A Guanosine-Derived Antitumor Supramolecular Prodrug.

The prodrug strategy for its potential to enhance the pharmacokinetic and/or pharmacodynamic properties of drugs, especially chemotherapeutic agents, has been widely recognized as an important means to improve therapeutic efficiency. Irinotecan's active metabolite, 7-ethyl-10-hydroxycamptothecin (SN38), a borate derivative, was incorporated into a G-quadruplex hydrogel (GB-SN38) by the ingenious and simple approach. Drug release does not depend on carboxylesterase, thus bypassing the side effects caused by ineffective activation, but specifically responds to the ROS-overexpressed tumor microenvironment by oxidative hydrolysis of borate ester that reduces serious systemic toxicity from nonspecific biodistribution of SN38. Comprehensive spectroscopy was used to define the structural and physicochemical characteristics of the drug-loaded hydrogel. The GB-SN38 hydrogel's high level of biosafety and notable tumor-suppressive properties were proven in in vitro and in vivo tests.

Computational design of ultrashort peptide inhibitors of the receptor-binding domain of the SARS-CoV-2 S protein.

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.

Guanosine Borate Hydrogel and Self-Assembled Nanostructures Capable of Enantioselective Aldol Reaction in Water.

A guanosine-based hydrogel formed by the self-assembly of guanosine and 4-((l-prolinamide)methyl)phenylboronic acid was constructed. The G quartets were selectively stabilized by K+ ions to form a self-supporting transparent hydrogel. These guanosine-derived assemblies were used to catalyze the aldol reaction in water without any additives, affording desirable conversion and enantioselectivity of the product. The controlled assays of small-molecule components indicated that the stable assemblies were the definite species that achieved high enantioselective catalysis. The current catalytic system can be readily recovered by simple extraction and still acquired good performance of the reaction after four cycles.

Smart Responsive Quercetin-Conjugated Glycol Chitosan Prodrug Micelles for Treatment of Inflammatory Bowel Diseases.

The incidence and progression of inflammatory bowel disease are closely related to oxidative stress caused by excessive production of reactive oxygen species (ROS). To develop an efficacious and safe nanotherapy against inflammatory bowel diseases (IBD), we designed a novel pH/ROS dual-responsive prodrug micelle GC-B-Que as an inflammatory-targeted drug, which was comprised by active quercetin (Que) covalently linked to biocompatible glycol chitosan (GC) by aryl boronic ester as a responsive linker. The optimized micelles exhibited well-controlled physiochemical properties and stability in a physiological environment. Time-dependent NMR spectra traced the changes in the polymer structure in the presence of H2O2, confirming the release of the drug. The in vitro drug release studies indicated a low release rate (<20 wt %) in physiological conditions, but nearly complete release (>95 wt % after 72 h incubation) in a pH 5.8 medium containing 10 µM H2O2, exhibiting a pH/ROS dual-responsive property and sustained release behavior. Importantly, the negligible drug release in a simulated gastric environment in 1 h allowed us to perform intragastric administration, which has potential to achieve the oral delivery by mature enteric-coating modification in future. Further in vivo activities and biodistribution experiments found that the GC-B-Que micelles tended to accumulate in intestinal inflammation sites and showed better therapeutic efficacy than the free drugs (quercetin and mesalazine) in a colitis mice model. Typical inflammatory cytokines including TNF-α, IL-6, and iNOS were significantly suppressed by GC-B-Que micelle treatment. Our work promoted inflammatory-targeted delivery and intestinal drug accumulation for active single drug quercetin and improved the therapeutic effect of IBD. The current study also provided an alternative strategy for designing a smart responsive nanocarrier for a catechol-based drug to better achieve the target drug delivery.

Guanosine-Based Self-Assembly as an Enantioselective Catalyst Scaffold.

A self-assembled G-quadruplex formed by guanosine and borate as the chiral scaffold was used to catalyze the asymmetric Friedel-Crafts reaction in water. Catalysis, depending on the self-assembly of guanosine and borate into a fibrillar structure in the presence of Cu2+ ions, can be modulated by the assembly concentration, temperature, and amount of Cu2+ ions. Detailed spectral experiments proved that the guanosine-based assembly in solution was responsible for the enantioselective catalysis, rather than small-molecule species. Some of the similar G-quartet assemblies were unable to promote the asymmetric reaction, implying unique properties of the current system, including excellent lifetime stability and supramolecular chiral structures. This work provided the first example of the self-assembled G-quadruplex achieving enantioselective catalysis and some perspective to better understand the design of nucleoside-based self-assemblies for an enantioselective reaction. In view of guanosine as a building block, these findings may be applied to discuss the prebiotic chiral catalyst preceded ribozymes.

