Azvudine is a thymus-homing anti-SARS-CoV-2 drug effective in treating COVID-19 patients.

Azvudine (FNC) is a nucleoside analog that inhibits HIV-1 RNA-dependent RNA polymerase (RdRp). Recently, we discovered FNC an agent against SARS-CoV-2, and have taken it into Phase III trial for COVID-19 patients. FNC monophosphate analog inhibited SARS-CoV-2 and HCoV-OC43 coronavirus with an EC50 between 1.2 and 4.3 µM, depending on viruses or cells, and selective index (SI) in 15-83 range. Oral administration of FNC in rats revealed a substantial thymus-homing feature, with FNC triphosphate (the active form) concentrated in the thymus and peripheral blood mononuclear cells (PBMC). Treating SARS-CoV-2 infected rhesus macaques with FNC (0.07 mg/kg, qd, orally) reduced viral load, recuperated the thymus, improved lymphocyte profiles, alleviated inflammation and organ damage, and lessened ground-glass opacities in chest X-ray. Single-cell sequencing suggested the promotion of thymus function by FNC. A randomized, single-arm clinical trial of FNC on compassionate use (n = 31) showed that oral FNC (5 mg, qd) cured all COVID-19 patients, with 100% viral ribonucleic acid negative conversion in 3.29 ± 2.22 days (range: 1-9 days) and 100% hospital discharge rate in 9.00 ± 4.93 days (range: 2-25 days). The side-effect of FNC is minor and transient dizziness and nausea in 16.12% (5/31) patients. Thus, FNC might cure COVID-19 through its anti-SARS-CoV-2 activity concentrated in the thymus, followed by promoted immunity.

A versatile cholera toxin conjugate for neuronal targeting and tracing.

Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by 'click' chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using 'click' chemistry.

Dual-responsive click-crosslinked micelles designed for enhanced chemotherapy for solid tumors.

The design of multiple stimuli-responsive, stable polymeric drug carriers is key for efficient drug release against solid tumors. Herein, core-crosslinked micelles were readily prepared from a pair of redox/pH-sensitive clickable copolymers. The two copolymers comprised the same poly(ethylene glycol) (PEG)-poly(ε-benzyloxycarbonyl-l-lysine) (PZLL) block but with either disulfide-linked azadibenzocyclooctyne (DBCO) or azide (AZ) group-tagged branched polyethylenimine (BPEI, 1.8 kDa). The data showed that an equivalent of the two copolymers could self-assemble into nanosized micelles with the crosslinked core via the DBCO-AZ click chemistry. The click-crosslinked micelles showed excellent size stability under multiple dilutions but destabilization in an acidic or reductive environment. Besides, they could load doxorubicin (DOX), an anticancer drug, and mediate slow drug release in a neutral environment but sufficient drug unloading under acidic plus reductive conditions. In vitro, DOX-loaded crosslinked micelles led to higher DOX accumulation in the cellular nucleus in comparison with non-crosslinked micelles from the PEG-PZLL-BPEI copolymer (PP), thus causing more marked cytotoxicity in SKOV-3 cells. In vivo, DOX-loaded crosslinked micelles caused significant growth inhibition of SKOV-3 tumors xenografted in BALB/c nude mice, and showed superior anticancer efficacy to non-crosslinked PP micelles. Chemotherapy with core-crosslinked micelles had no adverse side effects on the health (serum levels and body weight) of the mice. This study highlights the design of clickable block copolymers to easily construct core-crosslinked and multiple stimuli-responsive micelles for enhanced anticancer therapy.

End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity.

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. The 4, 5, 6 EGF-like domain of TM (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 is a promising anticoagulant candidate but has a very short half-life. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM456 modification. Recombinant TM456 with a C-terminal LPETG tag (rTM456-LPETG) was expressed in Escherichia coli for its end-point modification with NH2-diglycine-PEG5000-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific PEGylation of rTM456via CFCC. Both PEGylated rTM456 conjugates retained protein C activation activity as that of rTM456. Also, they were more stable than rTM456 in Trypsin digestion assay. Further, both PEGylated rTM456 conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.

Azido-galactose outperforms azido-mannose for metabolic labeling and targeting of hepatocellular carcinoma.

