Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs.

Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.

Solubility and Valence Variation of Ce in Low-Alkali Borosilicate Glass and Glass Network Structure Analysis.

Low-alkali borosilicate glass was used as the immobilization substrate, and Ce was used to replicate the trivalent and tetravalent actinides, in order to create simulated waste glass through melt heat treatment. The valence of Ce and solubility of CeO2 in waste glass were studied as well as its network structure and thermal and chemical stability. The solubility of Ce in waste glass was examined by XRD and SEM. The network structure was examined by Raman spectroscopy. The valence of Ce was determined by X-ray photoelectron spectroscopy. Thermal analysis and product consistency (PCT) were employed to determine the thermal and chemical stability of waste glasses. The results show that the solubility of cerium in low-alkali borosilicate glasses is at least 25.wt.% and precipitates a spherical CeO2 crystalline phase when it exceeds the solid solution limit; Ce is immobilized in the glass by entering the interstices of the glass network. Depolymerization and the transition from [BO3] to [BO4] occurs when CeO2 doping levels rise. About 60 percent of Ce4+ is converted to Ce3+, and the thermal stability of glass rises then falls with the increase of CeO2. All samples exhibit strong leaching resistance, with the average mass loss of Ce at 28 days being less than 10-4 gm-2d-1.

1-O-Octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-GS-441524 (V2043). Evaluation of Oral V2043 in a Mouse Model of SARS-CoV-2 Infection and Synthesis and Antiviral Evaluation of Additional Phospholipid Esters with Enhanced Anti-SARS-CoV-2 Activity.

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.

Enhancing the Mechanical Properties of Waterborne Polyurethane Paint by Graphene Oxide for Wood Products.

Water-based polyurethane paint is widely used for wood furniture by virtue of the eco-friendliness, rich gloss, and flexible tailorability of its mechanical properties. However, its low solution (water or alcohol) resistance and poor hardness and wear resistance limit its application. The emerging graphene oxide has a high specific surface area and abundant functional groups with excellent mechanical properties, endowing it with great potential to modify waterborne polyurethane as a nanofiller. In this study, graphene oxide prepared by Hummers' method is introduced in the chemosynthetic waterborne polyurethane through physical blending. The testing results demonstrate that the appropriate usage of graphene oxide at 0.1 wt% could obviously improve water absorption resistance and alcohol resistance, significantly enhancing the mechanical properties of waterborne polyurethane paint. The corresponding tensile strength, abrasion resistance, and pendulum hardness of the graphene oxide-modified paint film increase by 62.23%, 14.76%, and 12.7%, respectively, compared with the pristine paint film. Meanwhile, the composite paint film containing graphene oxide possesses superiority, including gloss, abrasion resistance, pendulum hardness, and tensile strength in contrast with the commercial paint. The use of graphene oxide to enhance the waterborne polyurethane possesses strong operability and practical value, and could provide useful reference for the modification of waterborne wood paint.

Effect of P2O5 and Na2O on the Solubility of Molybdenum and Structural Features in Borosilicate Glass.

In this paper, the effect of doping phosphorus in a borosilicate glass matrix to improve the solubility of Mo was investigated by X-ray diffraction (XRD), Raman, and solid-state nuclear magnetic resonance (NMR) spectroscopy, and the effectiveness of Na content on P species inhibiting the growth of the crystallization of Mo was assessed. The results indicate that phosphate-doped borosilicate glass can host 4 mol% of Mo, and that such a borosilicate glass matrix could only accommodate 1 mol% of Mo without phosphate doping. The effectiveness of phosphorus may be correlated with the Na content in borosilicate glass, and a high Na content borosilicate glass matrix requires more P doping to accommodate Mo. In addition, incorporating large amounts of P can compromise the aqueous durability of the glass matrix.

Synergistic inhibition of hepatitis C virus infection by a novel microtubule inhibitor in combination with daclatasvir.