Double-strand cleavage of DNA by a polyamide-phenazine-di-N-oxide conjugate.

Phenazine and its derivatives have been widely applied as nucleic acid cleavage agents due to active oxygen activating the C-H bond of the substrate. However, diffusion of oxygen radicals limits their potential applications in the DNA-targeted metal-free drug. Introduction of groove binder moiety such as polyamide enhanced the regional stability of radical molecules and reduced cytotoxicity of the drugs. In this work, we described the design and synthesis of a polyamide-modified phenazine-di-N-oxide as a DNA double-strand cleavage agent. The gel assays showed the hybrid conjugates can effectively break DNA double strands in a non-random manner under physiological conditions. The probable binding mode to DNA was investigated by sufficient spectral experiments, revealing weak interaction between hybrid ligand and nucleic acid molecules. The results of our study have implications on the design of groove-binding hybrid molecules as new artificial nucleases and may provide a strategy for developing efficient and safe DNA cleavage reagents.

Water-Soluble Cationic Polyphosphazenes Grafted with Cyclic Polyamine and Imidazole as an Effective Gene Delivery Vector.

Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. In this study, a series of polyphosphazenes (PPZ) bearing cyclic polyamine and imidazole groups were synthesized and investigated for gene delivery. Agarose gel electrophoresis assays showed that poly(imidazole/1,4,7,10-tetraazyclodocane)phosphazene (Im-PPZ-cyclen) had good binding ability with plasmid DNA (pDNA), yielding positively charged particles with a size around 120-140 nm from a ratio of 10:1 to 5:1 (Im-PPZ-cyclen/pDNA, w/w). The cytotoxicity of Im-PPZ-cyclen assayed by MTT was lower than that of PEI 25 kDa, and was similar to that reported for poly(di-2-dimethylaminoethylamine)phosphazene (poly(di-DMAEA)phosphazene) to some degree. The maximum transfection efficiency of Im-PPZ-cyclen/pDNA complexes against 293 T cells at the ratio of 5:1 (Im-PPZ-cyclen/pDNA, w/w) is close to that of Lipofectamine 2000. The present work may provide a strategy for the design of new cationic polymers with reduced cytotoxicity and be applied to gene delivery as an efficient nonviral vector.

Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies.

Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery.

An IDB-containing low molecular weight short peptide as an efficient DNA cleavage reagent.

Artificial nucleases have attracted significant interest due to their abilities in accelerating DNA cleavage, which results in the possibility of genome manipulation. However, compared with natural nucleases, the currently available artificial nucleases have low cleavage efficiency, especially metal-free artificial nucleases. Thus, it is still a challenge to develop highly efficient metal-free artificial nucleases via a non-oxidative pathway. We here designed and prepared a group of rigid bis-amine-grafted PASP conjugates (PASP-IDB), and investigated their abilities to induce DNA double-strand cleavage. The detailed assays showed that in the absence of metal ions, these short peptide conjugates can effectively break the phosphodiester linkage at a relatively low concentration and under physiological conditions through a hydrolytic process, giving the 10(7)-fold rate acceleration over uncatalyzed double-strand DNA. The probable mechanism verified by control experiments revealed that IDBs and free carboxyl groups in PASP synergically catalyzed DNA cleavage. In addition, the effects of degrees of substitution on the cleavage activity were studied, and the results indicated the existence of minimum building blocks of PASP-IDB for efficient DNA cleavage. The results of our study have implications on the design of short peptide-based molecules as new artificial nucleases and may provide a strategy for developing safe and efficient metal-free DNA cleavage reagents.

HPLC-ELSD determination of kanamycin B in the presence of kanamycin A in fermentation broth.

A novel method for the direct determination of kanamycin B in the presence of kanamycin A in fermentation broth using high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was developed. An Agilent Technologies C18 column was utilized, evaporation temperature of 40°C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water-acetonitrile (65:35, v/v), containing 11.6 mm heptafluorobutyric acid (isocratic elution with flow rate of 0.5 mL/min) with the gain 11. Kanamycin B was eluted at 5.6 min with an asymmetry factor of 1.827. The method showed good linearity over the concentration range of 0.05 to 0.80 mg/mL for the kanamycin B (r(2) = 0.9987). The intra-day and inter-day coefficients of variation obtained from kanamycin B were less than 4.3%. Mean recovery of kanamycin B from spiked fermentation broth was 95%. The developed method was applied to the determination of kanamycin B without any interference from other constituents in the fermentation broth. This method offers simple, rapid and quantitative detection of kanamycin B.

Electrospray ionization mass spectrometric studies on the characteristic fragmentation of Asp/cyclen conjugates.