Metabolic glycoengineering of unnatural monosaccharides provides a facile method to label cancer cells with chemical tags for glycan imaging and cancer targeting. Multiple types of monosaccharides have been utilized for metabolic cell labeling. However, the comparison of different types of monosaccharides in labeling efficiency and selectivity has not been reported. In this study, we compared N-azidoacetylgalactosamine (GalAz) and N-azidoacetylmannosamine (ManAz) for metabolic labeling of HepG2 hepatocellular carcinoma in vitro and in vivo. GalAz showed higher labeling efficiency at low concentrations, and outperformed ManAz in metabolic labeling of HepG2 tumors in vivo. GalAz mediated labeling of HepG2 tumors with azido groups significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 and DBCO-doxorubicin conjugate via efficient Click chemistry. This study, for the first time, uncovered the distinct labeling efficiency and selectivity of different unnatural monosaccharides in liver cancers.

Doxorubicin encapsulated clicked gold nanoparticle clusters exhibiting tumor-specific disassembly for enhanced tumor localization and computerized tomographic imaging.

Gold nanoparticles (AuNPs) and matrix metalloproteinase (MMP)-2 cleavable peptides are clicked into gold nanoparticle clusters (AuNCs) for enhanced drug localization and micro computerized tomography (µCT) theranostic of tumors. AuNPs are co-functionalized with doxorubicin (DOX) and an azide-terminated polymer (DOX/N3@AuNPs), and the DOX/N3@AuNPs are associated into DOX@AuNCs in the presence of an alkyne-terminated MMP-2 cleavable peptide (alkyne-peptide-alkyne; APA) by click chemistry. MMP-2-dependent dissociation shows that DOX@AuNCs are highly sensitive to the MMP-2 and are almost completed digested into single nanoparticles. DOX liberation shows that > 75% of the conjugated DOX is bursted out from the digested DOX@AuNCs while < 20% of DOX is released from the integrate DOX@AuNCs within 3 h in acidic conditions, suggesting that DOX is only liberated from dissociated DOX@AuNCs in acidic conditions. In vivo study shows that DOX@AuNCs accumulate in tumor ~ 150 times higher than DOX/N3@AuNPs do and efficiently suppress tumor growth. Mice administered with AuNCs shows clearer µCT images of tumors. Thus, DOX@AuNCs are expected promising carriers for both anticancer therapy and tumor imaging.

Structure-Activity Relationship Studies of ß-Lactam-azide Analogues as Orally Active Antitumor Agents Targeting the Tubulin Colchicine Site.

We have synthesized a series of new ß-lactam-azide derivatives as orally active anti-tumor agents by targeting tubulin colchicine binding site and examined their structure activity relationship (SAR). Among them, compound 28 exhibited the most potent antiproliferative activity against MGC-803 cells with an IC50 value of 0.106 µM by induction of G2/M arrest and apoptosis and inhibition of the epithelial to mesenchymal transition. 28 acted as a novel inhibitor of tubulin polymerization by its binding to the colchicine site. SAR analysis revealed that a hydrogen atom at the C-3 position of the ß-lactam was required for the potent antiproliferative activity of ß-lactam-azide derivatives. Oral administration of compound 28 also effectively inhibited MGC-803 xenograft tumor growth in vivo in nude mice without causing significant loss of body weight. These results suggested that compound 28 is a promising orally active anticancer agent with potential for development of further clinical applications.

Antibacterial Effects of Toothpastes Evaluated in an In Vitro Biofilm Model.

PURPOSE: To test the antibacterial effects of different toothpastes with the slurry method of toothpaste application in an in vitro oral biofilm model including relevant periodontal pathogens. MATERIALS AND METHODS: Four commercially available toothpastes, two containing sodium fluoride (NaF) at different concentrations (1450 and 2500 ppm), two NaF with either triclosan or stannous fluoride, and a control phosphate-buffered saline (PBS) were used. Multispecies biofilms containing 6 species of oral bacteria were grown on hydroxyapatite disks for 72 h and then exposed for 2 min to the toothpaste slurries or phosphate buffer saline (PBS) by immersion, under continuous agitation at 37°C. Biofilms were then analysed by means of real-time polymerase chain reaction (PCR), combined with propidium monoazide (PMA). Statistical evaluation was performed using ANOVA and Student's t-test, with Bonferroni correction for multiple comparisons. RESULTS: The toothpastes containing NaF and stannous fluoride demonstrated superior antimicrobial activity for A. actinomycetencomitans, P. gingivalis and F. nucleatum when compared to those containing NaF and triclosan, 1450 ppm NaF or 2500 ppm NaF in this multispecies biofilm model. CONCLUSION: The proposed model for the evaluation of toothpastes in the form of slurries detected significant differences in the antimicrobial effects among the tested NaF-containing toothpastes, with the stannous fluoride-based formulation achieving better results than the other formulations. The use of toothpaste as slurries and real-time PCR with PMA is an adequate method for comparing the in vitro antimicrobial effect of different toothpastes.