Even though substantial progress has been made in the treatment of hepatitis C virus (HCV) infection, viral resistance and relapse still occur in some patients and additional therapeutic approaches may ultimately be needed should viral resistance become more prevalent. Microtubules play important roles in several HCV life cycle events, including cell attachment, entry, cellular transportation, morphogenesis and progeny secretion steps. Therefore, it was hypothesized that microtubular inhibition might be a novel approach for the treatment of HCV infection. Here, the inhibitory effects of our recently developed microtubule inhibitors were studied in the HCV replicon luciferase reporter system and the infectious system. In addition, the combination responses of microtubule inhibitors with daclatasvir, which is a clinically used HCV NS5A inhibitor, were also evaluated. Our results indicated that microtubule targeting had activity against HCV replication and showed synergistic effect with a current clinical drug.

Convallatoxin suppresses osteosarcoma cell proliferation, migration, invasion, and enhances osteogenic differentiation by downregulating parathyroid hormone receptor 1 (PTHR1) expression and inactivating Wnt/ß-catenin pathway.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Convallatoxin, a natural cardiac glycoside, exhibits potent anti-tumor activities. Literature has confirmed that PTHR1 is highly expressed in OS tissues and cells and downregulation of PTHR1 could decrease the invasion and growth of OS cells and increase tumor differentiation. In addition, PTHR1 could activate Wnt signaling pathway to promote the malignant functions of OS. In the present study, MG63 and U2OS cells were treated with 0, 12.5, 25, and 50 nM convallatoxin in order to elucidate the precise function of convallatox on the malignant behaviors of OS cells. Moreover, MG63 and U2OS cells treated with convallatoxin were transfected with Ov-PTHR1 or sh-DKK1, aiming to explore whether convallatoxin impeded the malignant progression of OS by modulating PTHR1 and Wnt/ß-catenin pathway. CCK-8, wound healing and transwell assays were employed to assess the proliferation, migration, and invasion of OS cells. Differentiation markers (collagen 1, osteopontin, RANKL, Runx2, osteocalcin) were measured to evaluate OS cell differentiation. Results illuminated that convallatoxin suppressed proliferation, migration, and invasion as well as promoted osteogenic differentiation of OS cells. Besides, convallatoxin inhibited PTHR1 expression and inactivated Wnt/ß-catenin pathway and PTHR1 overexpression activated Wnt/ß-catenin pathway. Furthermore, PTHR1 overexpression or DKK1 knockdown reversed the suppressing effects of convallatoxin on OS cell proliferation, migration, and invasion, as well as the enhancing effect of convallatoxin on OS cell osteogenic differentiation. Collectively, convallatoxin may repress the malignant progression of OS by blocking PTHR1 and Wnt/ß-catenin pathway.

A fragment integrational approach to GPCR inhibition: Identification of a high affinity small molecule CXCR4 antagonist.

Targeting the protein-protein interactions involving CXCR4, a member of chemokine receptor family and G-protein-coupled receptor superfamily, has become an attractive therapeutic strategy for HIV-1 infection, hematopoietic stem cell mobilization, and cancer metastasis. As such, new small molecule CXCR4 antagonists are needed to offer therapeutic alternatives with enhanced clinical outcomes. Here, employing a fragment integrational approach we designed and synthesized a new and potent small molecule CXCR4 antagonist (named as HF51116), as well as a fluorescent (FITC)-labeled HF51116 (FITC-HF51116). HF51116 exhibited very high CXCR4 binding affinity with IC50 of 12 nM in competitive binding with a CXCR4 specific antibody 12G5, which is comparable to the wild type chemokines or synthetic peptides of much larger molecular sizes. Direct binding measurement using FITC-HF51116 further revealed the compound's high CXCR4 affinity. HF51116 strongly antagonized SDF-1α-induced cell migration, calcium mobilization, and CXCR4 internalization. Furthermore, HF51116 inhibited HIV-1 infection via CXCR4, demonstrating its antiviral therapeutic potential. The mechanism of HF51116-CXCR4 interaction was analyzed by site-directed mutagenesis and molecular modeling which suggested that the compound recognizes the minor and major subpockets of CXCR4. Its binding to CXCR4 was found to block G protein-dependent downstream signal pathways as detected by luciferase reporter assays. With its potent bioactivities and asymmetric structure amenable to chemical diversification, HF51116 may serve as a prototype for developing a new class of CXCR4-targeted therapeutics and proof of the concept of similar strategies for studying other GPCRs.

Effect of non-pharmacological interventions on anxiety, depression, sleep quality, and pain after orthopedic surgery: A protocol for systematic review and network meta-analysis.

