HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants

The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.     

Development and validation of a rapid ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of darunavir,ritonavir, and tenofovir in human plasma: Application to human pharmacokinetics

A sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of darunavir, ritonavir and tenofovir in human plasma. Sample preparation involved a simple liquid–liquid extraction using 200 μL of human plasma extracted with methyl tert‐butyl ether for three analytes and internal standard. The separation was accomplished on an Acquity UPLC BEH C₁₈ (50 mm x 2.1 mm, 1.7 μm) analytical column using gradient elution of acetonitrile/methanol (80:20, v/v) and 5.0 mM ammonium acetate containing 0.01% formic acid at a flow rate of 0.4 mL/min. The linearity of the method ranged between 20.0 and 12 000 ng/mL for darunavir, 2.0 and 2280 ng/mL for ritonavir, and 14.0 and 1600 ng/mL for tenofovir using 200 μL of plasma. The method was completely validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, stability, and dilution integrity. The extraction recoveries were consistent and ranged between 79.91 and 90.04% for all three analytes and internal standard. The method exhibited good intra‐day and inter‐day precision between 1.78 and 6.27%. Finally the method was successfully applied for human pharmacokinetic study in eight healthy male volunteers after the oral administration of 600 mg darunavir along with 100 mg ritonavir and 100 mg tenofovir as boosters.     

Estimation of effective Debye temperature from sound velocity of N-(p-n-alkoxybenzylidene)-p-n-alkylanilines, (nO.m) liquid crystalline compounds

The ratio of specific heats (γ), Poisson's ratio (σ), effective Debye temperature (θ_D) collision factor (S_m), etc., are estimated in different LC phases of N-(p-n-alkoxybenzylidene)-p-n-alkylanilines, (nO.m's) liquid crystalline compounds using the density and sound velocity data. The variation of these parameters in different mesomorphic phases with the chain length is studied. The effective Debye temperature (θ_D), the collision factor (S_m) exhibits a similar variation with the alkyl chain length. Further, the space filling factor (r_fm) estimated using Kittel's formula is constant for all the materials irrespective of the phase with a value of 0.416. The molecular radius shows an increment of 0.074, 0.072 and 0.068 ? per CH₂ group in isotropic, nematic and smectic-A phases respectively in 5O.m series. Further, the specific heat ratio (γ) value is found to be lower in isotropic phase than in liquid crystalline phases. The data is compared with the body of the data in the literature.     

Development and Validation of a New LC Method for Analysis of Brimonidine Tartrate and Related Compounds

A novel liquid chromatographic method for analysis three potential impurities in brimonidine tartrate drug substance has been developed and validated. Efficient chromatographic separation was achieved on a C₈ column (250 mm × 4.6 mm, 5-μm particles) with a simple mobile-phase gradient at a flow rate of 1.0 mL min^?1. Quantification was achieved by use of ultraviolet detection at 248 nm. Resolution between brimonidine tartrate and its three potential impurities was greater than 3.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for brimonidine and its three impurities. The method was capable of detecting all three impurities of brimonidine tartrate at levels below 0.07 μg in a test concentration of brimonidine tartrate of 1.0 mg mL^?1 and for an injection volume of 10 μL. A solution of brimonidine tartrate in acetonitrile–water 2:8 (v/v) was stable for 48 h. The drug was subjected to stress conditions as prescribed by the ICH. Degradation was found to occur slightly under oxidative stress conditions but the drug was stable to aqueous, acidic, and basic hydrolysis, and photolytic and thermal stress. The assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.     

Three-component, one-pot diastereoselective synthesis of 4-amidotetrahydropyrans via the Prins-Ritter reaction sequence

Three-component coupling of carbonyl compounds, homoallylic alcohols, and nitriles has been achieved using 20 mol % of phosphomolybdic acid (PMA) at ambient temperature via the Prins-Ritter sequence to furnish 4-amidotetrahydropyrans in high yields with all cis selectivity. Spirocyclic-4-amidotetrahydropyrans are obtained using cyclic ketones.     

Patterned silk films cast from ionic liquid solubilized fibroin as scaffolds for cell growth

Silk is an attractive biomaterial for use in tissue engineering applications because of its slow degradation, excellent mechanical properties, and biocompatibility. In this report, we demonstrate a simple method to cast patterned films directly from silk fibroin dissolved in an ionic liquid. The films cast from the silk ionic liquid solution were found to support normal cell proliferation and differentiation. The versatility of the silk ionic liquid solutions and the ability to process large amounts of silk into materials with controlled surface topography directly from the dissolved silk ionic liquid solution could enhance the desirability of biomaterials such as silk for a variety of applications.     

Paper like free-standing hybrid single-walled carbon nanotubes air electrodes for zinc–air batteries

Flexible electrode architectures based on non-functionalized (P2) and functionalized (P3) single-walled carbon nanotubes (SWNTs) were fabricated via a simple vacuum filtration process. A hybrid layer of various compositions of P2- and P3-SWNTs forms free-standing membranes (~80 μm in thickness), and their electrochemical performance was evaluated as an air electrode AEP_2/P3 in zinc–air batteries. Such bifunctionalized air electrodes showed uniform surface morphology with interconnected micron-sized porous structure with high porosity (~70%). The N₂ adsorption isotherms at 77 K are of type IV with BET-specific surface areas of AE_(60/40) and AE_(80/20) to be 130.54 and 158.76 m² g⁻¹, respectively, thus facilitates high active surface area for active oxygen reduction/evolution reactions. BJH pore size distribution of AE_(60/40) and AE_(80/20) shows maximum pores with diameter <15 nm. The zigzag interlaying of the SWNTs imparts mechanical stability and flexibility in zinc–air batteries. Zinc–air batteries with optimized compositions of P2- and P3-SWNTs in air electrode AE_(60/40) had ionic conductivity ~1 × 10⁻² S cm⁻¹ and delivered higher discharge capacity ~300 mAh g⁻¹ as compared to AE_(80/20) composition. The unique properties of AE_(P2/P3) studied in this work would enable flexible air electrode architectures in future metal–air batteries.