and A549 lung cancer cells 100 times more effectively than the S enantiomer (P2-6S). P2-6R accumulated in A549 cells significantly faster and produced 50-fold higher DNA adduct levels than P2-6S. Ligand similarity analysis suggests that only module 6R may be compatible with strainless monofunctional intercalative binding. NCI-60 screening and COMPARE analysis highlights the spectrum of activity and potential utility of P2-6R for treating NSCLC and other solid tumors.

Ultrasensitive Assay of Alkaline Phosphatase Based on the Fluorescent Response Difference of the Metal-Organic Framework Sensor.

The assay of alkaline phosphatase (ALP) is important in clinical diagnosis because the abnormal expression of this enzyme is related to many serious diseases. In this work, using a luminescent metal-organic framework (MOF) as the sensor, a fluorescent method was developed for the activity assay of ALP. With nanoscale particle size, the prepared MOF sensor exhibited good dispersability and stable photoluminescence in aqueous suspension. The emission of this MOF is inert to p-nitrophenylphosphate (NPP) but could be efficiently quenched by its dephosphorylated product, p-nitrophenol. Taking advantage of this feature, this MOF was added to the system of ALP-catalyzed NPP dephosphorylation to transduce the proceeding of the reaction real-timely to the fluorescent signal. The enzyme activity could be calculated based on the recorded kinetic trace. This method presented a low detection limit (2 × 10-3 U L-1) and a wide quantification range (0.6-90 U L-1) in our experiments, showing its quantification capability challenges the best of current ALP analytical methods. As a practical application, our method was successfully applied to the ALP analysis in human serum samples.

Cysteine-Directed Bioconjugation of a Platinum(II)-Acridine Anticancer Agent.

Classical maleimide Michael addition chemistry in conjunction with copper-free click chemistry was investigated as a synthetic strategy to attach cytotoxic platinum-acridine hybrid agents to carrier proteins. The structural integrity and selectivity of the model payloads, which were validated in human serum albumin (HSA) using mass spectrometric analysis and heteronuclear 2D 1H-15N HSQC NMR experiments, may have broad utility for the targeted delivery of highly cytotoxic platinum acridines and other nonclassical platinum containing anticancer agents.

MOF based fluorescent assay of xanthine oxidase for rapid inhibitor screening with real-time kinetics monitoring.

The activity assay of xanthine oxidase (XO) is of great application value in clinical diagnosis because the abnormal level of this enzyme is related to a series of pathological states. In this work, a Zr based metal-organic framework (BTB-MOF) with stable photoluminescence in pure water and buffer solution was synthesized. The examination about the fluorescent responses of this material to xanthine and its oxidation product, uric acid, showed that, although both of them affected the emission of BTB-MOF in quenching form, the efficiencies presented much difference. Taking advantage of this feature, a fluorescent method was developed for the activity assay of XO, that is, BTB-MOF was added to the enzymatic oxidation system as a sensor to transduce the proceeding of the reaction real-timely to the signal of fluorescent intensity change. Our method can work under the interference of normal biologically related species, and precisely reflect XO activity in the range of 0.2-40 U L-1 (detection limit = 0.004 U L-1). With consecutive fluorescence intensity scan, this assay could be applied as a high speed screening method of XO inhibitors with the testing time of 1 min. This work shows the wide potential application of MOFs in enzyme analysis.

Enzyme-Assisted Metal-Organic Framework Sensing System for Diethylstilbestrol Detection.

As novel fluorescent-sensing materials, metal-organic frameworks (MOFs) have shown great potential in environmental monitoring. However, most of the researches are limited to traditional pollutants, whereas the application of MOFs to the detection of the pollutants with more complicated structures, such as endocrine disrupting chemicals (EDCs), has rarely been explored. The difficulties faced in the sensing of EDCs include their electronic stability and the structural similarity among homologues, which could be overcome by the incorporation of enzymatic reaction. In this work, the first example of enzyme-assisted MOF-fluorescent-sensing was developed for the analysis of diethylstilbestrol (DES, a synthetic estrogen). In this system, DES is first oxidized by HRP/H2 O2 quantitatively to its quinone form, and then the quinone product is selectively captured by a stilbene based luminescent MOF to induce fluorescence response. By the tandem sensitization and filtration of enzymatic reaction and MOF adsorption, this method shows high sensitivity (DL=89 nm) and can distinguish DES from other similar-structured EDCs.

Design and cellular studies of a carbon nanotube-based delivery system for a hybrid platinum-acridine anticancer agent.

A three-component drug-delivery system has been developed consisting of multi-walled carbon nanotubes (MWCNTs) coated with a non-classical platinum chemotherapeutic agent ([PtCl(NH3)2(L)]Cl (P3A1; L=N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG). The optimized P3A1-MWCNTs are colloidally stable in physiological solution and deliver more P3A1 into breast cancer cells than treatment with the free drug. Furthermore, P3A1-MWCNTs are cytotoxic to several cell models of breast cancer and induce S-phase cell cycle arrest and non-apoptotic cell death in breast cancer cells. By contrast, free P3A1 induces apoptosis and allows progression to G2/M phase. Photothermal activation of P3A1-MWCNTs to generate mild hyperthermia potentiates their cytotoxicity. These findings suggest that delivery of P3A1 to cancer cells using MWCNTs as a drug carrier may be beneficial for combination cancer chemotherapy and photothermal therapy.