BACKGROUND: Patients after orthopedic surgery often experience the pain, anxiety, depression, and sleep disturbances, which can be greatly reduced by non-pharmacologic interventions as alternative therapies. Randomized controlled trials of nonpharmacologic interventions for anxiety, depression, sleep quality, and pain in patients after orthopedic surgery have been reported, but the results may be conflicting. Evidence to determine the optimal non-pharmacological intervention with a high efficacy is limited. This study aims to assess the effects of non-pharmacologic interventions on the bone anxiety, depression, sleep quality, and pain in patients after orthopedic surgery through a network meta-analysis, thus providing guidance in clinical application. METHODS: A systematic search of randomized controlled trials reporting the effects of non-pharmacological interventions on anxiety, depression, sleep quality and pain after orthopedic surgery published before October 2021 will be searched in Wanfang, VIP Information Chinese Journal Service Platform, China National Knowledge Infrastructure, Chinese BioMedicine Literature Database, Pubmed, Embase, Cochrane, and Web of science. Two reviewers will be independently responsible for study selection, quality appraisal, and data extraction. Stata 14.0 software will be used to perform the network meta-analysis. RESULTS: The findings of this research will be reported in a recognized journal. CONCLUSION: This meta-analysis will provide the stronger evidence for non-pharmacological interventions on alleviating bone anxiety, depression, sleep quality, and pain in patients after orthopedic surgery, which will help clinicians and decision makers in their choices.Open Science Framework registration number: DOI 10.17605/OSF.IO/2SCBD.

Borosilicate Glass-Ceramics Containing Zirconolite and Powellite for RE- and Mo-Rich Nuclear Waste Immobilization.

In order to increase the loading of rare earth- and molybdenum-rich high-level waste in the waste forms, zirconolite- and powellite-based multi-phase borosilicate glass-ceramics were synthesized via an in-situ heat treatment method. The effects of the CTZ (CaO, TiO2 and ZrO2) content on the crystallization, microstructure and aqueous durability of the multi-phase borosilicate glass-ceramics were studied. The results indicate that the increase of CTZ content can promote crystallization. The glass-ceramics presented even structures when the CTZ content was ≥ 40 wt%. For the glass-ceramic with 40 wt% CTZ, only zirconolite and powellite crystals were detected and powellite crystals were mainly distributed around zirconolite, whereas for the glass-ceramics with 50 wt% CTZ, perovskite was detected. Furthermore, the leaching rates of Na, Ca, Mo and Nd were in the ×10-3, ×10-4, ×10-3 and ×10-5 g·m-2·d·-1 orders of magnitude on the 28th leaching day, respectively.

Study on Structure and Properties of La2O3-Doped Basaltic Glasses for Immobilizing Simulated Lanthanides.

The La2O3-doped basaltic glass simulated high-level waste form (HLW) was prepared by the solid-state melt method. The simulated waste La2O3 maximum loading and the doping effect on structure, thermal stability, leaching behavior, density, and hardness of basaltic glasses were studied. XRD and SEM results show that the simulated waste loading of La2O3 in basaltic glass can be up to ~46 wt.%, and apatite (CaLa4(SiO4)3O) precipitates when the content of La2O3 reaches 56 wt.%. Raman results indicate that the addition of La2O3 breaks the Si-O-Si bond of large-membered and four-membered, but the number of A13+ involved in the formation of the network increase. Low content of La2O3 can help to repair the glass network, but it destroys the network as above 26 wt.%. DSC results show the thermal stability of simulated waste forms first increases and then decreases with the increase of La2O3 content. With the increase of La2O3 content, the density of the simulated waste form increases, and the hardness decreases. The leaching chemical stability of samples was evaluated by the ASTM Product Consistency Test (PCT) Method, which show that all the samples have good chemical stability. The leaching rates of La and Fe are three orders of magnitude lower than those of the other elements. Among them, L36 has the best comprehensive leaching performance.

Rethinking Remdesivir: Synthesis, Antiviral Activity, and Pharmacokinetics of Oral Lipid Prodrugs.

Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

Study on the Superhydrophobic Properties of an Epoxy Resin-Hydrogenated Silicone Oil Bulk Material Prepared by Sol-Gel Methods.