RATIONALE: Differentiation and structural characterization of Asp/cyclen conjugates by electrospray ionization tandem mass spectrometry (ESI-MS(n)) are significantly important for their biomedical application. Hence, the present study is conducted. METHODS: The fragmentations of Asp/cyclen conjugates generated by positive ion mode electrospray ionization were examined here by low-energy collision-induced dissociation (CID). ESI-MS(n) spectra of cyclen were acquired to confirm cyclen contraction products derived from the studied compounds. The fragments derived from the Asp/cyclen conjugates were proved by deuterium-exchange experiments. RESULTS: Asp/cyclen conjugates displayed characteristic dissociation pathways, including cleavages of amide bonds, loss of NH3 and cyclen contraction pathways. It was observed that cleavages of C-terminal amide bonds generated b2 and b2 + H2O ions from the protonated CyclenAspAspAsp and a b1 + H2O ion from the protonated CyclenAspAsp. In addition, various cyclen contraction products were also observed. CONCLUSIONS: In ESI-MS(n) spectra of studied compounds, fragments of bn-1 + H2O or cyclic anhydride were generated due to facile mobilization of C-terminal or side-chain COOH protons. In addition, the cyclen contraction products were detected. These results might provide sufficient information for the identification of Asp/cyclen conjugates by mass spectrometry.

Guanosine-Based Multidrug Strategy Delivery for Synergistic Anti-Inflammation.

The development of codelivery approaches for combination therapy is of great significance, especially for natural products that need to be combined to achieve therapeutic effects. Targeted delivery of multiple drugs through a single carrier remains a challenge. Here, a multi-drug-loaded hydrogel, incorporating quercetin, demethyleneberberine, and dencichine, based on a G4-quadruplex was designed and prepared. Catechol drugs were responsively released in a simulated inflammatory pathological environment by a borate ester linkage, while coagulating dencichine encapsulated in the hydrogel was released along with the degradation of assemblies. The multi-drug-loaded codelivery system is expected to enhance the treatment of inflammatory bowel disease through the synergistic effect of the components. The preparation, characteristic, and physicochemical properties of the multi-drug-loaded assembly were depicted by NMR, CD, and TEM. Degradation assays in vitro proved the good biocompatibility and safety of the hydrogel and a potential pathway to injectable administration. The assays of typical inflammatory cytokines, including TNF-α and IL-6, indicated that these can be significantly suppressed by the treatment of the hydrogel. The current work provided a simple strategy to construct a multi-drug-loaded hydrogel carrier, which facilitated synergistic therapy for natural products by a codelivery approach.

Machine Learning-Based Virtual Screening and Identification of the Fourth-Generation EGFR Inhibitors.

Epidermal growth factor receptor (EGFR) plays a pivotal regulatory role in treating patients with advanced nonsmall cell lung cancer (NSCLC). Following the emergence of the EGFR tertiary CIS C797S mutation, all types of inhibitors lose their inhibitory activity, necessitating the urgent development of new inhibitors. Computer systems employ machine learning methods to process substantial volumes of data and construct models that enable more accurate predictions of the outcomes of new inputs. The purpose of this article is to uncover innovative fourth-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) with the aid of machine learning techniques. The paper's data set was high-dimensional and sparse, encompassing both structured and unstructured descriptors. To address this considerable challenge, we introduced a fusion framework to select critical molecule descriptors by integrating the full quadratic effect model and the Lasso model. Based on structural descriptors obtained from the full quadratic effect model, we conceived and synthesized a variety of small-molecule inhibitors. These inhibitors demonstrated potent inhibitory effects on the two mutated kinases L858R/T790M/C797S and Del19/T790M/C797S. Moreover, we applied our model to virtual screening, successfully identifying four hit compounds. We have evaluated these hit ADME characteristics and look forward to conducting activity evaluations on them in the future to discover a new generation of EGFR-TKI.

Metal-free DNA linearized nuclease based on PASP-polyamine conjugates.

Genome manipulation controlled by small metal complexes has attracted extensive interest because of their potential application in the fields of molecular biotechnology and drug development. However, their medicinal application is still limited due to the distinct toxicity of the free radicals generated by partial metal complexes based on oxidative cleaving processes. Thus, it is still a challenge for us to use metal free agent to cleave DNA. In this work, we showed that a family of polyamine-grafted PASP (poly(aspartic acid)) conjugates is able to rapidly induce DNA cleavage in the absence of metal ions, and obtain a high-yield linearization product via a hydrolytic path. From the results of detailed control experiments, it was revealed that the formation of polyamine cation/phosphate anion pair and free ungrafted nucleophilic groups would be the key factors to improve DNA linearization. Constructing polyamine conjugates based on short peptide such as polyamine-grafted PASP, as achieved here, could provide an attractive strategy for developing mild and efficient artificial nucleases as well as researching catalytic mechanisms on DNA chemistry.