An in vivo strategy to counteract post-administration anticoagulant activity of azido-Warfarin.

Drugs, usually long acting and metabolically stable molecules, might be the source of adverse effects triggered by complex drug interactions, anaphylaxis and drug-induced coagulopathy. To circumvent this growing drug safety issue, we herein investigate the opportunity offered by bio-orthogonal chemistry for in vivo drug neutralization. We design a small-molecule anticoagulant drug (Warfarin) containing an azide group that acts as a safety pin. It allows drug deactivation and restoration of physiological coagulation via in vivo click reaction with a suitable cyclooctyne-based neutralizing agent. In this strategy, the new molecule formed by reaction of the drug and the antidote is deprived of biological activity and prone to fast renal clearance. This 'Click &Clear' approach lays ground for new strategies in designing drugs with switchable biophysical properties.

Targeted Ultrasound-Assisted Cancer-Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry.

Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4 ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.

Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice.

RATIONALE: There is an emerging body of evidence that implicates a crucial role of γ-aminobutyric acid subtype A (GABAA) receptors in modulating the rewarding effects of a number of abused drugs. Modulation of GABAA receptors may therefore represent a novel drug-class independent mechanism for the development of abuse treatment pharmacotherapeutics. OBJECTIVES: We tested the hypothesis that the GABAA receptor benzodiazepine-site (BDZ) negative modulator Ro15-4513 would reduce the reward-related effects of three pharmacologically dissimilar drugs; toluene vapor, d-methamphetamine, and diazepam using intracranial self-stimulation (ICSS) in mice. We also examined whether Ro15-4513 attenuated dopamine release produced by d-methamphetamine in an in vivo microdialysis procedure. RESULTS: Ro15-4513 abolished ICSS reward facilitation produced by all three abused drugs at Ro15-4513 doses which had no effect on ICSS when administered alone. In contrast, the BDZ antagonist flumazenil only attenuated the ICSS-facilitating effects of diazepam. Administration of the same dose of Ro15-4513 which abolished drug-facilitated ICSS produced a 58 % decrease in d-methamphetamine-stimulated dopamine in the nucleus accumbens of mice relative to d-methamphetamine alone. CONCLUSIONS: These results demonstrate that negative modulation of GABAA receptors can produce profound reductions in reward-related effects of a diverse group of drugs that activate the mesolimbic reward pathway through different mechanisms. These data suggest that pharmacological modulation of GABAA receptors may represent a viable pathway for the development of drug abuse pharmacotherapies.

Development of inhibitor-directed enzyme prodrug therapy (IDEPT) for prostate cancer.

Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies.

Cationic charged helical glycopolypeptide using ring opening polymerization of 6-deoxy-6-azido-glyco-N-carboxyanhydride.

Glycopolypeptides with a defined secondary structure are of significance in understanding biological phenomena. Synthetic glycopolypeptides, or polypeptides featuring pendant carbohydrate moieties, have been of particular interest in the field of tissue engineering and drug delivery. In this work, we have synthesized charged water-soluble glycopolypeptides that adopt a helical conformation in water. This was carried out by the synthesis of a glyco-N-carboxyanhydride (glyco-NCA) containing an azide group at the sixth position of the carbohydrate ring. Subsequently, the NCA was polymerized to obtain azide-containing glycopolypeptides having good control over molecular weight and polydispersity index (PDI) in high yields. We were also able to control the incorporation of the azide group by synthesizing random co-glycopolypeptide containing 6-deoxy-6-azido and regular 6-OAc functionalized glucose. This azide functionality allows for the easy attachment of a bioactive group, which could potentially enhance the biological activity of the glycopolypeptide. We were able to obtain water-soluble charged glycopolypeptides by both reducing the azide groups into amines and using CuAAC with propargylamine. These charged glycopolypeptides were shown to have a helical conformation in water. Preliminary studies showed that these charged glycopolypeptides showed good biocompatibility and were efficiently taken up by HepG2 cells.

Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide.

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.

Photosensitive Pt(IV)-azide prodrug-loaded nanoparticles exhibit controlled drug release and enhanced efficacy in vivo.