A superhydrophobic material was prepared by a simple and easily accessed sol-gel method using epoxy resin (E-51) and γ-aminopropyltriethoxysilane (KH-550) as the precursors, aqueous ammonia (NH4OH) as the catalyst and hydrogenated silicone oil (PMHS) as the hydrophobic modifier, and then pelleting the final product. The morphologies, surface chemical properties and thermal stability of the superhydrophobic bulk materials were characterized by scanning electron microscopy, Fourier infrared spectrometry and thermal analyzer. The hydrophobic properties and repairability of the as-prepared materials were also studied. The results showed that the prepared epoxy resin-hydrogenated silicone oil bulk materials were composed of tightly bound nanoparticles with a size of 50-100 nm in diameter. The material showed excellent superhydrophobic properties with a surface contact angle of 152°. The material also had good thermal resistance with a heat-resistant temperature of 300 °C and showed good repairability. The epoxy resin-hydrogenated silicone oil bulk superhydrophobic material exhibited excellent performance and showed wide application prospects.

Rethinking Remdesivir: Synthesis, Antiviral Activity and Pharmacokinetics of Oral Lipid Prodrugs.

Remdesivir (RDV, GS-5734) is currently the only FDA-approved antiviral drug for the treatment of SARS CoV-2 infection. The drug is approved for use in adults or children 12-years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2 infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed and several including molnupiravir and PF-07321332 are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of Remdesivir nucleoside (RVn, GS-441524) that are processed to RVn-monophosphate, the precursor of the active RVn-triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells and Huh7.5 cells. In Syrian hamsters oral treatment with ODBG-P-RVn was well tolerated and achieved therapeutic levels in plasma above the EC90 for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

Crystallization Kinetics of Modified Basalt Glass.

As the raw material for the production of basalt continuous fibers in Sichuan, basalt glass (BG) and modified basalt glass (MBG) were prepared by the melt quenching method with the basalt and chemically modified basalt, respectively. The crystallization kinetics of BG and MBG were investigated by differential scanning calorimetry (DSC) according to Kissinger methods. The results revealed that it is difficult for both glasses to crystallize at a high temperature. In addition, the crystallization activation energy of MBG is much higher than that of BG, which indicates that MBG is more difficult to crystallize than BG. The crystalline phases seemed to be formed from the surface of the two glasses. The morphologies and crystal structure of the crystalline phases in the heat-treated BG and MBG were analyzed by scanning electron microscope (SEM/EDX) and XRD. It was found that only a small amount of crystalline phase can be observed in the MBG, which indicates that the crystallization ability of the MBG was greatly suppressed. Results of this initial investigation indicate that chemical modification can effectively suppress the crystallization tendency of basalt glass and improve its thermal stability, which opens up an effective way for the industrial scale and stable production of basalt fiber.

Design, synthesis, and biological characterization of a new class of symmetrical polyamine-based small molecule CXCR4 antagonists.

CXCR4, a well-studied coreceptor of human immunodeficiency virus type 1 (HIV-1) entry, recognizes its cognate ligand SDF-1α (also named CXCL12) which plays many important roles, including regulating immune cells, controlling hematopoietic stem cells, and directing cancer cells migration. These pleiotropic roles make CXCR4 an attractive target to mitigate human disorders. Here a new class of symmetrical polyamines was designed and synthesized as potential small molecule CXCR4 antagonists. Among them, a representative compound 21 (namely HF50731) showed strong CXCR4 binding affinity (mean IC50 = 19.8 nM) in the CXCR4 competitive binding assay. Furthermore, compound 21 significantly inhibited SDF-1α-induced calcium mobilization and cell migration, and blocked HIV-1 infection via antagonizing CXCR4 coreceptor function. The structure-activity relationship analysis, site-directed mutagenesis, and molecular docking were conducted to further elucidate the binding mode of compound 21, suggesting that compound 21 could primarily occupy the minor subpocket of CXCR4 and partially bind in the major subpocket by interacting with residues W94, D97, D171, and E288. Our studies provide not only new insights for the fragment-based design of small molecule CXCR4 antagonists for clinical applications, but also a new and effective molecular probe for CXCR4-targeting biological studies.

Ni Hierarchical Structures Supported on Titania Nanowire Arrays as Efficient Nonenzymatic Glucose Sensor.