Rutin-Loaded Stimuli-Responsive Hydrogel for Anti-Inflammation.

An active flavonoid compound rutin was incorporated into a guanosine phenylborate hydrogel (GBR) by a stimuli-responsive borate ester linkage for the treatment of inflammatory bowel disease (IBD). The components and morphology of the drug delivery system were characterized by NMR, UV-vis spectroscopy, and AFM. Rheological measurements revealed the required injectability and self-healing ability, which contributed to its application in rectal administration. The cell assays proved the excellent compatibility and safety of the system, and a possible pathway to form multicellular aggregates. In vitro drug-release studies showed that the hydrogel exhibited good stability in physiological medium, and the drug was almost completely released (more than 90 wt % after 24 h of incubation) in acidic pH and excessive ROS-containing medium, realizing the dual-responsive release of pH/ROS. In vivo activities of the GBR hydrogel showed higher therapeutic efficacy than free rutin in a colitis mice model, and it could significantly inhibit overexpressed inflammatory cytokines, including TNF-α and IL-6. Degradation studies of the hydrogel provided further evidence for the safety of its in vivo application. The work provided a simple strategy to prepare a G-quadruplex drug carrier, which was expected to achieve multi-drug delivery.

Practical and Scalable Manufacturing Process for the Key Intermediate of Poly(ADP-Ribose) Polymerase Inhibitor Olaparib.

Olaparib (Lynparza) is a potent, highly selective inhibitor of poly(ADP-ribose)polymerase enzymes, approved by the U.S. FDA and EMA for the treatment of ovarian cancer. Herein, we report a practical, economical, and scalable process for the synthesis of 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid, a key intermediate for olaparib. The low-cost industrial byproduct phthalhydrazide was used as the starting material to construct the phthalazinone moiety, which allowed access to the key intermediate by the Negishi coupling reaction. Optimization of each step has enabled the development of an environmentally benign and robust process with effective control of impurities.

Computer-Aided Design of Lasso-like Self-Assembling Anticancer Peptides with Multiple Functions for Targeted Self-Delivery and Cancer Treatments.

Anticancer peptides are promising drug candidates for cancer treatment, but the short circulation time and low delivery efficiency limit their clinical applications. Herein, we designed several lasso-like self-assembling anticancer peptides (LASAPs) integrated with multiple functions by a computer-aided approach. Among these LASAPs, LASAP1 (CRGDKGPDCGKAFRRFLGALFKALSHLL, 1-9 disulfide bond) was determined to be superior to the others because it can self-assemble into homogeneous nanoparticles and exhibits improved stability in serum. Thus, LASAP1 was chosen for proving the design idea. LASAP1 can self-assemble into nanoparticles displaying iRGD on the surface because of its amphiphilic structure and accumulate to the tumor site after injection because of the EPR effect and iRGD targeting to αVß3 integrin. The nanoparticles could disassemble in the acidic microenvironment of the solid tumor, and cleaved by the overexpressed hK2, which was secreted by prostate tumor cells, to release the effector peptide PTP-7b (FLGALFKALSHLL), which was further activated by the acidic pH. Therefore, LASAP1 could target the orthotopic prostate tumor in the model mice after intraperitoneal injection and specifically inhibit tumor growth, with low systematic toxicity. Combining the multiple targeting functions, LASAP1 represents a promising design of self-delivery of peptide drugs for targeted cancer treatments.

Chitosan grafted with macrocyclic polyamines on C-2 and C-6 positions as nonviral gene vectors: preparation, characterization, and in vitro transfection studies.

Chitosan grafted with macrocyclic polyamines (Cs-g-MCPA) on the C-2 or the C-6 position was synthesized by a simple method. Four copolymers prepared were characterized by (1)H NMR, (13)C NMR, Fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), and gel permeation chromatography (GPC). Circular dichroism spectra (CD), fluorescence spectra and agarose gel electrophoresis assay showed that Cs-g-MCPA copolymers had good binding ability to DNA. By modification of the MCPAs, the copolymers showed low cytotoxicity and high transfection efficiency as new gene vectors. It was found that Cs-g-MCPA copolymers with different grafted positions showed different properties: copolymers grafted on the C-2 position showed higher cytotoxicity and higher transfection efficiency than those grafted on position C-6.

Artificial transcription factors which mediate double-strand DNA cleavage.

A new family of artificial transcription factor (ATF)-based conjugates have been designed and synthesized as potent chemical nucleases. Polyamides as the important and efficient ATFs were used to modify and activate several anchor compounds. The results demonstrate that the resulting conjugates remarkably promote the rate accelerations and non-random double-strand DNA cleavage activity. Interestingly, the cleavage activity of both the hydrolytic and oxidative agents was promoted efficiently through the modification of the ATFs.