Cisplatin has long been the first line of treatment for a variety of solid tumors. However, poor pharmacokinetics and high incidences of resistance in the clinic have motivated the production of numerous alternative Pt-based anticancer species. Recently, photosensitive Pt(IV) complexes have garnered much interest because they offer a method of selective induction of active Pt(II) at the tumor site by UVA irradiation. Here, we report the first synthesis, in vitro and in vivo characterization of a novel series of photosensitive Pt(IV)­azide prodrugs and micellar nanoparticle formulations thereof. Upon mild UVA irradiation, both free Pt(IV) complexes and micellar nanoparticles rapidly released biologically active Pt(II), capable of binding to 5'-GMP,while remaining extremely stable in the dark. In vitro, uptake of photosensitive Pt(IV) prodrugs by ovarian cancer SKOV-3 cells was greatly enhanced with the micellar nanoparticles compared to their free prodrug analogs, as well as cisplatin and oxaliplatin. Increased cytotoxicity was observed upon UVA treatment, with up to a 13-fold enhancement over oxaliplatin for the micellar nanoparticles. In vivo bioavailability of micellar nanoparticles was enhanced ~10 fold over free Pt(IV) prodrugs. Importantly, micellar nanoparticles demonstrated significantly improved efficacy against H22 murine hepatocarcinoma, showing decreased systemic toxicity and increased tumor growth inhibition relative to small molecule drugs. These findings establish that photosensitive Pt(IV) complexes, specifically when formulated into micellar nanoparticles, have the potential to offer a robust platform for the controlled delivery and selective activation of Pt-based anticancer therapeutics.

A histone deacetylase inhibitor YCW1 with antitumor and antimetastasis properties enhances cisplatin activity against non-small cell lung cancer in preclinical studies.

Many histone deacetylase (HDAC) inhibitors show limited therapeutic effects for solid tumors. Here, we develop a novel HDAC inhibitor YCW1 and verify the combination effect of YCW1 and cisplatin in lung cancer pre-clinical models. YCW1 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. YCW1 and cisplatin showed synergistic anti-tumor effects through impairment of DNA damage repair. YCW1 inhibited tumor growth in lung orthotopic and subcutaneously implanted xenograft models. YCW1 significantly suppressed lung metastases via inhibition of focal adhesion complex. Our findings suggested that YCW1 is a potential HDAC inhibitor for lung cancer treatment as single and in combination regimens with cisplatin.

Budesonide/formoterol maintenance and reliever therapy versus free-combination therapy for asthma: a real-life study.

BACKGROUND: This non-interventional real-life study (NCT00884689) compared budesonide/formoterol (Symbicort®) maintenance and reliever therapy (SMART™) with a free combination of inhaled corticosteroid (ICS) + long-acting ß2-agonist (LABA) (in separate inhalers) and as-needed short-acting ß2-agonist (SABA) in adult asthma patients with an exacerbation in the past 24 months. METHODS: Asthma patients received SMART™ or free-combination ICS + LABA and as-needed SABA for 6 months. Allocation of patients and doses prescribed were at physician's discretion. No other restrictions applied. Primary endpoint: mean no. of rescue medication puffs/day. RESULTS: 482 patients were included (SMART™ n = 310; free combination n = 172). SMART™ patients used less rescue medication vs the free-combination group (mean difference - 0.27 puffs/day; p = 0.013). Severe asthma exacerbations were rare (mean rate 0.20 vs 0.17/year for SMART™ vs free combination). The mean ICS dose was 615 µg in SMART™ and 678 µg in free-combination group. CONCLUSIONS: In this real-life setting, SMART™-treated patients used less rescue medication despite a lower ICS burden vs conventional asthma management with three different inhalers.

Direct modulation of microtubule stability contributes to anthracene general anesthesia.

Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40-47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region. The immobilization was often reversible, but it was characterized by a longer duration consistent with covalent attachment of the ligand to functionally important targets. IEF/SDS-PAGE examination of irradiated tadpole brain homogenate revealed labeled protein, identified by mass spectrometry as ß-tubulin. In vitro assays with aminoanthracene-cross-linked tubulin indicated inhibition of microtubule polymerization, similar to colchicine. Tandem mass spectrometry confirmed anthracene binding near the colchicine site. Stage 40-47 tadpoles were also incubated 1 h with microtubule stabilizing agents, epothilone D or discodermolide, followed by dosing with 1-aminoanthracene. The effective concentration of 1-aminoanthracene required to immobilize the tadpoles was significantly increased in the presence of either microtubule stabilizing agent. Epothilone D similarly mitigated the effects of a clinical neurosteroid general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are "on-pathway" targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics.

Cell-selective metabolic glycan labeling based on ligand-targeted liposomes.

A cell-specific metabolic glycan labeling strategy has been developed using azidosugars encapsulated in ligand-targeted liposomes. The ligands are designed to bind specific cell-surface receptors that are only expressed or up-regulated in target cells, which mediates the intracellular delivery of azidosugars. The delivered azidosugars are metabolically incorporated into cell-surface glycans, which are then imaged via a bioorthogonal reaction.