Developing new advanced nonenzymatic electrochemical nano-sensors for glucose detection has attracted intensive attraction. In this work, we designed a novel nanocomposite nonenzymatic glucose sensor by fabricating hierarchically nanostructured metal nickel on titania nanowire arrays, which was loaded on a transparent conductive substrate (i.e., fluorine-doped tin oxide, FTO) surface by mild hydrothermal method. Due to the large surface area of the hierarchically nanostructured Ni and fast electron transfer of the TiO2 nanowire arrays electrode, the nanocomposite electrode shows excellent electrochemical activity toward the oxidation of glucose. The electrode exhibits high sensitivity in detecting glucose concentration (1472 µA mM-1 cm-2) with a wide linear range from 2×10-4 M to 2×10-3 M, fast response time (within 5 s), and small detection limit (10 µM) (S/N = 3). The good analytical performance, low cost and simple preparation method make this novel electrode material promising for the development of effective glucose nonenzymatic glucose sensor.

Discovery of New Inhibitors of Hepatitis C Virus NS3/4A Protease and Its D168A Mutant.

Hepatitis C virus (HCV) is a human pathogen with high morbidity. The HCV NS3/4A protease is essential for viral replication and is one of the top three drug targets. Several drugs targeting the protease have been developed, but drug-resistant mutant strains emerged. Here, we screened a library and synthesized a novel class of small molecules based on a tryptophan derivative scaffold identified as HCV NS3/4A protease inhibitors that are active against both wild type and mutant form of the protease. Only the compounds with predicted binding poses not affected by the most frequent mutations in the active site were selected for experimental validation. The antiviral activities were evaluated by replicon and enzymatic assays. Twenty-two compounds were found to inhibit HCV with EC50 values ranging between 0.64 and 63 µM with compound 22 being the most active. In protease assays, 22 had a comparable inhibition profile for the common mutant HCV GT1b D168A and the wild-type enzyme. However, in the same assay, the potency of the approved drug, simeprevir, decreased 5.7-fold for the mutant enzyme relative to the wild type. The top three inhibitors were also tested against four human serine proteases and were shown to be specific to the viral protease. The fluorescence-based cell viability assay demonstrated a sufficient therapeutic range for the top three candidates.

Octadecyloxyethyl benzyl tenofovir: A novel tenofovir diester provides sustained intracellular levels of tenofovir diphosphate.

Pre-exposure prophylaxis (PrEP) with topically or systemically administered antiretroviral agents can prevent acquisition of human immunodeficiency virus type 1 (HIV-1) infection. However, in clinical trials using tenofovir-containing agents, HIV-1 acquisition is reduced but not eliminated. Incomplete adherence remains the major contributor to failure. Sustained release or long-acting antiretroviral agents may provide better HIV-1 protection by reducing the clinical impact of incomplete adherence. To reduce dosing frequency, we synthesized a novel tenofovir prodrug, octadecyloxyethyl benzyl tenofovir (ODE-Bn-TFV), that is designed to release TFV slowly in tissues, and showed potent anti-HIV activity in vitro (EC50 = 1.7 nM). In cells exposed to 14C labeled TFV, ODE-Bn-TFV or the quickly activated monoester ODE-TFV, rapid cellular uptake for both lipophilic analogs was noted, achieving 50-fold higher levels than unmodified TFV after 48 h. Following exposure to ODE-[8-14C]TFV, the intracellular diphosphate levels were approximately four-fold higher than with ODE-Bn-TFV. However, intracellular TFVpp drug levels fell rapidly yielding a half-life of about two days. TFVpp levels in ODE-Bn-TFV treated cells decreased much more slowly and reached half-maximal levels in about seven days. These results suggest early accumulation of ODE-Bn-TFV followed by sustained intracellular release following cleavage of the ester bonds linking the ODE and benzyl moieties to the active molecular precursor, thereby potentially allowing for less frequent administration than with more rapidly activated forms of tenofovir.

High affinity CXCR4 inhibitors generated by linking low affinity peptides.

G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model of GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and binding free energy calculation to determine how the peptide interacts with CXCR4 and inhibits its downstream signaling. These results demonstrate that this combinational approach is effective for generating nanomolar active inhibitors of CXCR4 and may be applicable to other GPCRs.